General Information

KP IX 1817

General Information

1. Capillary Electrophoresis ...................................................................................................... 1818

2. Determination of Bulk and Tapped Densities ..................................................................... 1824
3. Disinfection and Sterilization Methods ................................................................................ 1825
4. Guideline for Setting Dissolution Specification of Oral Dosage Forms ............................ 1827
5. Guideline to Pharmaceutical Quality Control Using Near Infrared (NIR)
Spectroscopy ........................................................................................................................... 1836
6. Guideline of Limits for Residual Solvents of Pharmaceuticals .......................................... 1850
7. Guideline of Validation of Analytical Procedures for Pharmaceuticals ........................... 1857
8. Isoelectric focusing ................................................................................................................. 1862
9. Particle Size Determination .................................................................................................. 1865
10. Powder Particle Density Determination .............................................................................. 1871
11. Preservatives-Effectiveness Tests ......................................................................................... 1872
12. Specific Surface Area Determination Method ..................................................................... 1874
13. Terminal Sterilization and Sterilization Indicators ............................................................ 1876
1818 General Information
1. Capillary Electrophoresis
Capillary electrophoresis is a physical method of
analysis based on the migration, inside a capillary, of
charged analytes dissolved in an electrolyte solution,
under the influence of a direct-current electric field.
The migration velocity of an analyte under an
electric field of intensity E, is determined by the elec-
trophoretic mobility of the analyte and the electro-
osmotic mobility of the buffer inside the capillary. The
electrophoretic mobility of a solute (µ ep) depends on
the characteristics of the solute (electric charge, molec-
ular size and shape) and those of the buffer in which
the migration takes place (type and ionic strength of
the electrolyte, pH, viscosity and additives). The elec-
trophoretic velocity (ep) of a solute, assuming a spher-
ical shape, is given by the following equation:
 q  V 
ep  ep  E    
 r   L 
 6
q: effective charge of the solute,
: viscosity of the electrolyte solution,
r: Stoke's radius of the solute,
V: applied voltage,
L: total length of the capillary.
When an electric field is applied through the ca-
pillary filled with buffer, a flow of solvent is generated
inside the capillary, called electro-osmotic flow. The
velocity of the electro-osmotic flow depends on the
electro-osmotic mobility (µ eo) which in turn depends
on the charge density on the capillary internal wall and
the buffer characteristics. The electro-osmotic velocity
(eo) is given by the following equation:
     V 
eo  eo  E      
   L
ε: dielectric constant of the buffer,
: zeta potential of the capillary surface .
The velocity of the solute () is given by:
  ep  eo
The electrophoretic mobility of the analyte and
the electro-osmotic mobility may act in the same direc-
tion or in opposite directions, depending on the charge
of the solute. In normal capillary electrophoresis, ani-
ons will migrate in the opposite direction to the electro-
osmotic flow and their velocities will be smaller than
the electro-osmotic velocity. Cations will migrate in
the same direction as the electro-osmotic flow and their
velocities will be greater than the electro-osmotic ve-
locity. Under conditions in which there is a fast electro-
osmotic velocity with respect to the electrophoretic
velocity of the solutes, both cations and anions can be
separated in the same run.
The time (t) taken by the solute to migrate the dis-
tance (l) from the injection end of the capillary to the
detection point (capillary effective length) is given by
the following expression:
l lL
t 
ep  eo ( ep  eo )  V
In general, uncoated fused-silica capillaries above
pH 3 have negative charge due to ionized silanol
groups in the inner wall. Consequently, the electro-
osmotic flow is from anode to cathode. The electro-
osmotic flow must remain constant from run to run if
good reproducibility is to be obtained in the migration
velocity of the solutes. For some applications, it may
be necessary to reduce or suppress the electro-osmotic
flow by modifying the inner wall of the capillary or by
changing the concentration, composition and/or pH of
the buffer solution. After the introduction of the sample
into the capillary, each analyte ion of the sample mi-
grates within the background electrolyte as an inde-
pendent zone, according to its electrophoretic mobility.
Zone dispersion, that is the spreading of each solute
band, results from different phenomena. Under ideal
conditions the sole contribution to the solute-zone
broadening is molecular diffusion of the solute along
the capillary (longitudinal diffusion). In this ideal case
the efficiency of the zone, expressed as the number of
theoretical plates (N), is given by the following equa-
tion:
( ep  eo )  V  l
N
2 D L
D: molecular diffusion coefficient of the solute in
the buffer.
In practice, other phenomena such as heat dissipa-
tion, sample adsorption onto the capillary wall, mis-
matched conductivity between sample and buffer,
length of the injection plug, detector cell size and un-
levelled buffer reservoirs can also significantly con-
tribute to band dispersion.
Separation between two bands (expressed as the
resolution, RS) can be obtained by modifying the elec-
trophoretic mobility of the analytes, the electro-
osmotic mobility induced in the capillary and by in-
creasing the efficiency for the band of each analyte,
according to the following equation:
N  ( epb  epa )
RS 
4  ( ep  eo )
µ epa and µ epb: electrophoretic mobilities of the two
analytes separated,

 ep : mean electrophoretic mobility of the two
analytes (ep  1  (epb  epa ))
2 used to increase the separation capacity of those sub-
Apparatus
An apparatus for capillary electrophoresis is com-
posed of :
(1) a high-voltage, controllable direct-current power
supply,
(2) two buffer reservoirs, held at the same level, con-
taining the prescribed anodic and cathodic solutions ,
(3) two electrode assemblies (the cathode and the an-
ode), immersed in the buffer reservoirs and connect-
ed to the power supply,
(4) a separation capillary (usually made of fused-silica)
which, when used with some specific types of detec-
tors, has an optical viewing window aligned with the
detector. The ends of the capillary are placed in the
buffer reservoirs. The capillary is filled with the so-
lution prescribed in the monograph,
(5) a suitable injection system,
(6) a detector able to monitor the amount of substances
of interest passing through a segment of the separa-
tion capillary at a given time. It is usually based on
absorption spectrophotometry (UV and visible) or
fluorometry, but conductimetric, amperometric or
mass spectrometric detection can be useful for spe-
cific applications. Indirect detection is an alternative
method used to detect non UV-absorbing and non-
fluorescent compounds,
(7) a thermostatic system able to maintain a constant
temperature inside the capillary is recommended to
obtain a good separation reproducibility,
(8) a recorder and a suitable integrator or a computer.
The definition of the injection process and its au-
tomation are critical for precise quantitative analysis.
Modes of injection include gravity, pressure or vacuum
injection and electrokinetic injection. The amount of
each sample component introduced electrokinetically
depends on its electrophoretic mobility, leading to pos-
sible discrimination using this injection mode.
Use the capillary, the buffer solutions, the precon-
ditioning method, the sample solution and the migra-
tion conditions prescribed in the monograph of the
considered substance. The employed electrolytic solu-
tion is filtered to remove particles and degassed to
avoid bubble formation that could interfere with the
detection system or interrupt the electrical contact in
the capillary during the separation run. A rigorous rins-
ing procedure should be developed for each analytical
method to achieve reproducible migration times of the
solutes.
1. Capillary Zone Electrophoresis
In capillary zone electrophoresis, analytes are
separated in a capillary containing only buffer without
any anti-convective medium. With this technique, sep-
aration takes place because the different components of
the sample migrate as discrete bands with different
velocities. The velocity of each band depends on the
KP X 1819
electrophoretic mobility of the solute and the electro-
osmotic flow in the capillary. Coated capillaries can be
stances adsorbing on fused-silica surfaces. Using this
mode of capillary electrophoresis, the analysis of both
small (Mr < 2000) and large molecules (2000 < Mr
<100000) can be accomplished. Due to the high effi-
ciency achieved in free solution capillary electrophore-
sis, separation of molecules having only minute differ-
ences in their charge to mass ratio can be effected. This
separation mode also allows the separation of chiral
compounds by addition of chiral selectors to the sepa-
ration buffer.
Optimization
Optimization of the separation is a complex pro-
cess where several separation parameters can play a
major role. The main factors to be considered in the
development of separations are instrumental and elec-
trolytic solution parameters.
Instrumental parameters
(1) Voltage A Joule heating plot is useful in optimiz-
ing the applied voltage and column temperature. Sepa-
ration time is inversely proportional to applied voltage.
However, an increase in the voltage used can cause
excessive heat production, giving rise to temperature
and, as a result thereof, viscosity gradients in the buffer
inside the capillary. This effect causes band broadening
and decreases resolution.
(2) Polarity Electrode polarity can be normal (anode
at the inlet and cathode at the outlet) and the electro-
osmotic flow will move toward the cathode . If the
electrode polarity is reversed, the electro-osmotic flow
is away from the outlet and only charged analytes with
electro-osmotic mobilities greater than the electro-
osmotic flow will pass to the outlet.
(3) Temperature The main effect of temperature is
observed on buffer viscosity and electrical conductivity,
and therefore on migration velocity. In some cases, an
increase in capillary temperature can cause a confor-
mational change in proteins, modifying their migration
time and the efficiency of the separation.
(4) Capillary The dimensions of the capillary (length
and internal diameter) contribute to analysis time, effi-
ciency of separations and load capacity. Increasing
both effective length and total length can decrease the
electric fields (working at constant voltage) which in-
creases migration time. For a given buffer and electric
field, heat dissipation, and hence sample band-
broadening, depend on the internal diameter of the ca-
pillary. The latter also affects the detection limit, de-
pending on the sample volume injected and the detec-
tion system employed. Since the adsorption of the
sample components on the capillary wall limits effi-
ciency, methods to avoid these interactions should be
considered in the development of a separation method.
In the specific case of proteins, several strategies have
been devised to avoid adsorption on the capillary wall.
Some of these strategies (use of extreme pH and ad-
1820 General Information
sorption of positively charged buffer additives) only
require modification of the buffer composition to pre-
vent protein adsorption. In other strategies, the internal
wall of the capillary is coated with a polymer, cova-
lently bonded to the silica, that prevents interaction
between the proteins and the negatively charged silica
surface. For this purpose, ready-to-use capillaries with
coatings consisting of neutral-hydrophilic, cationic and
anionic polymers are available.
Electrolytic solution parameters
(1) Buffer type and concentration Suitable buffers
for capillary electrophoresis have an appropriate buffer
capacity in the pH range of choice and low mobility to
minimize current generation. Matching buffer-ion mo-
bility to solute mobility, whenever possible, is im-
portant for minimizing band distortion. The type of
sample solvent used is also important to achieve on-
column sample focusing, which increases separation
efficiency and improves detection. An increase in buff-
er concentration (for a given pH) decreases electro-
osmotic flow and solute velocity.
(2) Buffer pH The pH of the buffer can affect sepa-
ration by modifying the charge of the analyte or addi-
tives, and by changing the electro-osmotic flow. In
protein and peptide separation, changing the pH of the
buffer from above to below the isoelectric point (pI)
changes the net charge of the solute from negative to
positive. An increase in the buffer pH generally in-
creases the electro-osmotic flow.
(3) Organic solvents Organic modifiers (methanol,
acetonitrile, etc.) may be added to the aqueous buffer
to increase the solubility of the solute or other additives
and/or to affect the degree of ionization of the sample
components. The addition of these organic modifiers to
the buffer generally causes a decrease in the electro-
osmotic flow.
(4) Additives for chiral separations For the separa-
tion of optical isomers, a chiral selector is added to the
separation buffer. The most commonly used chiral se-
lectors are cyclodextrins, but crown ethers, polysaccha-
rides and proteins may also be used. Since chiral
recognition is governed by the different interactions
between the chiral selector and each of the enantiomers,
the resolution achieved for the chiral compounds de-
pends largely on the type of chiral selector used. In this
regard, for the development of a given separation, it
may be useful to test cyclodextrins having a different
cavity size (-, -, or -cyclodextrin) or modified
cyclodextrins with neutral (methyl, ethyl, hydroxyalkyl,
etc.) or ionizable (aminomethyl, carboxymethyl,
sulfobutyl ether, etc.) groups. When using modified
cyclodextrins, batch-to-batch variations in the degree
of substitution of the cyclodextrins must be taken into
account since it will influence the selectivity. Other
factors controlling the resolution in chiral separations
are concentration of chiral selector, composition and
pH of the buffer and temperature. The use of organic
additives, such as methanol or urea can also modify the
resolution achieved.
2. Capillary Gel Electrophoresis
In capillary gel electrophoresis, separation takes
place inside a capillary filled with a gel that acts as a
molecular sieve. Molecules with similar charge to mass
ratios are separated according to molecular size since
smaller molecules move more freely through the net-
work of the gel and therefore migrate faster than larger
molecules. Different biological macromolecules (for
example, proteins and DNA fragments), which often
have similar charge to mass ratios, can thus be separat-
ed according to their molecular mass by capillary gel
electrophoresis.
Characteristics of Gels
Two types of gels are used in capillary electropho-
resis: permanently coated gels and dynamically coated
gels. Permanently coated gels, such as cross-linked
polyacrylamide, are prepared inside the capillary by
polymerization of the monomers. They are usually
bonded to the fused silica wall and cannot be removed
without destroying the capillary. If the gels are used for
protein analysis under reducing conditions, the separa-
tion buffer usually contains sodium dodecyl sulfate and
the samples are denatured by heating a mixture of so-
dium dodecyl sulfate and 2-mercaptoethanol or
dithiothreitol before injection. When non-reducing
conditions are used (for example, analysis of an intact
antibody), 2-mercaptoethanol and dithiothreitol are not
used. Separation in cross-linked gels can be optimized
by modifying the separation buffer (as indicated in the
capillary zone electrophoresis section) and controlling
the gel porosity during the gel preparation. For cross-
linked polyacrylamide gels, the porosity can be modi-
fied by changing the concentration of acrylamide
and/or the proportion of cross -linker. As a rule, a de-
crease in the porosity of the gel leads to a decrease in
the mobility of the solutes. Due to the rigidity of these
gels, only electrokinetic injection can be used.
Dynamically coated gels are hydrophilic polymers,
such as linear polyacrylamide, cellulose derivatives,
dextran, etc., which can be dissolved in aqueous sepa-
ration buffers giving rise to a separation medium that
also acts as a molecular sieve. These separation media
are easier to prepare than cross-linked polymers. They
can be prepared in a vial and filled by pressure in a
wall-coated capillary with no electroosmotic flow. Re-
placing the gel before every injection generally im-
proves the separation reproducibility. The porosity of
the gels can be increased by using polymers of higher
molecular mass (at a given polymer concentration) or
by decreasing the polymer concentration (for a given
polymer molecular mass). A reduction in the gel poros-
ity leads to a decrease in the mobility of the solute for
the same buffer. Since the dissolution of these poly-
mers in the buffer gives low viscosity solutions, both
hydrodynamic and electrokinetic injection techniques
can be used.
3. Capillary Isoelectric Focusing

In isoelectric focusing, the molecules migrate un-
der the influence of the electric field, so long as they
are charged, in a pH gradient generated by ampholytes
having pI values in a wide range (poly-
aminocarboxylic acids), dissolved in the separation
buffer.
The three basic steps of isoelectric focusing are
loading, focusing and mobilization.
(1) Loading step: Two methods may be employed:
(i) loading in one step: the sample is mixed with
ampholytes and introduced into the capillary either
by pressure or vacuum;
(ii) sequential loading: a leading buffer, then the
ampholytes, then the sample mixed with ampholytes,
again ampholytes alone and finally the terminating
buffer are introduced into the capillary. The volume
of the sample must be small enough not to modify
the pH gradient.
(2) Focusing step: When the voltage is applied,
ampholytes migrate toward the cathode or the anode,
according to their net charge, thus creating a pH gradi-
ent from anode (lower pH) to cathode (higher pH).
During this step the components to be separated mi-
grate until they reach a pH corresponding to their isoe-
lectric point (pI) and the current drops to very low val-
ues.
(3) Mobilization step: If mobilization is required for
detection, use one of the following methods. Three
methods are available:
(i) in the first method, mobilization is accomplished
during the focusing step under the effect of the elec-
tro-osmotic flow; the electro-osmotic flow must be
small enough to allow the focusing of the compo-
nents;
(ii) in the second method, mobilization is accom-
plished by applying positive pressure after the focus-
ing step;
(iii) in the third method, mobilization is achieved after
the focusing step by adding salts to the cathode res-
ervoir or the anode reservoir (depending on the di-
rection chosen for mobilization) in order to alter the
pH in the capillary when the voltage is applied. As
the pH is changed, the proteins and ampholytes are
mobilized in the direction of the reservoir which
contains the added salts and pass the detector. The
separation achieved, expressed as ΔpI, depends on
the pH gradient (dpH/dx), the number of ampholytes
having different pI values, the molecular diffusion
coefficient (D), the intensity of the electric field (E)
and the variation of the electrophoretic mobility of
the analyte with the pH (-dµ/dpH):
D  (dpH / dx)
pI  3 
E  (d / dpH )
Optimization
The main parameters to be considered in the de-
velopment of separations are:
KP X 1821
(1) Voltage Capillary isoelectric focusing utilizes
very high electric fields, 300 V/cm to 1000 V/cm in the
focusing step.
(2) Capillary The electro-osmotic flow must be re-
duced or suppressed depending on the mobilization
strategy (see above). Coated capillaries tend to reduce
the electro-osmotic flow.
(3) Solutions The anode buffer reservoir is filled
with a solution with a pH lower than the pI of the most
acidic ampholyte and the cathode reservoir is filled
with a solution with a pH higher than the pI of the most
basic ampholyte. Phosphoric acid for the anode and
sodium hydroxide for the cathode are frequently used.
Addition of a polymer, such as methylcellulose, in
the ampholyte solution tends to suppress convective
forces (if any) and electro-osmotic flow by increasing
the viscosity. Commercial ampholytes are available
covering many pH ranges and may be mixed if neces-
sary to obtain an expanded pH range. Broad pH ranges
are used to estimate the isoelectric point whereas nar-
rower ranges are employed to improve accuracy. Cali-
bration can be done by correlating migration time with
isoelectric point for a series of protein markers.
During the focusing step precipitation of proteins
at their isoelectric point can be prevented, if necessary,
using buffer additives such as glycerol, surfactants,
urea or zwitterionic buffers. However, depending on
the concentration, urea denatures proteins.
4. Micellar Electrokinetic Chromatography (MEKC)
In micellar electrokinetic chromatography, separa-
tion takes place in an electrolyte solution which con-
tains a surfactant at a concentration above the critical
micellar concentration (CMC). The solute molecules
are distributed between the aqueous buffer and the
pseudo-stationary phase composed of micelles, accord-
ing to the partition coefficient of the solute. The tech-
nique can therefore be considered as a hybrid of elec-
trophoresis and chromatography. MECK is a technique
that can be used for the separation of both neutral and
charged solutes, maintaining the efficiency, speed and
instrumental suitability of capillary electrophoresis.
One of the most widely used surfactants in MEKC is
the anionic surfactant sodium dodecyl sulfate, although
other surfactants, for example cationic surfactants such
as cetyltrimethylammonium salts, are also used.
The separation mechanism in MECK is as follows.
At neutral and alkaline pH, a strong electro-osmotic
flow is generated and moves the separation buffer ions
in the direction of the cathode. If sodium dodecyl sul-
fate is employed as the surfactant, the electrophoretic
migration of the anionic micelle is in the opposite di-
rection, towards the anode. As a result, the overall mi-
celle migration velocity is slowed down compared to
the bulk flow of the electrolytic solution.
In the case of neutral solutes, since the analyte can
partition between the micelle and the aqueous buffer,
and has no electrophoretic mobility, the analyte migra-
tion velocity will depend only on the partition coeffi-
cient between the micelle and the aqueous buffer. In
1822 General Information
the electropherogram, the peaks corresponding to each
uncharged solute are always between that of the
electroosmotic flow marker and that of the micelle (the
time elapsed between these two peaks is called the sep-
aration window). For electrically charged solutes, the
migration velocity depends on both the partition coef-
ficient of the solute between the micelle and the aque-
ous buffer, and on the electrophoretic mobility of the
solute in the absence of micelle.
Since the mechanism in MEKC of neutral or
weakly ionized solutes is essentially chromatographic,
migration of the solute and resolution can be rational-
ized in terms of the retention factor of the solute (k’),
also referred to as mass distribution ratio (Dm), which
is the ratio of the number of moles of solute in the mi-
celle to those in the mobile phase.
t R  t0 V length can decrease the electric fields (working at con-
k' K S
 tR  VM
t0  1  
 tmc 
tR: migration time of the solute ,
t0: analysis time of an unretained solute (determined
by injecting an electro-osmotic flow marker
which does not enter the micelle, for instance
methanol),
tmc: micelle migration time (measured by injecting a
micelle marker, such as Sudan III, which mi-
grates while continuously associated in the mi-
celle),
K: partition coefficient of the solute,
VS: volume of the micellar phase,
VM: volume of the mobile phase.
Likewise, the resolution between two closely migrating
solutes (RS) is given by the following equation:
t
1 ( 0 )
N   1 k 'b tmc
RS    
4  k 'b 1 1  ( t0 )  k '
a (3) Organic solvents To improve MEKC separation
tmc
N: number of theoretical plates for one of the so-
lutes,
: selectivity,
k’a, k’b: retention factors for both solutes, respec-
tively (k’b > k’a)
Similar, but not identical, equations give k’ and RS
values for electrically charged solutes.
Optimization
The main parameters to be considered in the de-
velopment of separations by MEKC are instrumental
and electrolytic solution parameters.
Instrumental parameters
(1) Voltage Separation time is inversely proportional
to applied voltage. However, an increase in voltage can
cause excessive heat production that gives rise to tem-
perature gradients and viscosity gradients of the buffer
in the cross-section of the capillary. This effect can be
significant with high conductivity buffers such as those
containing micelles. Poor heat dissipation causes band
broadening and decreases resolution.
(2) Temperature Variations in capillary temperature
affect the partition coefficient of the solute between the
buffer and the micelles, the critical micellar concentra-
tion and the viscosity of the buffer. These parameters
contribute to the migration time of the solutes. The use
of a good cooling system improves the reproducibility
of the migration time for the solutes.
(3) Capillary As in capillary zone electrophoresis,
the dimensions of the capillary (length and internal
diameter) contribute to analysis time and efficiency of
separations. Increasing both effective length and total
stant voltage), increase migration time and improve the
separation efficiency. The internal diameter controls
heat dissipation (for a given buffer and electric field)
and consequently the sample zone diffusion.
Electrolytic solution parameters
(1) Surfactant type and concentration The type of
surfactant, in the same way as the stationary phase in
chromatography, affects the resolution since it modi-
fies separation selectivity. Also, the log k’ of a neutral
compound increases linearly with the concentration of
surfactant in the mobile phase.
Since resolution in MEKC reaches a maximum when k’
approaches the value of t m t0 , modifying the concen-
tration of surfactant in the mobile phase changes the
resolution obtained.
(2) Buffer pH Although pH does not modify the
partition coefficient of non-ionized solutes, it can mod-
ify the electroosmotic flow in uncoated capillaries. A
decrease in the buffer pH decreases the electro-osmotic
flow and therefore increases the resolution of the neu-
tral solutes in MEKC, resulting in a longer analysis
time.
of hydrophobic compounds, organic modifiers, such as
methanol, propanol, acetonitrile, etc., can be added to
the electrolytic solution. The addition of these modifi-
ers usually decreases migration time and the selectivity
of the separation. Since the addition of organic modifi-
ers affects the critical micellar concentration, a given
surfactant concentration can be used only within a cer-
tain percentage of organic modifier before the
micellization is inhibited or adversely affected, result-
ing in the absence of micelles and, therefore, in the
absence of partition. The dissociation of micelles in the
presence of a high content of organic solvent does not
always mean that the separation will no longer be pos-
sible; in some cases the hydrophobic interaction be-
tween the ionic surfactant monomer and the neutral
solutes forms solvophilic complexes that can be sepa-
rated electrophoretically.

(4) Additives for chiral separations For the separa-
tion of enantiomers using MEKC, a chiral selector is
included in the micellar system, either covalently
bound to the surfactant or added to the micellar separa-
tion electrolyte. Micelles that have a moiety with chiral
discrimination properties include salts of N-
dodecanoyl-L -amino acids, bile salts, etc. Chiral reso-
lution can also be achieved using chiral discriminators,
such as cyclodextrins, added to the electrolytic solu-
tions which contain micellized achiral surfactants.
(5) Other additives Several strategies can be carried
out to modify selectivity, by adding chemicals to the
buffer. The addition of several types of cyclodextrins
to the buffer can also be used to reduce the interaction
of hydrophobic solutes with the micelle, thus increas-
ing the selectivity for this type of compound.
The addition of substances, capable of modifying
solute-micelle interactions by adsorption on the latter,
is used to improve the selectivity of the separations in
MEKC. These additives may be a second surfactant
(ionic or non-ionic) which gives rise to mixed micelles
or metallic cations which dissolve in the micelle and
form co-ordination complexes with the solutes.
Quantification
Peak areas must be divided by the corresponding
migration time to give the corrected area in order to:
(1) compensate for the shift in migration time from run
to run, thus reducing the variation of the response,
(2) compensate for the different responses of sample
constituents with different migration times.
Where an internal standard is used, verify that no
peak of the substance to be examined is masked by that
of the internal standard.
Calculations
From the values obtained, calculate the content of
the component or components being examined. When
prescribed, the percentage content of one or more
components of the sample to be examined is calculated
by determining the corrected area(s) of the peak(s) as a
percentage of the total of the corrected areas of all
peaks, excluding those due to solvents or any added
reagents. The use of an automatic integration system
(integrator or data acquisition and processing system)
is recommended.
System Suitability
In order to check the behavior of the capillary
electrophoresis system, system suitability parameters
are used. The choice of these parameters depends on
the mode of capillary electrophoresis used. They are:
retention factor (k’, only for micellar electrokinetic
chromatography), apparent number of theoretical plates
(N), symmetry factor (AS) and resolution (RS). In previ-
ous sections, the theoretical expressions for N and RS
have been described, but more practical equations that
allow these parameters to be calculated from the
electropherograms are given below.
KP X 1823
Apparent Number of Theoretical Plates
2
t 
N  5.54   R 
 wh 
tR: migration time or distance along the baseline
from the point of injection to the perpendicular
dropped from the maximum of the peak corre-
sponding to the component,
wh: width of the peak at half -height.
Resolution
The resolution (RS) between peaks of similar
height of two components may be calculated using the
following expression:
 1.18  (tR 2  t R1 ) 
RS    t R 2  t R1

 wh1  wh 2 
tR1, tR2: migration times or distances along the base-
line from the point of injection to the perpendicu-
lars dropped from the maxima of two adjacent
peaks,
wh1, wh2: peak widths at half -height.
When appropriate, the resolution may be calculat-
ed by measuring the height of the valley (Hv) between
two partly resolved peaks in a standard preparation and
the height of the smaller peak (Hp) and calculating the
peak-to-valley ratio:
Hp
p/v 
Hv
Symmetry Factor
The symmetry factor (AS) of a peak may be calcu-
lated using the following expression:
w0.05
AS 
2d
w0.05: width of the peak at 1/20 of the peak height,
d: distance between the perpendicular dropped from
the peak maximum and the leading edge of the
peak at 1/20 of the peak height .
Tests for area repeatability (standard deviation of
areas or of the area/migration-time ratio) and for mi-
gration time repeatability (standard deviation of migra-
tion time) are introduced as suitability parameters. Mi-
gration time repeatability provides a test for the suita-
bility of the capillary washing procedures. An alterna-
tive practice to avoid the lack of repeatability of the
migration time is to use migration time relative to an
internal standard.
A test for the verification of the signal-to-noise ra-
tio for a standard preparation (or the determination of
the limit of quantification) may also be useful for the
determination of related substances.
Signal-to-noise Ratio
1824 General Information

The detection limit and quantification limit corre-
spond to signal-to-noise ratios of 3 and 10 respectively.
The signal-to-noise ratio (S/N) is calculated using the
following expression:
2 H
S/N 
h
H: height of the peak corresponding to the compo-
nent concerned, in the electropherogram obtained
with the prescribed reference solution, measured
from the maximum of the peak to the extrapolat-
ed baseline of the signal observed over a distance
equal to twenty times the width at half height,
h: range of the background in an electropherogram
obtained after injection of a blank, observed over
a distance equal to twenty times the width at the
half height of the peak in the electropherogram
obtained with the prescribed reference solution
and, if possible, situated equally around the place
where this peak would be found .
2. Determination of Bulk and
Tapped Densities
Determination of Bulk and Tapped Densities is a
method to determine the bulk densities of powdered
drugs under loose and tapped packing conditions, re-
spectively. Loose packing is defined as the state ob-
tained by pouring a powder sample into a vessel with-
out any consolidation, and tapped packing is defined as
the state obtained when the vessel containing the pow-
der sample is to be repeatedly dropped a specified dis-
tance at a constant drop rate until the apparent volume
of sample in the vessel becomes almost constant. The
bulk density is expressed in mass per unit apparent
volume of powder (g/mL). Because the bulk density is
one of the measures of packing properties, compressi-
bility and flow properties, and is dependent on the ‘his-
tory’ of the powder, it is essential to document the bulk
density to specify how the determination was made.
Bulk density
The bulk density is an apparent density obtained
by pouring a powder sample into a vessel without any
consolidation. The determination of bulk density is
achieved by measuring the apparent volume of a pow-
der sample having a known mass in a graduated cylin-
der (Method 1) or by measuring the mass of powder in
a vessel having a known volume (Method 2).
read the unsettled apparent volume to the nearest grad-
uated unit and calculate the bulk density ρB by the fol-
lowing formula:
M
B 
V0
 B : Bulk density by constant mass method
(g/mL)
M : Mass of powder sample (g)
V0 : Apparent volume of powder sample (mL)
Record the average of 3 determinations and regard
the average value as the bulk density according to the
constant mass method. If a 30 g sample is too large to
determine, adjust the mass of sample so as to provide
an apparent volume of 60-100 mL.
Method 2 Constant volume method
Unless otherwise specified, pass a quantity of
sample sufficient to complete the test through a sieve
No. 16 (1000 µm) to break up agglomerates that may
have formed during storage. Allow an excess of sample
powder to pour into the measuring vessel having the
volume of V and mass of M0. Then, carefully scrape
excess powder from the top of the vessel using the
edge of a slide glass or other tool by smoothly moving
across it. Remove any material from the sides of the
vessel, and determine the total mass Mt and calculate
the bulk density ρB by the following formula:
(M t  M 0 )
B 
V
ρB: Bulk density by constant volume method (g/mL)
MT: Total mass of powder and measuring vessel (g)
M0: Mass of measuring vessel (g)
V: Volume of measuring vessel (mL)
Record the average of 3 determinations and regard
the average value as the bulk density according to the
constant volume method.

Method 1 Constant mass method

Unless otherwise specified, pass a quantity of
sample sufficient to complete the test through a sieve
No. 16 (1000 m) to break up agglomerates that may
have formed during storage. Weigh accurately about 30
g of test sample, and pour it into a dry 100 mL gradu- Measuring vessel
ated glass cylinder (readable to 1 mL). Carefully level
the powder without consolidation, if necessary, and

Supplementary cylinder
The figures are in mm.
This figure is one of the representations of 100 mL
vessel and supplementary cylinder.
Figure 1. Vessels for the determination of bulk density
by constant volume method and for the determination
tapped density
Tapped density
Tapped density is an apparent density obtained by
mechanically tapping a measuring vessel containing a
powder sample. The determination of tapped density is
achieved by measuring the apparent volume of a pow-
der sample having a known mass in a vessel after tap-
ping (Method 1) or by measuring the mass of powder
in a vessel having a known volume after tapping
(Method 2).
Method 1 Constant mass method
Unless otherwise specified, pass a quantity of
sample sufficient to complete the test through a sieve
No. 16 (1000 µm) or a sieve No. 22 (710 µm) to break
up agglomerates that may have formed during storage.
Weigh accurately about 100 g of test sample, and pour
it into a 250 mL graduated glass cylinder (readable to 2
mL) without consolidation. If the sample is not suffi-
cient, proceed according to the same procedure as that
described above by using a 100 mL graduated glass
cylinder (readable to 1 mL). It is essential to select
appropriate masses of the cylinder support, holder and
cylinder so as to ensure the dynamic stability of the
apparatus during tapping.
After attaching the glass cylinder containing the
powder sample to the tapping apparatus, carry out tap-
ping under the measuring conditions (tapping rate and
drop height) specified for each apparatus.
Unless otherwise specified, repeat increments of
50 taps or 1 minute until the difference between suc-
ceeding measurements is less than 2%, and determine
the final apparent volume, Vf and calculate the tapped
density ρT by the following formula:
M zation” and “Filtration Method” described in “Terminal
T 
Vf
KP X 1825
ρT: Tapped density by constant mass method (g/mL)
M: Mass of powder sample (g)
Vf: Final apparent volume of sample after tapping
(mL)
Record the average of 3 determinations and regard the
average value as the tap density according to the con-
stant mass method.
Method 2 Constant volume method
Unless otherwise specified, pass a quantity of
sample sufficient to complete the test through a sieve
No. 16 (1000 µm) to break up agglomerates that may
have formed during storage. Attach a supplementary
cylinder to the stainless steel measuring vessel having a
known mass of M0 and a volume of V (Fig. 1), and then
pour an excess of the sample into the vessel. After set-
ting up the vessel in an adequate tapping apparatus
with a fixed drop height, carry out tapping at the rate
and cumulative tap number specified for each appa-
ratus. Then remove the supplementary cylinder from
the vessel and carefully scrape excess powder from the
top of the vessel by smoothly moving across it the edge
of a slide glass or other tool. Remove any material
from the sides of the vessel, and determine the total
mass Mt. Calculate the tapped density ρT by the formu-
la:
(M t  M 0 )
T 
V
ρT: Tapped density by constant volume method
(g/mL)
Mt: Total mass of powder and measuring vessel (g)
M0: Mass of measuring vessel (g)
V: Volume of measuring vessel (mL)
Determine 3 measurements and calculate the av-
erage and the relative standard deviation using 3 differ-
ent powder samples. Regard the average value as the
tap density according to the constant volume method.
If the relative standard deviation is not less than 2%,
repeat the test with further tapping.
Note: Use balances readable to the nearest 0.1 g.
3. Disinfection and Sterilization
Methods
Disinfection and Sterilization Methods are applied
to kill microorganisms in processing equip-
ment/utensils and areas used for drug manufacturing,
as well as to perform microbiological tests specified in
the monographs, and so differ from “Terminal Sterili-
Sterilization and Sterilization Indicators”. The killing
effect on microorganisms or the estimated level of ste-
rility assurance is greatly variable, so the conditions for
1826 General Information
disinfection and sterilization treatment must be chosen
appropriately for each application. Generally, the fol-
lowing methods are to be used singly or in combination
after appropriate optimization of operation procedures
and conditions, in accordance with the kind and the
degree of the contaminating microorganisms and the
nature of the items to which the methods are applied.
The validation of sterilization in accordance with
Terminal Sterilization and Sterilization Indicators is
required when the methods are applied to the manufac-
turing process of drug products.
1. Disinfection methods
These methods are used to reduce the number of living
microorganisms, but do not always remove or kill all
microorganisms present. Generally, disinfection is
classified into chemical disinfection with the use of
chemical drugs (disinfectants) and physical disinfec-
tion with the use of moist heat, ultraviolet light, and
other agents.
1.1. Chemical disinfection
Microorganisms are killed with chemical drugs. The
killing effect and mechanisms of a chemical drug differ
depending on the type, applied concentration, action
temperature, and action time of the chemical drug used,
the degree of contamination on the object to be disin-
fected, and the series and state (e.g., vegetative bacteria
or spore bacteria) of microorganisms.
Therefore, in applying the method, full consideration is
required of the sterility and permissible storage period
of the prepared chemical drug, the possibility of re-
sistance of microorganisms at the site of application,
and the effect of residual chemical drug on the product.
In selecting a suitable chemical drug, the following
items should be considered in relation to the intended
use.
1) The antimicrobial spectrum
2) Action time for killing microorganisms
3) Action durability
4) Effect of the presence of proteins
5) Influence on the human body
6) Solubility in water
7) Influence on the object to be disinfected
8) Odor
9) Convenience of use
10) Easy disposability
11) Influence on the environment at disposal
12) Frequency of occurrence of resistance
1.2. Physical disinfection
Microorganisms are killed without a chemical drug.
(i) Steam flow method
Microorganisms are killed by direct application of
steam. This method is used for a product which may be
denatured by the moist heat method. As a rule, the
product is kept in flowing steam at 100 C for 30 to
60 minutes.
(ii) Boiling method
Microorganisms are killed by putting the object in boil-
ing water. This method is used for a product which
may be denatured by the moist heat method. As a rule,
the product is put in boiling water for 15 minutes or
more.
(iii) Intermittent method
Microorganisms are killed by heating for 30 to 60
minutes repeatedly, three to five times, once a day in
water at 80 to 100 C or in steam. This method is used
for a product which may be denatured by the moist
heat method. There is another method called the low
temperature intermittent method with repeated heating
at 60 to 80 C. During the intermission periods be-
tween heating or warming, a suitable temperature for
the growth of microorganisms of 20 C or higher, must
be maintained.
(iv) Ultraviolet method
As a rule, microorganisms are killed by irradiation with
ultraviolet rays as a wavelength of around 254 nm.
This method is used for products which are resistant to
ultraviolet rays, such as smooth-surfaced articles, facil-
ities, and equipment, or water and air. This method
does not suffer from the occurrence of resistance,
which is observed in chemical disinfection, and shows
a killing effect on bacteria, fungi, and viruses. It must
be taken into consideration that direct ultraviolet irra-
diation of the human body can injure the eyes and skin.
2. Sterilization methods
2.1. Heating methods
In these methods, the heating time before the tempera-
ture or pressure reaches the prescribed value differs
according to the properties of the product, the size of
the container, and the conditions. The duration of heat-
ing in conducting these methods is counted from the
time when all the parts of the product have reached the
prescribed temperature.
(i) Moist heat method
Microorganisms are killed in saturated steam at a suit-
able temperature and pressure. This method is general-
ly used for heat-stable substances, such as glass, porce-
lain, metal, rubber, plastics, paper, and fiber, as well as
heat-stable liquids, such as water, culture media, rea-
gents, test solutions, liquid samples, etc. As a rule, one
of the following conditions is used.
115 ~ 118 C for 30 minutes
121 ~ 124 C for 15 minutes
126 ~ 129 C for 10 minutes
(ii) Dry-heat method
Microorganisms are killed in dry-heated air. This
method is generally used for heat-stable substances,
such as glass, porcelain, and metal, as well as heat-
stable products, such as mineral oils, fats and oils,
powder samples, etc. This method is generally con-
ducted in the way of direct heating by gas or electricity
or circulating heated air. As a rule, one of the following
conditions is used.

160 ~ 170 C for 120 minutes
170 ~ 180 C for 60 minutes
180 ~ 190 C for 30 minutes
2.2. Irradiation methods
(i) Radiation method
Microorganisms are killed by gamma-rays emitted
from a radioisotope or electron beam and bremsstrah-
lung (X-ray) generated from an electron accelerator.
This method is generally used for radiation-resistant
substances such as glass, porcelain, metal, rubber, plas-
tics, fiber, etc. The dose is decided according to the
material properties, and the degree of contamination of
the product to be sterilized. Special consideration is
necessary of the possibility of qualitative change of the
product after the application of the method.
(ii) Microwave method
Microorganisms are killed by the heat generated by
direct microwave irradiation. This method is generally
used for microwave-resistant products such as water,
culture media, test solutions, etc. As a rule, microwave
radiation with a wavelength of around 2450±50 MHz is
used.
2.3. Gas methods
Microorganisms are killed by a sterilizing gas. Suitable
gases for killing microorganisms include ethylene ox-
ide gas, formaldehyde gas, hydrogen peroxide gas,
chlorine dioxide gas, etc. Temperature, humidity, the
concentration of gas, and the exposure time differ in
accordance with the species of gas used. As sterilizing
gases are generally toxic to humans, full consideration
is required of the environmental control for the use of
gases and the concentration of residual gas. In some of
the gas methods, it may be difficult to measure or esti-
mate quantitatively the killing of microorganisms.
2.4. Filtration method
Microorganisms are removed by filtration with a suita-
ble filtering device. This method is generally used for
gas, water, or culture media and test solutions contain-
ing a substance that is water-soluble and unstable to
heat. As a rule a filter having a pore size of 0.22 m or
smaller is used for the sterilization. However, in this
method, a filter with a pore size of 0.45 m of smaller
is permitted to be used.
4. Guideline for Setting
Dissolution Specification of Oral
Dosage Forms
1. Objective
This guideline is intended to provide specified ap-
proaches for setting dissolution specification of oral
solid dosage forms such as tablets and capsules. By
securing quality uniformity of drug product, this guide-
line contributes to production of qualified drug prod-
KP X 1827
ucts.
2. Significance of Dissolution Testing
Drug absorption from a solid dosage form, such as
the tablet and the capsule, after oral administration is
governed by a number of factors, including the release
properties of the drug substance from the drug product,
the dissolution or solubilization properties of the drug
under a given physiological condition and the permea-
bility across the intestinal epithelial barrier. A dissolu-
tion testing is a test that determines not only the release
of the drug substance but also the dissolution or
solubilization of the drug. Ultimately, a dissolution
testing can lead to the estimation of the performance of
the drug product in the biological system. Therefore,
when a dissolution testing is undertaken, it is advisable
to consider the solubility of the drug, the dissolution,
the intestinal permeability, the characteristics of the
dosage form, the pharmacokinetic properties as well as
physiological factors. In order to mimic physiological
conditions after the oral administration, the conditions
for dissolution testing vary, e.g., a various dissolution
media with varying pHs to represent the environments
in the gastrointestinal tract, a number of settings in the
rotational speed to simulate the peristaltic movement of
the gastrointestinal tract and addition of lipids, en-
zymes and/or surfactants to mimic the physiological
conditions in the tract. Sometimes the results from a
dissolution test may be used to predict the kinetics of
the absorption of the drug (viz, in vitro-in vivo correla-
tion). However, it is noted that the condition of dissolu-
tion testing does not always attempt to represent com-
pletely physiological conditions in the gastrointestinal
tract. That is, useful information on the quality control
and the quality assurance of a given drug product can
also be obtained from the dissolution test that does not
completely represent physiological conditions in hu-
man gastrointestinal tract.
A. Lot to lot uniformity in the quality
B. Development of composition of drug product and
of formulation
C. Understanding release mechanism of drug from
product
D. Determination of pharmaceutical equivalence dur-
ing storage
E. Determination of pharmaceutical equivalence of
post approval changes in drug substance, in com-
position, and in method, site and scale of manufac-
ture
3. Glossary
a. Conventional release dosage forms (also known as
immediate release dosage forms)
A conventional release dosage form or immediate
release dosage form is a dosage form in which the
dissolution profile is primarily governed by the
physicochemical property of the drug substance
and cannot be intentionally altered simply by
changing the amount of the drug substance in the
product or the method of manufacture.
1828 General Information

b. Delayed release dosage forms (also known as en-
teric coated dosage forms)
A delayed release dosage form or enteric coated
dosage form is a dosage form in which the dissolu-
tion of the drug is delayed for a certain period after
the administration so that the drug is not released
in the acidic condition of the stomach by means of
composing the product with specialized composi-
tions or specialized methods. Pharmacokinetic cri-
teria of the dosage form are such that the dissolu-
tion profile of the dosage form is comparable to its
conventional release dosage form counterpart ex-
cept that the release occurs after a certain time.
c. Prolonged release dosage forms (also known as
extended release dosage forms)
A prolonged release dosage form or extended re-
lease dosage form is a dosage form in which the
release of the drug substance is prolonged, com-
pared with that from the conventional release dos-
age form of the same route of administration, and
the frequency of administration is reduced com-
pared with that in the conventional forms. These
dosage forms are typically manufactured by means
of specialized compositions and/or specialized
methods.
rotary basket methods is recommended for cap-
sules, a dosage form that is not ease to sink, or
for slowly dissolving dosage forms. Although
dissolutions from tablets are typically tested
with the paddle method, the rotary basket meth-
od, rather than the paddle method, may be more
suitable in case where the disintegrated debris
are settled down at the bottom of the flask there-
by causing a delay in the dissolution. Also disso-
lution apparatuses for dissolution testing is rec-
ommended to accomplish the testing of Suitabil-
ity test for dissolution apparatus (see Appendix 1)
for an every certain period of time.
Apparatus 1 Apparatus 2
(basket) (paddle)

4. General consideration in setting the dissolution

specifications
a. Classification of dosage forms
In this guideline, dosage forms are classified into
conventional release dosage forms, delayed release
dosage forms and prolonged release dosage forms
as specified in section 3. Glossary. Apparatus 3 Apparatus 4
b. Apparatus for dissolution testing (flow-through cell) (reciprocating cylinder)
i. Apparatus
Currently, seven types of dissolution apparatuses
are specified in the official compendium and the
types are illustrated in Fig. 1. Depending on the
properties of the dosage form, a suitable appa-
ratus may be selected for the dissolution testing.
Two of the most frequently used methods are the
Method 1, the rotary basket method, and the
Method 2, the paddle method, both specified in
the Dissolution Test. The rotary basket method Apparatus 5 Apparatus 6
and the paddle method are both simple and ro- (paddle over disk) (cylinder)
bust testing procedure and are highly standard-
ized globally. Therefore, unless a special disso-
lution testing is required, the rotary basket
method and the paddle method are recommend-
ed. When a dissolution test is to be conducted by
the rotary basket method or the paddle method,
the apparatus should conform to the specifica-
tion of the Dissolution Test as defined in the
General Tests of the Korean Pharmacopoeia.
When a dissolution test is to be conducted for
the development of a new drug product, the se-
lection between the rotary basket method and
the paddle method depends on the consideration
of the in vitro dissolution pattern and in vivo
pharmacokinetic characteristics. In general, the

Acryl rod Angled disk
Teflon cylinder
Spring holder Reciprocating disk
Apparatus 7 (reciprocating holder)
Figure 1. Types of Dissolution Apparatuses
ii. Rotational speed
In general, the rotational speed is set at 50 rpm
for the paddle method and at 100 rpm for the ro-
tary basket method. A rotational speed faster
than 100 rpm is necessary for prolonged dosage
forms tested by the paddle method. However, it
is not always necessary to set the above men-
tioned speeds in a test. For example, in case
where 100% of the drug is dissolved within 10
minutes using the rotary basket method at the
rotational speed of 100 rpm for a dissolution test,
it is advisable to set a lower rotation speed
and/or to change the composition of the dissolu-
tion medium so that the dissolution pattern is
discernable. Therefore, the rotational speed of a
test should be set at a speed that is able to identi-
fy potential lack of pharmaceutical bioequiva-
lence of a drug product.
iii. Dissolution medium
1) General considerations
- Although the dissolution medium, having the
pH of 1.2, 4.0, 4.5, 6.8 or 7.4, is generally
used, the medium may be set at a different pH
and have a different composition, if necessary
because of the property of the dosage form
(e.g., the stability and/or the solubility of the
drug) or of a specific experimental purpose.
Under these circumstances, the properties, in-
cluding the stability and solubility, of the drug
substance must be considered.
- Select a dissolution medium that is able to
properly identify the difference in the dissolu-
tion rate between drug products or between
lots according to the factors that are composi-
KP X 1829
tion of drug product, manufacturing process,
equipment, drug substance, etc. In general, a
dissolution medium that results in a very rapid
dissolution is not suitable for the identification
and, thus, it is advisable to select a dissolution
medium that shows a relatively slower rate of
dissolution.
- In case where a given drug substance adsorbs
to the surface of dissolution apparatus, there-
by creating a problem in analyzing the results,
use a dissolution medium with a suitable
agent that prevents the adsorption.
- Suitable surfactants may be added to dissolu-
tion medium to enhance solubility of a very
poorly soluble drug. However, addition of or-
ganic solvent to medium is not recommended
for dissolution test.
- In case where the identical result cannot be
obtained for the test with the deaerated disso-
lution medium and for that with the non-
deaerated dissolution medium, then the
deaeration procedure is necessary (see Ap-
pendix II).
2) Characteristics of dissolution medium
a) Water
Since water does not have any buffer capac-
ity, the disadvantages of water as a dissolu-
tion medium include the potential changes
in the pH and in the surface tension of the
medium by the dissolution of drug and/or
the additives. Despite the disadvantages,
however, water is the most convenient me-
dium and ecologically-acceptable. There-
fore, water is the most widely used dissolu-
tion medium, in case where the dissolution
pattern from the formulation is not affected
by using water as the medium.
b) pH 1.2 Dissolution medium
Use the disintegration 1st fluid. This medi-
um is prepared using hydrochloric acid and
sodium chloride, and the concentration of
hydrochloric acid in the medium is set at
about 0.1 mol/L. In case where a particular
drug becomes unstable in the medium, it is
necessary that the testing condition is modi-
fied to maintain the stability. In fact, the
dissolutions from a number of formulations
are tested with the media having the con-
centration of hydrochloric acid ranging
from 0.001 mol/L to 0.1 mol/L.
c) pH 4.0/4.5 Dissolution medium
o pH 4.0 Dissolution medium: Use 0.05
mol/L sodium acetate buffer solution [a
mixture of 0.05 mol/L acetic acid and
0.05 mol/L sodium acetate (82:18)].
o pH 4.5 Dissolution medium: Use a buffer
solution prepared by dissolving 2.99 g of
sodium acetate trihydrate and 1.66 g of
glacial acetic acid in water to make 1000
mL.
1830 General Information
o In case where these buffer solutions in-
teract with a drug to create undesirable
problem, a buffer solution prepared from
citrate or phosphate may be alternatively
used.
d) pH 6.0 Dissolution medium
Use disodium phosphate and citric acid
buffer solution (pH 6.0) of the Korean
Pharmacopoeia.
e) pH 6.8 Dissolution medium
Use the disintegration 2nd fluid.
f) Simulated gastric fluid
Use simulated gastric fluid TS of USP.
g) Simulated intestinal fluid
Use simulated intestinal fluid TS of USP.
h) Neutral and basic dissolution medium
Although the dissolution medium, having
the pH of 1.2, 4.0, 4.5, 6.8 or 7.4, is gener-
ally used, the medium may be set at a dif-
ferent pH and have a different composition,
if necessary because of the property of the
dosage form (e.g., the stability and/or the
solubility of the drug) or of a specific exper-
imental purpose, and the dissolution is test-
ed. Examples of the special conditions in-
clude the cases when a particular drug is the
most stable in basic dissolution media,
when basic condition allows a discriminat-
ing analysis or when a given drug has a suf-
ficient solubility in basic media. When a
dissolution is tested for a product in basic
condition, coupled with the HPLC assay us-
ing C18 column, it may be necessary to neu-
tralize or sufficiently dilute the test solution
before the introduction to the column be-
cause the packing material tends to be un-
stable at high pHs.
3) Volume of dissolution medium
Although the volume of dissolution medium is
typically 900 mL in the test, it may be adjust-
ed to 500-1 L, depending on the testing condi-
tion. Dissolution test flask ranging from 150
mL to over 1 L in size is currently available.
General considerations for setting the volume
of dissolution medium are as follows;
a) Solubility of drug
Volume of dissolution medium is sufficient
enough to maintain a sink condition during
the testing. In general, a sink condition is a
condition where at least three times (gener-
ally 5-10 times) the volume for the com-
plete dissolution of a given amount of drug
is available. Therefore, under such condi-
tion, the concentration of the drug should
always be less than 30% of the intrinsic sol-
ubility when the drug product is completely
dissolved in the volume of medium used in
the test. In the dissolution test, it is neces-
sary to set the dissolution specification such
that the process of the dissolution of drug to
medium is not the rate limiting step in the
dissolution rate by maintaining a sink con-
dition in the test.
b) Use of surfactant
It is advisable to use a sufficient volume of
dissolution medium in the test when surfac-
tant is added to the medium to enhance the
solubility of a poorly water-soluble drug.
c) Ease of assay
For a dissolution test with a drug product
having a trace amount of drug, it is recom-
mended to use the possible minimum in
volume when the concentration of the dis-
solved drug may be lower than the limit of
quantification thereby creating an assay
problem.
d) Temperature of test medium
In general, maintain the temperature of the
medium at 37 ± 5 °C for the dissolution test
of oral solid dosage forms.
iv. Dissolution testing of drug product containing
poorly soluble drug
1) Determination of impact of medium pH on
dissolution rate
Conduct a series of dissolution tests with var-
ious media such as water, pH 1.2 dissolution
medium, pH 4.0/4.5 dissolution medium and
pH 6.8 dissolution medium. In these dissolu-
tion studies, use faster the rotational speed
(for example, the rotational speed of 120 rpm
for the rotary basket method and the rotational
speed of 75 rpm for the paddle method). Sam-
ple a portion of the medium at suitable times
after the initiation of the test, calculate the rate
of dissolution for the drug and graphically
present the results. Based on these results, the
dissolution medium is selected for subsequent
tests. In case where the dissolution rate of the
drug is below the range between 70 and 85%
of the drug, select a medium that shows the
most rapid dissolution rate and consider addi-
tion of surfactants to the medium.
2) Selection of suitable surfactant
Interaction between drug and surfactant is
governed by the physicochemical properties
of the drug and the surfactant. Therefore, for a
given drug, a various surfactants, e.g., non-
ionic, anionic and cationic surfactants, have to
be screened for the selection. In this case, the
concentration is recommended to set initially
at 2% and the suitability is tested for surfac-
tants such as;
o Anionic surfactant: Sodium lauryl
sulfate (SLS)
o Cationic surfactant: Cetyltrimethyl
ammonium bromide (CTAB)
o Non-ionic surfactant: Polysorbate 80,
40 and 20, lauryl dimethylamine N-
oxide (LDAO)
3) Determination of surfactant concentration

Use the lowest possible concentration of the
surfactant that shows a sufficient dissolution
rate within the specified time of the comple-
tion of the test (i.e., in 2 hours or 6 hours). To
accomplish this, study the dissolution with a
gradual decrease in the surfactant concentra-
tion such as from the initial 2%, then 1.5, 1.0,
0.75, 0.5 and finally to 0.25%.
v. Dissolution testing for delayed release dosage
forms
To confirm the function of enteric coating or the
diffusion control of the drug by coating, conduct
a series of dissolution tests first in an acidic
condition (i.e., acid stage) and then in buffer (i.e.,
buffer stage).
As an example for a dissolution test for a de-
layed release dosage form in the official com-
pendium, dissolution may be tested as directed
in the procedure for delayed release dosage
forms or first in 0.1 mol/L hydrochloric acid so-
lution, an acidic condition, and, then, in pH 6.8
phosphate buffer in the buffer condition. For the
dissolution testing via the two-stage approach,
there are next ways;
1) Dissolution is first tested for 2 hours in 750
mL of 0.1 mol/L hydrochloric acid solution at
37 ± 5 °C. After 2 hours, a portion of the dis-
solution medium is sampled for the quantifi-
cation of the drug in the medium. To the
above mentioned 0.1 mol/L hydrochloric acid
dissolution medium, add 250 mL of 0.2 mol/L
sodium phosphate solution, previously
warmed to 37 ± 5 °C. If necessary, the pH of
the medium may be adjusted with 2 mol/L hy-
drochloric acid or with 1 mol/L sodium hy-
droxide, and the dissolution further tested (the
adjustment of the pH should be carried out
within not more than 5 minutes of the addition
of 0.2 mol/L sodium phosphate solution).
2) Dissolution is first tested for 2 hours in 1 L
of 0.1 mol/L hydrochloric acid solution at 37
± 5 °C. After 2 hours, a portion of the dissolu-
tion medium is sampled and the remaining
medium is discarded. To the dissolution flask,
transfer 1 L of pH 6.8 phosphate buffer and
perform the Dissolution Test (prepared by
adding 750 mL of 0.1 mol/L hydrochloric acid
solution to 250 mL of 0.2 mol/L sodium phos-
phate solution, and, if necessary, adjusting the
pH to 6.8 by the addition of 2 mol/L hydro-
chloric acid or of 1 mol/L sodium hydroxide),
previously warmed to 37 ± 5 °C. Another way
is to transfer the paddle or the rotary basket,
containing the sample, from the apparatus of
the first dissolution study with 0.1 mol/L hy-
drochloric acid solution to a new dissolution
flask containing pH 6.8 phosphate buffer and
to continue to study the dissolution in the new
set up.
KP X 1831
vi. Two stage dissolution testing for gelatin cap-
sules
For the case of the dissolution test with hard- or
soft-gelatin capsules, a two-stage dissolution
testing is recommended. The first stage dissolu-
tion is tested using typical dissolution medium
as described in the previous section. When the
dissolution rate is too slow to be tested properly,
proceed to the second stage dissolution test with
an addition of enzyme to the test medium. Pep-
sin or pancreatin is typically used as the diges-
tive enzyme for the test, and the suitable enzyme
is selected depending on the pH of the medium.
When water or a dissolution medium having the
pH of less than 6.8 is used in the test, pepsin is
added with the activity of pepsin being not more
than 750000 units per 1 L of the dissolution me-
dium. In this case, the amount of pepsin addition
is not more than 3.2 g per 1 L of the medium.
For a dissolution medium having the pH not less
than 6.8, pancreatin is used as the enzyme with
the activity of pancreatin being not more than
1750 units per 1 L of the dissolution medium. In
this case, the amount of pancreatin addition is
not more than 50 mg per 1 L of the medium. In
case where water is used as the dissolution me-
dium at the first stage, 0.1 mol/L hydrochloric
acid solution containing pepsin or pH 6.8 phos-
phate buffer containing pancreatin may be used
as the second stage medium. However, if a high
concentration of surfactant, such as sodium
laury sulfate, is used in the first stage medium, a
care should be taken in the second stage test be-
cause the presence of surfactant may cause de-
naturation of the enzymes in the medium.
5. Dissolution Specification
a. Conventional release dosage forms
There are two approaches in setting the dissolution
specification for conventional release dosage
forms.
i. Singe-point specifications: This approach is typ-
ically applicable to drug products containing
highly soluble drug substances
(Biopharmaceutics Classification System(BCS)
Class I and III) in setting the dissolution specifi-
cation for the purpose of routine quality control.
(e.g., ‘Perform the test according to the next, the
dissolution rate of Acetaminophen in 30 minutes
should be not less than 80%.’) The single-point
specification may be set for the dissolution test
of a drug product in which not less than 80% of
the drug is dissolved within 20-30 minutes, even
in the presence of the lag time (typically found
in film coated formulations and capsules), of the
initiation of the test. It is not necessary to at-
tempt to construct in vitro-in vivo correlation for
such product in the development stage.
ii. Two-point specifications: This approach is typi-
cally applicable to drug products containing
1832 General Information
poorly soluble drug substances (BCS Class II
and IV) in setting the dissolution specification
for the purpose of routine quality control. In
general, the two-point specification is required
for drug products having the dissolution time is
not less than 45 minutes and is more useful than
the single-point specification.
b. Delayed release dosage forms
The dissolution specification is set at not more
than 10% for the acid stage and at the level speci-
fied in conventional release dosage forms for the
buffer stage.
c. Prolonged release dosage forms
Set the dissolution specification using at least three
time points. In this case, the sampling point should
be such that the dissolution from the samples rep-
resents the initial, middle and final stage of the
dissolution. The average rate of dissolution at the
final stage should reach as close as possible to
80%.
The initial stage time point (typically 1-2 hours of
the initiation of the test) is selected so that the
sample is able to identify the presence of initial
burst of dissolution from the drug product. In gen-
eral, 20-30% of the drug should be dissolved in the
initial stage. The middle point is selected to identi-
fy the dissolution of 50% of the drug and the final
point is selected to identify the dissolution of not
less than 80% of the drug thus confirming the dis-
solution of the majority of the drug by the final
sampling point. In case where the average rate of
dissolution does not reach 80% by the completion
of the test, however, the time of completion of the
test is set at a point when the rate of dissolution
does not change any more.
For conventional release dosage forms, delayed re-
lease dosage forms and prolonged release dosage
forms, the drug product should always conform to
the dissolution specification during the indicated
time of usage (i.e., within the expiration date).
6. Approaches in setting the dissolution specification
In general, the dissolution specification is set accord-
ing to the following approach. However, in case
where more appropriate method is available or other
suitable dissolution testing condition (e.g., the disso-
lution apparatus, the rotational speed and/or the dis-
solution medium etc.) that better represents the char-
acteristics of the dosage form, the other condition
may be used in the test.
a. Selection of test specimen
i. For a drug, select the lot(or equivalent) that has
been used for the clinical test or the bioavailabil-
ity test or the bioequivalence test.
ii. Select the lot that the difference between the
content of test specimen and its labeled amount
is not more than 5% and difference between
content of the test specimen and the content of
reference is not more than 5%.
b. The first sage (a preliminary test)
i. Number of test specimen
Proceed with 6 test specimens selected as di-
rected in the section 6-a and perform the disso-
lution test.
ii. Interval of sampling of dissolution solution
Sample the dissolution solution up to 2 hours of
the test at appropriate intervals (e.g., typically at
5, 10, 15, 30, 45, 60, 90 and 120 minutes) for
each dissolution medium. However, the test may
be terminated at a point when the final rate of
dissolution reaches not less than 85%.
iii. Conditions for dissolution testing
1) Dissolution apparatus
a) Use the paddle method in the Dissolution
Test of the Korean Pharmacopoeia as a pre-
ferred method.
b) Use other test method such as the rotary
basket method when, in the paddle method,
the disintegrated debris is settled down at
the bottom to form aggregates thereby inter-
fering the test.
c) A sinker may be used when the test speci-
men floats so that appropriately reproduci-
ble results cannot be obtained.
2) Dissolution medium
a) pH 1.2: Use the medium 1 of the dissolu-
tion test in the Korean Pharmacopoeia
b) pH 4.0/4.5: 0.05 mol/L sodium acetate
buffer solution*
0.05 mol/L sodium acetate buffer solution*:
a mixture of 0.05 mol/L acetic acid and 0.05
mol/L sodium acetate (82:28). But, the pH
and the composition of the test medium
may be modified depending on the pKa of a
given drug substance
c) pH 6.8: Use the medium 2 of the dissolu-
tion test in the Korean Pharmacopoeia
d) Water
e) Addition of surfactant to dissolution medi-
um: Use the medium in which the final rate
of dissolution is less than 85% in all medi-
um medium indicated in the section i)-iv) of
4-b.
3) Rotational speed
For the paddle method, initially use 50 rpm
and increase the speed to 75 rpm if the rate of
dissolution is slow. The rotational speed may
be occasionally set at 100 rpm, but the rota-
tional speed not less than 150 rpm is not used.
iv. Selection of dissolution test conditions
1) Conventional release dosage forms
Use the testing condition that results in 70-85%
of the rate of the dissolution. Since the testing
condition showing a rapid rate of dissolution
is not generally discriminating, select the dis-
solution medium and the rotational speed that
results in a relatively slow rate of dissolution
(not more than 1 hour).
2) Delayed release dosage forms
Proceed with the same testing condition in the

section 6-b-iv)-1) and consult to the condi-
tions in the section 5-b. In this case, use the
media of pH 1.2 and pH 6.8 as the test media.
3) Prolonged release dosage forms
Select at least three time points for sampling
such that a point of about 20-30% dissolution
of the drug is set as the initial point (1-2
hours), a point of about 50% dissolution of the
drug is set as the middle point and a point of
about 80% dissolution of the drug is set as the
final point.
c. The second stage (the dissolution test)
i. Proceed with the test conditions selected based
on the preliminary test and perform the test
ii. Number of test specimen
Proceed with the test specimens from three lots
having 12 test specimens per each lot and per-
form the test.
iii. Dissolution specification
1) Conventional release and delayed release
dosage forms
a) Time span of test: For conventional release
dosage forms, use not more than 1 hour as
the time span of the test. For delayed re-
lease dosage forms, use not more than 2
hours in the acid stage and not more than 1
hour in the buffer stage.
b) Setting specification of dissolution
Set the specification of the dissolution for
the test product at the value of about 10%
less than the average rate of dissolution at
the time point that reaches an almost plat-
eau in the graphical representation of the re-
sults for the dissolution test in the lot that
shows the medium rate of dissolution
among the three lots.
 Single-point specification
This specification is applicable to drug
product containing a highly soluble drug
substance (e.g., BCS Class I or III). Set the
specification as the lower boundary of the
dissolution rate at a time point that shows
the rate in the range of 70-85%.
 Two-point specification
This specification is applicable to drug
product containing gently dissoluble drug
substance (e.g., BCS Class II). 15 Minutes
is generally used as the first point and 30,
45 or 60 minutes as the second point for the
specification. For the drug product in which
a rapid dissolution may affect the efficacy
or the adverse reaction, or which has a nar-
row therapeutic index (see the Attachment 2
of the Minimum Requirements for Bioe-
quivalence Test), the specification is set us-
ing the two-point testing (if necessary, set
both upper and lower boundary values).
2) Prolonged release dosage forms
Select at least three time points for sampling
such that a point of about 20-30% dissolution
KP X 1833
of the drug is set as the initial point (1-2
hours), a point of about 50% dissolution of the
drug is set as the middle point, and a point of
about 80% dissolution of the drug or a point
when the rate of dissolution does not change
any more is set as a the final point. The rates
of dissolution for the initial point and the
middle point are set to ‘the average rate of
dissolution rate in ± 15%’ and the rate for the
final point being set to ‘the average rate of
dissolution in – 10%’.
3) Established test the dissolution according to
the preliminary specification using the refer-
ence drug and verify the validity of the speci-
fication, including the analytical procedure.
7. Preparation of validation data for dissolution test
a. Dissolution medium
Briefly summarize the reason for the selection of
the dissolution medium.
b. Dissolution test procedure
i. Describe the procedure for the dissolution test
and analysis according to the following items.
o Dissolution apparatus
o Preparation of the standard solution
o Preparation of the test solution: Sampling
time of the dissolution solution, dilution, fil-
tration (if there is a filtration procedure in-
volved, present the data for the recovery
evaluation)
o Analytical procedure for the quantification
in the dissolution solution (e.g., HPLC etc.)
o Formula for the calculation
o Dissolution specification
ii. Submit chromatograms or spectra of the blank
test solution, the standard solution and the test
solution.
c. Validation
Describe the validation data for the dissolution test
and the analytical procedure.
i. Data related to the stability of the test solution
during the analysis
ii. Analytical procedure
o Perform the test according to the ‘Guideline
of Validation of Analytical Procedures for
Pharmaceuticals’.
- Range
: Demonstrate linearity, accuracy and preci-
sion within the specification of the dissolution ±
20%.
(For example, for a delayed release dosage
form having the dissolution specification of
not more than 20% at 1 hour, not less than
90% at 9 hours, submit the data demonstrat-
ing the accuracy, precision and linearity in
the range of 0-110% of the labeled amount).
Appendix I
Suitability test for dissolution apparatus
1834 General Information
Suitability test has to be conducted to determine
whether a frequently used dissolution apparatus can be
properly used and the test is carried out by an adequate
test procedure. It is recommended that the suitability
test be conducted typically twice a year. In addition,
the test has to be performed when the dissolution appa-
ratus is moved or a significant change has been made
to the apparatus. Furthermore, the suitability test is
required when the test method is modified from the
rotary basket method to the paddle method and vice
versa. The suitability of a dissolution apparatus for the
rotary basket method or for the paddle method is tested
using two standard calibrators (USP dissolution cali-
brator: www.usp.org). The test is conducted using one
tablet of the non-dissolvable salicylic acid standard
tablet and one tablet of the dissolvable prednisone
standard tablet. For the case of the salicylic acid tablet,
50 mmol/L phosphate buffer, pH 7.4 is used as the dis-
solution medium and, for the case of the prednisone
tablet, water is used as the medium.
1. Suitability test for dissolution apparatus using the
300 mg salicylic acid standard tablet (lot number
O)
a. Use 900 mL of 50 mmol/L phosphate buffer
as the dissolution medium. In this case, the pH
of the buffer is adjusted to 7.4 ± 0.05 in the
room temperature.
b. To deaerate the dissolution medium, heat the
solution to 41 °C while stirring, and filter the
solution under vacuum using the filter paper
of the pore size of 0.45 µm. During the filtra-
tion procedure, continue stirring the filtrate.
After the filtration procedure, cap the flask
and continue the stirring for 5 additional
minutes under vacuum.
c. Transfer the dissolution medium to the flask
of the dissolution apparatus. During the trans-
fer, a special care should be taken not to trap
air bubbles in the medium. Allow the dissolu-
tion medium to reach an equilibrium tempera-
ture of 37 °C (it is not recommended that ro-
tate the paddle to facilitate the medium to
achieve the equilibrium).
d. When the temperature of the test medium
reaches at 37 °C, initiate the dissolution test
using the rotational speed of 100 rpm.
e. After 30 minutes of the initiation of the test,
sample a portion of the test medium, filter and
determine the absorbance at 296 nm and cal-
culate the rate of dissolution (%) of salicylic
acid. If analytically necessary, the filtrate may
be diluted with the dissolution medium.
f. If the rate of dissolution is within the range
specified in the table below, the dissolution
apparatus is regarded to be suitable.
Note)
Dissolve a portion of Salicylic acid RS in ethanol
to render the concentration of not more than 1%,
dilute the solution with the dissolution medium to
obtain a series of the standard solutions of known
concentration and use the standard solutions for
the analysis. In case where the test solution is fil-
tered, test for the potential adsorption of salicylic
acid to the filter medium.
Table 1. Dissolution specification of the salicylic acid
standard tablet (lot number O)
Type of dissolution ap- Rate of dissolution at 30
paratus minutes of the dissolu-
tion test (%)
Apparatus 1 (the rotary 23-29
basket method)
Apparatus 2 (the paddle 17-26
method)
2. Suitability test for dissolution apparatus using the
10 mg prednisone standard tablet (lot number N)
a. Use 500 mL of water as the dissolution medi-
um.
b. To deaerate the dissolution medium, heat the
solution to 41 °C while stirring, and filter the
solution under vacuum using the filter paper
of the pore size of 0.45 µm. During the filtra-
tion procedure, continue stirring the filtrate.
After the filtration procedure, cap the flask
and continue the stirring for 5 additional
minutes under vacuum.
c. Transfer the dissolution medium to the flask
of the dissolution apparatus. During the trans-
fer, a special care should be taken not to trap
air bubbles in the medium. Allow the dissolu-
tion medium to reach an equilibrium tempera-
ture of 37 °C (it is not recommended that ro-
tate the paddle to facilitate the medium to
achieve the equilibrium).
d. When the temperature of the test medium
reaches at 37 °C, initiate the dissolution test at
the rotational speed of 50 rpm.
e. After 30 minutes of the initiation of the test,
sample a portion of the test medium, filter and
determine the absorbance at 242 nm and cal-
culate the rate of dissolution (%) of salicylic
acid. If analytically necessary, the filtrate may
be diluted with the dissolution medium.
f. If dissolution rate(%) is within the next table,
it meets the requirements of the Suitability
test for dissolution apparatus.
Note)
Dissolve a portion of Prednisone RS in ethanol to
render the concentration of not more than 5%, di-
lute the solution with water to obtain a series of
the standard solutions of known concentration and
use the standard solutions for the analysis. In case
where the test solution is filtered, test for the po-
tential adsorption of prednisone to the filter medi-
um.
Table 2. Dissolution specification of the prednisone
standard tablet (lot number N)
Type of dissolution ap- Rate of dissolution at 30
 KP X 1835

paratus minutes of the dissolu- Tablet Clonazepam

tion test (%) Tablet Ergotamine tartrate
Apparatus 1 (the rotary 54-78 Capsule Etoposide
basket method) Capsule Isotretinoin
Apparatus 2 (the paddle 28-54 Tablet Meprobamate
method)
1. Deaeration method I
The values of rate of dissolution indicated tables 1 and - While stirring, heat the test medium to the
2 do not represent the average rate of dissolution but temperature of 41 °C and filter the medium
represents the rate of dissolution for individual 6 or 12 under a reduced pressure with the filter paper
dissolution testing apparatuses. In addition, the specific having the pore size of 0.45 µm. During the
value for the rate of dissolution changes with the lot filtration procedure, continue the stirring of
number of the standard tablets as indicated in table 3 the filtrate with a magnetic stirrer.
below. In addition, if the dissolution testing is solely - After the filtration procedure, continue the
conducted by, for example the paddle method, then the stirring for 5 additional minutes under vacuum
suitability test may be conducted for the paddle method (sonication may be used instead of the stir-
only. ring).
- Transfer the test medium to the dissolution
Table 3. Specification of suitability test for dissolution apparatus to render the temperature of 37 °C.
apparatus using the prednisone standard tablet (lot For the case of poorly soluble drugs, the test
number J) and the salicylic acid standard tablet (lot medium may contain surfactant such as
number K) polysorbate 80 or sodium lauryl sulfate. In
Calibrator Rate of dissolution at 30 minutes of the this case, foam may be formed during the
dissolution test (%) deaeration procedure, especially in the vacu-
Apparatus 1 (the Apparatus 2 (the um filtration step. Therefore, it is recom-
rotary basket paddle method) mended that the medium, in the absence of
method) surfactant, is first deaerated and, later, added
50 rpm 100 rpm 50 rpm 100 rpm with surfactant for the dissolution testing of
Predni- 6-23 43-63 46-59 58-69 poorly soluble drugs or sustained release dos-
sone age forms.
Salicylic 14-21 23-29 13-22 16-27
acid 2. Deaeration method II
The other typical method for the deaeration of the
Appendix II test medium is helium sparging. The main compo-
nent of air dissolved in the test medium is oxygen
Deaeration of test medium and nitrogen. Helium gas, which is more inert and
Deaeration of the test medium is not always necessary less water soluble than these gases, may be used to
for all drug products for the dissolution testing. In case purge oxygen and nitrogen from the test medium.
where the identical result is obtained for the test with In general, it is sufficient that 50 mL/min of the
the deaerated dissolution medium and for that with the purging rate is sufficient for the initial 10-30
non-deaerated dissolution medium, then the deaeration minutes and then, later, the rate may be reduced to
procedure is not necessary. However, under certain 5-10 mL/min. The deaeration by helium generally
cases, the air dissolved in the dissolution medium may takes 20-40 minutes for a completion. In fact,
participate in a chemical reaction with drug substance, when helium sparing deaeration is used, it is ex-
and air bubbles trapped in the medium may affect the pected that the relative standard deviation of the
dissolution of the drug. For example, for the case of rate of dissolution is lowered with the temperature
captoril, the air dissolved in the test medium may facil- equilibration being achieved sooner. Other
itate the oxidation of captopril thereby creating a stabil- deaeration method, involving a sonication of test
ity problem. In addition, a significant amount of air medium containing surfactant in a reduced pres-
bubbles trapped in the rotary basket generally decreas- sure, may also be used.
es significantly the rate of dissolution of drugs. There-
fore, in these cases, the air dissolved in the test medium Appendix III
has to be eliminated before the initiation of the test.
Table 4 lists typical drug products in which the Sinkers used in dissolution testing
deaeration is required for the dissolution test. When the dissolution is tested using the paddle method,
certain dosage forms, for example using capsules, do
Table 4. List of drug products in which deaeration is not settle down at the bottom of the flask but float to
required for dissolution testing the surface of the dissolution solution. In this case,
Dosage form Drug substance capsules may be placed in a sinker made of wire and
Tablet Captopril the dissolution is tested. In most cases, sinkers are
1836 General Information
made of wire helix and various products, but including
sinker and prolong sinker, may be used as specified in
the Dissolution Test.
Types of Specification
sinker
Basket type sinker (2.6 in radius ×
1.7 cm)
6 helix sinker (typically 2.5 × 1 cm)
4 helix sinker (typically 2.5 × 1 cm)
Sinker for controlled release formu-
lation (3.8 × 1 cm; the size of the
clamp of 5 mm × 7 mm)
3 extended type sinker from Vankel
Sinker specified in the Dissolution
Test (2.5-2.6 × 1.2 cm)
5. Guideline to Pharmaceutical
Quality Control Using Near
Infrared (NIR) Spectroscopy
I. Near Infrared Spectroscopy
Near infrared spectroscopy is a method very useful
method for identifying organic compounds by measur-
ing the transmittance or reflectance in a certain wave-
number or wavelength range, when the near infrared
light passes through the sample.
The range of near infrared is between 750 nm and
2500 nm. Even though the intensity of signal is weaker
than that in the mid-infrared region, the appearance of
overtones and combinations of the fundamental vibra-
tions of C-H, N-H, O-H, and S-H groups allows identi-
fication of materials, qualitative analysis and quantita-
tive analysis.
Spectrum is recorded as a graph with x-axis of
wavenumbers (or wavelength) and y-axis of transmit-
tance or absorbance, as is in mid-infrared region. When
a spectrum acquired with a sample, the spectrum can
be influenced by particle size, polymorphism, residual
solvents, or humidity and these influences can be re-
moved by applying a proper mathematical pretreatment.
1. Instrument
Usually, an instrument consists of a light source
compartment, a monochromator compartment, a detec-
tor compartment and a display unit.
- Light source compartment: a light source providing
a whole range or a part of the range of 750 nm to 2500
nm (13333 cm-1 to 4000 cm-1), or a laser providing
monochromic light is used.
- Monochromator compartment: a monochromator
such as diffraction grating, filter, or interference filter
is used.
- Detector compartment: it consists of a detector and
a signal processing system.
- Display unit: it consists of display, recorder, etc.
and mathematical pretreatment on a spectrum is possi-
ble.
2. Measurement
Proceed as one of the following methods:
1) Transmittance
Usually, this method is applied to liquids, diluted or
undiluted, and to solids in solution. The sample is
placed in an NIR transparent cell with a proper
pathlength (usually between 0.5 mm and 4 mm) or a
fiber optic probe is inserted into the sample. When
recording the spectrum of a liquid sample, any spectral
disturbances such as temperature dependent perturba-
tions should be taken into consideration, and in all cas-
es, compensation for background interferences should
be made. For example, a reference scan of air (for liq-
uids) or solvent (for solutions) may be subtracted from
the sample spectrum.
2) Diffused Reflectance
Usually, this method is applied to solids. Sample are
analyzed using a proper apparatus. When a fiberopric
probe is immersed into the sample to acquire spectrum,
a special care should be taken to get a reproducible
spectrum for each measurement. In all cases, compen-
sation for background interferences should be made.
For example, a reference scan of an internal or external
reflectance reference should be subtracted from the
sample spectrum. When the measurement is made, the
particle size, the state of hydration or of sovation
should also be taken into consideration.
3) Transflectance
Usually, this method is applied to liquids, diluted or
undiluted, to solids in solution or in suspension. The
measurement is made with the sample in a cell with a
proper diffuse reflector, made of either metal or of an
inert material (for example, titanium oxide), not exhib-
iting a spectrum in the near infrared range and intro-
duced behind the sample. The reflector should show
absorbance within the quantitative range.
II. Standards for Validation
This guideline is based on the contents of the ICH
general guideline to the analytical methods and is
adapted to allow the acquisition of comprehensive and
highly reliable information in applying analytical
methods using near infrared radiation, by examining
mainly specificity, linearity, range, accuracy and preci-
sion, which should be considered in using the near in-
frared spectroscopy.
1. Specificity

Specificity is an ability to analyze selectively the
analyte, despite of probable existence of interfering
substances. Generally, a tablet contains lots of sub-
stances in addition to the active ingredients. For exam-
ple, in the Ambroxol Tablet, the content of the princi-
pal ingredient, Ambroxol, is 12.5% as 30 mg out of
210 mg of total mass, and the remaining part, 87.5% of
the total mass may interfere with the analysis of
Ambroxol. These interfering substances are impurities,
degredation products, water, residual solvents, etc. and
a major part of exceipients. The influence of these sub-
stances can be reduced by applying chemometric ana-
lytical methods such as the principal component analy-
sis. The specificity can be proved by the following
methods.
First, acquire spectra of the active ingredients and all
of other related substances. If the characteristic wave-
lengths of the active ingredients do not overlap with
the wavelengths of peaks from other substances when
the original spectra without any pretreatment are exam-
ined, the method can be the easiest one to get the speci-
ficity in analyzing the active ingredients. But when this
kind of trend is not found with the original untreated
spectra, mathematical pretreatment methods or
chemometric resolution algorithms can be applied to
get the specificity by removing the interferences math-
ematically.
Second, by adding (fortifying the concentration)
probable interfering substances to a mixture before
tableting or powdered tablet, the interfering effect on
the analysis is identified. In other words, it should be
examined whether the wavelength showing variation
by the concentration of the additives overlaps with the
characteristic wavelengths of the active ingredients.
Third, in many cases, the contents of the principal
ingredient are in the range of not less than 95% and not
more than 105% of the labeled amounts. When the
linear relationship between the actual values and the
estimated values by near infrared spectroscopy of the
concentrations outside the allowed range of the labeled
amounts such as 90% and 110% can be obtained, this
can be the good method to confirm the specificity.
Fourth, the most reliable method approaching the
specificity is the identification and the qualification of
substances. The identification of substances is the
comparison of the spectra acquired with the test sam-
ples, with the spectra acquired with the (powdered)
library test samples having variations caused during the
production process. The qualification of substances is
the comparison of the mean spectrum of the test sam-
ples, with the library spectra. By these procedures, the
placebos can be identified.
The second derivative operation to spectral data has
several advantages for the analysis. The differentiation
between the peaks of the active ingredients and proba-
ble interfering substances can be achieved, and the
background perturbation can be removed. In addition
to this, peaks can be sharpened, and any variation
caused by the physical states of tablets can be removed,
resulting in the increased resolution. Usually, the sig-
KP X 1837
nal-to-noise ratio is lowered by 2 times by the first-
order derivative operation and by 4 times by the se-
cond-order derivative operation. Nonetheless, the sta-
bility of recent instruments is excellent and this effect
is not a big problem.
For the construction of calibration model, the use of
more than one wavelength for the analysis can remove
the effect caused by the substances other than the ac-
tive ingredients. An example of chemometric methods
is multiple linear regression, which uses more than one
wavelength. On the contrary, the methods which use
information of a whole range or a certain range of
wavelengths are principal component regression, par-
tial least squres regression, and so on.
The analysis of the principal components with con-
tents of less than 5% of the tablet is expected to be
problematic. Any method for increasing the specificity
by minimizing or removing the interference comprising
95% of the tablet may be studied further.
2. Linearity
The linearity of analytical process means the ability
to get the result directly propotional to the concentra-
tion (amount) of analyte in the sample. The linear re-
gression analysis is performed in the range of concen-
tration predictable by the calibration curve of near in-
frared spectroscopy. Usually, while the calibration
curve is obtained from the ratios of peak area in the
range of 0% to 150% in the methods of liquid chroma-
tography, the result of near infrared spectroscopy gives
the linear relationship between a set of reference values
in the narrower range of 90% to 110% found by the
conventional methods and a set of estimated values
found by near infrared spectroscopy. This linear rela-
tionship is evaluated by suitable statistical methods
such as calculating the regression line by the least
squares method. Data such as correlation coefficient of
the regression line, y-intercept, slope and sum of squres
of residuals are essential items of the documentation
for analyzing the regression data.
3. Range
Range is the interval between the maximum and the
minimum of concentrations used for measuring lineari-
ty, precision and accuracy during the analytical process.
The range of analysis is set to 2 times the allowed
range, for there are lots of difficulties in analyzing tab-
lets nondestructive and in collecting test samples for
calibration. For example, the allowed range of contents
of the principal ingredients is given to be 5%, consid-
ering the degradation of the principal ingredients dur-
ing the effective period of use and the error of analyti-
cal methods. In this case, the minimum range in ana-
lyzing the principal ingredients is 10% of the labeled
amount. The more accurate calibration be made if the
tablets are collected so that the range is widen to be
between 80% and 120% of the labeled amount. In re-
ality, the range suitable for the purpose of analysis is
preferred, considering the ranges of 80% to 120% for
the Assay of the drug substances or the preparations,
1838 General Information
70% to 130% for the Uniformity of Dosage Units and
20% of the range of specification for the Dissolution
Test.
4. Accuracy
The accuracy of analytical process is the measure
expressing the similarity between the values found by
the conventional analytical method (with proven accu-
racy) and the estimated values found by near infrared
spectroscopy. The accuracy should be proved in the
whole range specified by the analytical method. The
accuracy is a parameter evaluated only after establish-
ing precision, linearity and specificity.
The accuracy can be evaluated by at least 9 meas-
urements in the specified range. For example, the re-
sults measured by manipulating 3 levels of concentra-
tion and 3 repeatitions of each concentration level.
Three minimum levels of concentrations are selected as
the mean value and both of the maximum and the min-
imum. The accuracy is expressed as the recovery, when
the samples spiked with a known level of the analyte
are quantitated, or it is examined by comparison be-
tween the reference value and the mean value of meas-
urements in the confidence interval, when the compari-
son with the true value or the certified or agreed value
is made. In the quantitative analysis by near infrared
spectroscopy, the accuracy is expressed as the standard
error of prediction found with the validation set.
The calibration set should consist of the samples
with the concentrations of the active ingredients pro-
duced in the batch process and it should include the
whole range of the sample. The selected samples
should show the uniform distribution of probability,
not the normal distribution. The validation set should
consist of the samples with the same composition of
the active ingredients as in the calibration set.
The set of samples for the parallel test are selected
from the independent production batch and the analysis
is validated by the time. Therefore, the accuracy of the
analysis can be examined using the set completely in-
dependent from the calibration set and the validation
set of samples. This test is performed once a month
with independent set of samples after development of
the calibration model.
5. Precision
The precision of analytical process means the simi-
larity among the results obtained by a series of repeat-
ed analysis with the same sample under the specified
analytical conditions. The items of precision are re-
peatability, within-laboratory reproducibility and inter-
laboratory reproducibility. The precision is tested with
homogeneous and authentic samples. When the homo-
geneous and authentic samples are not easily available,
the test can be done with artificially synthesized sam-
ples or solutions. The precision is expressed as the rela-
tive standard deviation or the coefficient of variation
calculated from a series of measurements.
Repeatability is set to not more than 1.0% as the rel-
ative standard deviation (or CV) by measuring and
performing the whole procedure 6 times in a short pe-
riod time, as long as there is no thermal degradation of
the samples with concentration equivalent to 100% of
the tested concentration. The sample homogeneity and
the surface homogeneity should be taken into more
consideration especially when the contents of the prin-
cipal ingredients are low. In this case, the transmittance
is better than the reflectance, for the area of transmit-
tance is wider than that of reflectance. Meanwhile, the
transmittance may introduce more noise when the tab-
lets are thick and the light intensity reaching the detec-
tor is low.
The representative factors of variation which need
examination in within-laboratory reproducibility are
the date of experiment, the experimenter, and the in-
strument used and so on. The inter-laboratory repro-
ducibility is evaluated when the analytical method is
needed to be standardized for the collaborative experi-
ments between laboratories. If the inter-laboratory re-
producibility is proven, the within-laboratory reproduc-
ibility is not needed to be validated, but usually, the
within-laboratory reproducibility is mainly evaluated,
for the inter-laboratory reproducibility is hard to be
achieved.
III. Qualification of NIR Spectrophotometers
The purpose of qualification of NIR spectrophotom-
eters is to verify the suitability of the instrument for the
intended use by comparing with the specification of the
instrument. The qualification procedure includes De-
sign Qualification (DQ), Installation Qualification (IQ),
Operational Qualification (OQ) and Performance Qual-
ification (PQ).
1. Design Qualification (DQ)
The design qualification provides the evidence
whether the instrument is properly designed to be oper-
ational for the intended use. The various influencing
factors of the instrument are tested to verify whether
they correspond to the specifications of the instrument.
At least, it should be verified whether the factors corre-
spond the manufacturer’s specifications of the instru-
ment.
2. Installation Qualification (IQ)
The installation qualification verifies whether the in-
strument is installed according to the design and the
expressed specifications. Documentation of the serial
number of the hardware, the version of the software
and so on is included in the verification. And the envi-
ronment and the facilities of instrument installation are
are verified and the status of assembly, electric power
of the instrument and so on are also verified.
3. Operational Qualification (OQ)
The operational qualification verifies the instrument
once again by the methods used to select the spectro-
photometer, such as wavelength accuracy and precision,
linearity of the monochromator and noise.

4. Performance Qualification (PQ)
The performance qualification verifies whether the
instrument continues to be operational. Parts of the OQ
items are needed to be applied to verify the instrument
before the use or periodically (at least once every 6
months). The PQ should be performed when the
maintenance of the instrument is done or the lamp is
replaced.
IV. Quantitative Analysis
In the pharmaceutical industries, the quality of
pharmaceuticals are assessed by the repeatition of ex-
periments for several times with the drug substances
and the final pharmaceutical preparations. Nonetheless,
the conventional analytical methods for the quality
control cannot be applied to all of the produced phar-
maceuticals, for those methods are usually destructive.
Therefore, the weakness that only the representative
samples are managed can be a critical obstacle in the
matter of safety. Several principles of the analytical
process are needed for the optimal maintenance pro-
cess. The accuracy and the precision are necessary and
the sample preparation process is not required or is
minimal. Moreover, the simultaneous analysis of many
analytes should be possible and the rapid adjustment
during the production process should be possible. The
near infrared spectroscopy is one of the methods satis-
fying these principles. The near infrared spectroscopy
allows non-destructive, real-time total inspection of
tablets during the production process. The non-
destructive quantitative analysis of the tablets is to be
mentioned among the quantitative analyses of various
preparations and the general procedure is as follows:
• Feasibility test
• Reference data
• Selection of the samples
• Acquisition of sample spectra
• Construction of calibration model
• Validation of calibration model
• Validation of model performance
1. Feasibility test
The feasibility test is a procedure to check whether a
drug material or a pharmaceutical product can be ana-
lyzed quantitatively by NIR spectroscopy. The first
step is to observe the spectral response at the used level
of concentration of the analyte. After acquiring and
examining spectra of all the ingredients of a tablet, if
there are no significant differences among the ingredi-
ents, the second derivatives may be used to identify
characteristic peaks of each ingredient from the
derivatized spectra. In addition to this, when the spec-
tra are acquired at the different level of concentrations
of samples, any spectral changes caused by the
concentrationn are checked with the second derivatives
of those spectra.
KP X 1839
The feasibility test is also needed for the selection of
algorithm for the calibration. When the principal peaks
of the active ingredients do not overlap with those of
the excipients, the multiple linear regression can be
used. When there is a significant interference, the
pricipal component regression or the partial least
squares regression is more effective. Items that are
needed to be checked are whether to use transmittance
or reflectance, the use of fiber-optics, whether there are
reproducible results by repeated measurements of the
sample, whether there is robustness in the measurement
of the sample, and so on.
2. Reference data
The quantitative method by NIR spectroscopy is
based on the comparison of the reference data with
acquired spectral data. Therefore, if the reference data
are not correct, the estimated values by NIR spectros-
copy cannot be correct either. The reference data may
be obtained by the preparation of real reference stand-
ards or the application of reference analytical methods,
i.e. conventional analytical methods used for the evalu-
ation, such as gravimetry, chromatography, spectros-
copy, and so on, of which accuracy is proven. The lin-
earity, accuracy and precision of the reference analyti-
cal methods should be verified. The environmental
error can be minimized by concurrent use of the NIR
spectroscopy and the reference analytical method.
Three repeated measurements with the same sample by
the reference analytical method are made to get the
reference data
3. Selection of the samples
i) Calibration Test Set
The calibration test set is the standard for the quanti-
fication of the NIR spectral responses against the refer-
ence data. The sample should be selected carefully so
that the calibration test set includes the whole range of
possible concentrations of the analytes, and there is
robustness over the variation in the concentration of the
excipients, and the maximum variation of the analysis
is included. As other analytical method, interpolation
within the range of the calibration test set is possible
with the NIR spectroscopy, but extrapolation is not
possible.
ii) Validation Test Set
The construction of validation test set is the first step
of the validation. The validation test set is used to op-
timize the model and the concentration used should be
within the range of the calibration test set. If any varia-
tion outside the calibration test set is included, the cor-
rect validation values can be obtained.
Partitioning of the calibration test set and the valida-
tion test set is done as follows. With concurrent exper-
iments, the calibration test set and the validation test
set have the same instrumental error and the environ-
mental error.
1840 General Information
The test set is selected manually or by software
method so that the composition of the analytes is uni-
formly distributed.
The test set can be selected on the basis of the spec-
tral variation by software method.
Whichever method is used, the care should be taken
to have uniform distribution by the concentration range.
When the distribution is not uniform (for example,
concentrations at the center are used), there should be a
proper explanation.
The sample size measurable by NIR spectroscopy is
significantly smaller than those by the conventional
method. This is not because of the sample measure-
ment compartment but because of the sample area il-
luminated by the near infrared light. The infrared
measurement can detect the inhomogeneity even with
the mass of microgram level and a proper measurement
method is needed to accommodate this variation. This
property can be an advantage useful for testing the
Uniformity of Dosage Units, but in many applications,
averaging the signal from a larger area is needed.
Therefore, to get the averaged information, the sample
is moved transversely or rotated during the data acqui-
sition.
Among the various preparations, in cases of the tab-
lets, there are two cases: the case of using the products
as they are and the case of varying ratios of the active
ingredients to the excipients.
(1) The case of using the products as they are
First, this can be applied only when the transmit-
tance follows the Beer-Lambert’s law and it also ap-
plied to other tablets of the same composition with
different masses.
Second, this can be applied when the samples are
collected from the normal production and each tablet
has the concentration range needed for the usage.
(2) The case of varying ratios of the active ingredi-
ents to the excipients
First, the calibration test set is obtained by mixing
the products and the concentration expansion sam-
ples. Usually, the products lie between 95% and 105%
of the labeled amount, so to expand this range, tab-
lets with expanded concentration range are synthe-
sized in the laboratory. This concentration expansion
samples have different ratios of the active ingredi-
ents to the excipients and the ranges of between 90%
and 110% of the labeled amount or between 85%
and 115% of the labeled amount are used.
Second, the concentration is adjusted by adding the
active ingredients and the excipients to the powdered
products. If the active ingredients are low compared
to the total mass of the tablet, the better calibration
results can be acquired by adding the active ingredi-
ents to the powdered tablets.
Third, the concentration of the active ingredients is
varied using the sample synthesized in the laboratory.
This is similar to the use of the powdered tablet, but
the concentration range is wider (80% to 120%), for
the amount of the active ingredients can be varied
from the first stage of the sample preparation.
Fourth, the active ingredients and the excipients are
added to the powdered tablets and then the mixture
is tableted. But in this case, the total mass of the tab-
let is different from that of the products and it may
fail in terms of specificity.
Fifth, the model comprising all of the variation fac-
tor of the production process by using the global cal-
ibration set, which includes all of the existing varia-
tion factors to make the calibration models. This
method consists of 4 steps: laboratory composite
(prepared to have active ingredients with the concen-
tration level of 15% of the labeled amount), prepa-
ration of granules, tableting of core tablet, and coat-
ing the tablets.
4. Acquisition of sample spectra
The transmittance is applied to liquids, diluted or
undiluted and solids in the solution. The cells with
transmittance pathlength of 0.5 to 4 mm or dip-probes
are used and the compensation for background inter-
ferences should be made. On the contrary, the reflec-
tance and the diffused reflectance are applied mainly to
solids and the test is done to the sample placed in a
proper apparatus. When the fiber-optics are used, re-
producible spectra can be acquired by fixing the sam-
ple properly. As in the transmittance, compensation for
background interferences should be made.
In cases of solid particles, the physical differences
such as particle size, shape, compressibility, etc. can be
influential, so the particles are manipulated to be as
small and uniform in size as possible. Moreover, to
make the effect of the absorbed water and the residual
solvent constant, the samples are dried for a certain
length of time to have the same condition of the ab-
sorbed water and the residual solvent of the samples.
When the samples are analyzed by the NIR spectrosco-
py, the NIR spectra are influenced a lot by the ab-
sorbed water and the temperature, so it is desirable to
construct the environment of constant humidity and
constant temperature so that the experiment can be
done in the same environment. Moreover, the differ-
ence in the polymorphism of the sample or in the de-
gree of crystalization also can be influential to the
quantitative analysis, so the case should be taken.
After selecting a proper sample measurement meth-
od among various sample measurement apparatus by
considering the property, the shape, and the size of the
sample, the precision of the spectra acquired by repeat-
ed measurement is evaluated. When the measurement
is repeated 6 times, it meets the requirement if the
maximum value of the relative standard deviations of
whole range of wavelengths is not more than 1.0%.
5. Construction of calibration model
For the development of a calibration model, a proper
mathematical preptreatment is needed to be done, if
necessary. The pretreatment procedure is an important

step in chemometric analysis of the NIR data. The pre-
treatment is defined as a procedure in which the spec-
tral shape is enhanced by mathematical treatment of the
NIR data or unwanted variation is removed prior to the
development of a calibration model. A proper pre-
treatment method can be selected by testing the spec-
tral data prior to the data modeling. For example, vari-
ous methods of pretreatment are used in parallel and
evaluated and the best one is selected among them.
There are various methods of pretreatment. For ex-
ample, they are normalization, smoothing, baseline
correction, derivatives, mean centering, variance scal-
ing, autoscaling and so on.
After selecting a proper method of pretreatment, re-
gression analysis on the NIR data is done by applying
various quantitative algorithms. The calibration is a
procedure by which a mathematical model is con-
structed between the instrumental responses and the
properties of the sample (usually concentration). As
mentioned previously, the major algorithms for calibra-
tion are multiple linear regression (MLR), principal
component regression (PCR), partial least squares re-
gression (PLSR) and so on. The prediction is a proce-
dure of predicting the properties of the samples from
the instrumental signals using the developed model. In
a broad sense, there are two different approaches to
develop the calibration model and they are the
univariate analysis and the multivariate analysis. The
univariate analysis is the most commonly used method
in the conventional analysis, which relates the single
signal of the analytical instrument is related to the con-
centration of the single ingredient. This method is not
commonly used in the NIR spectroscopy. In the NIR
spectroscopy, many signals from the analytical instru-
ment are related to the various properties of the sam-
ples and the calibration model is constructed by the
multivariate analytical methods.
* For constructing a quantitative model, refer to the
Appendix I. The Appendix I explains mainly MLR,
PCR and PLSR, which are used generally in the NIR
spectroscopy.
6. Validation of calibration model
The quantitative model can be validated internally or
externally. Independent validation test set is used to get
the information on the predictability of the developed
model. The accuracy and the precision of the NIR
spectroscopy is compared with those of the reference
analytical method. Standard error of calibration (SEC)
and standard error of prediction (SEP) are used to
evaluate the quality of the quantitative model. A con-
ventional factor of model evaluation, the correlation
coefficient of regression is also used in the NIR spec-
troscopy, but it is not as important as in the conven-
tional univariate analysis.
When the content of a tablet is analyzed, the ratio (%)
of the amount of analyte to the mass of the tablet is
used as the value of concentration. When the calculated
SEP value derived as the relative error obtained by
KP X 1841
converting the amount (mg) of the analyte to 100 is not
exceeding the error range of the conventional method
of analysis, the use of the model is recommended.
7. Validation of model performance
Not just to evaluate simply the model, the perfor-
mance of the model is needed to be validated periodi-
cally to use that model continuously in the industrial
fields. The methods for validating model performance
can be classified into two methods, which are the
method of using the check samples and the method of
comparing with the reference analytical method. First,
to use the check samples, the check samples should be
stable over time and the short-term and long-term accu-
racy of the model should be evaluated with these sam-
ples. Second, to use the method of comparing with the
reference analytical method, the data by the NIR spec-
troscopy and the reference data are acquired periodical-
ly for n months (n=1,2,3, ) over n batches and a
paired t-test with the confidence level of 95% is per-
formed. When there is significant difference, the quan-
titative model should be reconstructed.
* Refer to the Appendix II for the validation of the
model.
* Refer to the Appendix III for the usual quantitative
analysis and the maintenance of the quantitative model.
V. Qualitative Analysis
The near infrared spectroscopy can be used for the
identification of substances and the qualitative analysis.
Identification : It is used when the chemical identifi-
cation of the substance is needed.
Qualitative analysis : After chemical identification
of the substance, the suitability of the sample to the
model of substances is measured. The model is devel-
oped from the samples representing various infor-
mation on the variation and it includes water, particle
size, solvent and other chemical and physical
informations.
Both of the identification and the qualitative analysis
enable the discrimination of substances in the library.
Typical qualitative application of the NIR spectroscopy
consists of the following procedures.
• Feasibility test
• Selection of the samples
• Acquisition of sample spectra
• Construction of library
• Validation of library
• Routine use
• Maintenance of library
1. Feasibility test
The feasibility test is performed as a first step, prior
to the development of model. For example, optimal
method of sample measurement, amount of the sample,
the minimum number of scans for effective analysis are
1842 General Information
examined. A prior knowledge on the organization of
samples in the library and the molecular structure is
useful in the analysis with the NIR spectroscopy. And
when the spectra of the representative samples are ac-
quired, it is checked whether the second derivative
spectra of the samples are different from each other.
2. Selection of the samples
The selection of the samples is an important proce-
dure for the successful qualitative analysis. The sample
set for developing the library and the independent set
for the purpose of validation are needed. All of the
samples used for building and validating the library are
needed to be authenticated. The level of authentication
of the sample is varied by the usage and the database
consists of the samples showing various source of vari-
ation.
The samples from the different batches are collected,
reflecting the changes in ingredients, suppliers, pro-
cesses, and storage conditions over a certain period of
time. If the chemical and physical stability is proven
over the storage period, the samples can be collected
regardless of the storage status.
The number of batches to be collected is dependent
on the complexity of analysis and the substances to be
analyzed should include the typical variation of the
system. The number of batches for the qualitative anal-
ysis is greater than that for the identification. There are
many kinds of the sample measurement apparatus, such
as cups, vials, fiber-optics and custom-made apparatus.
The selection of the apparatus is dependent on the us-
er’s needs and the validation of the apparatus is defined
in the stage of the design qualification (DQ). The sam-
ple measurement apparatus is the source of latent varia-
tions during the measurement. Therefore, it should be
validated continuously in terms of reproducibility, if
possible.
3. Acquisition of Sample Spectra
The transmittance is applied to liquids, diluted or
undiluted and solids in the solution. The cells with
transmittance pathlength of 0.5 to 4 mm or dip-probes
are used and the compensation for background inter-
ferences should be made. On the contrary, the reflec-
tance and the diffused reflectance are applied mainly to
solids and the test is done to the sample placed in a
proper apparatus. When the fiber-optics are used, re-
producible spectra can be acquired by fixing the sam-
ple properly. As in the transmittance, compensation for
background interferences should be made.
In cases of solid particles, the physical differences
such as particle size, shape, compressibility, etc. can be
influential, so the particles are manipulated to be as
small and uniform in size as possible. Moreover, to
make the effect of the absorbed water and the residual
solvent constant, the samples are dried for a certain
length of time to have the same condition of the ab-
sorbed water and the residual solvent of the samples.
When the samples are analyzed by the NIR spectrosco-
py, the NIR spectra are influenced a lot by the ab-
sorbed water and the temperature, so it is desirable to
construct the environment of constant humidity and
constant temperature so that the experiment can be
done in the same environment. Moreover, the differ-
ence in the polymorphism of the sample or in the de-
gree of crystalization also can be influential to the
quantitative analysis, so the case should be taken.
After selecting a proper sample measurement meth-
od among various sample measurement apparatus by
considering the property, the shape, and the size of the
sample, the precision of the spectra acquired by repeat-
ed measurement is evaluated. When the measurement
is repeated 6 times, it meets the requirement if the
maximum value of the relative standard deviations of
whole range of wavelengths is not more than 1.0%.
4. Construction of Library
The following procedures are used to develop the li-
brary.
• Definition of the purpose of developing the library
• Selection of the sample set for developing the li-
brary
• Data display
• Selection of the sample set for validating the library
• Data pretreatment/conversion
• Construction of the library
• Setting the threshold
1) Definition of the purpose of developing the library
Prior to the development of the library, it is im-
portant to set the effective range of the library accord-
ing to the intended use. This applies to the identifica-
tion and the qualitative analysis of the substances and it
includes checking the chemical similarity of the groups
to be analyzed and the number of groups.
2) Selection of the sample set for developing the li-
brary
For the development of the library, the variation
caused by the following factors should be considered.
The factors, especially important for the library for the
qualitative analysis are as follows:
• Moisture
• Particle size
• Residual solvents
• Degradation products
• Compositional change of formulated product
• Other chemical/physical properties
• Time
• Alternative sources of material
• Retained samples
• Temperature
• Operator
• Presentation, e.g. insertion of tube
• Between-instrument variation
• Others

The range of consideration over these factors is de-
pendent on the intended use and the needed ability of
classification.
3) Data display
The data display checks visually the sample showing
strange spectral features and identifies outliers. If pos-
sible, outliers should be identified and if there is rea-
sonable analytical explanation, they can be removed
and in that case, the clear reason should be documented.
4) Selection of the sample set for validating the li-
brary
There can be a situation, in which the representative
samples are needed to be selected from the large group.
In the simple situation, they can be selected manually,
but in more complex situation, they should be selected
after the determination of substance groups using a
proper selection tool (for example, principal compo-
nent analysis, cluster analysis, etc.).
The number of samples for each of the substance
groups is dependent on the qualitative algorithm and
the state of the system (how accurately the boundaries
of groups needed to be set).
5) Data pretreatment/conversion
Data are needed to be pretreated mathematically to
simplify the spectra more. For example, the algorithms
of derivatives and scatter correction can remove the
background variation caused by the physical difference.
The original untreated spectra are used when the in-
formation due to the physical status is important.
Mathematical transformation of spectra data can be
an artifact and can cause loss of important information,
so this should be taken into consideration. The algo-
rithms of data pretreatment and conversion should be
understood correctly and the theoretical basis always
should be provided whenever any conversion is per-
formed.
6) Construction of the library
The structure of the library is dependent on the limi-
tation of the software and the user’s needs. In the sim-
plest case, all of the substances are combined into one
library. On the contrary, it can be divided into sub-
libraries to secure the needed level of the specificity.
The mathematical transformation procedure may be
same for all of the substance groups in the principal
library. The transformation may be same within the
sub-library but different between the sub-libraries. For
example, this situation is met when the grade of the
lactose is to be determined after the sample is identi-
fied as lactose from the library of excipients. The spec-
tral range can be all of the available wavelengths or can
be limited to cetain wavelengths. The range of wave-
lengths can be limited when a certain measurement
apparatus is used or unnecessary spectral information
is needed to be removed (outside the dynamic range or
regions of high noise level). An analytical method of
partitioning the wavelength range is useful in removing
KP X 1843
unwanted effects or in bringing a small but important
difference into relief.
Like any other analytical techniques, the NIR spec-
troscopy cannot differentiate all of the substance
groups, especially very similar groups. In this case, two
groups are combined to one group or any other control
method is used to perform the identification and quali-
tative analysis.
There are many algorithms such as correlation, soft
independent modeling of class analogy (SIMCA),
Mahalanobis distance, SMV and so on. The user se-
lects a proper algorithm with the consideration of the
effective range of the library. When the discrimination
of the substances is each, it is recommended to use the
simplest algorithm. For example, when the purpose is
only the identification of the substance, the physical
factors are not needed to be considered, so the second
derivatives and the wavelength correlation algorithm
can be selected.
7) Setting the threshold
The internal validation is done with the value set by
the software itself or recommended by the manufactur-
er. The threshold of the library can be changed when
the library is validated internally or the sample is eval-
uated externally.
5. Validation of Library
The purpose of validation of an analytical procedure
is to check whether the analysis is suitable for the in-
tended use. Upon this purpose, the factors influencing
the needed validation should be determined.
1) Internal validation
The performance of the library should be evaluated
in the course of constructing any kind of spectral data-
base. This is based on the samples constructing the
library (whether these samples can be differenctiated).
The internal validation is done by the software and the
detailed procedure may be different by the used soft-
ware, but the basic procedure is as follows:
• The spectra used to construct the library are vali-
dated by a proper method (correlation or distance
method)
• It is checked whether the distribution of the sub-
stances in the library overlaps.
• The cross-validation is used for the construction of
the library.
2) External validation
When the internal validation is successful, the per-
formance of the database is validated using certified
samples not used for the database development.
Reproducibility: This is not commonly applied
method for the identification of substances. In the re-
producibility test for the qualitative analysis, the sub-
stances included in the library should be reliably dif-
1844 General Information
ferentiated from the substances outside the library,
using the threshold value.
Robustness: This category is dependent on the appli-
cation and the sample selection technique and the ef-
fect of delicate variation in the normal operating condi-
tion of the analysis is tested. The use of the design of
experiment can maximize the effect of the information
on the analyte. The following items are considered:
• The effect of environmental conditions (tempera-
ture, humidity) of the analysis
• The effect of temperature on the sample
• The location of sample
• The depth of probe and the degree of compres-
sion/filling of the substance
• Other effect of the sample measurement apparatus
• The effect of replacement of parts (lamp)
• The effect of pretreatment and parameters of the al-
gorithm used to construct the library (gap/segment
of the derivative operation, distance threshols, etc.)
6. Routine use
When the system is evaluated, it is adjusted to allow
the needed functions only. For example, the manager
of the NIR system or the library development expert
should evaluate the software thoroughly, but general
users are required to evaluate only the performance of
the general identification of substances
In the aspect of developing the library for the spec-
tral identification, the considerable part of the charac-
teristic variation within the substances are to be includ-
ed. But sometimes, this variation may not be included
in the sample set for the development of the library.
For example, when a test substance is analyzed to be
outside the boundary of one of the models in the library,
the test substance is expressed as to be “inadequate
NIR spectrum” for the NIR spectral model. In this case,
a proper alternative test is performed and the test sub-
stance is certified correctly before making decision that
the substance is within the model or inserting the spec-
trum into the library.
* Refer to the Appendix IV for the construction of
the library for the qualitative analysis.
7. Maintenance of library
1) Removal of existing substances
In normal situations, the removal of substances from
the library is not recommended, usually, but when the
selection of the samples is proven to be wrong, the
removal from the library is made and the library should
be evaluated once again.
2) Addition of new substances
To add new substances into the library, the test set of
substances is selected to satisfy the details of sample
selection parameters and the library is evaluated again
for the validation of the specificity, continuously.
3) Fixing the groups in the library
Sometimes, the sample set is fixed in the cases as
follows:
• Change in physical properties of the substance
• Change in supplier
• Inclusion of the wider range
In each case, new samples are certified by the meth-
od other than the NIR spectroscopy, before inserting
them in the model and the library is evaluated again for
the validation of the specificity, continuously.
* Refer to the Appendix 5 when there is any problem
in applying the library for the qualitative analysis.
VI. Terms Used
Calibration: a procedure of construction of a quanti-
tative model
Prediction: a procedure of estimating the concentra-
tion of unknown sample, using the constructed quanti-
tative model
Calibration set: a set of samples used for construct-
ing the quantitative model
Validation test set or validation set: a set of samples
used for validating the quantitative model
Multiplicative Scatter Correction: one of the mathe-
matical methods used for the correction of background
variation caused by the physical properties such as
particle size of the sample
Multiple Linear Regression (MLR): quantitative
model constructed with absorbance values at two or
more wavelengths.
Principal Component Regression (PCR): one of the
multivariate regression analysis using principal com-
ponent analysis, or factor analysis.
Partial Least Squares Regression (PLSR): one of the
multivariate regression analysis using principal com-
ponent analysis to which the concentration information
of the samples are included.
Soft Independent Modeling by Class Analogy
(SIMCA): a pattern recognition algorithm used for the
identification and the qualitative analysis of the sam-
ples.
NIST SRM: National Institute of Standards and
Technology Standard Reference Material
 KP X 1845

Appendix I. Selection of an algorithm for the construction of quantitative model.

1846 General Information

Appendix II. Validation and Evaluation of a Quantitative Model

 KP X 1847

Appendix III. Routine procedure of quantitative analysis and Maintenance of the quantitative model.
1848 General Information

Appendix IV. Construction of library needed for qualitative analysis

 KP X 1849

Appendix V. In case of failure of the library for qualitative analysis

1850 General Information
6. Guideline of Limits for
Residual Solvents of
Pharmaceuticals
1. INTRODUCTION
The objective of this guideline is to recommend ac-
ceptable amounts for residual solvents in pharmaceuti-
cals for the safety of the patient. The guideline recom-
mends use of less toxic solvents and describes levels
considered to be toxicologically acceptable for some
residual solvents.
‘Residual solvents’ in pharmaceuticals are defined
here as organic volatile chemicals that are used or pro-
duced in the manufacture of drug substances or excipi-
ents, or in the preparation of drug products. The sol-
vents are not completely removed by practical manu-
facturing techniques. Appropriate selection of the sol-
vent for the synthesis of drug substance may enhance
the yield, or determine characteristics such as crystal
form, purity, and solubility. Therefore, the solvent may
sometimes be a critical parameter in the synthetic pro-
cess. This guideline does not address solvents deliber-
ately used as excipients nor does it address solvates.
However, the content of solvents in such products
should be evaluated and justified.
Since there is no therapeutic benefit from residual
solvents, all residual solvents should be removed to the
extent possible to meet product specifications, good
manufacturing practices, or other quality-based re-
quirements. Drug products should contain no higher
levels of residual solvents than can be supported by
safety data. Some solvents that are known to cause
unacceptable toxicities (Class 1, Table 1) should be
avoided in the production of drug substances, excipi-
ents, or drug products unless their use can be strongly
justified in a risk-benefit assessment. Some solvents
associated with less severe toxicity (Class 2, Table 2)
should be limited in order to protect patients from po-
tential adverse effects. Ideally, less toxic solvents
(Class 3, Table 3) should be used where practical. The
complete list of solvents included in this guideline is
given in Appendix 1.
The lists are not exhaustive and other solvents can
be used and later added to the lists. Recommended
limits of Class 1 and 2 solvents or classification of sol-
vents may change as new safety data becomes availa-
ble. Supporting safety data in a marketing application
for a new drug product containing a new solvent may
be based on concepts in this guideline or the concept of
qualification of impurities as expressed in the guideline
for drug substance (Q3A, Impurities in New Drug Sub-
stances) or drug product (Q3B, Impurities in New Drug
Products), or all three guidelines.
2. SCOPE OF THE GUIDELINE
Residual solvents in drug substances, excipients, and
in drug products are within the scope of this guideline.
Therefore, testing should be performed for residual
solvents when production or purification processes are
known to result in the presence of such solvents. It is
only necessary to test for solvents that are used or pro-
duced in the manufacture or purification of drug sub-
stances, excipients, or drug product. Although manu-
facturers may choose to test the drug product, a cumu-
lative method may be used to calculate the residual
solvent levels in the drug product from the levels in the
ingredients used to produce the drug product. If the
calculation results in a level equal to or below that rec-
ommended in this guideline, no testing of the drug
product for residual solvents need be considered. If,
however, the calculated level is above the recommend-
ed level, the drug product should be tested to ascertain
whether the formulation process has reduced the rele-
vant solvent level to within the acceptable amount.
Drug product should also be tested if a solvent is used
during its manufacture.
This guideline does not apply to potential new drug
substances, excipients, or drug products used during
the clinical research stages of development, nor does it
apply to existing marketed drug products.
The guideline applies to all dosage forms and route
of administration. Higher levels of residual solvents
may be acceptable in certain cases such as short term
(30 days or less) or topical application. Justification for
these levels should be made on a case by case basis.
See Appendix 2 for additional background information
related to residual solvents.
3. GENERAL PRINCIPLES
3.1 Classification of Residual Solvents by Risk
Assessment
The term tolerable daily intake (TDI) is used by the
International Program on Chemical Safety (IPCS) to
describe exposure limits of toxic chemicals and ac-
ceptable daily intake (ADI) is used by the World
Health Organization (WHO) and other international
health authorities and institutions. The new term per-
mitted daily exposure (PDE) is defined in the present
guideline as a ‘pharmaceutically acceptable intake of
residual solvents’ to avoid confusion of differing val-
ues for ADI's of the same substance.
Residual solvents assessed in this guideline are listed
in Appendix 1 by common names and structures. They
were evaluated for their possible risk to human health
and placed into one of three classes as follows:
Class 1 solvents: Solvents to be avoided
Known human carcinogens or strongly sus-
pected human carcinogens, and environmental
hazards.
Class 2 solvents: Solvents to be limited
Non-genotoxic animal carcinogens or possible
causative agents of other irreversible toxicity
such as neurotoxicity or teratogenecity. Sol-
vents suspected of other significant but re-
versible toxicities.
 KP X 1851

Class 3 solvents: Solvents with low toxic potential permitted daily exposure to acetonitrile is 4.1 mg/day;
Solvents with low toxic potential to man; thus, the option 1 limit is 410 ppm.
no health-based exposure limit is needed. The maximum administered daily mass of a drug
Class 3 solvents have PDEs of 50 mg or more product is 5.0 g, and the drug product contains two
per day. excipients. The composition of the drug product and
the calculated maximum content of residual acetonitrile
3.2 Methods for Establishing Exposure Limits are given in the following table.
The method used to establish permitted daily expo-
sures for residual solvents is presented in Appendix 3. Compo- Amount in Acetonitrile Daily
Summaries of the toxicity data that were used to estab- nent formulation content exposure
lish limits are published in Pharmeuropa, Vol. 9, No. 1,
Supplement, April 1997. Drug
0.3 g 800 ppm 0.24 mg
substance
3.3 Options for Setting Limits of Class 2 Solvents Excipient 1 0.9 g 400 ppm 0.36 mg
Two options are available when setting limits for
Excipient 2 3.8 g 800 ppm 3.04 mg
Class 2 solvents.
Drug
5.0 g 728 ppm 3.64 mg
Option 1: The concentration limits in ppm stated in Product
Table 2 can be used. They were calculated us-
ing equation (1) below by assuming a product Excipient 1 meets the Option 1 limit, but the drug
mass of 10 g administered daily. substance, excipient 2, and drug product do not meet
the Option 1 limit. Nevertheless, the product meets the
Concentration (ppm) = 1000 PDE (1) Option 2 limit of 4.1 mg per day and thus conforms to
dose the recommendations in this guideline.
Consider another example using acetonitrile as re-
Here, PDE is given in terms of mg/day and dose is sidual solvent. The maximum administered daily mass
given in g/day. of a drug product is 5.0 g, and the drug product con-
These limits are considered acceptable for all sub- tains two excipients. The composition of the drug
stances, excipients, or products. Therefore this option product and the calculated maximum content of residu-
may be applied if the daily dose is not known or fixed. al acetonitrile are given in the following table.
If all excipients and drug substances in a formulation
meet the limits given in Option 1, then these compo- Compo- Amount in Acetonitrile Daily
nents may be used in any proportion. No further calcu- nent formulation content exposure
lation is necessary provided the daily dose does not
Drug
exceed 10 g. Products that are administered in doses 0.3 g 800 ppm 0.24 mg
substance
greater than 10 g per day should be considered under
Option 2. Excipient 1 0.9 g 2,000 ppm 1.80 mg
Excipient 2 3.8 g 800 ppm 3.04 mg
Option 2: It is not considered necessary for each com-
Drug
ponent of the drug product to comply with the 5.0 g 1,016 ppm 5.08 mg
Product
limits given in Option 1. The PDE in terms of
mg/day as stated in Table 2 can be used with
In this example, the product meets neither the Op-
the known maximum daily dose and equation
tion 1 nor the Option 2 limit according to this summa-
(1) above to determine the concentration of re-
tion. The manufacturer could test the drug product to
sidual solvent allowed in drug product. Such
determine if the formulation process reduced the level
limits are considered acceptable provided that
of acetonitrile. If the level of acetonitrile was not re-
it has been demonstrated that the residual sol-
duced during formulation to the allowed limit, then the
vent has been reduced to the practical mini-
manufacturer of the drug product should take other
mum. The limits should be realistic in relation
steps to reduce the amount of acetonitrile in the drug
to analytical precision, manufacturing capabil-
product. If all of these steps fail to reduce the level of
ity, reasonable variation in the manufacturing
residual solvent, in exceptional cases the manufacturer
process, and the limits should reflect contem-
could provide a summary of efforts made to reduce the
porary manufacturing standards.
solvent level to meet the guideline value, and provide a
risk-benefit analysis to support allowing the product to
Option 2 may be applied by adding the amounts of a
be utilized with residual solvent at a higher level.
residual solvent present in each of the components of
the drug product. The sum of the amounts of solvent
3.4 Analytical Procedures
per day should be less than that given by the PDE.
Residual solvents are typically determined using
Consider an example of the use of Option 1 and Op-
chromatographic techniques such as gas chromatog-
tion 2 applied to acetonitrile in a drug product. The
raphy. Harmonized procedures for determining levels
1852 General Information

of residual solvents as described in the pharmacopoeias Concentration

should be used, if feasible. Otherwise, manufacturers Solvent Note
limit (ppm)
would be free to select the most appropriate validated Carcino-
analytical procedure for a particular application. If only Benzene 2
gen
Class 3 solvents are present, a non-specific method Toxic and
such as loss on drying may be used. Carbon tetrachlo- environ-
Validation of methods for residual solvents should 4
ride mental
conform to the ICH guidelines Text on Validation of hazard
Analytical Procedures and Extension of the ICH Text 1,2-Dichloroethane 5 Toxic
on Validation of Analytical Procedures . 1,1-Dichloroethene 8 Toxic
Environ-
3.5 Reporting levels of residual solvents 1,1,1-
1500 mental
Manufacturers of pharmaceutical products need cer- Trichloroethane
hazard
tain information about the content of residual solvents
in excipients or drug substances in order to meet the 4.2 Solvents to Be Limited in Pharmaceutical
criteria of this guideline. The following statements are Products
given as acceptable examples of the information that Solvents in Table 2 should be limited in pharmaceu-
could be provided from a supplier of excipients or drug tical products because of their inherent toxicity. PDEs
substances to a pharmaceutical manufacturer. The sup- are given to the nearest 0.1 mg/day, and concentrations
plier might choose one of the following as appropriate: are given to the nearest 10 ppm. The stated values do
- Only Class 3 solvents are likely to be present. not reflect the necessary analytical precision of deter-
Loss on drying is less than 0.5%. mination. Precision should be determined as part of the
- Only Class 2 solvents X, Y, ... are likely to be validation of the method.
present. All are below the Option 1 limit. (Here
the supplier would name the Class 2 solvents Table 2. Class 2 solvents in pharmaceutical products.
represented by X, Y, ...)
PDE Concentration
- Only Class 2 solvents X, Y, ... and Class 3 sol- Solvent
(mg/day) limit (ppm)
vents are likely to be present. Residual Class 2
solvents are below the Option 1 limit and resid- Acetonitrile 4.1 410
ual Class 3 solvents are below 0.5%. Chlorobenzene 3.6 360
If Class 1 solvents are likely to be present, they Chloroform 0.6 60
should be identified and quantified. Cumene 0.7 70
"Likely to be present" refers to the solvent used in Cyclohexane 38.8 3880
the final manufacturing step and to solvents that are 1,2-Dichloroethene 18.7 1870
used in earlier manufacturing steps and not removed Dichloromethane 6.0 600
consistently by a validated process. 1,2-
1.0 100
If solvents of Class 2 or Class 3 are present at great- Dimethoxyethane
er than their Option 1 limits or 0.5%, respectively, they N,N-
10.9 1090
should be identified and quantified. Dimethylacetamide
N,N-
8.8 880
Dimethylformamide
4. LIMITS OF RESIDUAL SOLVENTS
1,4-Dioxane 3.8 380
2-Ethoxyethanol 1.6 160
4.1 Solvents to Be Avoided
Solvents in Class 1 should not be employed in the Ethyleneglycol 6.2 620
manufacture of drug substances, excipients, and drug Formamide 2.2 220
products because of their unacceptable toxicity or their Hexane 2.9 290
deleterious environmental effect. However, if their use Methanol 30.0 3000
is unavoidable in order to produce a drug product with 2-Methoxyethanol 0.5 50
a significant therapeutic advance, then their levels Methylbutyl ketone 0.5 50
should be restricted as shown in Table 1, unless other- Methylcyclohexane 11.8 1180
wise justified. 1,1,1-Trichloroethane is included in Ta- N-Methylpyrrolidone 48.4 4840
ble 1 because it is an environmental hazard. The stated Nitromethane 0.5 50
limit of 1500 ppm is based on a review of the safety Pyridine 2.0 200
data. Sulfolane 1.6 160
Tetrahydrofuran 7.2 720
Table 1. Class 1 solvents (solvents that should not be Tetralin 1.0 100
used in manufacture of pharmaceutical products). Toluene 8.9 890
1,1,2-
0.8 80
Trichloroethene
note 1)
Xylene 21.7 2170
note 1)
usually 60% m-xylene, 14% p-xylene, 9% o-
 KP X 1853

xylene with 17% ethyl benzene Isopropyl ether Trifluoroacetic acid

4.3 Solvents with Low Toxic Potential Furthermore, for the Class 2 or Class 3 solvent is used
Solvents in Class 3 (shown in Table 3) may be re- prior to the last step of a manufacturing process of drug
garded as less toxic and of lower risk to human health. substances, it is not necessary to establish the specifi-
Class 3 includes no solvent known as a human health cation of the residual solvents of the drug substances,
hazard at levels normally accepted in pharmaceuticals. in case of complying with the requirements of the re-
However, there are no long-term toxicity or carcino- sidual solvents that are not remain in the final active
genicity studies for many of the solvents in Class 3. drug substances.
Available data indicate that they are less toxic in acute (Examples of the recommended data) the content of 3
or short-term studies and negative in genotoxicity stud- consecutive batches of residual solvents that is not
ies. It is considered that amounts of these residual sol- more than acceptable concentration limit and the data
vents of 50 mg per day or less (corresponding to 5000 of detection limit. When Class 2 solvents are used, they
ppm or 0.5% under Option 1) would be acceptable should be controlled either in a suitable intermediate or
without justification. Higher amounts may also be ac- in the management data of final active substances de-
ceptable provided they are realistic in relation to manu- pending on the batch scale.
facturing capability and good manufacturing practice
(GMP). 5. GLOSSARY
 Genotoxic Carcinogens: Carcinogens which pro-
Table 3. Class 3 solvents, which should be limited by duce cancer by affecting genes or chromosomes.
GMP or other quality-based requirements, in  LOEL: Abbreviation for lowest-observed effect
pharmaceutical products. level.
Acetic acid Heptane
 Lowest-Observed Effect Level: The lowest dose
Acetone Isobutyl acetate
of substance in a study or group of studies that
Anisole Isopropyl acetate
produces biologically significant increases in fre-
1-Butanol Methyl acetate
quency or severity of any effects in the exposed
2-Butanol 3-Methyl-1-butanol
humans or animals.
Butyl acetate Methylethyl ketone
 Modifying Factor: A factor determined by profes-
tert-Butylmethyl ether Methylisobutyl ketone
sional judgment of a toxicologist and applied to
Dimethyl sulfoxide 2-Methyl-1-propanol
bioassay data to relate that data safely to humans.
Ethanol Pentane
Ethyl acetate 1-Pentanol  Neurotoxicity: The ability of a substance to cause
adverse effects on the nervous system.
Ethyl ether 1-Propanol
Ethyl formate 2-Propanol  NOEL: Abbreviation for no-observed-effect level.
Formic acid Propyl acetate  No-Observed-Effect Level: The highest dose of
substance at which there are no biologically sig-
4.4 Solvents for which No Adequate Toxicological nificant increases in frequency or severity of any
Data was Found effects in the exposed humans or animals.
The following solvents (Table 4) may be of inter-  PDE: Abbreviation for permitted daily exposure.
est to manufacturers of excipients, drug substances, or  Permitted Daily Exposure: The maximum ac-
drug products. However, no adequate toxicological ceptable intake per day of residual solvent in
data on which to base a PDE was found. Manufacturers pharmaceutical products.
should supply justification for residual levels of these  Reversible Toxicity: The occurrence of harmful
solvents in pharmaceutical products. effects that are caused by a substance and which
disappear after exposure to the substance ends.
Table 4. Solvents for which no adequate toxicologi-  Strongly Suspected Human Carcinogen: A sub-
cal data was found. stance for which there is no epidemiological evi-
dence of carcinogenesis but there are positive
Methylisopropyl ke- genotoxicity data and clear evidence of carcino-
1,1-Diethoxypropane genesis in rodents.
tone
1,1-Dimethoxymethane Methyltetrahydrofuran  Teratogenicity: The occurrence of structural mal-
formations in a developing fetus when a sub-
2,2-Dimethoxypropane Petroleum ether
stance is administered during pregnancy.
Isooctane Trichloroacetic acid

APPENDIX 1. LIST OF SOLVENTS INCLUDED IN THE GUIDELINE

Solvent Other Names Classification

Acetic acid Ethanoic acid Class 3
Acetone 2-Propanone; Propan-2-one Class 3
1854 Monographs, Part II

Acetonitrile Class 2
Anisole Methoxybenzene Class 3
Benzene Benzol Class 1
1-Butanol n-Butyl alcohol Class 3
Butan-1-ol
2-Butanol sec-Butyl alcohol; Butan-2-ol Class 3
Butyl acetate Acetic acid butyl ester Class 3
tert-Butylmethyl ether 2-Methoxy-2-methyl- propane Class 3
Carbon tetrachloride Tetrachloromethane Class 1
Chlorobenzene Class 2
Chloroform Trichloromethane Class 2
Cumene Isopropylbenzene; (1-Methyl)ethylbenzene Class 2
Cyclohexane Hexamethylene Class 2
1,2-Dichloroethane sym-Dichloroethane; Ethylene dichloride; Ethylene chloride Class 1
1,1-Dichloroethene 1,1-Dichloroethylene; Vinylidene chloride Class 1
1,2-Dichloroethene 1,2-Dichloroethylene; Acetylene dichloride Class 2
Dichloromethane Methylene chloride Class 2
1,2-Dimethoxyethane Ethyleneglycol dimethyl ether; Monoglyme; Dimethyl Cellosolve Class 2
N,N- DMA Class 2
Dimethylacetamide
N,N- DMF Class 2
Dimethylformamide
Dimethyl sulfoxide Methylsulfinylmethane; Methyl sulfoxide; DMSO Class 3
1,4-Dioxane p-Dioxane; [1,4]Dioxane Class 2
Ethanol Ethyl alcohol Class 3
2-Ethoxyethanol Cellosolve Class 2
Ethyl acetate Acetic acid ethyl ester Class 3
Ethyleneglycol 1,2-Dihydroxyethane; 1,2-Ethanediol Class 2
Ethyl ether Diethyl ether; Ethoxyethane; 1,1’-Oxybisethane Class 3
Ethyl formate Formic acid ethyl ester Class 3
Formamide Methanamide Class 2
Formic acid Class 3
Heptane n-Heptane Class 3
Hexane n-Hexane Class 2
Isobutyl acetate Acetic acid isobutyl ester Class 3
Isopropyl acetate Acetic acid isopropyl ester Class 3
Methanol Methyl alcohol Class 2
2-Methoxyethanol Methyl Cellosolve Class 2
Methyl acetate Acetic acid methyl ester Class 3
3-Methyl-1-butanol Isoamyl alcohol; Isopentyl alcohol; 3-Methylbutan-1-ol Class 3
Methylbutyl ketone 2-Hexanone; Hexan-2-one Class 2
Methylcyclohexane Cyclohexylmethane Class 2
Methylethyl ketone 2-Butanone; MEK; Butan-2-one Class 3
Methylisobutyl ketone 4-Methylpentan-2-one; 4-Methyl-2-pentanone; MIBK Class 3
2-Methyl-1-propanol Isobutyl alcohol; 2-Methylpropan-1-ol Class 3
N-Methylpyrrolidone 1-Methylpyrrolidin-2-one; 1-Methyl-2-pyrrolidinone Class 2
Nitromethane Class 2
Pentane n-Pentane Class 3
1-Pentanol Amyl alcohol; Pentan-1-ol; Pentyl alcohol Class 3
1-Propanol Propan-1-ol; Propyl alcohol Class 3
2-Propanol Propan-2-ol; Isopropyl alcohol Class 3
Propyl acetate Acetic acid propyl ester Class 3
Pyridine Class 2
Sulfolane Tetrahydrothiophene 1,1-dioxide Class 2
Tetrahydrofuran Tetramethylene oxide; Oxacyclopentane Class 2
Tetralin 1,2,3,4-Tetrahydro-naphthalene Class 2
Toluene Methylbenzene Class 2
1,1,1-Trichloroethane Methylchloroform Class 1

1,1,2-Trichloroethene Trichloroethene
Xylenenote) Dimethybenzene; Xylol
note)
usually 60% m-xylene, 14% p-xylene, 9% o-xylene with 17% ethyl benzene
APPENDIX 2. ADDITIONAL BACKGROUND
2.1. Environmental Regulation of Organic Vola-
tile Solvents
Several of the residual solvents frequently
used in the production of pharmaceuticals are listed
as toxic chemicals in Environmental Health Criteria
(EHC) monographs and the Integrated Risk Infor-
mation System (IRIS). The objectives of such
groups as the International Programme on Chemical
Safety (IPCS), the United States Environmental
Protection Agency (USEPA), and the United States
Food and Drug Administration (USFDA) include
the determination of acceptable exposure levels. The
goal is protection of human health and maintenance
of environmental integrity against the possible dele-
terious effects of chemicals resulting from long-
term environmental exposure. The methods in-
volved in the estimation of maximum safe exposure
limits are usually based on long-term studies. When
long-term study data are unavailable, shorter term
study data can be used with modification of the ap-
proach such as use of larger safety factors. The ap-
proach described therein relates primarily to long-
term or life-time exposure of the general population
in the ambient environment, i.e. ambient air, food,
drinking water and other media.
2.2. Residual Solvents in Pharmaceuticals
Exposure limits in this guideline are estab-
lished by referring to methodologies and toxicity
data described in EHC and IRIS monographs. How-
ever, some specific assumptions about residual sol-
vents to be used in the synthesis and formulation of
pharmaceutical products should be taken into ac-
count in establishing exposure limits. They are:
1) Patients (not the general population) use pharma-
ceuticals to treat their diseases or for prophylaxis to
prevent infection or disease.
2) The assumption of life-time patient exposure is
not necessary for most pharmaceutical products but
may be appropriate as a working hypothesis to re-
duce risk to human health.
3) Residual solvents are unavoidable components in
pharmaceutical production and will often be a part
of drug products.
4) Residual solvents should not exceed recommend-
ed levels except in exceptional circumstances.
5) Data from toxicological studies that are used to
determine acceptable levels for residual solvents
should have been generated using appropriate pro-
tocols such as those described for example by
OECD, EPA, and the FDA Red Book.
APPENDIX 3. METHODS FOR ESTABLISHING
KP X 1855
Class 2
Class 2
EXPOSURE LIMITS
The Gaylor-Kodell method of risk assessment
(Gaylor, D. W. and Kodell, R. L.: Linear Interpola-
tion algorithm for low dose assessment of toxic sub-
stance. J Environ. Pathology, 4, 305, 1980) is ap-
propriate for Class 1 carcinogenic solvents. Only in
cases where reliable carcinogenicity data are availa-
ble should extrapolation by the use of mathematical
models be applied to setting exposure limits. Expo-
sure limits for Class 1 solvents could be determined
with the use of a large safety factor (i.e., 10,000 to
100,000) with respect to the no-observed-effect lev-
el (NOEL). Detection and quantitation of these sol-
vents should be by state-of-the-art analytical tech-
niques.
Acceptable exposure levels in this guideline for
Class 2 solvents were established by calculation of
PDE values according to the procedures for setting
exposure limits in pharmaceuticals (Pharmacopeial
Forum, Nov-Dec 1989), and the method adopted by
IPCS for Assessing Human Health Risk of Chemi-
cals (Environmental Health Criteria 170, WHO,
1994). These methods are similar to those used by
the USEPA (IRIS) and the USFDA (Red Book) and
others. The method is outlined here to give a better
understanding of the origin of the PDE values. It is
not necessary to perform these calculations in order
to use the PDE values tabulated in Section 4 of this
document.
PDE is derived from the no-observed-effect level
(NOEL), or the lowest-observed effect level (LOEL)
in the most relevant animal study as follows:
NOEL x Weight Adjustment (1)
PDE 
F1 x F2 x F3 x F4 x F5
The PDE is derived preferably from a NOEL. If no
NOEL is obtained, the LOEL may be used. Modify-
ing factors proposed here, for relating the data to
humans, are the same kind of "uncertainty factors"
used in Environmental Health Criteria (Environ-
mental Health Criteria 170, World Health Organiza-
tion, Geneva, 1994), and "modifying factors" or
"safety factors" in Pharmacopeial Forum. The as-
sumption of 100% systemic exposure is used in all
calculations regardless of route of administration.
The modifying factors are as follows:
F1 = A factor to account for extrapolation between
species
F1 = 5 for extrapolation from rats to humans
F1 = 12 for extrapolation from mice to humans
F1 = 2 for extrapolation from dogs to humans
F1 = 2.5 for extrapolation from rabbits to hu-
mans
F1 = 3 for extrapolation from monkeys to hu-
1856 Monographs, Part II

mans adjustment for a lower body weight would be ap-

F1 = 10 for extrapolation from other animals to
humans
F1 takes into account the comparative surface area:
body weight ratios for the species concerned and for
man. Surface area (S) is calculated as:
S = kM 0.67 (2)
propriate.
As an example of the application of the equation (1),
consider a toxicity study of acetonitrile in mice that
is summarized in Pharmeuropa, Vol. 9, No. 1, Sup-
plement, April 1997, page S24. The NOEL is calcu-
lated to be 50.7 mg kg-1 day-1. The PDE for ace-
tonitrile in this study is calculated as follows:

in which M = body mass, and the constant k has 50.7 mg kg -1 day -1  50 kg

been taken to be 10. The body weights used in the PDE   4.2 mg day -1
12 10  5 11
equation are those shown below in the Table 1.
In this example,
F2 = A factor of 10 to account for variability F1 = 12 to account for the extrapolation from
between individuals mice to humans
A factor of 10 is generally given for all F2 = 10 to account for differences between indi-
organic solvents, and 10 is used consist- vidual humans
ently in this guideline. F3 = 5 because the duration of the study was
F3 = A variable factor to account for toxicity only 13 weeks
studies of short-term exposure F4 = 1 because no severe toxicity was encoun-
F3 = 1 for studies that last at least one half life- tered
time (1 year for rodents or rabbits; 7 years F5 = 1 because the no effect level was deter-
for cats, dogs and monkeys). mined
F3 = 1 for reproductive studies in which the
whole period of organogenesis is covered. Table 1. Values used in the calculations in this
F3 = 2 for a 6-month study in rodents, or a 3.5- guideline.
year study in non-rodents.
F3 = 5 for a 3-month study in rodents, or a 2- rat body weight 425 g
year study in non-rodents. pregnant rat body weight 330 g
F3 = 10 for studies of a shorter duration.
In all cases, the higher factor has been used for mouse body weight 28 g
study durations between the time points, e.g. a fac- pregnant mouse body weight 30 g
tor of 2 for a 9-month rodent study.
guinea pig body weight 500 g
F4 = A factor that may be applied in cases of severe
toxicity, e.g. non-genotoxic carcinogenicity, neuro- Rhesus monkey body weight 2.5 kg
toxicity or teratogenicity. In studies of reproductive rabbit body weight
toxicity, the following factors are used: 4 kg
(pregnant or not)
F4 = 1 for fetal toxicity associated with mater-
beagle dog body weight 11.5 kg
nal toxicity
F4 = 5 for fetal toxicity without maternal toxici- rat respiratory volume 290 L/day
ty
mouse respiratory volume 43 L/day
F4 = 5 for a teratogenic effect with maternal
toxicity rabbit respiratory volume 1440 L/day
F4 = 10 for a teratogenic effect without mater- guinea pig respiratory volume 430 L/day
nal toxicity
F5 = A variable factor that may be applied if the no- human respiratory volume 28,800 L/day
effect level was not established dog respiratory volume 9,000 L/day
When only an LOEL is available, a factor of up to
10 could be used depending on the severity of the monkey respiratory volume 1,150 L/day
toxicity. mouse water consumption 5 mL/day
The weight adjustment assumes an arbitrary adult
human body weight for either sex of 50 kg. This rat water consumption 30 mL/day
relatively low weight provides an additional safety rat food consumption 30 g/day
factor against the standard weights of 60 kg or 70 kg
that are often used in this type of calculation. It is
recognized that some adult patients weigh less than The equation for an ideal gas, PV = nRT, is used to
50 kg; these patients are considered to be accom- convert concentrations of gases used in inhalation
modated by the built-in safety factors used to de- studies from units of ppm to units of mg/L or mg/m3.
termine a PDE. If the solvent was present in a for- Consider as an example in which the rat reproduc-
mulation specifically intended for pediatric use, an tive toxicity study by inhalation of carbon tetrachlo-
ride (molecular weight 153.84) is summarized in

Pharmeuropa, Vol. 9, No. 1, Supplement, April 1997,
page S9.
1
n P 00 x 10 atm x 15 8 0 mg mol
RT 0 08 L atm 1 mol 1 x 8
15 mg
1 8 mg L
5L
The relationship 1000 L = 1 m3 is used to convert to
mg/ m3.
7. Guideline of Validation of
Analytical Procedures for
Pharmaceuticals
I. OBJECTIVE
The purpose of this guideline is to provide the de-
tailed guidance on how to conduct the validation of
the analytical procedures necessary for application
(approval) for manufacture · import of pharmaceu-
ticals · quasi-drugs according to notifications of
the Korean Food and Drug Administration, such as
‘Regulation of Pharmaceuticals Approval, Notifi-
cation and Review’, and for quality control.
II. INTRODUCTION
The objective of validation of an analytical pro-
cedure for pharmaceuticals is to demonstrate that
the procedure, applied in the quality control of the
pharmaceutical, is suitable for the intended pur-
pose.
The purpose is to direct how to establish relevant
validation parameters for each analytical procedure.
The scope of analytical procedures that are sub-
jected to the present guideline is as follows:
1) Identification tests in the specification for
pharmaceuticals
2) Purity tests: quantitative tests and limit tests
for the impurity in a sample
3) Quantification tests: assay for the active
component of drug substance or drug prod-
uct, for other selected component(s) in drug
products, for Uniformity of Dosage Units
test or for dissolution test
The objective of the analytical procedure should
be clear since the procedure will govern the valida-
tion characteristics which need to be evaluated.
Typical validation parameters include specificity,
accuracy, precision, detection limit, quantification
limit, linearity, range, and robustness. Appropriate
validation parameters are selected and evaluated
depending on the objective of a given analytical
procedure.
Furthermore, revalidation may be necessary when
there are change in the synthesis of the drug sub-
stance, change in the composition of the finished
product, and change in the analytical procedure.
KP X 1857
The degree of revalidation required depends on the
nature of the changes.
In addition, validation methods, other than those
described in this guideline, may be used, although
the appropriateness of the alternative method has
to be justified.
III. GLOSSARY
1. Analytical Procedure
The analytical procedure refers to the way of per-
forming the analysis. It should describe in detail
the steps necessary to perform each analytical test.
The analytical procedure includes but is not lim-
ited to: the analyte, the sample, the reference
standard, the standard reagents, the use of analyti-
cal instruments, the use of the calibration curve,
the use of formulae for the calculation in the iden-
tification test, purity test or assay.
- The term ‘validation of analytical proce-
dure’ is defined as the process of confirming
that the analytical procedure for a quality
control test of pharmaceuticals is suitable for
its intended use.
- Identification Test refers to ensure the iden-
tity of an analyte, and generally compare the
physicochemical characteristics (spectrum,
information from chromatographic methods,
and chemical reactivity, etc) of a sample with
those of a reference material.
- Purity Test is to ensure that all the analytical
procedures performed allow an accurate
statement of the content of impurities of an
analyte, i.e. related substances test, heavy
metals, residual solvents content, etc. Testing
for impurities can be either a quantification
test or a limit test for the impurity in a sample.
- Assay (Content or Potency) is to provide an
exact result which allows an accurate state-
ment on the content or potency of the analyte
in a sample. The assay represents a quantita-
tive measurement of the major component(s)
or other selected component(s) (i.e., stabilizer,
additive) in the drug substance or in the drug
product. The validation principles are also
applicable to assay for dissolution test.
2. Specificity
Specificity is the ability to assess unequivocally
the analyte in the presence of components, e.g.,
impurities, degradation products, matrix, etc,
which may be expected to be present. Lack of
specificity of an individual analytical procedure
may be compensated by other supporting analyti-
cal procedure(s).
3. Accuracy
The accuracy of an analytical procedure express-
es the closeness of agreement between the value
which is accepted either as a conventional true
value or an accepted reference value and the value
1858 Monographs, Part II
found.
4. Precision
The precision of an analytical procedure express-
es the closeness of agreement (degree of scatter)
between a series of measurements obtained from
homogeneous sample under the prescribed condi-
tions. Precision may be considered at three levels:
repeatability, intermediate precision and reproduc-
ibility.
- Repeatability expresses the precision of
measurements obtained under the same oper-
ating conditions (same analyst, same labora-
tory, same equipment, same sample, etc) over
a short interval of time. Repeatability is also
termed intra-assay precision.
- Intermediate Precision expresses within-
laboratories variations: different days, differ-
ent analysts, different equipment, etc.
- Reproducibility expresses the precision of
observed values (collaborative studies, usual-
ly applied to standardization of methodology)
obtained from a homogenous sample in dif-
ferent laboratories.
5. Detection Limit
The detection limit of an individual analytical pro-
cedure is the lowest amount of analyte in a sample
which can be detected but not necessarily quanti-
fied as an exact value.
6. Quantitation Limit
The quantitation limit of an individual analytical
procedure is the lowest amount of analyte in a
sample which can be quantitatively determined
with suitable precision and accuracy. The quantita-
tion limit is a parameter of quantitative assays for
low levels of compounds in sample matrices, and
is used particularly for the determination of impu-
rities and/or degradation products.
7. Linearity
The linearity of an analytical procedure is its abil-
ity to obtain test results which are directly propor-
tional to the amount (concentration) of analyte in
the sample.
8. Range
The range of an analytical procedure is the inter-
val between the upper and lower concentration
(amounts) of analyte in the sample (including these
concentrations) for which it has been demonstrated
that the analytical procedure has a suitable level of
precision, accuracy and linearity.
9. Robustness
The robustness of an analytical procedure is a
measure of its capacity to remain unaffected by
small, but deliberate variations in method parame-
ters. It provides an indication of its reliability dur-
ing normal usage.
IV. VALIDATION OF ANALYTICAL PROCE-
DURES
For validation of analytical procedures, the vali-
dation procedure and protocol most suitable for
their product must be first selected. Then, all data
and validation parameters collected during the val-
idation process are used to evaluate the appropri-
ateness. The relevant data collected during valida-
tion and formulae used for calculating validation
parameters should be presented and justified as
appropriate. The validation parameters applicable
to the identification test, the control of impurities
and the assay procedures is summarized in Appen-
dix 1.
Well-characterized reference materials, which
documented purity as well as physicochemical and
biological characteristics, should be used through-
out the validation study. The degree of purity nec-
essary depends on the intended use.
1. Specificity
An investigation of specificity should be con-
ducted during the validation of identification tests,
the control of impurities and the assay procedures.
The procedures used to demonstrate specificity
will depend on the intended objective of the ana-
lytical procedure.
It is not always possible to demonstrate that an
analytical procedure is specific for a particular
analyte with complete discrimination. In this case,
a combination of two or more analytical proce-
dures is recommended to achieve the necessary
level of discrimination.
A. Identification
Suitable identification tests should be able to dis-
criminate between compounds of closely related
structures which are likely to be present. The dis-
crimination of a procedure may be confirmed by
obtaining positive results, based on the comparison
with a known reference material, from samples
containing the analyte, coupled with negative re-
sults from samples which do not contain the
analyte. In addition, the identification test may be
applied to materials structurally similar to or close-
ly related to the analyte to confirm that a positive
response is not obtained. The choice of such poten-
tially interfering materials should be based on
sound scientific judgement with a consideration of
the interferences that could occur.
B. Assay and Impurity Test(s)
For chromatographic procedures, representative
chromatograms should be presented to demon-
strate specificity and individual components
should be appropriately labeled. Similar consid-
erations should be given to other separation tech-
niques.

Critical separations in chromatography should
be investigated at an appropriate level to demon-
strate that the components are adequately sepa-
rated. For critical separations, specificity can be
demonstrated by the resolution of the two com-
ponents which elute closest to each other.
In cases where a non-specific assay is used, oth-
er supporting analytical procedures should be
used to demonstrate overall specificity. For ex-
ample, where a titration is adopted to assay the
drug substance for release, the combination of the
assay and a suitable test for impurities can be
used.
The approach is similar for both assay and im-
purity tests.
(1) Impurities are available
For the assay, this should involve demonstration
of the discrimination of the analyte in the pres-
ence of impurities and/or excipients; practically,
this can be done by spiking pure substances (drug
substance or drug product) with appropriate lev-
els of impurities and/or excipients and demon-
strating that the assay result is unaffected by the
presence of these materials (by comparison with
the assay result obtained on unspiked samples).
For the impurity test, the discrimination may be
established by spiking drug substance or drug
product with appropriate levels of impurities and
demonstrating the separation of these impurities
individually and/or from other components in the
sample matrix.
(2) Impurities are not available
If impurity or degradation products standards
are unavailable, specificity may be demonstrated
by comparing the test results of samples contain-
ing impurities or degradation products to a se-
cond well-characterized procedure. In this ap-
proach, analytical procedure having known vali-
dation parameter includes pharmacopoeial meth-
od, other validated analytical procedure e.g.:
pharmacopoeial method of other validated analyt-
ical procedure (independent procedure). As ap-
propriate, this should include samples stored un-
der relevant stress conditions light, heat, humidity,
acid/base hydrolysis and oxidation.
- For the assay, the two results should be com-
pared.
- For the impurity tests, the impurity profiles
should be compared.
Peak purity tests may be useful to show that the
analyte chromatographic peak is not attributable
to more than one component (e.g., diode array,
mass spectrometry).
2. Linearity
A linear relationship should be evaluated across
KP X 1859
the range (see section 3) of the analytical proce-
dure. It may be demonstrated directly on the drug
substance (by dilution of a standard stock solution)
and/or separate weighings of synthetic mixtures of
the drug product components, using the proposed
procedure. The latter aspect can be studied during
investigation of the range.
Linearity should be evaluated by visual inspec-
tion of a plot of signals as a function of analyte
concentration or content. If there is a linear rela-
tionship, test results should be evaluated by appro-
priate statistical methods, for example, by calcula-
tion of a regression line by the method of least
squares. In some cases, to obtain linearity between
assays and sample concentrations, the test data
may need to be subjected to a mathematical trans-
formation prior to the regression analysis. Data
from the regression line itself may be helpful to
provide mathematical estimates of the degree of
linearity.
The correlation coefficient, y-intercept, slope of
the regression line and residual sum of squares
should be submitted. A plot of the data should be
included. In addition, an analysis of the deviation
of the actual data points from the regression line
may also be helpful for evaluating linearity.
Some analytical procedures, such as immunoas-
says, do not demonstrate linearity after any trans-
formation. In this case, the analytical response
should be described by an appropriate function of
the concentration (amountly or empiraclly) of the
concentration (or content) of an analyte in a sam-
ple.
For the establishment of linearity, a minimum of
5 concentrations is recommended. Other approach-
es should be justified.
3. Range
The specified range is normally derived from lin-
earity studies and depends on the intended applica-
tion of the procedure. It is established by confirm-
ing that the analytical procedure provides an ac-
ceptable degree of linearity, accuracy and precision
when applied to samples containing amounts of
analyte within or at the extremes of the specified
range of the analytical procedure.
The following minimum specified ranges should
be considered:
A. Assay of a drug substance or a drug product
Normally from 80 to 120 percent of the test con-
centration.
B. Content uniformity
Covering a minimum of 70 to 130 percent of the
test concentration, unless a wider more appropriate
range, based on the nature of the dosage form such
as metered dose inhalers, is justified
C. Dissolution testing
±20 % over the specified range indicated in disso-
lution specification of the Specifications and Test
1860 Monographs, Part II
Procedures of the drug product.
For example, if the specifications for a controlled
released product cover a region from 20%, after 1
hour, up to 90%, after 24 hours, the validated
range would be 0-110% of the labeled content.
D. Assay of related substance
From the reporting level of an impurity to 120%
of the specification.
For impurities known to be unusually potent or to
produce toxic or unexpected pharmacological ef-
fects, the detection/quantitation limit should be
commensurate with the level at which the impuri-
ties must be controlled. For validation of impurity
test procedures carried out during development, it
may be necessary to consider the range around a
suggested (probable) limit.
E. If assay and purity are performed together as
one test and only a 100% standard is used, lineari-
ty should cover the range from the reporting level
of the impurities to 120% of the assay specifica-
tion.
4. Accuracy
Accuracy should be established across the speci-
fied range of the analytical procedure.
A. Assay
(1) Drug Substance
Several methods of determining accuracy are
available:
(a) When the purity is known
Application of an analytical procedure
to an analyte of known purity (e.g.,
reference material);
(b) When other analytical procedure
with the known accuracy is availa-
ble
Comparison of the results of the pro-
posed analytical procedure with those
of a second well characterized proce-
dure, the accuracy of which is stated
and/or defined (independent proce-
dure);
(c) Accuracy may be inferred once preci-
sion, linearity and specificity have
been established.
(2) Drug Product
Several methods for determining accuracy are
available:
(a) Application of the analytical proce-
dure to synthetic mixtures of the drug
product components to which known
quantities of the drug substance to be
analyzed have been added;
(b) In cases where it is impossible to ob-
tain samples of all drug product com-
ponents, it may be acceptable either
1) to add known quantities of the analyte
to the drug product, or
2) to compare the results obtained from a
second well-characterized procedure,
the accuracy of which is stated and/or
defined (independent procedure).
(c) Accuracy may be inferred once preci-
sion, linearity and specificity have
been established.
B. Quantification of Impurities
Accuracy should be assessed on samples (drug
substance/drug product) spiked with known
amounts of impurities.
In cases where it is impossible to obtain samples
of certain impurities, and/or degradation products,
it is considered acceptable to compare results ob-
tained by an independent procedure. The response
factor of the drug substance can be used.
It should be clear how the individual or total im-
purities are to be determined, e.g., weight/weight
or area percent, in all cases with respect to the ma-
jor analyte.
C. Recommended Data
Accuracy should be assessed using a minimum of
9 determinations over a minimum of 3 concentra-
tion levels covering the specified range (e.g., 3
concentrations/3 replicates each of the total analyt-
ical procedure).
Accuracy should be reported as percent recovery
by the assay of known added amount of analyte in
the sample or as the difference between the mean
and the accepted true value together with the con-
fidence intervals.
5. Precision
Validation of tests for assay and for quantitative
determination of impurities includes an investiga-
tion of precision.
A. Repeatability
Repeatability should be assessed using:
(1) a minimum of 9 determinations covering the
specified range for the procedure (e.g., 3 con-
centrations/3 replicates each), or
(2) a minimum of 6 determinations at 100% of
the test concentration.
B. Intermediate precision
The extent to which intermediate precision should
be established depends on the circumstances under
which the procedure is intended to be used. The
applicant should establish the effects of random
events on the precision of the analytical procedure.
Typical variations to be studied include days, ana-
lysts, equipment, etc. The use of an experimental
design (matrix) is encouraged.
C. Reproducibility
Reproducibility is assessed by means of an inter-
laboratory trial. Reproducibility should be consid-
ered in case of the standardization of an analytical

procedure, for instance, for inclusion of procedures
in pharmacopoeias. These data are not part of the
marketing authorization dossier.
D. Recommended Data
The standard deviation, relative standard deviation
(coefficient of variation) and confidence interval
should be reported for each type of precision in-
vestigated.
6. Detection limit
Several approaches for determining the detection
limit are possible, depending on whether the pro-
cedure is a non-instrumental or instrumental. Ap-
proaches other than those listed below may be ac-
ceptable.
A. Based on Visual Evaluation
Visual evaluation may be used for non-
instrumental methods but may also be used with
instrumental methods.
The detection limit is determined by the analysis
of samples with known concentrations of analyte
and by establishing the minimum level at which
the analyte can be reliably detected.
B. Based on Signal-to-Noise
This approach can only be applied to analytical
procedures which exhibit baseline noise. Determi-
nation of the signal-to-noise ratio is performed by
comparing measured signals from samples with
known low concentrations of analyte with those of
blank samples and establishing the minimum con-
centration at which the analyte can be reliably de-
tected. A signal-to-noise ratio between 3 or 2:1 is
generally considered acceptable for estimating the
detection limit.
C. Based on the Standard Deviation of the Re-
sponse and the Slope
The detection limit (DL) may be expressed as:
DL = . * σ / S
where
σ = the standard deviation of the response
S = the slope of the calibration curve
The slope S may be estimated from the calibra-
tion curve of the analyte. The estimate of σ
may be carried out in a variety of ways for ex-
ample:
(1) Based on the Standard Deviation of the
Blank
Measurement of the magnitude of analytical
background response is performed by analyz-
ing an appropriate number of blank samples
and calculating the standard deviation of the-
se responses.
(2) Based on the Calibration Curve
KP X 1861
A specific calibration curve should be stud-
ied using samples containing an analyte in the
range of DL. The residual standard deviation
of a regression line or the standard deviation
of y-intercepts of regression lines may be
used as the standard deviation.
D. Recommended Data
The detection limit and the method used for de-
termining the detection limit should be presented.
If DL is determined based on visual evaluation or
based on signal-to-noise ratio, the presentation of
the relevant chromatograms is considered accepta-
ble for justification.
In cases where an estimated value for the detec-
tion limit is obtained by calculation or extrapola-
tion, this estimate may subsequently be validated
by the independent analysis of a suitable number
of samples known to be near or prepared at the de-
tection limit.
7. Quantification limit
Several approaches for determining the quantifi-
cation limit are possible, depending on whether the
procedure is non-instrumental or instrumental.
Approaches other than those listed below may be
acceptable.
A. Based on Visual Evaluation
Visual evaluation may be used for non-
instrumental methods but may also be used with
instrumental methods.
The quantification limit is generally determined
by the analysis of samples with known concentra-
tions of analyte and by establishing the minimum
level at which the analyte can be quantified with
acceptable accuracy and precision.
B. Based on Signal-to-Noise Approach
This approach can only be applied to analytical
procedures that exhibit baseline noise. Determina-
tion of the signal-to-noise ratio is performed by
comparing measured signals from samples with
known low concentrations of analyte with those of
blank samples and by establishing the minimum
concentration at which the analyte can be reliably
quantified. A typical signal-to-noise ratio is 10:1.
C. Based on the Standard Deviation of the Re-
sponse and the Slope
The quantification limit (QL) may be expressed
as:
QL = 10 * σ / S
where
σ = the standard deviation of the response
S = the slope of the calibration curve
The slope S may be estimated from the calibra-
1862 Monographs, Part II
tion curve of the analyte. The estimate of σ
may be carried out in a variety of ways for ex-
ample:
(1) Based on Standard Deviation of the Blank
Measurement of the magnitude of analytical
background response is performed by analyz-
ing an appropriate number of blank samples
and calculating the standard deviation of the-
se responses.
(2) Based on the Calibration Curve
A specific calibration curve should be stud-
ied using samples, containing an analyte in
the range of QL. The residual standard devia-
tion of a regression line or the standard devia-
tion of y-intercepts of regression lines may be
used as the standard deviation.
D. Recommended Data
The quantification limit and the method used for
determining the quantification limit should be pre-
sented.
The limit should be subsequently validated by the
analysis of a suitable number of samples known to
be near or prepared at the quantification limit.
8. Robustness
The evaluation of robustness should be consid-
ered during the development phase and depends on
the type of procedure under study. It should show
the reliability of an analysis with respect to delib-
erate variations in method parameters.
If measurements are susceptible to variations in
analytical conditions, the analytical conditions
should be suitably controlled or a precautionary
statement should be included in the procedure. One
consequence of the evaluation of robustness
should be that a series of system suitability param-
eters (e.g., resolution test) is established to ensure
that the validity of the analytical procedure is
maintained whenever used.
A. Typical variation factors common for all an-
alytical procedures
- stability of analytical solutions,
- extraction time
B. Typical variation factors for liquid chroma-
tography
- influence of variations of pH in a mobile
phase,
- influence of variations in mobile phase com-
position,
- different columns (different lots or suppliers),
- temperature,
- flow rate
C. Typical variation factors for gas chromatog-
raphy
- change in columns (different lots and/or sup-
pliers),
- temperature,
- flow rate
9. System suitability testing
System suitability testing is an integral part of
many analytical procedures. The tests are based on
the concept that the equipment, electronics, analyt-
ical operations and samples to be analyzed consti-
tute an integral system that can be evaluated as
such. System suitability test parameters to be es-
tablished for a particular procedure depend on the
type of procedure being validated. See General
Tests for additional information.
Appendix 1) Validation characteristics applicable
to analytical procedures
- signifies that this characteristic is not normal-
ly evaluated
+ signifies that this characteristic is normally
evaluated
(1) in cases where reproducibility (see glossary)
has been performed, intermediate precision is
not needed
(2) lack of specificity of one analytical procedure
could be compressed by other supporting
analytical procedure(s)
(3) may be needed in some cases
8. Isoelectric focusing
General Principles
Isoelectric focusing (IEF) is a method of electro-
phoresis that separates proteins according to their isoe-
lectric points. Separation is carried out in a slab of
polyacrylamide or agarose gel that contains a mixture
of amphoteric electrolytes (ampholytes). When sub-
jected to an electric field, the ampholytes migrate in
the gel to create a pH gradient. In some cases, gels
containing an immobilized pH gradient, prepared by
incorporating weak acids and bases to specific regions
of the gel network during the preparation of the gel,
are used. When the applied proteins reach the gel frac-
tion that has a pH that is the same as their isoelectric
point (pI), their charge is neutralized and migration
ceases. Gradients can be made over various ranges of
pH, according to the mixture of ampholytes chosen.
Theoretical Aspects
When a protein is at the position of its isoelectric
point, it has no net charge and cannot be moved in a
gel matrix by the electric field. It may, however, move
from that position by diffusion. The pH gradient forc-
es a protein to remain in its isoelectric point position,
thus concentrating it; this concentrating effect is called
“focusing”. The applied voltage is limited by the heat
generated, which must be dissipated. The use of thin
gels and an efficient cooling plate controlled by a
thermostatic circulator prevents the burning of the gel
whilst allowing sharp focusing. The separation is es-
 KP X 1863

timated by determining the minimum pI difference The time required for completion of focusing in
(ΔpI), which is necessary to separate 2 neighboring thin-layer polyacrylamide gels is determined by plac-
bands: ing a colored protein (e.g. hemoglobin) at different
positions on the gel surface and by applying the elec-
D  (dpH / dx) tric field: the steady state is reached when all applica-
R  pI  3  tions give an identical band pattern. In some protocols
E  (d / dpH ) the completion of the focusing is indicated by the time
elapsed after the sample application. The IEF gel can
D: Diffusion coefficient of the protein be used as an identity test when the migration pattern
dpH/dx: pH gradient on the gel is compared to a suitable standard prepara-
E: Intensity of the electric field, in volts per centi- tion and IEF calibration proteins, the IEF gel can be
meter used as a limit test when the density of a band on IEF
-dµ/dpH: Variation of the solute mobility with the is compared subjectively with the density of bands
pH in the region close to the pI appearing in a standard preparation, or it can be used
as a quantitative test when the density is measured
ASSAY using a densitometer or similar instrumentation to
Type of
Testing for - dissolu- determine the relative concentration of protein in the
analytical
impurities tion bands subject to validation.
procedure
(meas-
Identifi-
uremen Apparatus
cation
quan t only) An apparatus for IEF consists of :
characteris- - content
tific limit A controllable generator for constant potential, current
tics / po-
ation and power. Potentials of 2500 V have been used and
tency are considered optimal under a given set of operating
Accuracy - + - + conditions. Supply of up to 30 W of constant power is
Precision recommended,
Repeata- - + - + A rigid plastic IEF chamber that contains a cooled
bility - +(1) - +(1) plate, of suitable material, to support the gel,
Interm.Pr + + + + A plastic cover with platinum electrodes that are con-
ecision - -(3) + - nected to the gel by means of paper wicks of suitable
Specifici- - + - - width , length and thickness , impregnated with solu-
ty(2) - + - + tions of anodic and cathodic electrolytes .
Detection - + - +
Limit Isoelectric Focusing in Polyacrylamide Gels: De-
Quantifica- tailed Procedure
tion Limit The following method is a detailed description of
Linearity an IEF procedure in thick polyacrylamide slab gels,
Range which is used unless otherwise stated in the mono-
graph.
Since D and -dµ/dpH for a given protein cannot
be altered, the separation can be improved by using a Preparation of the Gels
narrower pH range and by increasing R, the intensity Mould The mould (see Figure) is composed of
of the electric field. Resolution between protein bands a glass plate (A) on which a polyester film (B) is
on an IEF gel prepared with carrier ampholytes can be placed to facilitate handling of the gel, one or more
quite good. Improvements in resolution may be spacers (C), a second glass plate (D) and clamps to
achieved by using immobilized pH gradients where hold the structure together .
the buffering species, which are analogous to carrier 7.5% Polyacrylamide gel Dissolve 29.1 g of
ampholytes, are copolymerized within the gel matrix. acrylamide and 0.9 g of N,N’-methylenebisacrylamide
Proteins exhibiting pIs differing by as little as 0.02 pH in 100 mL of water. To 2.5 volumes of this solution,
units may be resolved using a gel prepared with carrier add the mixture of ampholytes specified in the mono-
ampholytes while immobilized pH gradients can re- graph and dilute to 10 volumes with water. Mix care-
solve proteins differing by approximately 0.001 pH fully and degas the solution.
units.

Practical Aspects
Special attention must be paid to sample charac-
teristics and/or preparation. Having salt in the sample
can be problematic and it is best to prepare the sample,
if possible, in de-ionized water or 2% ampholytes,
using dialysis or gel filtration if necessary.
1864 Monographs, Part II

procedure may be made subject to validation. These

include:
(1) the use of commercially available pre-cast gels and
of commercial staining and destaining kits,
D (2) the use of immobilized pH gradients,
C (3) the use of disk gels,
B (4) the use of gel cassettes of different dimensions,
A including ultra-thin (0.2 mm) gels,
(5) variations in the sample application procedure,
including different sample volumes or the use of
Preparation of the mould Place the polyester
sample application masks or wicks other than paper ,
film on the lower glass plate, apply the spacer, place
(6) the use of alternate running conditions, including
the second glass plate and fit the clamps. Place 7.5%
variations in the electric field depending on gel di-
polyacrylamide gel prepared before use on a magnetic
mensions and equipment, and the use of fixed mi-
stirrer, and add 0.25 volumes of a solution of ammo-
gration times rather than subjective interpretation of
nium persulfate (1 in 10) and 0.25 volumes of
band stability ,
N,N,N’,N’-tetramethylethylenediamine. Immediately
(7) the inclusion of a pre-focusing step,
fill the space between the glass plates of the mould
(8) the use of automated instrumentation,
with the solution.
(9) the use of agarose gels.
Method
Dismantle the mould and, making use of the pol-
Validation of Iso-Electric Focusing Procedures
yester film, transfer the gel onto the cooled support,
Where alternative methods to the detailed proce-
wetted with 2-3 mL of a suitable liquid, taking care to
dure are employed they must be validated. The follow-
avoid forming air bubbles. Prepare the test solutions
ing criteria may be used to validate the separation:
and reference solutions as specified in the monograph.
(1) formation of a stable pH gradient of desired char-
Place strips of paper for sample application, about 10
acteristics, assessed for example using colored pH
mm × 5 mm in size, on the gel and impregnate each
markers of known isoelectric points ,
with the prescribed amount of the test and reference
(2) comparison with the electropherogram provided
solutions. Also apply the prescribed quantity of a solu-
with the chemical reference substance for the prepa-
tion of proteins with known isoelectric points as pH
ration to be examined,
markers. In some protocols, the gel has precast slots
(3) any other validation criteria as prescribed in the
where a solution of the sample is applied instead of
monograph.
using impregnated paper strips. Cut 2 strips of paper
to the length of the gel and impregnate them with the
Specified Variations to the General Method
electrolyte solutions (acid for the anode and alkaline
Variations to the general method required for the
for the cathode). The compositions of the anode and
analysis of specific substances may be specified in
cathode solutions are given in the monograph. Apply
detail in monographs. These include:
these paper wicks to each side of the gel several mil-
(1) the addition of urea in the gel (3 mol/L concentra-
limeters from the edge. Fit the cover so that the elec-
tion is often satisfactory to keep protein in solution but
trodes are in contact with the wicks (respecting the
up to 8mol/L can be used): some proteins precipitate
anodic and cathodic poles). Proceed with the isoelec-
at their isoelectric point. In this case, urea is included
tric focusing by applying the electrical parameters
in the gel formulation to keep the protein in solution.
described in the monograph. Switch off the current
If urea is used, only fresh solutions should be used to
when the migration of the mixture of standard proteins
prevent carbamylation of the protein,
has stabilized. Using forceps, remove the sample ap-
(2) the use of alternative staining methods,
plication strips and the 2 electrode wicks. Immerse the
(3) the use of gel additives such as non-ionic deter-
gel in ‘fixing solution for isoelectric focusing in poly-
gents (e.g. octylglucoside) or zwitterionic detergents
acrylamide gel’. Incubate with gentle shaking at room
[e.g., 3-[(3- cholamido propyl)dimethylammonio]-1-
temperature for 30 minutes. Drain off the solution and
propane sulfonate (CHAPS) or 3-[(3-
add 00 mL of ‘destaining solution’. Incubate with
cholamidopropyl)dimethyl ammonio]-2-hydroxy-1-
shaking for 1hour. Drain the gel, add ‘coomassie
propanesulfate (CHAPSO)], and the addition of
staining TS’. Incubate for 30 minutes. Destain the gel
ampholyte to the sample, to prevent proteins from
by passive diffusion with ‘destaining solution’ until
aggregating or precipitating.
the bands are well visualized against a clear back-
Points to Consider
ground. Locate the position and intensity of the bands
Samples can be applied to any area on the gel,
in the electropherogram as prescribed in the mono-
but to protect the proteins from extreme pH environ-
graph.
ments samples should not be applied close to either
Variations to the Detailed Procedure (Subject to Vali-
electrode. During method development the analyst can
dation)
try applying the protein in 3 positions on the gel (i.e.
Where reference to the general method on isoe-
middle and both ends); the pattern of a protein applied
lectric focusing is made, variations in methodology or

at opposite ends of the gel may not be identical.
A phenomenon known as ‘cathodic drift’, where
the pH gradient decays over time, may occur if a gel is
focused too long. Although not well understood,
electroendoosmosis and absorption of carbon dioxide
may be factors that lead to cathodic drift. Cathodic
drift is observed as focused protein migrating off the
cathode end of the gel. Immobilized pH gradients may
be used to address this problem.
Efficient cooling (approximately 4 °C) of the bed
that the gel lies on during focusing is important. High
field strengths used during isoelectric focusing can
lead to overheating and affect the quality of the fo-
cused gel.
Reagents and Solutions
Fixing solution for isoelectric focusing in poly-
acrylamide gel Dissolve 35 g of 5-sulfosalicylic
acid dihydrate and 100 g of trichloroacetic acid in
water to make 1000 mL.
Coomassie staining TS Dissolve 125 mg of
coomassie brilliant blue R-250 in 100 mL of a mixture
of water, methanol and acetic acid (100) (5:4:1), and
filter.
Destaining solution A mixture of water, metha-
nol and acetic acid (100) (5:4:1).
9. Particle Size
Determination
Particle Size Determination is a method to deter-
mine directly or indirectly morphological appearance,
shape, size and its distribution of powdered pharma-
ceutical drugs and excipients to examine their
micromeritic properties. Optical microscopy or analyt-
ical sieving method is used according to the measuring
purpose and the properties of the test specimen.
Method 1. Optical Microscopy
The optical microscopy is used to observe the mor-
phological appearance and shape of individual particle
either directly with the naked eye or by using a micro-
scopic photograph, in order to measure the particle
size. The particle size distribution can also be deter-
mined by this method. It is also possible with this
method to measure the size of the individual particle
even then different kinds of particles mingle if they
are optically distinguishable. Data processing tech-
niques, such as image analysis, can be useful for de-
termining the particle size distribution.
This method for particle characterization can gener-
ally be applied to particles not less than 1 μm. The
lower limit is imposed by the resolving power of the
microscope. The upper limit is less definite and is de-
termined by the increased difficulty associated with
the characterization of large particles. Various alterna-
tive techniques are available for particle characteriza-
tion, outside the applicable range of optical microsco-
KP X 1865
py. Optical microscopy is particularly useful for char-
acterizing particles that are not spherical. This method
may also serve as a base for the calibration of faster
and more routine methods that may be developed.
Apparatus - Use a microscope that is stable and pro-
tected from vibration. The microscope magnification
(product of the objective magnification, ocular magni-
fication, and additional magnifying components) must
be sufficient to allow adequate characterization of the
smallest particles to be classified in the test specimen.
The greatest numerical aperture of the objective
should be sought for each magnification range. Polar-
izing filters may be used in conjunction with suitable
analyzers and retardation plates. Color filters of rela-
tively narrow spectral transmission should be used
with achromatic objectives and are preferable with
apochromats and are required for appropriate color
rendition in photomicrography. Condensers corrected
for at least spherical aberration should be used in the
microscope substage and with the lamp. The numeri-
cal aperture of the substage condenser should match
that of the objective under the condition of use, in the
other words this is affected by the actual aperture of
the condenser diaphragm and the presence of immer-
sion oils.
Adjustment - The precise alignment of all elements
of the optical system and proper focusing are essential.
The focusing of the elements should be done in ac-
cordance with the recommendations of the microscope
manufacturer. Critical axial alignment is recommend-
ed.
Illumination - A requirement for good illumination
is a uniform and adjustable intensity of light over the
entire field of view; Kohler illumination is preferred.
With colored particles, choose the color of the filters
used so as to control the contrast and detail of the im-
age.
Visual Characterization - The magnification and
numerical aperture should be sufficiently high to allow
adequate resolution of the images of the particles to be
characterized. Determine the actual magnification
using a calibrated stage micrometer to calibrate an
ocular micrometer. Errors can be minimized if the
magnification is sufficient that the image of the parti-
cle is at least 10 ocular divisions. Each objective must
be calibrated separately. The ocular scale, the stage
micrometer scale and the ocular scale should be
aligned to calibrate. In this way, a precise determina-
tion of the distance between ocular stage divisions can
be made.
When the particle size is measured, an ocular mi-
crometer is inserted at the position of the ocular dia-
phragm, and a calibrated stage micrometer is placed at
the center of the microscope stage and fixed in place.
The ocular is attached to the lens barrel and adjusted
to the focus point of the stage micrometer scale. Then,
the distance between the scales of the two microme-
ters is determined, and the sample size equivalent 1
division of the ocular scale is calculated using the fol-
1866 Monographs, Part II
lowing formula:
The particle size equivalent 1 division on the ocular
scale (m) = Length on the stage micrometer
(m)/Number of scale divisions on the ocular mi-
crometer
The stage micrometer is removed and the test speci-
men is placed on the microscope stage. After adjusting
the focus, the particle sizes are determined from the
number of scale divisions read through the ocular.
Several different magnifications may be necessary to
characterize materials having a wide particle size dis-
tribution.
Photographic Characterization - If particle size is
to be determined by photographic methods, take care
to ensure that the object is sharply focused at the plane
of the photographic emulsion. Determine the actual
magnification by photographing a calibrated stage
micrometer, using photographic film of sufficient
speed, resolving power, and contrast. Exposure and
processing should be identical for photographs of both
the test specimen and the determination of magnifica-
tion. The apparent size of a photographic image is
influenced by the exposure, development, and printing
processes as well as by the resolving power of the
microscope.
Preparation of the Mount - The mounting medi-
um will be selected according to the physical proper-
ties of the test specimen. Sufficient, but not excessive,
contrast between the specimen and the mounting me-
dium is required to ensure adequate detail of the spec-
imen edge. The particles should rest in one plane and
be adequately dispersed to distinguish individual par-
ticles of interest. Furthermore, the particles must be
representative of the distribution of sizes in the mate-
rial and must not be altered during preparation of the
mount. Selection of the mounting medium must in-
clude a consideration of the analyte solubility.
Crystallinity characterization - The crystallinity
of a material may be characterized to determine com-
pliance with the crystallinity requirement where stated
in the individual monograph of a drug substance. Un-
less otherwise specified in the individual monograph,
mount a few particles of the specimen in mineral oil
on a clean glass slide. Examine the mixture using a
polarizing microscope: the particles show birefrin-
gence (interference colors) and extinction positions
when the microscope stage is revolved.
Limit Test of Particle Size by Microscopy -
Weigh a suitable quantity of the power to be examined
(for example, 10 to 100 mg), and suspend it in 10 mL
of a suitable medium in which the power does not
dissolve, adding, if necessary, a wetting agent. A ho-
mogeneous suspension of particles can be maintained
by suspending the particles in a medium of similar or
matching density and by providing adequate agitation.
Introduce a portion of the homogeneous suspension
into a suitable counting cell, and scan under a micro-
scope an area corresponding to not less than 10 g of
the powder to be examined. Count all the particles
having a maximum dimension greater than the pre-
scribed size limit. The size limit and the permitted
number of particles exceeding the limit are defined for
each substance.
Particle Size Characterization - The measurement
of particle size varies in complexity depending on the
shape of the particle and the number of particles char-
acterized must be sufficient to insure an acceptable
level of uncertainty in the measured parameters1). For
irregular particles, a variety of definitions of particle
size exist. In general, for irregularly shaped particles,
characterization of particle size must also include in-
formation on the type of diameter measured as well as
information on particle shape. Several commonly used
measurements of particle size are defined below (see
Fig. 1):
Fig. 1 Commonly used measurements of particle size
Feret's Diameter - The distance between imaginary
parallel lines tangent to a randomly oriented particle
and perpendicular to the ocular scale.
Martin's Diameter - The diameter of the particle at
the point that divides a randomly oriented particle into
two equal projected areas.
Projected area Diameter - The diameter of a circle
that has the same projected are as the particle.
Length - The longest dimension from edge to edge of
a particle oriented parallel to the ocular scale.
Width - The longest dimension of the particle meas-
ured at right angles to the length.
Particle Shape Characterization - For irregularly
shaped particles, characterization of particle size must
also include information on particle shape. The homo-
geneity of the powder should be checked using appro-
priate magnification. The following defines some
commonly used descriptors of particle shape (see Fig.
2):

Fig. 2 Commonly used descriptions of particle shape
Acicular - Slender, needle-like particle of similar
width and thickness.
Columnar - Long, thin particle with a width and
thickness that are greater than those of an acicular
particle.
Flake - Thin, flat particle of similar length and width.
Plate - Flat particles of similar length and width but
with greater thickness than flakes.
Lath - Long, thin, and blade-like particle
Equant - Particles of similar length, width and thick-
ness; both cubical and spherical particles are included.
General Observations - A particle is generally
considered to be the smallest discrete unit. A particle
may be a liquid or semisolid droplet; a single crystal
or polycrystalline; amorphous or an agglomerate. Par-
ticles may be associated. This degree of association
may be described by the following terms:
Lamellar - Stacked plates.
Aggregate - Mass of adhered particles.
Agglomerate - Fused or cemented particles.
Conglomerate - Mixture of two or more types of par-
ticles.
Spherulite - Radical cluster.
Drusy - Particle covered with tiny particles.
Particle condition may be described by the following
terms
Edges - Angular, rounded, smooth, sharp, fractured.
Optical - Color (using proper color balancing filters),
transparent, translucent, opaque.
Defects - Occlusions, inclusions.
Surface characteristics may be described as:
Cracked - Partial split, break, or fissure.
Smooth - Free of irregularities, roughness, or projec-
tions.
Porous - Having openings or passageways.
Rough - Bumpy, uneven, not smooth.
Pitted - Small indentations.
Method 2. Analytical Sieving Method
The analytical sieving method is a method to esti-
mate the particle size distribution of powered pharma-
ceutical drugs by sieving. The particle size determined
by this method is shown as the size of a minimum
sieve opening through which the particle passes.
Sieving is one of the oldest methods of classifying
powders and granules by particle size distribution.
KP X 1867
When using a woven sieve cloth, the sieving will es-
sentially sort the particles by their intermediate size
dimension (i.e., breadth or width). Mechanical sieving
is most suitable where the majority of the particles are
larger than about 75 m. For smaller particles, the
light weight provides insufficient force during sieving
to overcome the surface forces of cohesion and adhe-
sion that cause the particles to stick to each other and
to the sieve, and thus cause particles that would be
expected to pass through the sieve to be retained. For
such materials other means of agitation such as air-jet
sieving or sonic sifting may be more appropriate.
Nevertheless, sieving can sometimes be used for some
powders or granules having median particle sizes
smaller than 75 m where the method can be validated.
In pharmaceutical terms, sieving is usually the method
of choice for classification of the coarser grades of
single powders or granules. It is a particularly attrac-
tive method in that powders and granules are classi-
fied only on the basis of particle size, and in most cas-
es the analysis can be carried out in the dry state.
Among the limitations of sieving method are the
need for an appreciable amount of sample (normally at
least 25 g, depending on the density of the powder or
granule, and the diameter of test sieves) and difficulty
in sieving oily or other cohesive powders or granules
that tend to clog the sieve openings. The method is
essentially a two-dimensional estimate of size because
passage through the sieve aperture is frequently more
dependent on maximum width and thickness than on
length.
This method is intended for estimation of the total
particle size distribution of a single material. It is not
intended for determination of the proportion of parti-
cles passing or retained on one or two sieves.
Estimate the particle size distribution as described
under Dry Sieving Method, unless otherwise specified
in the individual monograph. Where difficulty is expe-
rienced in reaching the endpoint (i.e., material does
not readily pass through the sieves) or when it is nec-
essary to use the finer end of the sieving range (below
75 m), serious consideration should be given to the
use of an alternative particle-sizing method.
Sieving should be carried out under conditions that
do not cause the test sample to gain or lose moisture.
The relative humidity of the environment in which the
sieving is carried out should be controlled to prevent
moisture uptake or loss by the sample. In the absence
of evidence to the contrary, analytical test sieving is
normally carried at ambient humidity. Any special
conditions that apply to a particular material should be
detailed in the individual monograph.
Principles of Analytical Sieving - Analytical test
sieves are constructed from a woven-wire mesh,
which is of simple weave that is assumed to give near-
ly square apertures and is sealed into the base of an
open cylindrical container. The basic analytical meth-
od involves stacking the sieves on top of one another
in ascending degrees of coarseness, and then placing
the test powder on the top sieve.
1868 Monographs, Part II
The nest of sieves is subjected to a standardized peri-
od of agitation, and then the weight of material re-
tained on each sieve is accurately determined. The test
gives the weight percentage of powder in each sieve
size range.
This sieving process for estimating the particle size
distribution of a single pharmaceutical powder is gen-
erally intended for use where at least 80 % of the par-
ticles are larger than 75 m. The size parameter in-
volved in determining particle size distribution by
analytical sieving is the length of the side of the min-
imum square aperture through which the particle will
pass.
TEST SIEVES
Unless otherwise specified in the monograph, use the
sieves listed in the Table 1.
Sieves are selected to cover the entire range of parti-
cle sizes present in the test specimen. A nest of sieves
having a 2 progression of the area of the sieve
openings is recommended. The nest of sieves is as-
sembled with the coarsest screen at the top and the
finest at the bottom. Use micrometers or millimeters in
denoting test sieve openings. [Note - Mesh numbers
are provided in the table for conversion purposes only.]
Test sieves are made from stainless steel or, less pref-
erably, from brass or other suitable non-reactive wire.
Calibration and recalibration of test sieves is in ac-
cordance with the most current edition of ISO 3310-
12). Sieves should be carefully examined for gross
distortions and fractures, especially at their screen
frame joints, before use. Sieves may be calibrated op-
tically to estimate the average opening size, and open-
ing variability, of the sieve mesh. Alternatively, for
the valuation of the effective opening of test sieves in
the size range of 212 to 850 m, Standard Glass
Spheres are available. Unless otherwise specified in
the individual monograph, perform the sieve analysis
at controlled room temperature and at ambient relative
humidity.
Cleaning Test Sieves - Ideally, test sieves should
be cleaned using only an air jet or a liquid stream. If
some apertures remain blocked by test particles, care-
ful gentle brushing maybe used as a last resort.
Test Specimen - If the test specimen weight is not
given in the monograph for a particular material, use a
test specimen having a weight between 25 and 100 g,
depending on the bulk density of the material, and test
sieves having a 200 mm diameter. For 76 mm sieves
the amount of material that can be accommodated is
approximately 1/7th that which can be accommodated
on a 200 mm sieve. Determine the most appropriate
weight for a given material by test sieving accurately
weighed specimens of different weights, such as 25,
50, and 100 g, for the same time period on a mechani-
cal shaker. [Note - If the test results are similar for the
25 g and 50 g specimens, but the 100 g specimen
shows a lower percentage through the finest sieve, the
100-g specimen size is too large.] Where only a spec-
imen of 10 to 25 g is available, smaller diameter test
sieves conforming to the same mesh specifications
(table 1) may be substituted, but the endpoint must be
re-determined. The use of test samples having a small-
er mass (e. g. down to 5 g) may be needed. For mate-
rials with low apparent particle density, or for materi-
als mainly comprising particles with a highly iso-
diametrical shape, specimen weights below 5 g for a
200 mm screen may be necessary to avoid excessive
blocking of the sieve. During validation of a particular
sieve analysis method, it is expected that the problem
of sieve blocking will have been addressed.
If the test material is prone to picking up or losing
significant amounts of water with varying humidity,
the test must be carried out in an appropriately con-
trolled environment. Similarly, if the test material is
known to develop an electrostatic charge, careful ob-
servation must be made to ensure that such charging is
not influencing the analysis. An antistatic agent, such
as colloidal silicon dioxide and/or aluminum oxide,
may be added at a 0.5 percent (m/m) level to minimize
this effect. If both of the above effects cannot elimi-
nated, an alternative particle-sizing technique must be
selected.
Agitation Methods - Several different sieve and
powder agitation devices are commercially available,
all of which may be used to perform sieve analyses.
However, the different methods of agitation may give
different results for sieve analyses and endpoint de-
terminations because of the different types and magni-
tude of the forces acting on the individual particles
under test. Methods using mechanical agitation or
electromagnetic agitation, and that can include either a
vertical oscillation or a horizontal circular motion, or
tapping or a combination of both tapping and horizon-
tal circular motion are available. Entrainment of the
particles in an air stream may also be used. The results
must indicate which agitation method was used and
the agitation parameters used (if they can be varied).
End point Determination - The test sieving analy-
sis is complete when the weight on any of the test
sieves does not change by more than 5 % or 0.1 g (10 %
in the case of 76 mm sieves) of the previous weight on
that sieve. If less than 5 % of the total specimen
weight is present on a given sieve, the endpoint for
that sieve is increased to a weight change of not more
than 20 % of the previous weight on that sieve. If
more than 50 % of the total specimen weight is found
on any one sieve, unless this is indicated in the mono-
graph, the test should be repeated, but with the addi-
tion to the sieve nest of a more coarse sieve intermedi-
ate between that carrying the excessive weight and the
next coarsest sieve in the original nest, i.e., addition of
the ISO series sieve omitted from the nest of sieves.
SIEVING METHODS
Mechanical agitation
Dry Sieving Method - Tare each test sieve to the
nearest 0.1 g. Place an accurately weighed quantity of

test specimen on the top (coarsest) sieve, and replace
the lid. Agitate the nest of sieves for 5 minutes. Then
carefully remove each from the nest without loss of
material. If there is some fine powder on the down
surface of each sieve, take if off by the brush gently,
and combine it with the sieve fraction retained on each
next down sieve. Reweigh each sieve, and determine
the weight of material on each sieve. Determine the
weight of material in the collecting pan in a similar
manner. Reassemble the nest of sieves, and agitate for
5 minutes. Remove and weigh each sieve as previous-
ly described. Repeat these steps until the endpoint
criteria are met (see Endpoint Determination under
Test Sieves). Upon completion of the analysis, recon-
cile the weights of material. Total losses must not ex-
ceed 5 % of the weight of the original test specimen.
Repeat the analysis with a fresh specimen, but using
a single sieving time equal to that of the combined
times used above. Confirm that this sieving time con-
forms to the requirements for endpoint determination.
When this endpoint has been validated for a specific
material, then a single fixed time of sieving may be
used for future analyses, providing the particle size
distribution falls within normal variation.
If there is evidence that the particles retained on any
sieve are aggregates rather than single particles, the
use of mechanical dry sieving is unlikely to give good
reproducibility, a different particle size analysis meth-
od should be used.
Air Entrainment Methods
Air jet and Sonic Shifter Sieving - Different types
of commercial equipment that use a moving air cur-
rent are available for sieving. A system that uses a
single sieve at a time is referred to as air jet sieving. It
uses the same general sieving methodology as that
described under the Dry Sieving Method, but with a
standardized air jet replacing the normal agitation
mechanism. It requires sequential analyses on individ-
ual sieves starting with the finest sieve to obtain a par-
ticle size distribution. Air jet sieving often includes the
use of finer test sieves than used in ordinary dry siev-
ing. This technique is more suitable where only over-
size or undersize fractions are needed.
ISO Nominal Aperture
US Sieve
Principal sizes Supplementary sizes No.
R 20/3 R 20 R 40/3
11.20 mm 11.20 mm 11.20 mm
10.00 mm
9.50 mm
9.00 mm
8.00 mm 8.00 mm 8.00 mm
7.10 mm
6.70 mm
6.30 mm
5.60 mm 5.60 mm 5.60 mm
5.00 mm
4.75 mm
KP X 1869
In the sonic sifting method a nest of sieves is used,
and the test specimen is carried in a vertically oscillat-
ing column of air that lifts the specimen and then car-
ries it back against the mesh openings at a given num-
ber of pulses per minute. It may be necessary to lower
the sample amount to 5 g, when sonic shifting is em-
ployed.
The air jet sieving and sonic sieving methods may be
useful for powders or granules when mechanical siev-
ing techniques are incapable of giving a meaningful
analysis.
These methods are highly dependent upon proper
dispersion of the powder in the air current. This re-
quirement may be hard to achieve if the method is
used at the lower end of the sieving range (i. e., below
75m), when the particles tend to be more cohesive,
and especially if there is any tendency for the material
to develop an electrostatic charge. For the above rea-
sons endpoint determination is particularly critical,
and it is very important to confirm that the oversize
material comprises single particles and is not com-
posed of aggregates.
INTERPRETATION
The raw data must include the weight of test speci-
men, the total sieving time, and the precise sieving
methodology and the set values for any variable pa-
rameters, in addition to the weights retained on the
individual sieves and in the pan. It may be convenient
to convert the raw data into a cumulative weight dis-
tribution in terms of a cumulative weight undersize,
the range of sieves used should include a sieve
through which all the material passes. If there is evi-
dence on any of the test sieves that the material re-
maining on it is composed of aggregates formed dur-
ing the sieving process, the analysis is invalid.
1)
Additional information on particle size measurement,
sample size, and data analysis is available, for exam-
ple, in ISO 9276.
2)
International Organization for Standardization (ISO)
Specification ISO 3310-1; Test sieves-Technical re-
quirements and testing
Recommended
European Korean
USP
Sieve No. Sieve No.
Sieves(mesh)
11200
5600 3.5
4
1870 Monographs, Part II

4.50 mm
4.00 mm 4.00 mm 4.00 mm 5 4000 4000 4.7
3.55 mm
3.35 mm 6 5.5
3.15 mm
2.80 mm 2.80 mm 2.80 mm 7 2800 2800 6.5
2.50 mm
2.36 mm 8 7.5
2.24 mm
2.00 mm 2.00 mm 2.00 mm 10 2000 2000 8.6
1.80 mm
1.70 mm 12 10
1.60 mm
1.40 mm 1.40 mm 1.40 mm 14 1400 1400 12
1.25 mm
1.18 mm 16 14
1.12 mm
1.00 mm 1.00 mm 1.00 mm 18 1000 1000 16
900 m
850 m 20 18
800 m
710 m 710 m 710 m 25 710 710 22
630 m
600 m 30 26
560 m
500 m 500 m 500 m 35 500 500 30
450 m
425 m 40 36
400 m
355 m 355 m 355 m 45 355 355 42
315 m
300 m 50 50
280 m
250 m 250 m 250 m 60 250 250 60
224 m
212 m 70 70
200 m
180 m 180 m 180 m 80 180 180 83
160 m
150 m 100 100
140 m
125 m 125 m 125 m 120 125 125 119
112 m
106 m 140 140
100 m
90 m 90 m 90 m 170 90 90 166
80 m
75 m 200 200
71 m
63 m 63 m 63 m 230 63 63 235
56 m
53 m 270 282
50 m
45 m 45 m 45 m 325 45 45 330
40 m
38 m 38 391

10. Powder Particle Density
Determination
The Powder Particle Density Determination is a
method to determine particle density of powdered
pharmaceutical drugs or raw materials of drugs, and
the gas displacement pycnometer is generally used.
The powder density by this method is determined with
an assumption that the volume of the gas displaced by
the powder in a closed system is equal to the volume
of the powder. The bulk density at loose packing or
the tapped density at tapping express the apparent
densities of the powder, since interparticular void vol-
ume of the powder is considered to be a part of the
volume of the powder. On the contrary, the
pycnometric particle density expresses the powder
density nearly equal to the crystal density, since the
volume of the powder, that is deducted with void vol-
ume of open pores accessible to gas, is counted.
Powder particle density is expressed in mass per
unit volume (kg/m3), and generally expressed in g/cm3.
Apparatus
The schematic diagram of particle density appa-
ratus for gas displacement pycnometric measurement
is shown in Figure. The apparatus consists of a test
cell in which the sample is placed, a reference cell and
a manometer.
Generally, helium is used as the measurement gas.
The apparatus has to be equipped with a system capa-
ble of pressuring the test cell to the defined pressure
through the manometer.
Calibration of apparatus The volumes of the
test cell (Vc) and the reference cell (Vr) must be accu-
rately determined to the nearest 0.001 cm3. In order to
assure accuracy of the results of volume obtained,
calibration of the apparatus is carried out as follows
using a calibration ball of known volume for particle
density measurement. The final pressure (Pf) are de-
termined for the initial empty test cell followed by the
test cell placed with the calibration ball for particle
density measurement in accordance with the proce-
dures, and Vc and Vr are calculated using the equation
described in the section of Procedure. Calculation can
be made taking into account that the sample volume
(Vs) is zero in the first run.
Vr: Reference cell volume (cm3)
Vc: Test cell volume (cm3)
Vs: Sample volume (cm3)
M : Manometer (cm3)
Figure. Schematic diagram of a gas
displacement pycnometer used to
determine powder particle density
Procedure
The measurement of the particle density is carried
KP X 1871
out between 15 and 30 C, and the temperature must
not vary by more than 2 C during the course of
measurement.
Firstly, weigh the mass of the test cell and record it.
After weighing out the amount of the sample as de-
scribed in the individual monograph and placing it in
the test cell, seal the cell in the pycnometer. Secondly,
introduce the measurement gas (helium) into the test
cell, in order to remove volatile contaminants in the
powder. If necessary, keep the sample powder under
reduced pressure to remove the volatile contaminants
in advance and use it as the test sample for measure-
ment.
Open the valve which connects the reference cell
with the test cell, confirm with manometer that the
pressure inside the system is stable, and then read the
system reference pressure (Pr). Secondly, close the
valve that connects to the two cells, and introduce the
measurement gas into the test cell to achieve positive
pressure. Confirm with the manometer that the pres-
sure inside is stable, and then read the initial pressure
(Pi). Open the valve to connect test cell with the refer-
ence cell. After confirming that the indicator of the
manometer is stable, read the final pressure (Pf). Cal-
culate the sample volume (Vs) with the following
equation.
Vr
Vs  Vc 
Pi  Pr
1
Pf  Pr
Vr: Reference cell volume (cm3)
Vc: Test cell volume (cm3)
Vs: Sample volume (cm3)
Pi: Initial pressure (kPa)
Pf: Final pressure (kPa)
Pr: Reference pressure (kPa)
Repeat the measurement sequence for the same
powder sample until consecutive measurements of the
sample volume agree to within 0.5 %, and calculate
the mean of sample volumes (Vs). Finally, unload the
test cell, weigh the mass of test cell, and calculate the
final sample mass by deducing the empty cell mass
from the test cell mass. The powder particle density ρ
is calculated by the following equation.
m

Vs
ρ: Powder particle density (g/ cm3)
m: Final sample mass (g)
Vs: Sample volume (cm3)
1872 Monographs, Part II
11. Preservatives-Effectiveness
Tests
The purpose of the Preservatives-Effectiveness
Tests is to assess microbiologically the preservative
efficacy, either due to the action of product compo-
nents themselves or any added preservatives for mul-
tiple containers. The efficacy of the preservatives is
assessed by direct inoculation and mixing of the test
strains in the product, and titration of survival of the
test strains with time.
Preservatives must not be used solely to comply
with GMP for drugs or to reduce viable aerobic counts.
In fact, preservatives themselves are toxic substances.
Therefore, preservatives must not be added to prod-
ucts in amounts which might jeopardize the safety of
human beings, and consideration must be given to
minimizing the amounts of preservatives used. These
tests are commonly used to verify that products main-
tain their preservative effectiveness at the design
phase of formulation or in the case of periodic moni-
toring. Although these tests are not performed for lot
releasing testing, the efficacy of the preservative pre-
sent in the product packaged in the final containers
should be verified throughout the entire dating period.
1. Products and Their Categories
The products have been divided into two catego-
ries for these tests. Category I products are those made
with aqueous bases or vehicles, and Category II prod-
ucts are those made with nonaqueous bases or vehicles.
Oil-in-water emulsions are considered Category I
products, and water-in-oil emulsions Category II
products. Category I is further divided into three sub-
types depending on the dosage form.
Category IA: Injections and other sterile
parenterals
Category IB: Nonsterile parenterals
Category IC: Oral products made with aqueous
bases (including syrup products to be dissolved or
suspended before use)
Category II: All the dosage forms listed under Cat-
egory I made with nonaqueous bases or vehicles.
2. Test Microorganisms and Culture Media
The following strains or those considered to be
equivalent are used as the test microorganisms.
Escherichia coli ATCC 8739, NBRC 3972, KCTC
2571
Pseudomonas aeruginosa ATCC 9027, NBRC
13275, KCTC 2513
Staphylococcus aureus ATCC 6538, NBRC 13276,
KCTC 1916, 1927 or 3881
Candida albicans ATCC 10231, NBRC 1594,
JCM 2085, KCTC 7965
Asperigillus niger ATCC 16404, NBRC 9455,
KCTC 6196 or 6317
These test microorganisms are representative of
those that might be found in the environment in which
the product is manufactured, used or stored, and they
are also recognized as opportunistic pathogens. In
addition to these strains designated as test microorgan-
isms, it is further recommended to use strains that
might contaminate the product and grow on or in it,
depending on its characteristics. For the test microor-
ganisms received from coordinated collections of mi-
croorganisms, one passage is defined as the transfer of
microorganisms from an established culture to a fresh
medium, and microorganisms subjected to not more
than 5 passages should be used for the tests. Single-
strain challenges rather than mixed cultures should be
used. The test strains can be harvested by growth on
solid agar or liquid media.
Culture on agar plate media: Inoculate each of
the five test strains on the surface of agar plates or
agar slants. For growth of bacteria, use Soybean-
Casein Digest Agar Medium, and for yeasts and
moulds, use Sabouraud Agar, Glucose-Peptone Agar
or Potato Dextrose Agar Medium. Incubate bacterial
cultures at 30 C to 35 C for 18 to 24 hours, the cul-
ture of C. albicans at 20 C to 25 C for 40 to 48
hours and the culture of A. niger at 20 C to 25 C for
one week or until good sporulation is obtained. Har-
vest these cultured cells aseptically using a platinum
loop, etc. Suspend the collected cells in sterile physio-
logical saline or in 0.1 % peptone water and adjust the
viable cell count to about 108 microorganisms per mL.
In the case of A. niger, suspend the cultured cells in
sterile physiological saline or 0.1 % peptone water
containing 0.05 w/v% of polysorbate 80 and adjust the
spore count to about 108 per mL. Use these suspen-
sions as the inocula.
Liquid cultures: After culturing each of the four
strains except for A. niger in a suitable medium, re-
move the medium by centrifugation. Wash the cells in
sterile physiological saline or 0.1 % peptone water and
resuspend them in the same solution with the viable
cell or spore count of the inoculum adjusted to about
108 per mL.
When strains other than the five listed above are
cultured, select a culture medium suitable for growth
of the strain concerned. The cell suspension may also
be prepared by a method suitable for that strain. Use
the inoculum suspensions within 24 hours after they
have been prepared from the cultivations on agar plate
media or in liquid media. Store the inoculum suspen-
sions in a refrigerator if it is not possible to inoculate
them into the test specimen within 2 hours. Titrate the
viable cell count of the inocula immediately before
use, and then calculate the theoretical viable cell count
per mL (g) of the product present just after inoculation.
3. Test Procedure
3.1. Category I products
Inject each of the cell suspensions aseptically into
five containers containing the product and mix uni-
formly. When it is difficult to inject the cell suspen-
 KP X 1873

sion into the container aseptically or the volume of the miscibility between the liquid medium and semisolid
product in each container is too small to be tested, ointments or oils in which test microorganisms were
transfer aseptically a sufficient volume of the product inoculated. Sometimes, these agents also serve to inac-
into each of alternative sterile containers, and mix the tivate or neutralize many of the most commonly used
inoculum. When the product is not sterile, incubate preservatives.
additional containers containing the uninoculated
product as controls and calculate their viable cell 4. Interpretation
counts (the viable counts of bacteria and those of Interpret the preservative efficacy of the product
yeasts and moulds). A sterile syringe, spatula or glass according to Table 1. When the results described in
rod may be used to mix the cell suspension uniformly Table 1 are obtained, the product examined is consid-
in the product. The volume of the suspension mixed in ered to be effectively preserved. There is a strong pos-
the product must not exceed 1/100 of the volume of sibility of massive microbial contamination having
the product. Generally, the cell suspension is inoculat- occurred when microorganisms other than the inocu-
ed and mixed so that the concentration of viable cells lated ones are found in the sterile product to be exam-
is 105 to 106 cells per mL or per gram of the product. ined, and caution is required in the test procedures
Incubate these inoculated containers at 20 C to 25 C and/or the control of the manufacturing process of the
with protection from light, and calculate the viable cell product. When the contamination level in a non-sterile
count of 1 mL or 1 g of the product taken at 0, 14 and product to be examined exceeds the microbial enu-
28 days subsequent to inoculation. Record any marked meration limit specified in “Microbial
changes (e.g., changes in color or the development of
a bad odor) when observed in the mixed samples dur- Table 1. Interpretation criteria by product category
ing this time. Such changes should be considered
when assessing the preservative efficacy of the prod- Interpretation criteria
Prod Microor-
uct concerned. Express sequential changes in the via- After 14 days After 28 days
cate ganisms
ble counts as percentages, with the count at the start of
the test taken as 100. Titration of the viable cell counts 0.1 % of in- Same or less
is based, in principle, on the Pour Plate Methods in Cat- Bacteria oculum count than level after
“Microbial Limit Test”. In this case, confirm whether egory or less 14 days
any antimicrobial substance is present in the test spec- IA Yeasts Same or less Same or less
imen. If a confirmed antimicrobial substance needs to /moulds than inoculum than inoculum
be eliminated, incorporate an effective inactivator of 1count
% of inocu- Same or less
count
the substance in the buffer solution or liquid medium Cat- Bacteria lum count or than level after
to be used for dilution of the test specimen, as well as egory less 14 days
in the agar plate count medium. However, it is neces- IB Yeasts Same or less Same or less
sary to confirm that the inactivator has no effect on the /moulds than inoculum than inoculum
growth of the microorganisms. When the occurrence count
10 % of inocu- count
Same or less
of the preservative or the product itself affects titration Bacteria lum count or than level after
Cat-
of the viable cell count and there is no suitable less 14 days
egory
inactivator available, calculate the viable cell counts
IC Yeasts Same or less Same or less
by the Membrane Filtration method in “Microbial
/moulds than inoculum than inoculum
Limit Test”.
Bacteria countor less
Same count
Same or less
3.2. Category II products
The procedures are the same as those described for Cat- than inoculum than inoculum
Category I products. However, special procedures and egory count count
considerations are required for both uniform disper- II Yeasts Same or less Same or less
sion of the test microorganism in the product and titra- /moulds than inoculum than inoculum
tion of viable cell counts in the samples. countPharmaceutical
Attributes of Non-sterile count
Products” in
For semisolid ointment bases, heat the sample to General Information, caution is also required in the
45 C to 50 C until it becomes oily, add the cell sus- test procedures and/or the control of the manufactur-
pension and disperse the inoculum uniformly with a ing process of the product.
sterile glass rod or spatula. Surfactants may also be
added to achieve uniform dispersion, but it is neces- 5. Culture media
sary to confirm that the surfactant added has no effect Culture media and buffer solutions used for Pre-
on survival or growth of the test microorganisms and servatives-Effectiveness Tests are described below.
that it does not potentiate the preservative efficacy of Other media may be used if they have similar nutritive
the product. For titration of the viable cell count, a ingredients and selective and growth-promoting prop-
surfactant or an emulsifier may be added to disperse erties for the microorganisms to be tested.
the product uniformly in the buffer solution or liquid Soybean Casein Digest Agar Medium
medium. For instance, sorbitan monooleate, Casein peptone 15 g
polysorbate 80 or lecithin may be added to improve Soybean peptone 5.0 g
1874 Monographs, Part II

Sodium chloride 5.0 g can be measured by a volumetric or continuous flow

Agar 15.0 g procedure.
Water 1000 mL
Mix all of the components and sterilize at 121 C Multi-Point Measurement
for 15 ~ 20 minutes in an autoclave. The medium pH When the gas is physically adsorbed by the pow-
after sterilization: 7.1 ~ 7.3. der sample, the following relationship (Brunauer,
Sabouraud  Glucose Agar Medium Emmett and Teller (BET) adsorption isotherm) holds
Peptone (animal tissue an casein) 10.0 g when the relative pressure (P/P0) is in the range of
Glucose 40.0 g 0.05 to 0.30 for pressure P of the adsorbate gas in
Agar 15.0 g equilibrium for the volume of gas adsorbed, Vα.
Water 1000 mL
Mix all of the components and sterilize at 121 C 1 (C  1) P 1
   (1)
for 15 ~ 20 minutes in an autoclave. The medium pH P Vm C P0 Vm C
after sterilization: 5.4 ~ 5.8. Va ( 0  1)
P
Glucose  Peptone (GP) Agar Medium
Glucose 20.0 g P: Patial vapour pressure of adsorbate gas in equilib-
Yeast extract 2.0 g rium with the surface at -195.8 ºC (b.p. of liquid
Magnesium sulfate heptahydrate 0.5 g nitrogen) (Pa)
Peptone 5.0 g P0: Saturated pressure of adsorbate gas (Pa)
Monobasic potassium phosphate 1.0 g Va: Volume of gas adsorbed at saturated temperature
Agar 15.0 g and pressure (STP) [0 ºC and atmospheric pressure
Water 1000 mL (1.013 × 105 Pa)] (mL)
Mix all of the components and sterilize at 121 C Vm: Volume of gas adsorbed at STP to produce ap-
for 15 ~ 20 minutes in an autoclave. The medium pH parent monolayer on the sample surface (mL)
after sterilization: 5.6 ~ 5.8. C: Dimensionless constant that is related to the en-
Potato  Dextrose Agar Medium thalpy of adsorption of adsorbate gas on the pow-
Potato extract 4.0 g der sample.
Glucose 20.0 g
Agar 15.0 g A value of Va is measured at each of not less than
Water 1000 mL 3 values of P/P0. Then the BET value, 1/[Va{P0/P-1}],
Mix all of the components and sterilize at 121 C is plotted against P/P0 according to equation (1). This
for 15 ~ 20 minutes in an autoclave. The medium pH plot should yield a straight line usually in the approx-
after sterilization: 5.4 ~ 5.8 imate relative pressure range 0.05 to 0.30. The data
0.1% Peptone Water are considered acceptable if the correlation coefficient,
Peptone 1.0 g r, of the linear regression is not less than 0.9975; that
Sodium chloride 8.0 g is, r2 is not less than 0.995. From the resulting linear
Water 1000 mL plot, the slope, which is equal to (C-1)/VmC, and the
Mix all of the components and sterilize at 121 C intercept, which is equal to 1/VmC, are evaluated by
for 15 ~ 20 minutes in an autoclave. The medium pH linear regression analysis. From these values, Vm is
after sterilization: 7.2 ~ 7.4. calculated as 1/(slope + intercept), while C is calculat-
ed as (slope/intercept) + 1. From the value of Vm so
determined, the specific surface area, S (m2/g) is cal-
12. Specific Surface Area culated by the equation:

Determination Method (V m Na)

S (2)
m  22400
The specific surface area determination method is
a method to determine specific surface area (the total N: Avogadro constant (6.022 × 1023 /mol)
surface area of powder per unit mass) of a pharmaceu- a: Effective cross-sectional area of one adsorbate
tical powder sample by using gas adsorption method. molecule (m2)
The specific surface area of a powder is determined by N2: 0.162 × 10-18
physical adsorption of a gas on the surface of the solid Kr: 0.195 × 10-18
and by calculating the amount of adsorbate gas corre- m: mass of test powder (g)
sponding to a monomolecular layer on the surface.
Physical adsorption results from relatively weak forc- Single-point measurement
es (van der Waals forces) between the adsorbate gas Normally, at least 3 measurements of Va each at
molecules and the adsorbent surface of the test powder. different values of P/P0 are required for the determina-
The determination is usually carried out at the temper- tion of specific surface area by the dynamic flow gas
ature of liquid nitrogen. The amount of gas adsorbed adsorption technique (Method I) or by volumetric gas
 KP X 1875

adsorption (Method II). However, under certain cir- samples, other outgassing methods such as the desorp-
cumstances described below, it may be acceptable to tion-adsorption cycling method may be employed.
determine the specific surface area of a powder from a The standard technique is the adsorption of nitro-
single value of Va measured at a single value of P/P0 gen at liquid nitrogen temperature.
such as 0.30, using the following equation for calcu- For powders of low specific area (< 0.2 m2/g) the
lating Vm: proportion adsorbed is low. In such cases the use of
krypton at liquid nitrogen temperature is preferred
P because the low vapor pressure exerted by this gas
Vm  Va (1  ) (3)
greatly reduces error.
P0
All gases used must be free from moisture.
Accurately weigh a quantity of the test powder
The single-point method may be employed di- such that the total surface of the sample is at least 1 m2
rectly for a series of powder samples of a given mate- when the adsorbate is nitrogen and 0.5 m2 when the
rial for which the material constant C is much greater adsorbate is krypton. Lower quantities of sample may
than 1. Close similarity between the single-point val- be used after appropriate validation.
ues and multiple-point values suggests that 1/C ap- Adsorption of gas should be measured either by
proaches zero. The error on Vm can be reduced by us- Method I or Method II.
ing the multiple-point method to evaluate C for one of
the samples of the series on which the material con- Method I: the dynamic flow method
stant C is expected to be large. Then Vm is calculated In the dynamic flow method (see Fig. 1), the rec-
from the single value of Va measured at a single value ommended adsorbate gas is dry nitrogen or krypton,
of P/P0 by the equation: while helium is employed as a diluents gas, which is
not adsorbed under the recommended conditions. A
P  1 C  1 P  minimum of 3 mixtures of the appropriate adsorbate
Vm  Va  0  1    (4)
P  C C P0  with helium are required within the P/P0 range 0.05 to
0.30.
Sample preparation
Before the specific surface area of the sample can
be determined, it is necessary to remove gases that
may have become physically adsorbed onto the sur-
face during storage and handling. If outgassing is not
achieved, the specific surface area may be reduced or
may be variable because an intermediate area of the
surface is covered with molecules of the previously
adsorbed gases or vapors. The outgassing conditions
are critical for obtaining the required precision and
accuracy of specific surface area measurements on
pharmaceuticals because of the sensitivity of the sur-
face of the materials. The outgassing conditions must
be demonstrated to yield reproducible BET plots, a
A: Flow control valve
constant weight of test powder, and no detectable
B: Differential flow controller
physical or chemical changes in the test powder.
C: On-off valve
The outgassing conditions defined by the temper-
D: Gas inlet
ature, pressure and time should be so chosen that the
E: O ring seals
original surface of the solid is reproduced as closely as
F: Cold trap
possible.
G: Thermal equilibration tube
Outgassing of many substances is often achieved
H: Detector
by applying a vacuum, by purging the sample in a
I: Digital display
flowing stream of a non-reactive, dry gas, or by apply-
J: Calibration septum
ing a desorption-adsorption cycling method. In either
K: Sample cell
case, elevated temperatures are sometimes applied to
L: Self seals quick connection
increase the rate at which the contaminants leave the
M: Short path ballast
surface. Caution should be exercised when outgassing
N: Detector
powder samples using elevated temperatures to avoid
O: Path selection valve
affecting the nature of the surface and the integrity of
P: Long pass ballast
the sample.
Q: Flow meter
If heating is employed, the recommended tem-
R: Outgassing station
perature and time of outgassing are as low as possible
S: Diffusion baffle
to achieve reproducible measurement of specific sur-
T: Vent
face area in an acceptable time. For heating sensitive
1876 Monographs, Part II
Fig. 1. Schematic diagram of the dynamic flow
method apparatus
The gas detector-integrator should provide a sig-
nal that is approximately proportional to the volume of
the gas passing through it under defined conditions of
temperature and pressure. For this purpose, a thermal
conductivity detector with an electronic integrator is
one among various suitable types. A minimum of 3
data points within the recommended range of 0.05 to
0.30 for P/P0 is to be determined.
A known mixture of the gases, usually nitrogen
and helium, is passed through a thermal conductivity
cell and then to a recording potentiometer.
Immerse the sample cell in liquid nitrogen, then
the sample adsorbs nitrogen from the mobile phase.
This unbalances the thermal conductivity cell, and a
pulse is generated on a recorder chart.
Remove from the coolant; this gives a desorption
peak equal in area and in the opposite direction to the
adsorption peak.
Since this is better defined than the adsorption
peak, it is the one used for the determination.
To effect the calibration, inject a known quantity
of adsorbate into the system, sufficient to give a peak
of similar magnitude to the desorption peak and obtain
the proportion of gas volume per unit peak area.
Method II: the volumetric method
In the volumetric method (see Figure 2), the rec-
ommended adsorbate gas is nitrogen. And it is admit-
ted into the evacuated space above the previously out-
gassed powder sample to give a defined equilibrium
pressure, P, of the gas. The use of a diluent gas, such
as helium, is therefore unnecessary, although helium
may be employed for other purposes, such as to meas-
ure the dead volume.
A: Vacuum gauge
B: Nitrogen reservoir
C: Helium reservoir
D: Vapor pressure manometer
E: Vacuum and air
F: Cold traps and vacuum pumps
Fig. 2. Schematic diagram of the volumetric method
apparatus
Admit a small amount of dry nitrogen into the sample
tube to prevent contamination of the clean surface of
sample, remove the sample tube, insert the stopper,
and weigh it. Calculate the weight of the sample. At-
tach the sample tube to the volumetric apparatus. Cau-
tiously evacuate the sample down to the specified
pressure (e.g. between 2 Pa and 10 Pa).
If the principle of operation of the instrument requires
the determination of the dead volume in the sample
tube, this procedure is carried out at this point, fol-
lowed by evacuation of the sample. Raise a Dewar
vessel containing liquid nitrogen up to a defined point
on the sample cell. Admit a sufficient volume of
adsorbate gas to give the lowest desired relative pres-
sure. Measure the volume adsorbed, Va. For multipoint
measurements, repeat the measurement of Va at suc-
cessively higher P/P0 values. When nitrogen is used as
the adsorbate gas, P/P0 values of 0.10, 0.20, and 0.30
are often suitable.
Reference materials
Periodically verify the functioning of the apparatus
using appropriate reference materials of known sur-
face area, such as α-alumina for specific surface area
determination, which should have a specific surface
area similar to that the sample to be examined.
13. Terminal Sterilization and
Sterilization Indicators
Sterilization is a process whereby the killing or
removal of all forms of viable microorganisms in sub-
stances is accomplished. It is achieved by terminal
sterilization or a filtration method. For substances to
which terminal sterilization can be applied, an appro-
priate sterilization method should be selected in ac-
cordance with the properties of the product, including
the packaging, after full consideration of the ad-
vantages and disadvantages of each sterilization meth-
od, from the heat method, irradiation method and gas
method. After installation of the sterilizer (including
design and development of the sterilization process),
validation is required to confirm that the sterilization
process is properly performing its designed function,
under conditions of loading and unloading of the
product, on the basis of sufficient scientific evidence.
After the process has been validated and the steriliza-
tion of the product commenced, the process must be
controlled correctly, and qualification tests of the
equipment and procedures must be performed regular-
ly. The bioburden per product, prior to terminal sterili-
zation, must be evaluated periodically or on the basis
of batches. Refer to the ISO standard (ISO 11737-1)
relevant to bioburden estimation. For a substance to
which terminal sterilization can be applied, generally
use sterilization conditions such that a sterility assur-
ance level of less than 10-6 can be obtained. The prop-

erty of the sterilization should be judged by employing
an appropriate sterilization process control, with the
use of a suitable sterilization indicator, and if neces-
sary, based on the result of the sterility test.
The filtration procedure is used for the sterilization of
a liquid product, to which terminal sterilization cannot
be applied. Concerning the disinfection and/or sterili-
zation necessary for processing equipment and areas
of pharmaceutical products, and performing microbio-
logical tests specified in the monographs, see Disin-
fection and Sterilization Methods.
1. Definitions
The definitions of the terms used in this text are
as follows.
Terminal sterilization: A process whereby a prod-
uct is sterilized in its final container or packaging, and
which permits the measurement and evaluation of
quantifiable microbial lethality.
Product: A generic term used to describe raw ma-
terials, intermediate products, and finished products,
to be sterilized.
Bioburden: Numbers and types of viable micro-
organisms in a product to be sterilized.
Sterility assurance level (SAL): Probability of a
viable microorganism being present in a product unit
after exposure to the proper sterilization process, ex-
pressed as 10-n.
Integrity test: A non-destructive test which is
used to predict the functional performance of a filter
instead of the microorganism challenge test.
D value: The value which shows the exposure
time (decimal reduction time) or absorbed dose (deci-
mal reduction dose) required to cause a 1-logarithm or
90 % reduction in the population of test microorgan-
isms under stated exposure conditions.
Sterilization indicator: Indicators used to monitor
the sterilization process, as an index of sterility, in-
cluding biological indicators (BI), chemical indicators
(CI), dosimeters and the like.
2. Sterilization
2.1 Heat method
In the heat method, microorganisms are killed by heat-
ing.
(i) Moist heat method
Microorganisms are killed in saturated steam un-
der pressure. In this method, factors which may
affect the sterilization include temperature, steam
pressure and exposure time. Therefore, in routine
sterilization process control, it is required to
monitor continuously the temperature, steam
pressure and exposure time, and they should be
included in the specifications of the sterilizer.
(ii) Dry heat method
Microorganisms are killed in dry heated air. This
method is usually conducted in a batch-type dry
heat sterilizer or a funnel-type dry heat sterilizer.
In this method, factors which may affect the ster-
ilization include temperature and exposure time.
KP X 1877
Therefore, in routine sterilization process control,
it is required to monitor continuously the tem-
perature and exposure time, and they should be
included in the specifications of the sterilizer.
2.2 Irradiation method
Microorganisms are directly killed by ionizing radia-
tion, or by the heat generated by microwave radiation.
(i) Radiation method
Ionizing radiations which may be used are gam-
ma () rays emitted from a radioisotope such as 60C,
an electron beam and bremsstrahlung (X rays) gener-
ated from an electron accelerator. Although any pro-
cedure can be applied to thermally unstable products
with no radioactivity residue, it is necessary to consid-
er the possibility of material degradation. Although a
25 kGy dose is traditionally used as a sterilization
dose, there are some ways to calculate the dose as
follows: the bioburden of the substance to be sterilized
is measured and the sterilization dose is calculated
based on the mean bioburden and the standard re-
sistance distribution (Method I in ISO 11137), the
dose is calculated from the fraction positive infor-
mation from a sterility test in which representative
product samples are exposed to a substerilizing dose
(Method 2 in ISO 11137), or the dose is calculated
based on the bioburden and D value of the most re-
sistant microorganisms (Log method) (see 5-3). In the
case of the radiation sterilization procedure, factors
which may affect the sterilization include dose (ab-
sorbed dose) and exposure time. Therefore, in  ray
sterilization process control, it is required to determine
the dose (the absorbed dose) at appropriate intervals
and to monitor continuously the exposure time in
terms of the operation parameters (the conveyer speed
and the cycle time). The dose control mechanism
should be included in the specifications of the steriliz-
er. In the case of electron beam or bremsstrahlung
irradiation, it is required to monitor the acceleration
voltage, the beam current and beam scanning width
besides the above-mentioned items.
(ii) Microwave method
Microorganisms are killed by the heat generated
by microwave radiation, usually at the frequency of
2450 50 MHz. This method is applied to liquids or
water-rich products in sealed containers. Since a glass
or plastic container may be destroyed or deformed due
to the rise of the inner pressure, the containers must be
certified to be able to withstand the heat and the inner
pressure generated during microwave sterilization.
Leakage of electromagnetic radiation must be at a
sufficiently low level to cause no harm to humans and
no interference with radio communications and the
like. In this method, factors which may affect the steri-
lization include temperature, processing time and mi-
crowave output power. Therefore, in routine steriliza-
tion process control, it is required to monitor continu-
ously the temperature, time and the microwave output
power, and they should be included in the specifica-
tions of the sterilizer.
2.3 Gas Method
1878 Monographs, Part II
Ethylene oxide (EO) is widely used as a steriliza-
tion gas. Since EO gas has an explosive nature, a 10 –
30 % mixture with carbon dioxide is commonly used.
Also, as EO gas is a strong alkylating agent, it cannot
be applied to the products which are likely to react
with or absorb it. Furthermore, because EO gas is tox-
ic, the residual concentration of EO gas and other sec-
ondary generated toxic gases in products sterilized
with EO gas must be reduced to less than the safe lev-
els thereof by means of aeration and the like before
the product is shipped. In this method, factors which
may affect the sterilization include temperature, gas
concentration (pressure), humidity and exposure time.
Therefore, in routine sterilization process control, it is
required to monitor continuously the temperature, gas
concentration (pressure), humidity and exposure time,
and they should be included in the specifications of
the sterilizer.
3. Filtration method
Microorganisms are removed by using a steriliz-
ing filter made of an appropriate material. However,
this method is not intended for microorganisms small-
er than bacteria, Generally, a sterilizing filter chal-
lenged with more than 107 microorganisms of a strain
Brevundimonas diminuta (ATCC 1914, NBRC 14213,
JCM 2428), cultured under the appropriate conditions,
per square centimeter of effective filter area should
provide a sterile effluent. In this method, factors
which may affect the sterilization include pressure,
flow rate, filter unit characteristics and the like. In
routine filtration process control, it is required to per-
form integrity tests of the sterilizing filter after each
filtration process (also prior to the filtration process, if
necessary).
4. Sterilization Indicators
4.1 Biological Indicator (BI)
A BI is prepared from specific microorganisms
resistant to the specified sterilization process and is
used to develop and/or validate a sterilization process.
The dry type BI is classified into two kinds. In one,
bacterial spores are added to a carrier such as filter
paper, glass or plastic and then the carrier are dried
and packaged. In the other, bacterial spore are added
to representative units of the product to be sterilized or
to simulated products. Packaging materials of the BI
should show good heat penetration in dry heat sterili-
zation and good gas or steam penetration in ethylene
oxide and moist heat sterilizations. It should be con-
firmed that any carrier does not affect the D value of
the spores. In the case of a liquid product, the spores
may be suspended in the same solution as the product
or in a solution showing an equivalent effect in the
sterilization of biological indicator. However, when
the spores are suspended in liquid, it is necessary to
ensure that the resistance characteristics of the spores
are not affected due to germination. Typical examples
of biological indicator are shown in Table 1.
Table 1. Typical examples of biological indicator
Representative micro-
Sterilization Strain name
organisms*
ATCC 7953,
NBRC 13737,
JCM 9488,
Moist heat Geobacillus KCTC 2107
method stearothermophilus ATCC 12980,
NBRC 12550,
JCM 2501,
KCTC 1752
ATCC 9372,
Dry heat
Bacillus atrophaeus NBRC 13721
method
KCTC 1022
ATCC 9372,
Gas method Bacillus atrophaeus NBRC 13721
KCTC 1022
* In addition to these microorganisms, other microor-
ganisms with the greatest resistance to the sterilization
procedure concerned, found in the bioburden, can be
used as the biological indicator.
4.1.1 D value of BI
Methods for determination of the D value include
the survival curve method and the fraction negative
method (Stumbo, Murphy & Cochran method, Lim-
ited Spearman-Karber method and the like). In using
marketed BIs, it is usually unnecessary to determine
the D value before use if the D value indicated on the
label has been determined by a standardized biological
indicator evaluation resistometer (BIER) under strictly
prescribed conditions in accordance with ISO 11138-1.
It is acceptable that the D value indicated on the label
shows a scattering of not more than 30 seconds.
4.1.2 Setting up procedure of BI
(i) In the case of dry materials
A dry type BI is placed at predetermined cold
spots in the product to be sterilized or a suitable prod-
uct showing an equivalent effect in the sterilization.
The BIs are usually primary packaged in the same way
as the product, including a secondary packaging, if
applicable.
(ii) In the case of wet materials
Spores are suspended as the BI in the same solu-
tion as the product or in an appropriate similar solu-
tion, and should be placed at cold spots in the sterilizer.
4.1.3 Culture conditions of BI
Soybean casein digest medium is generally used.
General culture conditions are at 55 ~ 60 ℃ for 7 days
in the case of G. stearothermophilus and at 30 ~ 35 ℃
for 7 days in the case of B. atrophaeus.
4.2 Chemical indicator (CI)
CI is an indicator which shows a color change of
a substance applied to a paper slip, etc. as a result of
physical and/or chemical change due to exposure to
heat, gas or radiation. The CI can be classified into
three types. The first is employed to identify whether
or not sterilization has already been implemented, the
 KP X 1879

second is employed to control the sterilization process
(for example, its color changes after sterilization for a
sufficient time), and the third is the Bowie & Dick
type used to evaluate the effectiveness of air removal
during the pre-vacuum phase of the pre-vacuum steri-
lization cycle.
4.3 Dosimeter
In the radiation (-ray) method, the sterilization
effect depends on the absorbed radiation dose, so the
sterilization process control is mainly performed by
measuring the dose. A dosimeter is installed at a posi-
tion corresponding to the minimum dose region of an
exposed container or a position where the dose is in a
known relation to that in the above region. Measure-
ment should be done for each radiation batch. If there
are many containers in the same batch, dosimeters
should be employed so that more than one dosimeter
is always installed at the effective radiation section of
the irradiation chamber. It should be noted that dosim-
eters may be affected by environmental conditions
(temperature, humidity, ultraviolet light, time to read-
ing, etc.) before and during irradiation. Practical do-
simeters for –ray and bremsstrahlung sterilization
include the dyed polymethylmethacrylate dosimeter,
clear polymethymethacrylate dosimeter, ceric-cerous
sulfate dosimeter, alanine-EPR dosimeter and the like.
A dosimeter for gamma radiation cannot generally be
used for sterilization process control by the electron
beam energy less than 3 MeV. Dosimeters for electron
beam sterilization include the cellulose acetate dosim-
eter, radiochromic film dosimeter and the like. A prac-
tical dosimeter must be calibrated against an appropri-
ate national or international standard dosimetry system.
extensive bioburden estimation is considered as the
maximum bioburden count, and the sterilization time
(or radiation dose) is calculated with the bioburden
count based on an objective sterility assurance level.
When this procedure is used, it is required to deter-
mine the resistance of the bioburden to the steriliza-
tion as well as the bioburden count in the product be-
ing sterilized. If a more resistant microorganism than
the BI spore is found in the bioburden estimation, it
should be used as the BI.
N0
Sterilization time(or radiation dose)  D  log( )
N
D : D value of the BI
N: Sterility assurance level
N0: Maximum bioburden count in the product
5.4 Absolute bioburden method
The sterilization conditions are determined by
employing the D value of the most resistant microor-
ganism found in the product or environment by the
resistant estimation and being based on the bioburden
count in the product. Generally, a count of mean
bioburden added three times of its standard deviation
obtained through the extensive bioburden estimation is
employed as the bioburden count. When this proce-
dure is used, it is required to make frequent counting
and resistance determination of microorganisms in
daily bioburden estimation

5. Determination of sterilization conditions using

microorganism as an indicator
Taking account of the characteristics upon the
sterilization concerned, bioburden, etc., of a product to
be sterilized, choose a suitable method from the fol-
lowings and determine the conditions.
5.1 Half-cycle method
In this method, a sterilization time of twice as
long as that required to inactivate all of 106 counts of
BI placed in the product is used, regardless of
bioburden count in the product being sterilized or the
resistance of the objective microorganisms to the steri-
lization.
5.2 Overkill method
In this method, a sterilization condition giving a
sterility assurance level of not more than 10-6 counts is
used, regardless of bioburden count in the product
being sterilized or the resistance of the objective mi-
croorganisms to the sterilization. Generally, a steriliza-
tion condition providing 12 logarithmic reduction
(12D) of known count of BI of more than 1.0 D value
is used.
5.3 Combination of BI and bioburden
Generally, a count of mean bioburden added
three times of its standard deviation obtained by an