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Introduction

Quality control
The pharmaceutical quality control laboratory serves one of the most important functions
in pharmaceutical production and control. A significant portion of the CGMP regulations
pertains to the quality control laboratory.
Quality control in pharmaceutical laboratory includes a series of analytical measurements
used to monitor the quality of the analytical data.
Quality control is useful for:
• Guiding formulation development
• Comparing finished products with different formulations.

• Confirming the acceptability of changes to manufacturing procedures during


scale-up or post-marketing changes.

Quality control ensures that the quality of the end product is acceptable to regulatory
authorities such as FDA & it is truly required for pharmaceutical products as patients (not
the general population) use pharmaceuticals to treat their diseases or for prophylaxis to
prevent infection or disease. [5]

In the pharmaceutical quality control laboratory, the stability of the pharmaceutical


products such as solid/liquid dosage forms is studied under various conditions. Complete
analysis of the pharmaceutical products include the following tests:

• Dissolution testing

• Content Uniformity testing.

• Impurity profiling.

In dissolution testing, the release rate of an active ingredient in a pharmaceutical product


is measured. It should be within the acceptable limits specified by the regulatory
authorities. Content uniformity testing deals with the quantification of the active

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ingredient in a pharmaceutical product. Similarly impurity profiling, as the name
suggests, deals with the detection and the quantification of the impurities present in a raw
material or a finished product.

These tests are dealt in detail in the following chapters. These tests are performed on raw
materials & on finished products during manufacture, scaling up and after a batch is
ready to be marketed. These tests are done by using protocols such as those described, for
example, by United States phamacopoeia (USP), British Phamacopoeia (BP) or Indian
Pharmacopoeia. The pharmaceutical products for which the Q.C. protocols are available
in USP/IP/BP are called official articles and for the newly identified drug molecules
analytical methods are developed by the manufacturer, the methods are validated and
submitted to regulatory authorities.

Although all the quality control tests have different applications, all of them involve
qualitative as well as quantitative analysis of the pharmaceutical product and in many
analytical laboratories HPLC is used for such analyses & now a days ultra fast liquid
chromatographs are used to save precious time of analysis. So, before understanding the
actual quality control methods it is required to study the instrumentation, principle and
working of HPLC and UFLC and to study the analytical method development for HPLC
& UFLC.

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High performance liquid chromatography

High-pressure liquid chromatography (HPLC), sometimes called “high-performance


liquid chromatography”, is a separation technique based on a solid stationary phase and a
liquid mobile phase.

Separations are achieved by partition, adsorption, or ion-exchange processes, depending


upon the type of stationary phase used. HPLC has distinct advantages over gas
chromatography for the analysis of organic compounds. Compounds to be analyzed are
dissolved in a suitable solvent, and most separations take place at room temperature.
Thus, most drugs, being nonvolatile or thermally unstable compounds, can be
chromatographed without decomposition or the necessity of making volatile derivatives.
Most pharmaceutical analyses are based on partition chromatography

Principle of HPLC

The basic operating principle of HPLC is to force the analyte through a column of the
stationary phase (usually a tube packed with small spherical particles with a certain
surface chemistry) by pumping a liquid (mobile phase) at high pressure through the
column. The sample to be analyzed is introduced in small volume to the stream of mobile
phase and is retarded by specific chemical or physical interactions with the stationary
phase as it traverses the length of the column. The amount of retardation depends on the
nature of the analyte, stationary phase and mobile phase composition. The time at which
a specific analyte elutes (comes out of the end of the column) is called the retention time
and is considered a reasonably unique identifying characteristic of a given analyte. The
use of pressure increases the linear velocity (speed) giving the components less time to
diffuse within the column, leading to improved resolution in the resulting chromatogram.
Common solvents used include any miscible combinations of water or various organic
liquids (the most common are methanol and acetonitrile). Water may contain buffers or
salts to assist in the separation of the analyte components, or compounds such as
Trifluoroacetic acid which acts as an ion pairing agent. [3,11,12]

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A further refinement to HPLC has been to vary the mobile phase composition during the
analysis; this is known as “gradient elution”. A normal gradient for reversed phase
chromatography might start at 5 % methanol and progress linearly to 50 % methanol over
25 minutes, depending on how hydrophobic the analyte is. The gradient separates the
analyte mixtures as a function of the affinity of the analyte for the current mobile phase
composition relative to the stationary phase. This partitioning process is similar to that
which occurs during a liquid-liquid extraction but is continuous, not step-wise. In this
example, using a water/methanol gradient, the more hydrophobic components will elute
(come off the column) under conditions of relatively high methanol; whereas the more
hydrophilic compounds will elute under conditions of relatively low methanol. The
choice of solvents, additives and gradient depend on the nature of the stationary phase
and the analyte. Often a series of tests are performed on the analyte and a number of
generic runs may be processed in order to find the optimum HPLC method for the analyte
- the method which gives the best separation of peaks. [3]

Distribution of analytes between phases

The distribution of analytes between phases can often be described quite simply. An
analyte is in equilibrium between the two phases;

Amobile Astationary

The equilibrium constant, K, is termed the “partition coefficient”; defined as the molar
concentration of analyte in the stationary phase divided by the molar concentration of the
analyte in the mobile phase.

The time between sample injection and an analyte peak reaching a detector at the end of
the column is termed the “retention time (tR)”. Each analyte in a sample will have a
different retention time. The time taken for the mobile phase to pass through the column
is called tM.

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A term called the “retention factor”, k', is often used to describe the migration rate of an
analyte on a column. You may also find it called the capacity factor. The retention factor
for analyte A is defined as;

k'A = ( t R – t M ) / tM

t R and tM are easily obtained from a chromatogram. When an analytes retention factor is
less than one, elution is so fast that accurate determination of the retention time is very
difficult. High retention factors (greater than 20) mean that elution takes a very long time.
Ideally, the retention factor for an analyte is between one and five.

We define a quantity called the “selectivity factor”, α, which describes the separation of
two species (A and B) on the column;

α = k 'B / k 'A

When calculating the selectivity factor, species A elutes faster than species B. The
selectivity factor is always greater than one. [2]

Band broadening and column efficiency

To obtain optimal separations, sharp, symmetrical chromatographic peaks must be


obtained. This means that band broadening must be limited. It is also beneficial to
measure the efficiency of the column. [3]

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The Theoretical Plate Model of Chromatography

The plate model supposes that the chromatographic column contains a large number of
separate layers, called theoretical plates. Separate equilibrations of the sample between
the stationary and mobile phase occur in these "plates". The analyte moves down the
column by transfer of equilibrated mobile phase from one plate to the next.

It is important to remember that the plates do not really exist; they are a figment of the
imagination that helps us understand the processes at work in the column. They also
serve as a way of measuring column efficiency, either by stating the number of
theoretical plates in a column, N (the more plates the better), or by stating the plate
height; the Height Equivalent to a Theoretical Plate (the smaller the better).

If the length of the column is L, then the HETP is

HETP = L / N

The number of theoretical plates that a real column possesses can be found by examining
a chromatographic peak after elution;

where w1/2 is the peak width at half-height.

As can be seen from this equation, columns behave as if they have different numbers of
plates for different solutes in a mixture. [2]

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The Rate Theory of Chromatography

A more realistic description of the processes at work inside a column takes account of the
time taken for the solute to equilibrate between the stationary and mobile phase (unlike
the plate model, which assumes that equilibration is infinitely fast). The resulting band
shape of a chromatographic peak is therefore affected by the rate of elution. It is also
affected by the different paths available to solute molecules as they travel between
particles of stationary phase. If we consider the various mechanisms, which contribute to
band broadening, we arrive at the Van Deemter equation for plate height;

HETP = A + B / u + C u

where u is the average velocity of the mobile phase. A, B, and C are factors which
contribute to band broadening.

A - Eddy diffusion
The mobile phase moves through the column, which is packed with stationary phase.
Solute molecules will take different paths through the stationary phase at random. This
will cause broadening of the solute band, because different paths are of different lengths.

B - Longitudinal diffusion
The concentration of analyte is less at the edges of the band than at the center. Analyte
diffuses out from the center to the edges. This causes band broadening. If the velocity of
the mobile phase is high then the analyte spends less time on the column, which
decreases the effects of longitudinal diffusion.

C - Resistance to mass transfer


The analyte takes a certain amount of time to equilibrate between the stationary and
mobile phase. If the velocity of the mobile phase is high, and the analyte has a strong
affinity for the stationary phase, then the analyte in the mobile phase will move ahead of
the analyte in the stationary phase. The band of analyte is broadened. The higher the
velocity of mobile phase, the worse the broadening becomes.

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Van Deemter plots
A plot of plate height vs. average linear velocity of mobile phase.

Such plots are of considerable use in determining the optimum mobile phase flow rate.

Resolution

Although the selectivity factor, α, describes the separation of band centres, it does not
take into account peak widths. Another measure of how well species have been separated
is provided by measurement of the resolution. The resolution of two species, A and B, is
defined as

Baseline resolution is achieved when R = 1.5

It is useful to relate the resolution to the number of plates in the column, the selectivity
factor and the retention factors of the two solutes;

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To obtain high resolution, the three terms must be maximized. An increase in N, the
number of theoretical plates, by lengthening the column leads to an increase in retention
time and increased band broadening - which may not be desirable. Instead, to increase the
number of plates, the height equivalent to a theoretical plate can be reduced by reducing
the size of the stationary phase particles.

It is often found that by controlling the capacity factor, k', separations can be greatly
improved. This can be achieved by changing the composition of the mobile phase [7,1]

The selectivity factor, α, can also be manipulated to improve separations. When α is close
to unity, optimizing k' and increasing N is not sufficient to give good separation in a
reasonable time. In these cases, k' is optimized first, and then a is increased by one of the
following procedures:

1. Changing mobile phase composition


2. Changing column temperature
3. Changing composition of stationary phase

Using special chemical effects (such as incorporating a species which complexes with
one of the solutes into the stationary phase) [4]

Types of HPLC

(A) Normal phase chromatography

Normal phase HPLC (NP-HPLC) was the first kind of HPLC chemistry used, and
separates analytes based on polarity. This method uses a polar stationary phase and a
non-polar mobile phase, and is used when the analyte of interest is fairly polar in nature.
The polar analyte associates with and is retained by the polar stationary phase.
Adsorption strengths increase with increase in analyte polarity, and the interaction
between the polar analyte and the polar stationary phase (relative to the mobile phase)
increases the elution time. The interaction strength not only depends on the functional
groups in the analyte molecule, but also on steric factors and structural isomers are often
resolved from one another. Use of more polar solvents in the mobile phase will decrease

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the retention time of the analytes while more hydrophobic solvents tend to increase
retention times. Particularly polar solvents in a mixture tend to deactivate the column by
occupying the stationary phase surface. This is somewhat particular to normal phase
because it is most purely an adsorptive mechanism (the interactions are with a hard
surface rather than a soft layer on a surface)..

NP-HPLC had fallen out of favor in the 1970's with the development of reversed-phase
HPLC because of a lack of reproducibility of retention times as water or protic organic
solvents changed the hydration state of the silica or alumina chromatographic media.
Recently it has become useful again with the development of HILIC bonded phases
which utilize a partition mechanism which provides reproducibility.

(B) Reversed phase chromatography

Reversed phase HPLC (RP-HPLC) consists of a non-polar stationary phase and an


aqueous, moderately polar mobile phase. One common stationary phase is a silica which
has been treated with RMe2SiCl, where R is a straight chain alkyl group such as C18H37 or
C8H17. The retention time is therefore longer for molecules which are more non-polar in
nature, allowing polar molecules to elute more readily. Retention Time (RT) is increased
by the addition of polar solvent to the mobile phase and decreased by the addition of
more hydrophobic solvent. Reversed phase chromatography (RPC) is so commonly used
that it is not uncommon for it to be incorrectly referred to as "HPLC" without further
specification. The pharmaceutical industry regularly employs RPC to qualify drugs
before their release.

RPC operates on the principle of hydrophobic interactions, which result from repulsive
forces between a polar eluent, the relatively non-polar analyte, and the non-polar
stationary phase. The binding of the analyte to the stationary phase is proportional to the
contact surface area around the non-polar segment of the analyte molecule upon
association with the ligand in the aqueous eluent. This solvophobic effect is dominated by
the force of water for "cavity-reduction" around the analyte and the C18-chain versus the

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complex of both. The energy released in this process is proportional to the surface tension
of the eluent (water: 73 erg/cm², methanol: 22 erg/cm²) and to the hydrophobic surface of
the analyte and the ligand respectively. The retention can be decreased by adding less-
polar solvent (MeOH, ACN) into the mobile phase to reduce the surface tension of water.
Gradient elution uses this effect by automatically changing the polarity of the mobile
phase during the course of the analysis.

Structural properties of the analyte molecule play an important role in its retention
characteristics. In general, an analyte with a larger hydrophobic surface area (C-H, C-C,
and generally non-polar atomic bonds, such as S-S and others) results in a longer
retention time because it increases the molecule's non-polar surface area, which is non-
interacting with the water structure. On the other hand, polar groups, such as -OH, -NH2,
COO- or -NH3+ reduce retention as they are well integrated into water. Very large
molecules, however, can result in an incomplete interaction between the large analyte
surface and the ligands alkyl chains can have problems entering the pores of the
stationary phase.

RT increases with hydrophobic - non-polar - surface area. Branched chain compounds


elute more rapidly than their corresponding linear isomers because the overall surface
area is decreased. Similarly organic compounds with single C-C-bonds elute later than
the ones with a C=C or C-C-triple bond, as the double or triple bond is shorter than a
single C-C-bond.

Aside from mobile phase surface tension (organizational strength in eluent structure),
other mobile phase modifiers can affect analyte retention. For example, the addition of
inorganic salts causes a moderate linear increase in the surface tension of aqueous
solutions (ca. 1.5 erg/cm² pro Mol for NaCl, 2.5 erg/cm² pro Mol for (NH4)2SO4), and
because the entropy of the analyte-solvent interface is controlled by surface tension, the
addition of salts tend to increase the retention time. This technique is used for mild
separation and recovery of proteins and protection of their biological activity in protein
analysis (hydrophobic interaction chromatography, HIC).

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Another important component is the influence of the pH since this can change the
hydrophobicity of the analyte. For this reason most methods use a buffering agent, such
as sodium phosphate, to control the pH. A volatile organic acid such as formic acid or
most commonly trifluoroacetic acid is often added to the mobile phase, if mass
spectrometry is applied to the eluent fractions. The buffers serve multiple purposes: they
control pH, neutralize the charge on any residual exposed silica on the stationary phase
and act as ion pairing agents to neutralize charge on the analyte. The effect varies
depending on use but generally improve the chromatography.

Reversed phase columns are quite difficult to damage compared with normal silica
columns, however, many reversed phase columns consist of alkyl derivatized silica
particles and should never be used with aqueous bases as these will destroy the
underlying silica particle. They can be used with aqueous acid, but the column should not
be exposed to the acid for too long, as it can corrode the metal parts of the HPLC
equipment. The metal content of HPLC columns must be kept low if the best possible
ability to separate substances is to be retained. A good test for the metal content of a
column is to inject a sample which is a mixture of 2,2'- and 4,4'- bipyridine. Because the
2,2'-bipyridine can chelate the metal, the shape of the peak for the 2,2'-bipy will be
distorted (tailed) when metal ions are present on the surface of the silica.

(C) Size exclusion chromatography

Size exclusion chromatography (SEC), also known as gel permeation chromatography or


gel filtration chromatography, separates particles on the basis of size. It is generally a low
resolution chromatography and thus it is often reserved for the final, "polishing" step of
purification. It is also useful for determining the tertiary structure and quaternary
structure of purified proteins.

This technique is widely used for the molecular weight determination of polysaccharides.
SEC is the official technique (suggested by European pharmacopeia) for the molecular
weight comparison of different commercially available low-molecular weight heparins.

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(D) Ion exchange chromatography.

In Ion-exchange chromatography, retention is based on the attraction between solute ions


and charged sites bound to the stationary phase. Ions of the same charge are excluded.
Some types of Ion Exchangers include: (1) Polystyrene resins- allows cross linkage
which increases the stability of the chain. Higher cross linkage reduces swerving, which
increases the equilibration time and ultimately improves selectivity. (2) Cellulose and
dextran ion exchangers (gels)-These possess larger pore sizes and low charge densities
making them suitable for protein separation.(3) Controlled-pore glass or porous silica.

In general, ion exchangers favor the binding of ions of higher charge and smaller radius.

An increase in counter ion (with respect to the functional groups in resins) concentration
reduces the retention time. An increase in pH reduces the retention time in cation
exchange while a decrease in pH reduces the retention time in anion exchange.

This form of chromatography is widely used in the following applications: In purifying


water, preconcentration of trace components, Ligand-exchange chromatography, Ion-
exchange chromatography of proteins, High-pH anion-exchange chromatography of
carbohydrates and oligosaccharides, etc.

(E) Bioaffinity chromatography

This chromatographic process relies on the property of biologically active substances to


form stable, specific, and reversible complexes. The formation of these complexes
involves the participation of common molecular forces such as the Van der Waals
interaction, electrostatic interaction, dipole-dipole interaction, hydrophobic interaction,
and the hydrogen bond. An efficient, biospecific bond is formed by a simultaneous and
concerted action of several of these forces in the complementary binding sites.[1,3]

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Types of flow

Isocratic flow and gradient elution

With regard to the mobile phase, a composition of the mobile phase that remains constant
throughout the procedure is termed isocratic.

In contrast to this is the so called "gradient elution", which is a separation where the
mobile phase changes its composition during a separation process. One example is a
gradient in 20 min starting from 10 % Methanol and ending up with 30 % Methanol.
Such a gradient can be increasing or decreasing. The benefit of gradient elution is that it
helps speed up elution by allowing components that elute more quickly to come off the
column under different conditions than components which are more readily retained by
the column. By changing the composition of the solvent, components that are to be
resolved can be selectively more or less associated with the mobile phase. As a result, at
equilibrium they spend more time in the solvent and less time in the stationary phase, and
therefore they elute faster.[10]

Other parameters

(A) Internal diameter

The internal diameter (ID) of an HPLC column is a critical aspect that determines
quantity of analyte that can be loaded onto the column and also influences sensitivity.
Larger columns are usually seen in industrial applications such as the purification of a
drug product for later use. Low ID columns have improved sensitivity and lower solvent
consumption at the expense of loading capacity.

• Larger ID columns (over 10 mm) are used to purify usable amounts of material
because of their large loading capacity.
• Analytical scale columns (4.6 mm) have been the most common type of columns,
though smaller columns are rapidly gaining in popularity. They are used in

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traditional quantitative analysis of samples and often use a UV-Vis absorbance
detector.
• Narrow-bore columns (1-2 mm) are used for applications when more sensitivity is
desired either with special UV-vis detectors, fluorescence detection or with other
detection methods like liquid chromatography-mass spectrometry
• Capillary columns (under 0.3 mm) which are used almost exclusively with
alternative detection means such as mass spectrometry. They are usually made
from fused silica capillaries, rather than the stainless steel tubing that larger
columns employ.

(B) Particle size

Most traditional HPLC is performed with the stationary phase attached to the outside of
small spherical silica particles (very small beads). These particles come in a variety of
sizes with 5μm beads being the most common. Smaller particles generally provide more
surface area and better separations, but the pressure required for optimum linear velocity
increases by the inverse of the particle diameter squared. This means that changing to
particles that are half as big, keeping the size of the column the same, will double the
performance, but increase the required pressure by a factor of four. Larger particles are
more often used in non-HPLC applications such as solid-phase extraction.

(C) Pore size

Many stationary phases are porous to provide greater surface area. Small pores provide
greater surface area while larger pore size has better kinetics especially for larger
analytes. For example a protein which is only slightly smaller than a pore might enter the
pore but not easily leave once inside.

(D) Pump pressure

Pumps vary in pressure capacity, but their performance is measured on their ability to
yield a consistent and reproducible flow rate. Pressure may reach as high as 6000 lbf/in2
(~40 MPa, or about 400 atmospheres). Modern HPLC systems have been improved to

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work at much higher pressures, and therefore be able to use much smaller particle sizes in
the columns (< 2 micrometres). These "Ultra High Performance Liquid Chromatography"
systems or UHPLCs can work at up to 15,000 lbf/in² (~ 100 MPa or about 1000
atmospheres).[3]

Manufacturers of HPLC chromatographs

• Agilent Technologies
• Beckman Coulter, Inc.
• Hitachi
• PerkinElmer, Inc.
• Shimadzu Scientific Instruments
• Thermo Electron Corporation
• Varian, Inc.
• Waters Corporation

Manufacturers of HPLC columns and accessories

• Agilent Technologies
• Beckman Coulter, Inc.
• Merck KGaA
• Phenomenex
• Shimadzu Scientific Instruments
• Sigma-Aldrich
• Thermo Electron Corporation
• Tosoh Corporation
• Varian, Inc.
• Waters Corporation

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HPLC instrumentation:

Apparatus:

A liquid chromatograph consists of a reservoir containing the mobile phase, a pump to


force the mobile phase through the system at high pressure, an injector to introduce the
sample into the mobile phase, a chromatographic column, a detector, and a data
collection device such as a computer, integrator, or recorder. Short, small-bore columns
containing densely packed particles of stationary phase provide for the rapid exchange of
compounds between the mobile and stationary phases. In addition to receiving and
reporting detector output, computers are used to control chromatographic settings and
operations, thus providing for long periods of unattended operation. [2]

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Pumping Systems:

HPLC pumping systems deliver metered amounts of mobile phase from the solvent
reservoirs to the column through high-pressure tubing and fittings. Modern systems
consist of one or more computer-controlled metering pumps that can be programmed to
vary the ratio of mobile phase components, as is required for gradient chromatography, or
to mix isocratic mobile phases (i.e., mobile phases having a fixed ratio of solvents).
However, the proportion of ingredients in premixed isocratic mobile phases can be more
accurately controlled than in those delivered by most pumping systems. Operating
pressures up to 5000 psi or higher, with delivery rates up to about 10 mL per minute are
typical. Pumps used for quantitative analysis should be constructed of materials inert to
corrosive mobile phase components and be capable of delivering the mobile phase at a
constant rate with minimal fluctuations over extended periods of time. [3]

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Injectors:

After dissolution in mobile phase or other suitable solution, compounds to be


chromatographed are injected into the mobile phase, either manually by syringe or loop
injectors, or automatically by autosamplers. The latter consist of a carousel or rack to
hold sample vials with tops that have a pierceable septum or stopper and an injection
device to transfer sample from the vials to a loop from which it is loaded into the
chromatograph. Some autosamplers can be programmed to control sample volume, the
number of injections and loop rinse cycles, the interval between injections, and other
operating variables.

A syringe can be used for manual injection of samples through a septum when column
head pressures are less than 70 atmospheres (about 1000 psi). At higher pressures an
injection valve is essential. Some valve systems incorporate a calibrated loop that is filled
with test solution for transfer to the column in the mobile phase. In other systems, the test
solution is transferred to a cavity by syringe and then switched into the mobile phase. [3]

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Columns:

For most pharmaceutical analyses, separation is achieved by partition of compounds in


the test solution between the mobile and stationary phases. Systems consisting of polar
stationary phases and nonpolar mobile phases are described as normal phase, while the
opposite arrangement, polar mobile phases and nonpolar stationary phases, are called
reverse-phase chromatography. Partition chromatography is almost always used for
hydrocarbon-soluble compounds of molecular weight less than 1000. The affinity of a
compound for the stationary phase, and thus its retention time on the column, is
controlled by making the mobile phase more or less polar. Mobile phase polarity can be
varied by the addition of a second, and sometimes a third or even a fourth, component.

Stationary phases for modern, reverse-phase liquid chromatography typically consist of


an organic phase chemically bound to silica or other materials. Particles are usually 3 to
10 µm in diameter, but sizes may range up to 50 µm or more for preparative columns.
Small particles thinly coated with organic phase provide for low mass transfer resistance
and, hence, rapid transfer of compounds between the stationary and mobile phases.
Column polarity depends on the polarity of the bound functional groups, which range
from relatively nonpolar octadecyl silane to very polar nitrile groups. Liquid, nonbound
stationary phases must be largely immiscible in the mobile phase. Even so, it is usually
necessary to presaturate the mobile phase with stationary phase to prevent stripping of the
stationary phase from the column. Polymeric stationary phases coated on the support are
more durable.

Columns used for analytical separations usually have internal diameters of 2 to 5 mm;
larger diameter columns are used for preparative chromatography. Columns may be
heated to give more efficient separations, but only rarely are they used at temperatures
above 60 because of potential stationary phase degradation or mobile phase volatility.
Unless otherwise specified in the individual monograph, columns are used at ambient
temperature Ion exchange chromatography is used to separate water-soluble, ionizable
compounds of molecular weight less than 1500. The stationary phases are usually
synthetic organic resins; cation-exchange resins contain negatively charged active sites

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and are used to separate basic substances such as amines, while anion-exchange resins
have positively charged active sites for separation of compounds with negatively charged
groups, such as phosphate, sulfonate, or carboxylate groups. Water-soluble ionic or
ionizable compounds are attracted to the resins, and differences in affinity bring about the
chromatographic separation. The pH of the mobile phase, temperature, ion type, ionic
concentration, and organic modifiers affect the equilibrium, and these variables can be
adjusted to obtain the desired degree of separation.

In size-exclusion chromatography, columns are packed with a porous stationary phase.


Molecules of the compounds being chromatographed are filtered according to size. Those
too large to enter the pores pass unretained through the column. Smaller molecules enter
the pores and are increasingly retained as molecular size decreases. These columns are
typically used to measure aggregation and degradation of large molecules. [3]

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Detectors:

Many HPLC methods require the use of spectrophotometric detectors. Such a detector
consists of a flow-through cell mounted at the end of the column. A beam of UV
radiation passes through the flow cell and into the detector. As compounds elute from the
column, they pass through the cell and absorb the radiation, resulting in measurable
energy level changes. [3]

Fixed, variable, and multi-wavelength detectors are widely available. Fixed


wavelength detectors operate at a single wavelength, typically 254 nm, emitted by a low-
pressure mercury lamp. Variable wavelength detectors contain a continuous source, such
as a deuterium or high-pressure xenon lamp, and a monochromator or an interference
filter to generate monochromatic radiation at a wavelength selected by the operator. The
wavelength accuracy of a variable-wavelength detector equipped with a monochromator
should be checked by the procedure recommended by its manufacturer; if the observed
wavelengths differ by more than 3 nm from the correct values, recalibration of the
instrument is indicated. Modern variable wavelength detectors can be programmed to
change wavelength while an analysis is in progress. Multi-wavelength detectors measure
absorbance at two or more wavelengths simultaneously. In diode array multi-wavelength
detectors, continuous radiation is passed through the sample cell, then resolved into its
constituent wavelengths, which are individually detected by the photodiode array. These
detectors acquire absorbance data over the entire UV-visible range, thus providing the
analyst with chromatograms at multiple, selectable wavelengths and spectra of the eluting
peaks. Diode array detectors usually have lower signal-to-noise ratios than fixed or
variable wavelength detectors, and thus are less suitable for analysis of compounds
present at low concentrations.

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Differential refractometer detectors measure the difference between the refractive
index of the mobile phase alone and that of the mobile phase containing
chromatographed compounds as it emerges from the column. Refractive index detectors
are used to detect non-UV absorbing compounds, but they are less sensitive than UV
detectors. They are sensitive to small changes in solvent composition, flow rate, and
temperature, so that a reference column may be required to obtain a satisfactory baseline.

Fluorometric detectors are sensitive to compounds that are inherently fluorescent or that
can be converted to fluorescent derivatives either by chemical transformation of the
compound or by coupling with fluorescent reagents at specific functional groups. If
derivatization is required, it can be done prior to chromatographic separation or,
alternatively, the reagent can be introduced into the mobile phase just prior to its entering
the detector.

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Potentiometric, voltametric, or polarographic electrochemical detectors are useful
for the quantitation of species that can be oxidized or reduced at a working electrode.
These detectors are selective, sensitive, and reliable, but require conducting mobile
phases free of dissolved oxygen and reducible metal ions. A pulseless pump must be
used, and care must be taken to ensure that the pH, ionic strength, and temperature of the
mobile phase remain constant. Working electrodes are prone to contamination by reaction
products with consequent variable responses.

Electrochemical detectors with carbon-paste electrodes may be used advantageously to


measure nanogram quantities of easily oxidized compounds, notably phenols and
catechols.

New detectors continue to be developed in attempts to overcome the deficiencies of those


being used.[10]

Data Collection Devices

Modern data stations receive and store detector output and print out chromatograms
complete with peak heights, peak areas, sample identification, and method variables.
They are also used to program the liquid chromatograph, controlling most variables and
providing for long periods of unattended operation.

Data also may be collected on simple recorders for manual measurement or on stand-
alone integrators, which range in complexity from those providing a printout of peak
areas to those providing chromatograms with peak areas and peak heights calculated and
data stored for possible subsequent reprocessing. [10]

Procedure:

The mobile phase composition significantly influences chromatographic performance


and the resolution of compounds in the mixture being chromatographed. For accurate
quantitative work, high-purity reagents and “HPLC grade” organic solvents must be used.

24
Water of suitable quality should have low conductivity and low UV absorption,
appropriate to the intended use.

Reagents used with special types of detectors (e.g., electrochemical, mass spectrometer)
may require the establishment of additional tolerances for potential interfering species.
Composition has a much greater effect than temperature on the capacity factor, k’

In partition chromatography, the partition coefficient, and hence the separation, can be
changed by addition of another component to the mobile phase. In ion-exchange
chromatography, pH and ionic strength, as well as changes in the composition of the
mobile phase, affect capacity factors. The technique of continuously changing the solvent
composition during the chromatographic run is called gradient elution or solvent
programming. It is sometimes used to chromatograph complex mixtures of components
differing greatly in their capacity factors. Detectors that are sensitive to change in solvent
composition, such as the differential refractometer, are more difficult to use with the
gradient elution technique.

The detector must have a broad linear dynamic range, and compounds to be measured
must be resolved from any interfering substances. The linear dynamic range of a
compound is the range over which the detector signal response is directly proportional to
the amount of the compound. For maximum flexibility in quantitative work, this range
should be about three orders of magnitude. HPLC systems are calibrated by plotting peak
responses in comparison with known concentrations of a reference standard, using either
an external or an internal standardization procedure.

Reliable quantitative results are obtained by external calibration if automatic injectors or


autosamplers are used. This method involves direct comparison of the peak responses
obtained by separately chromatographing the test and reference standard solutions. If
syringe injection, which is irreproducible at the high pressures involved, must be used,
better quantitative results are obtained by the internal calibration procedure where a
known amount of a noninterfering compound, the internal standard, is added to the test

25
and reference standard solutions, and the ratios of peak responses of drug and internal
standard are compared.

Because of normal variations in equipment, supplies, and techniques, a system suitability


test is required to ensure that a given operating system may be generally applicable. [10]

Trouble shooting

Start up - Preliminary checks

Problem Possible cause Solution


Detector off Check detector
Broken connections
Check connections
No peaks or to recorder
very small No sample/Wrong Check sample. Be sure it is not deteriorated.
peaks sample Check for bubbles in the vials
Wrong settings on
Check attenuation. Check gain
recorder or detector
Pump off Start Pump
Check reservoirs. Check position of the inlet
tubing. Check loop for obstruction or air.
Flow interrupted
Check degasing of mobile phase. Check
compatibility of the mobile phase components.
No Flow
Check fittings. Check pump for leaks and
Leak
precipitates. Check pump seals.
Disconnect column and prime pump. Flush
Air trapped in the
system with 100% methanol or isopropanol.
system
Contact servicing if necessary.

Column and Fittings Leaks

26
Problem Possible cause Solution
Loose fitting Tighten or replace fitting
Column end
White powder at Cut tubing and replace ferrule; disassemble
leaks
loose fitting fitting, rinse and reassemble.

Leak at detector Detector-seal failure Replace detector seal or gaskets.

Leak at injection Worn or scratched


Replace valve rotor
valve valve rotor
Replace pump seal; check piston for scratches
Leak at pump Pump seal failure
and, if necessary, replace

Change in Retention time

Problem Possible cause Solution


Changing Use buffer with concentration
Buffer retention times
Retention Times greater than 20 mM.
Flush column occasionally with
Contamination buildup
strong solvent
Equilibration time Pass at least 10 column volumes
insufficient for gradient run through the column for gradient
or changes in isocratic regeneration or after solvent
mobile phase changes
First few injections - active Condition column by injecting
sites concentrated sample
Ensure gradient system is
delivering a constant composition;
Inconsistent on-line mobile-
compare with manually prepared
phase mixing
mobile phase; partially premix
mobile phase
Selective evaporation of Cover solvent reservoirs; use less-

27
vigorous helium purging; prepare
mobile-phase component
fresh mobile phase
Thermostat or insulate column;
Varying column
ensure laboratory temperature is
temperature
constant.
Use mobile-phase modifier,
Active sites on column competing base (basic compounds),
packing or increase buffer strength; use
higher coverage column packing.
Column overloaded with Decrease sample amount or use
Decreasing sample larger-diameter column.
Retention Times Increasing flow rate Check and reset pump flow rate.
Loss of bonded stationary Use mobile-phase pH between pH
phase or base silica 2 and pH 8
Thermostat or insulate column;
Varying column
ensure laboratory temperature is
temperature
constant
Check and reset pump flow rate;
check for pump cavitation; check
Decreasing flow rate
for leaking pump seals and other
leaks in system
Increasing
Cover solvent reservoirs; ensure
Retention Times Changing mobile-phase
that gradient system is delivering
composition
correct composition.
Loss of bonded stationary Use mobile-phase pH between pH
phase 2 and pH 8
Reversed phase ion pairing
Slow column
- long chain ion pairing Use ion-pairing reagent with
equilibration
reagents require longer shorter alkyl chain length
time
equilibration time

28
Baseline

Problem Possible cause Solution


Air bubbles in mobile Degas or use back pressure restrictor on
phase detector
Void Time Positive-negative -
noise difference in refractive Normal with many samples; use mobile
index of injection solvent phase as sample solvent
and mobile phase
Negative direction Use non-UV absorbing mobile phase
(gradient elution) - solvents; use HPLC grade mobile phase
absorbance of mobile- solvents; add UV absorbing compound to
phase A mobile phase B.
Use higher UV absorbance detector
Positive direction (gradient wavelength; use non-UV absorbing
elution) - absorbance of mobile phase solvents; use HPLC grade
Drifting mobile phase B mobile phase solvents; add UV absorbing
baseline compound to mobile phase A.
Positive direction -
Flush column with strong solvent; clean
contamination buildup and
up sample; use HPLC grade solvents
elution
Monitor and control changes in room
Wavy or undulating -
temperature; insulate column or use
temperature changes in
column oven; cover refractive index
room
detector and keep it out of air currents.
Baseline Continuous - detector lamp Replace UV lamp( each should last 2000
noise problem or dirty cell h; clean and flush flow cell.
Use proper mixing device; check
Gradient or isocratic
proportioning precision by spiking one
proportioning - lack of
solvent with UV absorbing compound and
solvent mixing
monitor UV absorbance detector output
Gradient or isocratic Clean or replace proportioning precision

29
proportioning -
malfunctioning valves; partially remix solvents.
proportioning valves
Occasional sharp spikes -
Use voltage stabilizer for LC system; use
external electrical
independent electrical circuit.
interference
Service or replace pulse damper; purge air
Periodic - pump pulses
from pump; clean or replace check valves.
Random - contamination Flush column with strong solvent; clean
buildup up sample; use HPLC grade solvent
Degas mobile phase; use backpressure
Spikes - bubble in detector
restrictor at detector outlet.
Spikes - column
temperature higher than Use lower column temperature.
boiling point of solvent

Pressure

Problem Possible cause Solution


Loosen cap on mobile phase
Insufficient flow from pump
reservoir
Leak in hydraulic lines from Tighten or replace fittings;
pump to column tighten rotor in injection valve
Decreasing Leaking pump check valve or Replace or clean check valves;
Pressure seals replace pump seals.
Degas solvent; check for
obstruction in line from solvent
Pump cavitation
reservoir to pump; replace inlet-
line frit
Fluctuating Degas solvent; purge solvent with
Bubble in pump
pressure helium
Leaking pump check valve or Replace or clean check valves;

30
seals replace pump seals
High Back Improve sample cleanup; use
Pressure Column blocked with guard column; reverse-flush
irreversibly adsorbed sample column with strong solvent to
dissolve blockage
Column particle size too small Use larger particle size (for
(for example 3 micrometers) example 5 micrometer)
Use at least 10% organic modifier
in mobile phase; use fresh buffer
daily; add 0.02% sodium azide to
Microbial growth on column
aqueous mobile phase; store
column in at least 25% organic
solvent without buffer
Use lower viscosity solvents or
Mobile phase viscosity too high
higher temperature
Plugged frit in in-line filter or
Replace frit or guard column
guard column
Replace end fitting or frit
Plugged inlet frit
assembly
Use correct solvent with column;
change to proper solvent
Polymeric columns - solvent compositional consult
change causes swelling of manufacturer's solvent-
packing compatibility chart use a column
with a higher percentage of cross-
linking
Salt precipitation (especially in Ensure mobile phase
reversed-phase chromatography compatibility with buffer
with high concentration of concentration; decrease ionic
organic solvent in mobile phase) strength and water-organic
concentration of organic solvent solvent ratio; premix mobile

31
in mobile phase) phase
When injector disconnected from
Clean injector or replace rotor
column - blockage in injector
Systematically disconnect
components from detector end to
Blocked flow lines column end to find blockage;
replace or clean blocked
component
Filter sample; use .5 micrometer
Increasing Particulate buildup at head of
in-line filter; disconnect and back
Pressure column
flush column; replace inlet frit
Ensure mobile phase
compatibility with buffer
Water-organic solvent systems -
concentration; decrease ionic
buffer precipitation
strength or water organic solvent
ratio

Peaks

Problem Possible cause Solution


Broad Analytes eluted early
Dilute sample 1:10 and re-inject
peaks due to sample overload
Use smallest possible cell volume
Detector-cell volume
consistent with sensitivity needs; use
too large
detector with no heat exchanger in system
Decrease solvent strength of injection
Injection volume too
solvent to focus solute; inject smaller
large
volume
Large extra column Use low- or zero-dead-volume end
volume fittings and connectors; use smallest
possible diameter of connecting tubing
(<0.10 in. i.d.); connect tubing with

32
matched fittings
Mobile-phase solvent Increase column temperature; change to
viscosity too high lower viscosity solvent
Decrease injector sample loop size;
Peak dispersion in
introduce air bubble in front and back of
injector valve
sample in loop
Use smaller-particle-diameter packing,
Poor column efficiency lower-viscosity mobile phase, higher
column temperature, or lower flow rate
Use gradient elution or stronger isocratic
Retention time too long
mobile phase
Sampling rate of data
Increase sampling frequency.
system too low
Slow detector time
Adjust time constant to match peak width
constant
Some peaks broad - late
Flush column with strong solvent at end
elution of analytes
of run; end gradient at higher solvent
retained from previous
concentration
injection
Ghost Flush column to remove contaminants in
Contamination
peaks it; use HPLC-grade solvent
Elution of analytes Flush column with strong solvent at end
retained from previous of run; end gradient at higher solvent
injection concentration
Ion-pair
Prepare sample in mobile phase; reduce
chromatography - upset
injection volume
equilibrium
Oxidation of
Prepare trifluoroacetic acid solutions
trifluoroacetic acid in
fresh daily; use antioxidant
peptide mapping
Reversed-phase Check suitability of water by running

33
different amounts through column and
measure peak height of interferences as
chromatography -
function of enrichment time; clean water
contaminated water
by running it through old reversed-phase
column; use HPLC-grade water.
Unknown interferences Use sample cleanup or prefractionation
in sample before injection.
Refractive index
detection - refractive
Reverse polarity to make peak positive
index of solute less than
Negative that of mobile phase
peaks UV-absorbance Use mobile phase with lower UV
detection - absorbance absorbance; if recycling solvent, stop
of solute less than that recycling when recycled solvent affects
of mobile phase detection

34
Peaks continued

Problem Possible cause Solution


Replace or clean frit; install 0.5-um porosity in-
line filter between pump and injector to eliminate
Blocked Frit
mobile-phase contaminants or between injector
and column to eliminate sample contaminants
Co elution of Use sample cleanup or prefractionation; adjust
interfering selectivity by changing mobile or stationary
compound phase
Co elution of
interfering Flush column with strong solvent at end of ran;
compound from end gradient at higher solvent concentration
previous injection
Peak
Use higher-capacity stationary phase; increase
Doubling Column overloaded
column diameter; decrease sample amount
Replace column, or, if possible, open top end
Column void or
fitting and clean and fill void with glass beads or
channeling
same column packing; repack column
Injection solvent Use weaker injection solvent or stronger mobile
too strong phase
Use injection volume equal to one-sixth of
Sample volume too
column volume when sample prepared in mobile
large
phase for injection
Unwept injector
Replace injector rotor
flow path
Channeling in
Replace or repack column
Peak column
Fronting Use higher-capacity stationary phase; increase
Column overloaded
column diameter; decrease sample amount
Tailing Basic solutes - Use competing base such as triethylamine; use a

35
stronger mobile phase; use base-deactivated
silanol interactions silica-based reversed-phase column; use
polymeric column
Beginning of peak
See peak doubling
doubling
Use high purity silica-based column with low
Chelating solutes -
trace-metal content; add EDTA or chelating
trace metals in base
compound to mobile phase; use polymeric
silica
column
Silica-based
Use polymeric, sterically protected, or high-
column -
coverage reversed-phase column; install silica gel
degradation at high
saturator column between pump and injector
pH
Silica-based
Peaks
column -
Reduce temperature to less than 50 C
degradation at high
temperature
Silica-based Decrease mobile-phase pH to suppress silanol
column - silanol ionization; increase buffer concentration;
interactions derivatize solute to change polar interactions
Minimize number of connections; ensure injector
Unwept dead
rotor seal is tight; ensure all compression fittings
volume
are correctly seated
Replace column, or, if possible, open top end
fitting and clean and fill in void with glass beads
Void formation at
or same column packing; rotate injection valve
head of column
quickly; use injection valve with pressure bypass;
avoid pressure shock
Spikes Bubbles in mobile Degas mobile phase; use back-pressure restrictor
phase at detector outlet; ensure that all fittings are tight
Column stored Store column tightly capped; flush reversed-

36
without caps phase columns with degassed methanol

37
Ultra Fast Liquid Chromatography (UFLC)
High Performance Liquid Chromatography, HPLC, is a proven technique that has been
used in laboratories worldwide over the past 30-plus years. One of the primary derives
for the growth of technique has been evolution of packaging materials used to effect the
separation. The underlying principles of this evolution are governed by Van Deemter
equation, which is the empirical formula that describes the relationship between linear
velocity (flow rate) and plate height (HETP or efficiency) since particle size is one of the
variable, a Van Deemter curve can be used to investigate column performance.
According to Van Deemter equation, as the particle size decreases to less than 2.5
micrometer, not only is there a significant gain in efficiency, but also the efficiency does
not diminish at increased flow rates or linear velocity. By using smaller particles, speed
and peak capacity (number of peaks resolved per unit time in gradient separations) can be
extended to new limits. This is the underlying principle of Ultra Fast Liquid
Chromatography.
Going back to the concept of resolution,

To obtain high resolution, number of theoretical plates must be maximized. An increase


in N, the number of theoretical plates, by lengthening the column leads to an increase in
retention time and increased band broadening - which may not be desirable. Instead, to
increase the number of plates, the height equivalent to a theoretical plate can be reduced
by reducing the size of the stationary phase particles. This is the underlying principle of
UFLC.

38
Differences between HPLC and UFLC

Item/ Way to UFLC Conventional UFLC


Parameter 5 μm 2.2 μm
4.6 mm 3.0 mm i.d.×75 mm
i.d.×150 mm Column vol: 0.5 mL
Column vol.:
2.5 mL
1 Tube ID Proportional to column 0.3 mm 0.1 mm
section area ratio [0.3×(3.0/4.6)2 ]
2 Mixer Proportional to column 1.5 mL 0.3 mL (including
volume volume ratio (sample loop (including loop)
volume should be loop) [1.5×(0.5/2.5)]
included)
3 Flow rate Opt. flow rate for each 1.0 mL/min 1.2 mL/min
column
4 Gradient Proportional to (column A/B=70/30 at A/B=70/30 at 0 min
time volume ratio) / (flow rate 0 min A/B=30/70 at 5 min
program ratio) A/B=30/70 at [30×(0.5/2.5)/(1.2/1)]
30 min
5 Column No change 40℃ 40℃
temp
6 Response (col. vol. ratio) / (flow 500 ms ≈ 100 ms
rate ratio) [500×(0.5/2.5)/
*Sampling rate is also (1.2/1)]
changed
7 Injection Proportional to column 10 µ L 4µ L
volume cross section area ratio [10×(3.0/4.6)2 ]

1. Column internal diameter

39
Internal Diameter Optimal Flow Rate
Application
(mm ID) (ml/min)

2.0 0.4-0.5 For fast semi micro analysis with LC-MS

3.0 0.9-1.2 For fast analysis in general purpose HPLC

4.6 2.0-2.5 For fast analysis with large sample load

2. Column length

Length Application

30mm For ultra high-speed analysis

50mm For general purpose fast analysis

To shorten analysis time from that obtained with 5µm particle diameter, 150mm
75mm
column.

100mm For high-speed, high-separation analysis

40
41
Method development for HPLC
The 3 critical components for an HPLC method are:
• Sample preparation
• HPLC analysis
• Standardization (calculations).

During the preliminary method development stage, all individual components should be
investigated before the final method optimization. This gives the scientist a chance to
critically evaluate the method performance in each component and streamline the final
method optimization. Sample preparation for chromatography is as important as the
chromatographic conditions.

Developing an HPLC method involves, understanding the chemistry of analytes and the
drug products. The intended use of the method should be known to the scientist. During
time management for HPLC method development around 10% of the time is given for
understanding the chemistry of the analyte and the drug product. [6]
The next step is to develop a preliminary HPLC conditions to achieve minimally
acceptable separations. These methods are used during entire method development
procedure. Around 20% of the total time is given for the development of preliminary
method.
The solubility of the analyte is then studied with different solvents to develop a suitable
sample preparation scheme for the drug product. 10% of the total time is given for the
sample preparation. The method is then standardized by varying the chromatographic
conditions such as mobile phase composition, temperature of the column etc.10% of the
total time is given to the standardization step.
Finally the robustness of the method is checked by repeated injections of the analyte.
Relative standard deviation for the analyte peak is tried to be kept minimum. Method
performance under different conditions – different instruments, different samples is
studied. 20% of the time is given to this step. [8]

The developed method is then validated according to ICH guidelines. It is a time


consuming step and takes about 30% of the required time. There’s no specific end to the

42
method development procedure. According to the objective of the method being
developed there are some points to be taken care of:

For a related substance method, determining the “significant and relevant” related
substances is very critical. With limited experience with the drug product, a good way to
determine the significant related substances is to look at the degradation products
observed during stress testing. Significant degradation products observed during stress
testing should be investigated in the method development..
Based on the current ICH guidelines on specifications, the related substances method for
active pharmaceutical ingredients (API) should focus on both the API degradation
products and synthetic impurities, while the same method for drug products should focus
only on the degradation products. In general practice, unless there are any special
toxicology concerns, related substances below the limit of quantitation (LOQ) should not
be reported and therefore should not be investigated.
In this stage, relevant related substances should be separated into 2 groups:
• Significant related substances: Linearity, accuracy and response factors should be
established for the significant related substances during the method validation. To
limit the workload during method development, usually 3 or less significant
related substances should be selected in a method
• Other related substances: These are potential degradation products that are not
significant in amount. The developed HPLC conditions only need to provide good
resolution for these related substances to show that they do not exist in significant
levels.[7,10]

Resolution (Rs)
A stability indicating method must resolve all significant degradation products from each
other. Typically the minimum requirement for baseline resolution is 1.5. This limit is
valid only for 2 Gaussian-shape peaks of equal size. In actual method development, Rs =
2.0 should be used as a minimum to account for day-to-day variability, non-ideal peak
shapes and differences in peak sizes. [3]

43
Limit of Quantitation (LOQ)
The desired method LOQ is related to the ICH reporting limits. If the corresponding ICH
reporting limit is 0.1%, the method LOQ should be 0.05% or less to ensure the results are
accurate up to one decimal place. However, it is of little value to develop a method with
an LOQ much below this level in standard practice because when the method is too
sensitive, method precision and accuracy are compromised. [3]

Precision, Accuracy
Expectations for precision and accuracy should be determined on a case-by-case basis.
For a typical related substance method, the RSD of 6 replicates should be less than 10%.
Accuracy should be within 70 % to 130% of theory at the LOQ level. [3]

Analysis time
A run time of about 5-10 minutes per injection is sufficient in most routine related
substance analyses. Unless the method is intended to support a high-volume assay,
shortening the run time further is not recommended as it may compromise the method
performance in other aspects (e.g., specificity, precision and accuracy.) [8]

Adaptability for Automation


For methods that are likely to be used in a high sample volume application, it is very
important for the Method to be “automatable”. The manual sample preparation procedure
should be easy to perform. This will ensure the sample preparation can be automated in
common sample preparation workstations. [3]

Understand the Chemistry


Similar to any other research project, a comprehensive literature search of the chemical
and physical properties of the analytes (and other structurally related compounds) is
essential to ensure the success of the project.

Chemical Properties
Most sample preparations involve the use of organic-aqueous and acid-base extraction

44
techniques. Therefore it is very helpful to understand the solubility and pKa of the
analytes. Solubility in different organic or aqueous solvents determines the best
composition of the sample solvent. pKa determines the pH in which the analyte will exist
as a neutral or ionic species. This information will facilitate an efficient sample extraction
scheme and determine the optimum pH in mobile phase to achieve good separations. [7]

Potential Degradation Products


Subjecting the API or drug product to common stress conditions provides insight into the
stability of the analytes under different conditions. The common stress conditions include
acidic pH, basic pH, neutral pH, different temperature and humidity conditions,
oxidation, reduction and photo-degradation. These studies help to determine the
significant related substances to be used in method development, and to determine the
sample solvent that gives the best sample solution stability.
In addition, the structures of the analytes will indicate the potential active sites for
degradation. Knowledge from basic organic chemistry will help to predict the reactivity
of the functional groups. For example, some excipients are known to contain trace level
of peroxide impurities. If the analyte is susceptible to oxidation, these peroxide impurities
could possibly produce significant degradation products. [2]

Sample Matrix
Physical (e.g., solubility) and chemical (e.g., UV activity, stability, pH effect) properties
of the sample matrix will help to design an appropriate sample preparation scheme. For
example, Hydroxypropyl Methylcellulose (HPMC) is known to absorb water to form a
very viscous solution; therefore it is essential to use mostly organic solvents in sample
preparation. [2]

Preliminary HPLC Conditions


In order to develop preliminary HPLC conditions in a timely fashion, scientists should
use artificial mixtures of active pharmaceutical ingredients and related substances at
relatively high concentrations (e.g., 1-2% of related substance relative to API) to develop

45
the preliminary HPLC conditions. The concentration ratio between API and the related
substances should be maintained to ensure the chromatography represents that of a real
sample. Alternatively, a highly stressed sample (e.g., 5% degradation) can also be used at
this stage. With the known composition and high levels of degradation products in the
sample, one can evaluate the chromatography to determine whether there are adequate
separations for all analytes. The high concentrations of related substances are used to
ensure all peaks will be detected.
Computer assisted method development can be very helpful in developing the
preliminary HPLC conditions quickly. Since the objective at this stage is to quickly
develop HPLC conditions for subsequent method development experiments, scientists
should focus on the separation of the significant related substances (section 3.1.1) instead
of trying to achieve good resolution for all related substances. These significant related
substances should be baseline resolved from each other with Rs > 2.0. After the
preliminary method development the HPLC conditions can be further fine-tuned at a later
stage to achieve the required specificity for the other related substances. [2]

Aged HPLC Column


An aged HPLC column should be used to develop the initial HPLC conditions. Usually it
is more difficult to achieve the required resolution with an aged column (e.g., column
with about 200 injections). This will reflect the worst-case scenario likely to be
encountered in actual method uses, and help the long-term method robustness.
In general, develop all methods with HPLC columns from the same vendor. The
preferred brand of HPLC column should be selected primarily based on the long term
stability and lot to lot reproducibility. [2]

Sample Preparation
• Selection of Sample Solvent
This stage focuses on the selection of the sample solvent (for extraction) and the proper
sample preparation procedures. Investigate the effect of sample solvents of different %
organic, pH, extraction volume and extraction procedure on accuracy, precision,

46
sensitivity (LOQ) and the changes in the chromatography (e.g., peak shape, resolution).
Whenever possible use the mobile phase in the sample preparation. This will ensure that
there will not be any compatibility issues between the sample solution and the HPLC
conditions. [6,8]
• Accuracy:
To investigate the accuracy in sample preparation (i.e., extraction efficiency), prepare a
spiked solution by adding known amounts of related substances into a sample matrix.
Compare responses of the spike solutions and the neat standard solutions to assess the
recovery from the sample preparation. In this stage, since only one particular step is being
investigated (i.e., sample preparation), close to theoretical recovery should be observed at
this point (e.g., 90-110%). [6,8]

• Precision:
Use the stressed sample to represent the worst-case scenario and perform replicate
sample preparations from the same sample composite. Investigate the consistency of the
related substance profile (i.e., any missing peaks?) and the repeatability results from these
preparations. Another objective is to determine the sample concentration that gives an
acceptable LOQ (Signal to Noise ratio, S/N) in low-level spike concentrations. The
sample concentration should be low enough to maintain linearity and precision, but high
enough to achieve the desired LOQ. For example, if the ICH reporting limit for this drug
product is 0.1%, the LOQ of the method should be less than 0.05% (i.e., desired LOQ, in
%). By using spike sample solutions of very diluted concentrations for the significant
related substances, estimate the concentrations that give a S/N of about 10 for the
significant related substances. This estimated concentration is the approximate LOQ
concentration (i.e., estimated LOQ concentration, in ∝g/mL). The following equation can
be used to estimate the target sample concentration for the method
Target sample concentration =estimated LOQ concentration (∝g/mL) x 1/desired LOQ
(%) x 100% [6,8]

Standardization

47
• Area % method
If the response of the active pharmaceutical ingredient is linear from LOQ to the nominal
sample concentration, use the % area approach where the related substance is reported as
% area. This is the most straightforward approach, and doesn’t require the preparation of
standard solutions. It also has the highest precision since preparation to preparation
variation will not affect the results. However, in order to ensure the concentration is
linear within this range, the sample concentration is usually limited and this will reduce
the method sensitivity (i.e., increase LOQ) In general, use this approach as long as the
desired LOQ can be achieved. [10]
• External Standard method
Use the external standard method if the response of the active pharmaceutical ingredient
is not linear throughout the whole range, or the desired LOQ cannot be achieved by the
area % method. The concentration of standard solution should be high enough to ensure
the standard solution can be prepared accurately and precisely on a routine basis, it
should be low enough to approximate the concentration of related substance in the
sample solution. In general, the standard concentration should correspond to about 5 % of
related substances. [10]
• Wavelength Selection and Relative Response Factor
Generate the linearity plot of API and related substances at different wavelengths. At this
point, Photodiode Array Detector can be used to investigate the linearity of the active
pharmaceutical ingredient and related substances in the proposed concentration range. By
comparing the linearity slopes of the active pharmaceutical ingredient and the related
substances, one can estimate the relative response factors of the related substances at
different wavelengths. Disregard of whether Area % or External Standard approach is
used, if the relative response factors of some significant related substances are far from
unity, a response factor correction must be applied.
The optimum wavelength of detection is the wavelength that gives the highest sensitivity
for the significant related substances and minimizes the difference in response factors
between those of the active pharmaceutical ingredient and the related substances.

48
After the optimum wavelength is determined, use a highly stressed sample (e.g., 5%
degradation) to verify that the selected wavelength will give the highest % related
substance results. [6]

Overall accuracy
A final check of the method performance is to determine the overall accuracy of the
method. Unlike the accuracy from sample preparation (section 6.1.1), which simply
compares the response of the analyte with and without spiking with matrix, the overall
accuracy compares the % related substances calculated from an accuracy solution with
that of the theoretical value. The accuracy solutions are the solutions spiked with known
concentrations of related substances and matrix. Since the extraction efficiency, choice of
wavelength and the bias in standardization influence the calculated related substance
result, this is the best way to investigate the accuracy of the method. Overall accuracy
reflects the true accuracy of the method [6]

Method Optimization
Robustness
After the individual components of the method are optimized, perform the final
optimization of the method to improve the accuracy, precision and LOQ. Use an
experimental design approach to determine the experimental factors that have significant
impact on the method. This is very important in determining what factors need to be
investigated in the robustness testing during the method validation. To streamline the
method optimization process, use Plackett Burmann Design (or similar approach) to
simultaneously determine the main effects of many experimental factors.
Some of the typical experimental factors that need to be investigated are:
HPLC conditions: % organic, pH, flow rate, temperature, wavelength, and column age.
Sample preparation: % organic, pH, shaking/sonication, sample size, and sample age.
Calculation/standardization: integration, wavelength, standard concentration, response
factor correction.
Typical responses that need to be investigated are:

49
Results: precision (%RSD), % related substance of significant related substances, total
related substances. [8]

50
Method validation
Validation can be defined as “ establishing documented evidence which provides a high
degree of assurance that specific process will consistently produce a product meeting its
predetermined specification and quality attributes.” Method validation comes into play
after method development and begins with installation and qualification of instruments.
Since HPLC methods are used for different purposes, the method validation may also be
different. For e.g. several publications outline guides to validate pharmaceutical methods
such as USP, ICH ,FDA guidelines
The first step in development and validation of HPLC method should be to set clear
understandable minimum requirements that are acceptable to the chromatographer and
the end user.
Complete list of criteria should be made and evaluated before the method is validated.
The Statistics generated from validation studies should be similar and predictive of the
range of values gathered from real sample analysis. Validtion is of three types:
• Full validation
• Partial validation
• Cross validation
Full validation
Full validation is important while developing and implementing an analyitical method for
the first time. It is important for a new drug entity. A full validation of the revised assay
is important if metabolites are added to an existing assay for quantification.

Partial Validation:
Partial validations are modifications of already validated analytical methods. Typical
analytical method changes that fall into this category include,
• Change in analytical methodology (e.g. change in detection system).
• Change in sample processing procedures.
• Change in relevant concentration range.
• Change in instruments or software.

51
Cross Validation.
Cross validation is a comparison of validation parameters when two or more analytical
methods are used to generate data within the same study or across different studies.
An example of cross validation would be a situation where an original validated
analytical method serves as the reference and the revised analytical method is the
comparator. The comparisons should be done both ways. [7]

PARAMETERS FOR VALIDATION OF HPLC METHODS


• Selectivity
• Specificity
• Linearity
• Accuracy
• Precision
• Detection limit [LOD]
• Limit of quantification
• Range
• Ruggedness
• Robustness

Selectivity:
Selectivity is the ability of an analytical method to differentiate and quantify the analyte
in the presence of other components in the sample. Each blank sample should be tested
for interference and selectivity should be ensured at the Lower Limit Of Quantification
(LLOQ).
Before any sample is introduced into a chromatographic system, the appropriate
resolution criteria must be outlined and satisfied. Generally the ability to resolve
individual components is generally a limiting factor for number of analytes that can be
measured using a single procedure. If appropriate resolution cannot be achieved, the
unresolved components at their maximum expected concentration should be validated to
demonstrate that these components would not affect the final result.

52
Specificity:
Specificity is a measure of the capability of the analytical method to be perfectly selective
for an analyte or group of similar analystes..

Linearity:
Validation requires linearity to be established to verify that the analyte response is
linearly proportional to the concentration range of interest. A linearity study is generally
performed by preparing analyte solutions at various concentration levels and these
solutions should be prepared and analyzed at least three times.

Accuracy:
The accuracy of an analytical method describes the closeness of mean test results
obtained by the method to the true value (concentration) of the analyte. Accuracy is
determined by replicate analysis of samples containing known amounts of the analyte.
A minimum of 3 concentrations in the range of expected concentrations is recommended.
The mean value should be within 15% of the actual value except at LLOQ, where it
should not deviate by more than 20%. The deviation of the mean from the true value
serves as the measure of accuracy.

Precision:
The precision of an analytical method describes the closeness of individual measures of
an analyte when the procedure is applied repeatedly to multiple aliquots of a single
homogenous volume of the sample
Precision should be measured using a minimum of 5 determinations per concentration
& minimum of 3 concentrations in the range of expected concentration is recommended.
The precision determined at each concentration level should not exceed 15% of the co
efficient of variation (CV) expect for the LLOQ, where it should not exceed 20% of the
CV.
According to ICH guidelines the measured standard deviation is subdivided into three
categories: repeatability, intermediate precision and reproducibility.

53
Repeatability of a method is obtained if the analysis is carried out in one laboratory by
one operator using the same equipment over a relatively short period of time.
Intermediate precision is measured in one laboratory but over several days and/or using
different analysts.
Reproducibility is defined as the variability of the measurement process in different
laboratories with different instruments.

Limit of detection:
It is the lowest conc. of the analyte that can be detected, but not necessarily quantifies, in
chromatography the detection limit is the injected amount that results in a peak with a
height at least twice as high as baseline noise. It is determined experimentally.

Limit of quantitation:
The limit of quantitation is the injected amount, which results in a reproducible
measurement of peak areas (equivalent amounts). Peak heights are typically required to
be about 10 to 20 times higher than the baseline noise.

Range of the method:


The working range of the method generally gives an optimum concentration range for
quantitative analyses. In practice, the linear range is generally determined by analysis or
samples of varying concentrations of the analyte of interest and plotting concentration
versus detector response.

Ruggedness:
Ruggedness is a measure of reproducibility of the results under normal, expected
operational conditions from laboratory to laboratory and from analyst to analyst i.e. the
chromatographer must be certain that the new method holds up under other conditions for
which the method has been validated.

Robustness:

54
It is the ability of the method to allow the analyte to remain unaffected by small
changes in the parameters such as ionic strength of the sample, detector temperature,
temperature of the sample and injection volume.
For the determination of a method’s robustness pH, flow rate, column temperature,
injection volume, detection wavelength or mobile phase composition is varied within a
realistic range and the quantitative influence of the parameter is within the specified
tolerance, the parameter is said to be within the method’s robustness range. [8]

55
Quality control parameters analyzed using HPLC

Dissolution
Dissolution testing is used to measure the release rate of an active component from a
solid dosage form under controlled conditions. This technique is used to assess the
performance of tablets, capsules and other solids.

Dissolution testing is useful for:

• Guiding formulation development

• Assessing the quality of a sample by determining whether the release of active


ingredient from the formulation is within acceptable limits (often used for release
and stability testing)

• Comparing finished products with different formulations

• Confirming the acceptability of changes to manufacturing procedures during


scale-up or post-marketing changes [9]

Dissolution testing involves dissolution of a solid dosage form in an appropriate


solvent and the conc. Of the active ingredient is measured at regular intervals of
time using HPLC. Two different types of apparatus are used for dissolution
testing. These are as described below:
Apparatus 1— The assembly consists of the following:
A covered vessel made of glass or other inert, transparent material; a motor; a metallic
drive shaft; and a cylindrical basket. The vessel is partially immersed in a suitable water
bath of any convenient size or placed in a heating jacket. The water bath or heating jacket
permits holding the temperature inside the vessel at 37 ± 0.5 during the test and keeping
the bath fluid in constant, smooth motion. No part of the assembly, including the
environment in which the assembly is placed, contributes significant motion, agitation, or
vibration beyond that due to the smoothly rotating stirring element. Apparatus that
permits observation of the specimen and stirring element during the test is preferable. The
vessel is cylindrical, with a hemispherical bottom and with one of the following

56
dimensions and capacities: for a nominal capacity of 1 L, the height is 160 mm to 210
mm and its inside diameter is 98 mm to 106 mm; for a nominal capacity of 2 L, the
height is 280 mm to 300 mm and its inside diameter is 98 mm to 106 mm; and for a
nominal capacity of 4 L, the height is 280 mm to300 mm and its inside diameter is 145
mm to 155 mm. Its sides are flanged at the top. A fitted cover may be used to retard
evaporation. The shaft is positioned so that its axis is not more than 2 mm at any point
from the vertical axis of the vessel and rotates smoothly and without significant wobble.
A speed-regulating device is used that allows the shaft rotation speed to be selected and
maintained at the rate specified in the individual monograph, within ±4%. Shaft and
basket components of the stirring element are fabricated of stainless steel. The entire
assembly is as shown in Figure 1

57
Fig. 1. Basket Stirring Element
Unless otherwise specified in the individual monograph, use 40-mesh cloth. A basket
having a gold coating 0.0001 inch (2.5 µm) thick may be used. The dosage unit is placed
in a dry basket at the beginning of each test. The distance between the inside bottom of
the vessel and the basket is maintained at 25 ± 2 mm during the test. [10]
Apparatus 2— the assembly from Apparatus 1 is used, except that a paddle formed from
a blade and a shaft is used as the stirring element. The shaft is positioned so that its axis is
not more than 2 mm at any point from the vertical axis of the vessel and rotates smoothly
without significant wobble. The vertical centerline of the blade passes through the axis of
the shaft so that the bottom of the blade is flush with the bottom of the shaft. The paddle
conforms to the specifications shown in Figure 2.

58
Fig. 2. Paddle Stirring element

The distance of 25 ± 2 mm between the blade and the inside bottom of the vessel is
maintained during the test. The metallic or suitably inert, rigid blade and shaft comprise a
single entity. A suitable two-part detachable design may be used provided the assembly
remains firmly engaged during the test. The paddle blade and shaft may be coated with a
suitable inert coating. The dosage unit is allowed to sink to the bottom of the vessel
before rotation of the blade is started. A small, loose piece of nonreactive material such
as not more than a few turns of wire helix may be attached to dosage units that would
otherwise float. Other validated sinker devices may be used. [10]

Dissolution Testing Apparatus

59
Impurity profiling

Impurities In Official articles

Concepts about purity change with time are inseparable from developments in analytical
chemistry. If a material previously considered to be pure can be resolved into more than
one component, that material can be redefined into new terms of purity and impurity.
Inorganic, organic, biochemical, isomeric, or polymeric components can all be
considered impurities. Microbiological species or strains are sometimes described in
similar terms of resolving into more than one component.

Monographs on bulk pharmaceutical chemicals usually cite one of three types of purity
tests: (1) a chromatographic purity test coupled with a nonspecific assay; (2) a
chromatographic purity-indicating method that serves as the assay; or (3) a specific test
and limit for a known impurity, an approach that usually requires a reference standard for
that impurity. Modern separation methods clearly play a dominant role in scientific
research today because these methods simultaneously separate and measure components
and fulfill the analytical ideal of making measurements only on purified specimens.. The
purity profile of a specimen that is constructed from the results of experiments using a
number of analytical methods is the ultimate goal.

Definitions

Foreign Substances

Foreign substances, which are introduced by contamination or adulteration, are not


consequences of the synthesis or preparation of compendial articles and thus cannot be
anticipated when monograph tests and assays are selected. Examples of foreign
substances include ephedrine in Ipecac or a pesticide in an oral liquid analgesic.

Residual Solvents

60
Residual solvents are defined as organic volatile chemicals that are used or produced in
the manufacture of drug substances or in the preparation of drug products. The solvents
are not completely removed by practical manufacturing techniques. Appropriate selection
of the solvent for the synthesis of a drug substance may enhance the yield or determine
characteristics such as crystal form, purity, and solubility and, as such, may be a critical
parameter in the synthetic process. Because there is no therapeutic benefit from residual
solvents, they should be removed to the extent possible to meet product specifications,
good manufacturing practices, or other quality-based requirements. Drug products should
contain no higher levels of residual solvents than can be supported by safety data.

Toxic Impurities

Toxic impurities have significant undesirable biological activity, even as minor


components, and require individual identification and quantitation by specific tests. These
impurities may arise out of the synthesis, preparation, or degradation of compendial
articles. Based on validation data, individualized tests and specifications are selected.
These feature comparison to a Reference Standard of the impurity, if available. It is
incumbent on the manufacturer to provide data that would support the classification of
such impurities as toxic impurities.

Concomitant Components

Concomitant components are characteristic of many bulk pharmaceutical chemicals and


are not considered to be impurities in the Pharmacopeial sense. Examples of concomitant
components are geometric and optical isomers (or racemates) and antibiotics that are
mixtures. Any component that can be considered a toxic impurity because of significant
undesirable biological effect is not considered to be a concomitant component.

Signal Impurities

Signal impurities are distinct from ordinary impurities in that they require individual
identification and quantitation by specific tests. Based on validation data, individualized
tests and specifications are selected. These feature a comparison to a reference standard
of the impurity, if available.

61
Signal impurities may include some process-related impurities or degradation products
that provide key information about the process, such as diazotizable substances in
thiazides. It is incumbent on the manufacturer to provide data that would support the
classification of such impurities as signal impurities rather than ordinary impurities.

Ordinary Impurities

Ordinary impurities are those species in bulk pharmaceutical chemicals that are
innocuous by virtue of having no significant, undesirable biological activity in the
amounts present. These impurities may arise out of the synthesis, preparation, or
degradation of compendial articles. Tests for related substances or chromatographic
purity might also control the presence of ordinary impurities.

The value of 2.0% was selected as the general limit on ordinary impurities in monographs
where documentation did not support adoption of other values.

Related Substances

Related substances are structurally related to a drug substance. These substances may be
identified or unidentified degradation products or impurities arising from a manufacturing
process or during storage of a material.

Process Contaminants

Process contaminants are identified or unidentified substances (excluding related


substances and water), including reagents, inorganics (e.g., heavy metals, chloride, or
sulfate), raw materials, and solvents. These substances may be introduced during
manufacturing or handling procedures. [11]

62
Materials and methods

Objective 1:

Complete analysis of Enalapril Maleate as per USP


Materials required:
Dissolution testing:
• Dissolution testing apparatus: apparatus 2
• Dissolution Medium: phosphate buffer pH 6.8
• Glass wares- Measuring cylinder – 1000mL, test tubes, pipettes (not required if
the dissolution tester has an auto sampler), 100mL volumetric flask, agilent 1200
series sample vials with septa
• Sonicator
Requirements for chromatography:
• Standard preparation: 10 mg of USP Enalapril Maleate RS was transferred to a
100 mL volumetric flask & diluted using pH 6.8 phosphate buffer. The final
concentration thus became 0.1 mg of USP Enalpril Maleate per mL.
• Test preparation: filtered portion of the dissolved tablet was used as the test
preparation.
• Buffer solution: 1.38 gms of monobasic sodium phosphate was dissolved in about
800 mL water,pH was adjusted to 2.2 & the solution was diluted to 1000mL with
water.
• Mobile phase: a filtered and degassed mixture of buffer solution and acetonitrile-
75:25 was made and used as the mobile phase.
• Chromatographic system:
Instrument: Agilent 1200 series HPLC
Column: 4.6mm x 25 cm x 5 packing L7
Temperature: 50
Flow rate: 2mL/minute

63
Content uniformity testing:
• Glass wares- test tubes, 100mL volumetric flasks 2, agilent 1200 series sample
vials with septa
• Sonicator
Requirements for chromatography:
• Buffer solution: 1.38 gms of monobasic sodium phosphate was dissolved in about
800 mL water,pH was adjusted to 2.2 & the solution was diluted to 1000mL with
water.
• Standard preparation: 10 mg of USP Enalapril Maleate RS was transferred to a
100 mL volumetric flask & diluted buffer solution. The final concentration thus
became 0.1 mg of USP Enalpril Maleate per mL.
• Test preparation: one tablet of 10 mg enalapril maleate was transferred to a 100
mL volumetric flask & volume was adjusted with buffer solution.
• Mobile phase: a filtered and degassed mixture of buffer solution and acetonitrile-
75:25 was made and used as the mobile phase.
• Chromatographic system:
Instrument: Agilent 1200 series HPLC
Column: 4.6mm x 25 cm x 5 packing L7
Temperature: 50
Flow rate: 2mL/minute

Assay:
• Glass wares- test tubes, 100 mL beaker, 100mL volumetric flasks 2, 25 mL
volumetric flask, agilent 1200 series sample vials with septa
• Sonicator
Requirements for chromatography:
• Buffer solution: 1.38 gms of monobasic sodium phosphate was dissolved in about
800 mL water, pH was adjusted to 2.2 & the solution was diluted to 1000mL with
water.

64
• Enalaprilat standard solution: 40 mg of USP Enalarilat RS was taken in a 100 mL
volumetric flask and the volume was adjusted using distilled water. The conc of
the solution thus became 0.4 mg of USP Enalaprilat RS per mL..
• Enalprilapril diketopiperazine solution: 20 mg of USP enalapril Maleate RS was
placed in a100 mL beaker to form a mound on the bottom of the beaker. The
beaker was then placed on a hot plate at 70-80c to melt the solid. When melting
was observed, the beaker was immediately removed from the hot plate and
allowed to cool. To the cooled residue, 50 mL of acetonitrile was added and the
solution was sonicated to dissolve the residue. The solution typically contained, in
each mL, between 0.2 mg & 0.4 mg of enalapril diketopiperazine.
• Standard preparation: 20 mg of USP Enalapril Maleate RS was transferred to 100
mL volumetric flask.0.5 mL of Enalaprilat standard solution was added to the
flask & volume was adjusted with buffer solution. The solution had a known
conc. of about 0.2 mg of USP Enalapril Maleate RS per mL and 0.002 mg of USP
Enalaprilat RS per mL.
• Assay preparation: 10 tablets of enalapril maleate were transferred to 500 mL
volumetric flask and the vol. was adjusted with buffer solution.
• System suitability solution: 0.5 mL of enalapril diketopiperazine solution was
transferred to a 25 mL volumetric flask and diluted with standard preparation to
volume.
• Mobile phase: a filtered and degassed mixture of buffer solution and acetonitrile-
75:25 was made and used as the mobile phase.
• Chromatographic system:
Instrument: Agilent 1200 series HPLC
Column: 4.6mm x 25 cm x 5 packing L7
Temperature: 50
Flow rate: 2mL/minute

Related substances:
• Glass wares- test tubes, 100 mL beaker, 100mL volumetric flasks 3, agilent 1200
series sample vials with septa

65
• Sonicator
Requirements for chromatography:
• Buffer solution: 1.38 gms of monobasic sodium phosphate was dissolved in about
800 mL water, pH was adjusted to 2.2 & the solution was diluted to 1000mL with
water.
• Enalaprilat standard solution: 40 mg of USP Enalarilat RS was taken in a 100 mL
volumetric flask and the volume was adjusted using distilled water. The conc of
the solution thus became 0.4 mg of USP Enalaprilat RS per mL..
• Enalprilapril diketopiperazine solution: 20 mg of USP enalapril Maleate RS was
placed in a100 mL beaker to form a mound on the bottom of the beaker. The
beaker was then placed on a hot plate at 70-80c to melt the solid. When melting
was observed, the beaker was immediately removed from the hot plate and
allowed to cool. To the cooled residue, 50 mL of acetonitrile was added and the
solution was sonicated to dissolve the residue.the solution typically contained, in
each mL, between 0.2 mg & 0.4 mg of enalapril diketopiperazine.
• Standard preparation: 20 mg of USP Enalapril Maleate RS was transferred to 100
mL volumetric flask.0.5 mL of Enalaprilat standard solution was added to the
flask & volume was adjusted with buffer solution. The solution had known conc.
of about 0.2 mg of USP Enalapril Maleate RS per mL and 0.002 mg of USP
Enalaprilat RS per mL.
• Related compounds standard solution: 1 mL of standard preparation was
transferred to 100 mL volumetric flask and diluted with buffer solution to volume.
• Test preparation: assay preparation was used as the test sample.
• Mobile phase: a filtered and degassed mixture of buffer solution and acetonitrile-
75:25 was made and used as the mobile phase.
• Chromatographic system:
Instrument: Agilent 1200 series HPLC
Column: 4.6mm x 25 cm x 5 packing L7
Temperature: 50
Flow rate: 2mL/minute

66
Objective 2:

To study separation pattern of Parabens mixture (methyl, ethyl, butyl


parabens) using conventional HPLC and to transfer the method to
UFLC and study the separation pattern.

Materials required:
• Glass wares: 100mL volumetric flask.
• Sonicator:
Requirements for conventional liquid chromatography:
• Sample preparation: 5mg each of methyl, ethyl and butyl Parabens is dissolved in
100 mL water.
• Mobile phase: methanol: water (60:40)
• Chromatographic system:
Instrument: Agilent 1200 series HPLC
Column: Shimpack XR-ODS (C18) (4.66mm. x 150mm x 2.2 µm)
Temperature: 30℃
Flow rate: 1.00 mL/min
Requirements for UFLC
• Sample preparation: 5mg each of methyl, ethyl and butyl Parabens is dissolved in
100 mL water.
• Mobile phase: methanol: water (60:40)
• Chromatographic system:
Instrument: Shimadzu prominence UFLC
Column: Shimpack XR-ODS, 3mm x 50mm x 1µm
Temperature: 30℃
Flow rate: 1.00 mL/min

67
Methods:

Objective 1:

Complete analysis of Enalapril Maleate as per USP


Dissolution testing:
1000 mL of dissolution med. was placed in the vessel of apparatus 2. The dissolution
med. was equilibrated to 37 ± 0.5. One tablet was placed in the apparatus taking care to
exclude air bubbles from the surface of the tablet and the apparatus was operated at 50
rpm for 30 minutes. After 30 minutes a specimen from a zone midway between the
surface of the dissolution medium and the top of the vessel was withdrawn and used as a
test sample after filtration. The conc of enalapril maleate was then determined using
HPLC.
About 50µl of the test preparation and the standard preparation were separately injected
in the chromatograph. The column temperature was maintained at 50℃ and the flow rate
was about 2mL/min. The responses for major peaks were then recorded. The quantity of
enalapril maleate in each tablet was calculated by the formula;
(TC/D)(rU / rS),
in which T is the labeled quantity, in mg, of enalapril maleate in the Tablet; C is
the concentration, in mg per mL, of USP Enalapril Maleate RS in the Standard
preparation; D is the concentration, in mg per mL, of enalapril maleate in the Test
preparation, based upon the labeled quantity per Tablet and the extent of dilution; and rU
and rS are the enalapril peak responses obtained from the Test preparation and the
Standard preparation, respectively.

Content uniformity testing:


About 50µl of the test preparation and the standard preparation were separately
injected in the chromatograph. The column temperature was maintained at 50℃ and the
flow rate was about 2mL/min .The responses for major peaks were then recorded. The
quantity of enalapril maleate in each tablet was calculated by the formula;
(TC/D)(rU / rS),

68
in which T is the labeled quantity, in mg, of enalapril maleate in the Tablet; C is
the concentration, in mg per mL, of USP Enalapril Maleate RS in the Standard
preparation; D is the concentration, in mg per mL, of enalapril maleate in the Test
preparation, based upon the labeled quantity per Tablet and the extent of dilution; and rU
and rS are the enalapril peak responses obtained from the Test preparation and the
Standard preparation, respectively.

Assay:
About 50µl of the assay preparation and the standard preparation were separately injected
in the chromatograph. The column temperature was maintained at 50℃ and the flow rate
was about 2mL/min .The responses for major peaks were then recorded. The quantity of
enalapril maleate in each tablet was calculated by the formula;
(CV/N)(rU / rS),
in which C is the concentration, in mg per mL, of USP Enalapril Maleate RS in the
Standard preparation; V is the nominal capacity, in mL, of the volumetric flask
containing the Assay preparation; N is the number of Tablets taken for the Assay
preparation; and rU and rS are the enalapril peak responses obtained from the Assay
preparation and the Standard preparation, respectively.

Related substances:
About 50µl of the test preparation, the standard preparation, the related compounds
standard solution and the buffer solution were separately injected in the chromatograph.
The column temperature was maintained at 50℃ and the flow rate was about 2mL/min.
The responses for all the peaks greater than 0.1 % of the enalapril peak that were not
observed in the buffer solution were measured. The percentage of anhydrous enalaprilat
present in the portion of tablets was calculated by the formula;
(492.52/348.39)(CV/N)(rU / rS)(100/L),
in which 492.52 and 348.39 are the molecular weights of enalapril maleate and
anhydrous enalaprilat, respectively; C is the concentration, in mg per mL, of USP
Enalaprilat RS in the Standard preparation; V is the nominal capacity, in mL, of the
volumetric flask containing the Test preparation; N is the number of Tablets taken for the

69
Test preparation; rU and rS are the enalaprilat peak responses obtained from the Test
preparation and the Standard preparation, respectively; and L is the labeled amount of
enalapril maleate in theTablet.
the percentage of enalapril diketopiperazine (as enalapril maleate) present in the portion
of Tablets was calculated from the formula,
(492.52/358.44)(C¢V/N)(rU /1.25 rS)(100/L),
in which 492.52 and 358.44 are the molecular weights of enalapril maleate and enalapril
diketopiperazine, respectively; C¢is the concentration, in mg per mL, of USP Enalapril
Maleate RS in the Related compounds standard solution; V is the nominal capacity, in
mL, of the volumetric flask containing the Test preparation; N is the number of Tablets
taken for the Test preparation; rU is the enalapril diketopiperazine peak response
obtained from the Test preparation; 1.25 is the response for enalapril diketopiperazine
relative to that for enalapril maleate; rS is the enalapril peak response obtained from the
Related compounds standard solution; and L is the labeled amount, in mg, of enalapril
maleate in the Tablet.
the percentage of any other related compound was calculated by the formula:
(C¢V/N)(rR / rS)(100/L),
in which rR is the sum of the responses of any related compound, other than
those from maleic acid, enalapril, enalaprilat, and enalapril diketopiperazine obtained
from the Test preparation; rs is the enalapril peak response obtained from the Related
compounds standard solution; and C¢, V, N, and L are as defined above: the sum of all
related compounds including those from enalaprilat and enalapril diketopiperazine is not
greater than 5.0%.

70
Objective 2:

To study separation pattern of Parabens mixture (methyl, ethyl, butyl


Parabens) using conventional HPLC and to transfer the method to
UFLC and study the separation pattern.

Procedure:
For conventional HPLC:
About 15µl of the sample preparation was injected in the chromatograph. The column
temperature was maintained at 30℃ and the flow rate is about 1mL/min. The responses
for major peaks were then recorded.
For UFLC:
About 8µl of the sample preparation was injected in the chromatograph. The column
temperature was maintained at 30℃ and the flow rate is about 1mL/min. The responses
for major peaks were then recorded.
The changes in the separation pattern of the same sample i.e. parabens mixture were
studied by comparing the chromatograms of HPLC and UFLC.

71
Results and calculations

Objective 1:

Complete analysis of Enalapril Maleate tablets 10 mg using HPLC.

Dissolution testing:
Chromatogram for the standard preparation:
Data file: C:\EZchrom Elite\Data\August2007\Enalapril Maleate tablets
10mg\10807_001.dat
Method: C:\EZchrom Elite\Data\August2007\Enalapril Maleate tablets 10mg\RS.met
Acquired: 1/08/07 12:10:01PM
Name: standard
Vial no: 1
Injection vol: 50µL
User: TGK

Results:

Retention Name Area Area Theoretical Asymmetry


Time % plates
1 1.710 11740 0.194 522 0.9
2 2.132 184 0.003 10877 1.0
3 8.790 ENALAPRIL MALEATE 6038812 99.80 870 1.3

72
Chromatogram for the test preparation:
Data file: C:\EZchrom Elite\Data\August2007\Enalapril Maleate tablets
10mg\10807_003.dat
Method: C:\EZchrom Elite\Data\August2007\Enalapril Maleate tablets 10mg\RS.met
Acquired: 1/08/07 12:10:01PM
Name: test 2
Vial no: 3
Injection vol: 50µL
User: TGK

Results:

Retention Name Area Area % Theoretical Asymmetry


Time plates
1 1.474 2299296 79.151 2789 0.8
2 1.769 11458 0.394 16345 1.1
3 2.158 198 0.006 2675 1.0
4 8.689 ENALAPRIL MALEATE 593963 20.447 1018 1.2

73
Calculations:
The amount of enalapril maleate in mg released in the dissolution medium is given by the
formula:
(TC/D)(rU / rS),
in which T is the labeled quantity, in mg, of enalapril maleate in the Tablet; C is
the concentration, in mg per mL, of USP Enalapril Maleate RS in the Standard
preparation; D is the concentration, in mg per mL, of enalapril maleate in the Test
preparation, based upon the labeled quantity per Tablet and the extent of dilution; and rU
and rS are the enalapril peak responses obtained from the Test preparation and the
Standard preparation, respectively.
Substituting the values from the chromatogram, we get,

Amt of enalapril maleate released = 10 X 0.1/0.01 X 593963/6038812


= 9.835 mg

According to USP not less than 80% of the labeled amount of the tablet should be
dissolved in 30 minutes and from the calculations it can be seen that 9.85 mg of the 10
mg i.e. 98.5% of the labeled quantity is released in the dissolution medium after 30
minutes. Thus the batch passes the dissolution test.

74
Content uniformity testing:
Since the buffer solution and the mobile phase for dissolution testing and content
uniformity testing is same, no separate injection of the standard preparation is required.
The chromatogram for the test preparation is given below:

Data file: C:\EZchrom Elite\Data\August2007\Enalapril Maleate tablets


10mg\10807_002.dat
Method: C:\EZchrom Elite\Data\August2007\Enalapril Maleate tablets 10mg\RS.met
Acquired: 1/08/07 12:36:07PM
Name: test 1
Vial no: 2
Injection vol: 50µL
User: TGK

Results:

Retention Name Area Area % Theoretical Asymmetry


Time plates
1 1.467 2299267 79.167 2569 0.8
9
2 1.780 11578 0.039 16329 1.0
3 2.111 175 0.0006 2696 1.2
4 8.780 ENALAPRIL MALEATE 6038808 20.793 1016 1.2

75
Calculations:
Amount of Enalapril maleate, in mg, per tablet is given by the formula:
(TC/D)(rU / rS),
in which T is the labeled quantity, in mg, of enalapril maleate in the Tablet; C is
the concentration, in mg per mL, of USP Enalapril Maleate RS in the Standard
preparation; D is the concentration, in mg per mL, of enalapril maleate in the Test
preparation, based upon the labeled quantity per Tablet and the extent of dilution; and rU
and rS are the enalapril peak responses obtained from the Test preparation and the
Standard preparation, respectively.
Substituting the values from the chromatogram, we get,

Amt of enalapril maleate per tablet = 10 X 0.1/0.1 X 6038798/6038812


= 9.999 mg
According to USP the tablets should contain not less than 90% and more than 110% of
the labeled amount of enalapril maleate.
Since the calculations indicate that the tablets contain 9.999 mg i.e. 99.99% of the labeled
amount of enalapril maleate, the batch passes the content uniformity testing.

76
Assay:
Chromatogram for the standard preparation:
Data file: C:\EZchrom Elite\Data\August2007\Enalapril Maleate tablets
10mg\10807_005.dat
Method: C:\EZchrom Elite\Data\August2007\Enalapril Maleate tablets 10mg\RS.met
Acquired: 1/08/07 1:47:03PM
Name: assay std
Vial no: 5
Injection vol: 50µL
User: TGK

Results:

Retention Name Area Area Theoretical Asymmetry


Time % plates
1 1.689 24148 0.396 15896 1.2
2 2.213 8107 0.133 3698 1.3
3 2.487 ENALAPRILAT 6733 0.110 2698 1.9
4 3.341 1134 0.019 7569 1.2
5 4.146 234 0.003 9658 1
6 4.710 271 0.004 16598 1.4
7 5.418 709 0.012 7698 1.0
8 8.451 ENALAPRIL MALEATE 6050899 99.321 968 1.2

77
Chromatogram for assay preparation:

Data file: C:\EZchrom Elite\Data\August2007\Enalapril Maleate tablets


10mg\10807_007.dat
Method: C:\EZchrom Elite\Data\August2007\Enalapril Maleate tablets 10mg\RS.met
Acquired: 1/08/07 03:10:01PM
Name: test 1
Vial no: 7
Injection vol: 50µL
User: TGK

Results:

Retention Name Area Area Theoretical Asymmetry


Time % plates
1 1.467 2148250 77.920 2516 0.7
6
2 1.710 22388 0.081 16329 1.1
3 2.041 7406 0.027 2923 1.3
4 2.595 ENALAPRILAT 9526 0.035 2013 2.1
5 3.333 1059 0.004 4250 2.2
6 4.446 245 0.001 14494 0.8
7 4.602 262 0.001 13766 1.4
8 5.384 726 0.003 7786 1.4
9 8.353 ENALAPRIL MALEATE 6038798 21.905 866 1.3

78
10 12.867 DIKETOPIPERAZINE 5035 0.018 6230 1.2

Calculations:
The quantity of enalapril maleate in each tablet is calculated by the formula;
(CV/N)(rU / rS),
in which C is the concentration, in mg per mL, of USP Enalapril Maleate RS in the
Standard preparation; V is the nominal capacity, in mL, of the volumetric flask
containing the Assay preparation; N is the number of Tablets taken for the Assay
preparation; and rU and rS are the enalapril peak responses obtained from the Assay
preparation and the Standard preparation, respectively.
Substituting the values from the chromatogram we get,
Amt of enalapril maleate per tablet = 0.2 X (500/10) X (6038798/6050899)
= 9.980mg
According to USP the tablets should contain not less than 90% and more than 110% of
the labeled amount of enalapril maleate.
Since the calculations indicate that the tablets contain 9.980 mg i.e. 99.80 % of the
labeled amount of enalapril maleate, the batch passes the content uniformity testing.

79
80
Related substances:
Chromatogram for the blank solution:
Data file: C:\EZchrom Elite\Data\August2007\Enalapril Maleate tablets
10mg\10807_004.dat
Method: C:\EZchrom Elite\Data\August2007\Enalapril Maleate tablets 10mg\RS.met
Acquired: 1/08/07 01:10:01PM
Name: blank
Vial no: 4
Injection vol: 50µL
User: TGK

Results:

Retention Name Area Area Theoretical Asymmetry


Time % plates
1 1.695 22741 68.584 17588 1.1
2 2.012 7698 23.216 3658 1.3
3 3.128 1235 3.725 7856 2.2
4 4.487 198 0.597 10256 0.8
5 4.692 305 0.919 15256 1.4
6 5.394 850 2.563 7896 1.4
7 23.693 131 0.395 0 1.7

81
Since the buffer solution and the mobile phase for assay and related substances testing is
same, no separate injection of the standard preparation is required.
The chromatogram for the related compounds standard preparation is given below:

Data file: C:\EZchrom Elite\Data\August2007\Enalapril Maleate tablets


10mg\10807_006.dat
Method: C:\EZchrom Elite\Data\August2007\Enalapril Maleate tablets 10mg\RS.met
Acquired: 1/08/07 2:17:03PM
Name: reference
Vial no: 6
Injection vol: 50µL
User: TGK

Results:

Retention Name Area Area Theoretical Asymmetry


Time % plates
1 1.670 21270 27.381 15785 1.0
2 2.211 8813 11.345 3647 1.2
3 2.510 ENALAPRILAT 689 0.886 2698 1.6
4 3.180 1041 1.340 7125 1.0
5 4.210 310 0.399 9741 1.1
6 4.711 280 0.360 15963 1.2
7 5.532 678 0.873 7752 1.1
8 8.472 ENALAPRIL MALEATE 44602 57.415 903 1.0

Analyst:

82
Chromatogram for the test preparation:
Data file: C:\EZchrom Elite\Data\August2007\Enalapril Maleate tablets
10mg\10807_007.dat
Method: C:\EZchrom Elite\Data\August2007\Enalapril Maleate tablets 10mg\RS.met
Acquired: 1/08/07 03:10:01PM
Name: test 1
Vial no: 7
Injection vol: 50µL
User: TGK

Results:

Retention Name Area Area Theoretical Asymmetry


Time % plates
1 1.467 2148250 77.920 2516 0.7
6
2 1.710 22388 0.081 16329 1.1
3 2.041 7406 0.027 2923 1.3
4 2.595 ENALAPRILAT 9526 0.035 2013 2.1
5 3.333 1059 0.004 4250 2.2
6 4.446 245 0.001 14494 0.8
7 4.602 262 0.001 13766 1.4
8 5.384 726 0.003 7786 1.4
9 8.353 ENALAPRIL MALEATE 6038798 21.905 866 1.3
10 12.867 DIKETOPIPERAZINE 5035 0.018 6230 1.2

83
Calculations:

The percentage of anhydrous enalaprilat present in the portion of tablets is calculated by


the formula;
(492.52/348.39)(CV/N)(rU / rS)(100/L),
in which 492.52 and 348.39 are the molecular weights of enalapril maleate and
anhydrous enalaprilat, respectively; C is the concentration, in mg per mL, of USP
Enalaprilat RS in the Standard preparation; V is the nominal capacity, in mL, of the
volumetric flask containing the Test preparation; N is the number of Tablets taken for the
Test preparation; rU and rS are the enalaprilat peak responses obtained from the Test
preparation and the Standard preparation, respectively; and L is the labeled amount of
enalapril maleate in theTablet.
Substituting the values from the chromatogram we get,
percentage of anhydrous enalaprilat
= (492.52/348.39) X 0.002 X (500/10) X (9526/6733) X10
=2%
The percentage of enalapril diketopiperazine (as enalapril maleate) present in the portion
of Tablets is calculated from the formula,
(492.52/358.44)(C¢V/N)(rU /1.25 rS)(100/L),
in which 492.52 and 358.44 are the molecular weights of enalapril maleate and enalapril
diketopiperazine, respectively; C¢is the concentration, in mg per mL, of USP Enalapril
Maleate RS in the Related compounds standard solution; V is the nominal capacity, in
mL, of the volumetric flask containing the Test preparation; N is the number of Tablets
taken for the Test preparation; rU is the enalapril diketopiperazine peak response
obtained from the Test preparation; 1.25 is the response for enalapril diketopiperazine
relative to that for enalapril maleate; rS is the enalapril peak response obtained from the
Related compounds standard solution; and L is the labeled amount, in mg, of enalapril
maleate in the Tablet.
Substituting the values from the chromatogram we get,
The percentage of enalapril diketopiperazine

84
= (492.52/358.44) X 0.002 X (500/10) X 3246/(1.25 X 44602)
= 0.008 %
No other impurity was found in the tablet as seen from comparison of chromatograms of
blank & test preparation.

85
Objective 2:

To study separation pattern of Parabens mixture (methyl, ethyl, butyl


Parabens) using conventional HPLC and to transfer the method to
UFLC and study the separation pattern.
Separation pattern of parabens using conventional HPLC:

Separation pattern of parabens using UFLC:

86
Peak table (HPLC):

Peak table (UFLC):

87
Conclusion

Objective 1:

Complete analysis of Enalapril Maleate as per USP


Complete analysis of Enalapril Maleate was done using HPLC as per USP.
Results of the complete quality analysis are summarized in the following table:
Parameters Acceptance criteria as Values obtained after Conclusion
per USP analysis of the tablets.
Dissolution testing Not less than 80% of Amt of enalapril Batch passes
the labeled amount of maleate released in 30
the tablet should be minutes = 9.835 mg =
dissolved in 30 98.35 % of the labeled
minutes amount
Content uniformity The tablets should Amt of enalapril Batch passes.
contain not less than maleate per tablet
90% and more than = 9.999 mg
110% of the labeled = 99.99% of the
amount of enalapril labeled amount
maleate.

Assay The tablets should Amt of enalapril Batch passes.


contain not less than maleate per tablet
90% and more than = 9.980mg = 99.80 %
110% of the labeled of the labeled amount
amount of enalapril
maleate.

Contd…

88
The percentage of
Related substances The sum of all the anhydrous enalaprilat Batch passes.
related compounds is =2%
not greater than 5.0 % The percentage of
enalapril
diketopiperazine
= 0.008 %
Since no other
impurity is present the
sum of all the related
substance becomes
2.008 %

Thus the actual procedure of quality control of pharmaceutical products using HPLC was
studied.

Objective 2:

To study separation pattern of parabens mixture (methyl, ethyl, butyl


parabens) using conventional HPLC and to transfer the method to
UFLC and study the separation pattern.
By comparing the separation patterns of parabens mixture using conventional HPLC and
UFLC it was found that the stationary phase chemistry affects the speed of separation and
it was seen that the decrease in the size of the stationary phase particles decreases the
time required for separation with improvement in resolution.
As the analysis time was reduced to 3.0 minutes with UFLC from 21.0 minutes in the
conventional HPLC, UFLC can be considered to be better than HPLC for pharmaceutical
analyses.

89
References

1. D. A. Skoog, J. J. Leary – “ Principles of instrumental analysis”, 6th edition,


Indian reprint 2007,copyright © 2007 by Thomson Brooks/Cole, a part of the
Thomson corporation.

2. Phyllis Brown, DeAntonois, Prentice Hall – “Handbook of Instrumental


techniques for analytical chemistry”, 1997.

3. L. R. Snyder and J. J. Kirkland – “Introduction to Modern Liquid


Chromatography”, 2nd edition. John Wiley & Sons, 1979.

4. LC-GC Magazine, Advanstar Communications.

5. Sal Emmanuel, Jean-Pierre Fontelle – “Quality Assurance and Quality control in


pharmaceutical Industry”, 4th edition, 2004.

6. Craig S. Young and Raymond J. Weigand – “An Efficient Approach to HPLC


Method Development “, 1999.

7. V P shah, guidance for industry - “Bioanalytical method development and


validation” May 2001.

8. J. Mark Green – “A Practical Guide to Analytical Method Validation” 1996, (68)


305A-309A Copyright © 1996 by the American Chemical Society

9. Dissolution testing of immediate release and sustained release solid dosage forms.
U.S. Department of Health and Human Services,
Food and Drug Administration,

90
Center for Drug Evaluation and Research (CDER),
August 1997.

10. General chapters - The United States Pharmacopoeia 29

11. General chapters - The Indian Pharmacopoeia

12. General chapters - British Pharmacopoeia

91