CHAPTER I

Introduction of HPLC, LC-MS/MS and aim of the work

13
 GENERAL INTRODUCTION

Quality control and quality assurance of pharmaceutical chemicals and their

formulations are essential for ensuring the availability of safe and effective drug
formulations to the consumers. Pharmaceutical analysis is indispensable in the process
of quality control of drugs for statutory certification of drugs and their formulations
either by the industry or by the regulatory authorities. Constant development of new
and improved analytical methods is essential for accurate determination of drugs in
biological fluids. These methods further have applications in quality assurance,
pharmacokinetic, bioequivalence and toxicological studies.

High performance liquid chromatography with UV / mass spectroscopic

detection is the fastest growing analytical technique for accurate analysis of drugs in
various forms. Its simplicity and wide range of sensitivity and short analysis time
makes it ideal for analysis of many drugs in both biological fluids and dosage forms.
With the development of more sophisticated instrumentation and efficient column
materials the HPLC & LC-MS/MS techniques have now become more accurate and
reliable.

The present study incorporated in the thesis was taken up by the author with an
aim to develop more efficient and validated new high performance liquid
chromatographic methods with UV / mass spectrometric detection for estimation of
some important drugs namely felbamate, gemfibrozil, linezolid, pioglitazone and their
metabolites and lamivudine, zidovudine individually or in combination in human
plasma. The study design involves the development of new reverse phase HPLC & LC-
MS/MS methods for estimation of the selected drugs, validation of the methods thus
developed and testing their suitability for estimation of the drugs in plasma samples.
Out of a total of five methods proposed three were carried out by adopting reverse
phase HPLC technique and the remaining by LC-MS/MS technique. The methods were
validated as per FDA as well as ICH guidelines.
14
 A literature survey on the analytical methods of felbamate, gemfibrozil,
linezolid, pioglitazone and their metabolites and lamivudine, zidovudine revealed that
some HPLC & LC-MS/MS methods are available for their estimation in plasma and
other biological fluids. Some of these methods have certain drawbacks like low
resolution, lesser sensitivity, long run time, incomplete recovery, large volumes of
plasma sample for the extraction technique and large volume of injection which results
in less number of injections on the column etc. Furthermore, some methods were
partially validated and not as per the desired guidelines. Hence, the author had
attempted to develop simple, faster, more reproducible methods for the determination
of these drugs, using low volumes of plasma samples for the extraction and injection
thereby ensuring longer column life. The methods proposed by the author are less
tedious and economical. The proposed methods can be used as alternative methods to
those reported by the earlier workers and provide good choice for the routine
determination of the chosen drugs in their plasma samples for their clinical,
pharmacokinetic, bioavailability and in bioequivalence studies.

The thesis has been presented in five chapters. Chapter-I describes the
introductory information about HPLC & LC-MS/MS and their techniques. This is
followed by the general guidelines and methodology to be followed for developing
methods for estimation of the drugs by HPLC & LC-MS/MS. Later, the procedures
adopted to determine various parameters for validation of the methods have been
reported.

Chapter- II, III and IV deals the HPLC method development for the assay of
three drugs namely felbamate, gemfibrozil, and linezolid in human plasma. The
method development is followed by the determination of various validation
parameters.

15
Chapter- IV and V describe’s the details of the authors experimentation and results
obtained in the LC-MS/MS method development for the assay of Pioglitazone and their
metabolites (Keto-pioglitazone and Hydroxy-pioglitazone), and simultaneously
determination of Lamivudine & Zidovudine in human plasma. The method
development is followed by the determination of various validation parameters.
1) Drug profile
2) Past work on the analytical aspects of the drug
3) Experimentation and results

A) Materials
i) Instrumentation
ii) Drug and internal standard
iii) Chemicals and solvents
iv) Dilutions for Calibration Curve standards and quality control samples
v) Calibration curve plasma standards and quality control plasma samples

B) Method development and optimization of the chromatographic conditions

i) Selection of the column and detection wave length
ii) Composition of the mobile phase; Flow rate
iii) Extraction process of plasma samples and their drying

C) Method validation
Auto sampler carry over test, screening of plasma lots and specificity, linearity,
Precision & accuracy, recovery, stability of drugs in stock solution, stability of
drugs in biological matrix, freeze-thaw stability, bench top stability, in-injector
stability, dry extract stability, long term stability, dilution integrity etc.

4) Summary of the results and discussion

16
5) References

The results obtained in these experiments have been thoroughly discussed at the
end of each part. The references cited in the body of the thesis have been given at the
end of each part.

17
INTRODUCTION TO HPLC TECHINQUE

The development of any new or improved method for the analysis of an analyte
usually tailors the existing analytical approaches and instrumentation. Method
development usually requires selecting the method requirements and deciding on the
type of instrumentation1. In the development stage of an HPLC method, decision
regarding the choice of column, mobile phase, detector and method of quantitation
must be addressed.

Once the instrumentation has been selected, it is important to determine the

chromatographic parameters for the analyte of interest. It is necessary to consider the
properties of the analyte(s) that may be advantageous to select the nature of the column
to be used, establish the approximate composition and pH of the mobile phase for
separation, wave length to be employed or mass/charge ratio to be scanned at for
detection of the compound, the concentration range to be followed and choice of a
suitable internal standard for quantification purpose etc. such information may be
already available in the literature for the analyte or related compounds.

This is followed by optimization and preliminary evaluation of the method.

Optimization criteria must be determined with cognizance of the goals common to any
new method. Initial analytical parameters of merit like sensitivity (measured as
response per amount injected), limit of detection, limit of quantitation and linearity of
calibration plots. As a precautionary measure, it is important that method development
to be performed using only the analytical standards that have been well identified and
characterized and whose purity is known.

During the optimization stage, the initial sets of conditions that have evolved
from the first stages of development are improved or optimised in terms of resolution,
peak shape, plate counts, peak asymmetry, capacity, elution time, detection limits, limit

18
of quantitation, and overall ability to quantify the specific analyte of interest. Results
obtained during optimization must be evaluated against the goals of the analysis set
forth by the analytical figures of merit. This evaluation may reveal that additional
improvement and optimization are needed to meet some of the initial method
requirements.
Optimization of the method should yield maximum sensitivity, good peak symmetry,
minimum detection and quantitation levels, a wide linearity range, and a high degree of
accuracy and precision. Other potential optimization goals include baseline resolution
of the analyte of interest from other sample components, unique peak identification,
online demonstration of purity and interfacing of computerized data for routine sample
analysis. Absolute quantitation should use simplified methods that require minimal
sample handling and analysis time.

Optimization of the method can follow either manual or computer driven

approaches. The manual approach involves varying one experimental condition at a
time, while holding all others constant and recording changes in response. The variables
might include flow rate, mobile or stationary phase composition, temperature, detection
wavelength, and pH. This univariate approach to system optimization is slow, time
consuming and expensive. However, it may provide a much better understanding of the
principle involved and of the interactions of the variables. In computer-driven
automated method development, efficiency is optimized while experimental input is
minimized. Computer-driven automated approaches can be applied to many
applications. In addition, they are capable of significantly reducing the time, energy,
and cost of analysis.

19
SYSTEMATIC APPROACH TO THE REVERSE PHASE CHROMATOGRAPHIC
SEPARATION OF PHARMACEUTICAL COMPOUNDS
Classifying the sample

The first step in the method development is to characterize the drug

whether it is regular or special. The regular compounds are those that are neutral or
ionic. The inorganic ions, bio-molecules, carbohydrates, isomers, enantiomers and
synthetic polymers etc. are called special compounds. The selection of initial conditions
for regular compounds depends on the sample type. The general approach for the
reverse phase chromatographic method development is based on the following
considerations.

The regular samples like pharmaceuticals (either ionic or neutral) respond

in predicable fashion to changes in solvent strength (%B) and type (e.g. acetonitrile or
methanol) or temperature. A 10% decrease in %B increases retention by about three fold
and selectivity usually changes as either %B or solvent type is varied. An increase in
temperature causes a decrease in retention as well as changes in selectivity. It is possible
to separate many regular samples just by varying solvent strength and type.
Alternatively, varying solvent strength and temperature can separate many ionic
samples and some non-ionic samples.

The Column and Flow rate

To avoid problems from irreproducible sample retention during method

development, it is important that columns be stable and reproducible. A C8 or C18
column made from specially purified less acidic silica and designed specifically for the
separation of basic compounds is generally suitable for all samples and is strongly
recommended. If temperatures >50 0 C are used at low pH, sterically protected bonded-
phase column packing are preferred. The column should provide reasonable resolution
in initial experiments, short run times and an acceptable pressure drop for different
mobile phases. A 5µ, 150 X 4.6 mm column with a flow rate of 2 mL/min is good for

20
different mobile phases as initial choice. These conditions provide reasonable plate
number (N=8000), a run time of < 15 min for a capacity factor k < 20 and a maximum
pressure drop < 440 kgf for any mobile phase made from mixtures of water, acetonitrile
or methanol.

Mobile phase

The preferred organic solvent (B) for the mobile phase mixture is acetonitrile (ACN)
because of its favorable UV transmittance and low viscosity. However, methanol
(MeOH) is a reasonable alternative. Amine modifiers like tetra hydro furan (THF) are
less desirable because they may require longer column equilibration times, which can
be a problem in method development and routine use of the method. They may
occasionally introduce additional problems like erratic base line and poor peak shape.
However, some samples may require the use of amine modifiers when poor peak
shapes or low plate number are encountered.

The pH of the mobile phase should be selected with two important

considerations. A low pH that protonates column silanols and reduces their
chromatographic activity is generally preferred. A low pH (<3) is usually quite different
from the pKa values of common acidic and basic functional groups. Therefore, at low
pH the retention of these compounds will not be affected by small changes in pH and
the reverse phase liquid chromatographic method will be more rugged. For columns
that are stable at low pH i.e. is pH of 2 to 2.5 is recommended. For less stable columns, a
pH of 3.0 is a better choice.

Separation temperature

Mostly the temperature controllers operate best above ambient (>300C). Higher
temperature operation also gives lower operating pressures and higher plate numbers,
because of decrease in mobile phase viscosity. A temperature of 30-400 C is usually a

21
good starting point. However, ambient temperature is required if the method will be
used in laboratories that lack column thermostating.

Sample size

Initially, a 10-50 µL injection (25-50 µg) can be used for maximum detection
sensitivity. Smaller injection volumes are required for column diameters of below 4.5
mm and /or particles smaller than 5 µm. The sample should be dissolved initially in
water (1mg/mL) or dilute solution of acetonitrile in water. For the final method
development stage, the best sample solvent is the mobile phase. The samples which
cannot be dissolved in water or the mobile phase should be dissolved initially in either
acetonitrile or methanol and then diluted with water or mobile phase before injection.

Equilibration of the column with the mobile phase

The analytical column is completely equilibrated with the mobile phase before
injecting the sample for analysis and retention data are collected for interpretation. This
is done for ensuring accurate retention data. Equilibration is required whenever the
column, mobiles phase or temperature is changed during method development; usually
by flow rate at least 10 column volumes of the new mobile phase before the first
injection. Some mobile phases may require a much longer column equilibration time
(e.g. mobile phases that contain THF amine modifiers such as tri ethylamine and tetra
butylamine and any ion pair reagent).

Column equilibration and reproducible data can be confirmed by first washing

the column with at least 10 columns volumes of the new mobile phase and injecting the
sample and then a second washing with at least 5 column volumes of the new mobile
phase and reinjection of the sample. If the column is equilibrated, the retention times
should not change by more than 0.02 min between the two runs.

INTRODUCTION TO LC-MS/MS TECHINQUE

22
 A mass spectrometer is an instrument that measures the masses of electrically
charged molecules, or ions. Mass spectrometer5 (MS) is an analytical technique that is
used for the identification of unknown compounds, the quantitation of known
compounds, and the elucidation of structural information and chemical properties of
molecules, works by using magnetic and electric fields to exert forces on charged
particles (ions) in vacuum. Therefore, a compound must be charged or ionized to be
analyzed by a mass spectrometer. Furthermore, the ions must be introduced in the gas
phase into the vacuum system of the mass spectrometer. This is easily done for gaseous
or heat-volatile samples. However, many (thermally labile) analytes decompose upon
heating. These kinds of samples require either desorption or desolvation methods if
they are to be analyzed by mass spectrometry. Although ionization and
desorption/desolvation are usually separate processes, the term "ionization method" is
commonly used to refer to both ionization and desorption (or desolvation) methods.
The choice of ionization method depends on the nature of the sample and the type of
information required from the analysis. So-called 'soft ionization' methods such as field
desorption and electrospray ionization tend to produce mass spectra with little or no
fragment-ion content. Mass spectrometers measure the mass-to-charge (m/z) ratios of
gas phase ions. Creating gas phase ions is the role of the ionization method. Ionization
methods available on the instruments within the MS Facility are described below. Use
this as a guide to determine which ionization method is best suited for your sample.

23
 Fig-1: Schematic diagram of Mass Spectrometer

Electrospray Ionisation (ESI) is one of the Atmospheric Pressure Ionisation (API)

techniques and is well-suited to the analysis of polar molecules ranging from less than
50 Da to more than 1,000,000 Da in molecular mass.

Standard electrospray ionisation source (Platform II)

During standard electrospray ionization the sample is dissolved in a polar,
volatile solvent and pumped through a narrow, stainless steel capillary (75 - 150
micrometers i.d.) at a flow rate of between 5 µL/min and 2 mL/min. A high voltage of 3
to 4 kV is applied to the tip of the capillary, which is situated within the ionization
source of the mass spectrometer, and as a consequence of this strong electric field, the
sample emerging from the tip is dispersed into an aerosol of highly charged droplets, a
process that is aided by a co-axially introduced nebulising gas flowing around the
outside of the capillary. This gas, usually nitrogen, helps to direct the spray emerging
from the capillary tip towards the mass spectrometer. The charged droplets diminish in
size by solvent evaporation, assisted by a warm flow of nitrogen known as the drying
gas which passes across the front of the ionisation source. Eventually charged sample
ions are released from the droplets. Some of which pass through a sampling cone or
orifice into an intermediate vacuum region and from there through a small aperture

24
into the analyser of the mass spectrometer, which is held under high vacuum. The lens
voltages are optimised individually for each sample.

PARAMETERS AND ION MOVEMENT

Source-dependent parameters, compound-dependent parameters, and detector
parameters are all configured in the analyst software and applied at specific points to
the mass filter rail (ion path). Understanding what each parameter controls and how it
affects resolution, intensity, and peak shape will ensure optimal results during sample
analysis. You should also consider how changing the value of one parameter can affect
another parameter further along the ion path.

Source-Dependent Parameters

Optimal source-dependent parameter values depend on the LC conditions.

Source-dependent parameters should be optimized at or near the desired LC flow
conditions using split infusion or FIA. The positioning of the probe in the source can
have a significant impact on the sensitivity of the analysis. For more information on
how to optimize the position of the probe, refer to the appropriate source operators
manual. These parameters may change depending on the source using.

Nebulizer Gas (Neb): The Neb parameter controls the nebulizer gas. The nebulizer gas
helps generate small droplets of sample flow and affects spray stability and sensitivity.
(This parameter is called Gas 1 for Q TRAP™, 4000 Q TRAP™, API 2000™, API 3200
and API 4000™ systems).

GS1: The GS1 parameter controls the nebulizer gas. The nebulizer gas helps generate
small droplets of sample flow and affects spray stability and sensitivity. (This parameter
is called nebulizer gas for API 3000™ systems.)

25
GS2: The GS2 parameter controls the auxiliary or turbo gas. It is used to help evaporate
(This parameter is called auxiliary, or turbo, gas for API 3000 systems) the spray
droplets and prevent solvent from entering the instrument.

Auxiliary Gas (Aux): The Aux parameter controls the auxiliary or turbo gas. It is used
to help evaporate the spray droplets and prevent solvent from entering the instrument.
(This parameter is called Gas 2 for Q TRAP, 4000 Q TRAP, API 2000, and API 4000
systems.)
Temperature: The temeperrature parameter controls the temperature of the turbo gas in
the Turbo Ion Spray™ source or the temperature of the probe in the heated nebulizer
(or APCI) source. It is used to help evaporate the solvent to produce gas phase sample
ions.

Curtain Gas (CUR): The CUR parameter controls the Curtain Gas™, which flows
between the curtain plate and the orifice. Curtain Gas™ prevents solvent droplets from
entering and contaminating the ion optics. The Curtain Gas™ should be maintained as
high as possible without losing sensitivity.

Ion Spray Voltage (IS): The IS parameter controls the voltage applied to the needle that
ionizes the sample in the ion source. It depends on the polarity, and affects the stability
of the spray and the sensitivity. If you are using the PhotoSpray™ source on an API
2000, API 3200, API 4000, API 5000™, Q TRAP, or 4000 Q TRAP instrument, this
parameter is called Ion Transfer Voltage.
Interface Heater (ihe): The ihe parameter switches the interface heater on and off.
Heating the interface helps maximize the ion signal and prevents contamination of the
ion optics. For API 4000, API4000 Q TRAP and API3200 systems with the Turbo V
source, the interface plate is heated to 100 °C. For the API 2000 and Q TRAP systems,
the interface plate is heated to 100 °C. (This parameter does not apply to API 150EX and
API 3000 systems.) It also does not apply to QSTAR systems.

26
Interface Heater Temperature (IHT): The IHT parameter controls the temperature of
the NanoSpray interface heater and is only available if the NanoSpray source and
interface are installed. The temperature can be adjusted up to 250 °C. (This parameter
applies to the API 4000 and 4000 Q TRAP instruments.)

Nebulizer or Needle Current (NC): The NC parameter controls the current applied to
the corona discharge needle in the APCI (atmospheric pressure chemical ionization)
probe, used in the Turbo V™ source. The discharge ionizes solvent molecules, which in
turn ionize the sample molecules.

Compound-Dependent Parameters
The available compound-dependent parameters vary with instrument type. They
consist mostly of lens elements in the ion path. Optimal values for compound-
dependent parameters do not depend on LC flow conditions. Therefore, the parameters
can be optimized using any sample introduction technique. The parameters listed here
are generally the only ones that need to be optimized. For information on working with
other parameters, refer to the online Help.
Compound-Dependent Parameters for Both Quadrupole- and LIT-mode Scans The
following parameters are available for optimization if you are running a quadrupole-
mode scan or an LIT-mode scan.

De clustering Potential (DP): The DP parameter controls the potential difference

between ground (Skimmer) and the orifice plate. It is used to minimize solvent cluster
ions, which may attach to the sample. The higher the voltage, the greater the amount of
fragmentation or de clustering. If the de clustering potential is too high, the sample ion
itself may fragment.

27
Collisionally Activated Dissociation Gas (CAD): The CAD parameter controls the
pressure of collision gas in the collision cell during Q2 MS, MS/MS, and LIT scans. For
Q3 MS scans, the collision gas helps to focus the ions as they pass through the collision
cell. For MS/MS scans, the collision gas acts as a target to fragment the precursor ions.
When the parent ions collide with the collision gas, they can dissociate to fragment ions.
Although this parameter is on the Source/Gas tab, this parameter is compound-
dependent and not dependent on the sample flow.
Focusing Potential (FP): The FP parameter controls the voltage applied to the focusing
ring lens. The focusing potential helps focus the ions through the skimmer region of the
mass spectrometer interface. It can induce fragmentation in the interface area, similar to
the de clustering potential. (This parameter does not apply to the API 4000, or 4000 Q
TRAP, 5000 systems.)

Entrance Potential (EP): The EP parameter controls the potential difference between the
voltage on Q0 and ground. The entrance potential guides and focus the ions through
the high pressure Qo region, EP effects the value of all the ion path voltage. The
Entrance Potential uses the Denotes ID EP.
Collision Cell Entrance Potential (CEP): The CEP parameter controls the collision cell
entrance potential, which is the potential difference between Q0 and IQ2. It focus ions in
to Q2 (collision cell). The optimal CEP gives the greatest intensity for the ions of
interest. For MS type scans, the default value is appropriate. For MS/MS scans,
optimize CEP for the precursor ion. This is used to focus and accelerate the ions into the
collision cell (Q2). It is used in Q1 and MS/MS type scans and is mass dependent. (This
parameter applies only to API 2000, 3200 and Q TRAP systems.)

Collision Cell Exit Potential (CXP): The CXP parameter controls the collision cell exit
potential, which is used to focus and accelerate the ions out of the collision cell (Q2). It
is used in Q3 and MS/MS type scans. In API 3000, API3200, API 4000, and 4000 Q
TRAP systems, CXP is the potential difference between RO2 and ST3 (the stubby lens

28
between Q2 and Q3), and is not mass-dependent. In API 2000 and Q TRAP systems,
CXP is the potential difference between RO2 and IQ3 (interquad lens 3) and is mass
dependent. The Collision Cell Exit Potential parameter uses the Access ID CXP. It is also
displayed by its Parameter ID ST3.

Rod Offset 2 (RO2): The RO2 parameter controls the potential applied to the collision
cell, Q2. In Q1 and Q3 scans, RO2 is used to focus and transmit the ions. In MS/MS type
scans, Q2 is accessed as CE.

Collision Energy (CE): The CE parameter controls the collision energy, which is the
potential difference between Q0 and Q2 for MS/MS-type scans. This is the amount of
energy that the precursor ions receive as they are accelerated into the Q2 collision cell,
where they collide with gas molecules and fragment.
Collision Cell Rod Offset: The RO2 parameter is also referred to as the Collision
Energy (CE). This parameter controls the potential applied to the collision cell (Q2). In
Q1 and Q3 scans, RO2 is used to focus and transmit the ions. In MS/MS scans CE is the
potential difference between Q0 and Q2. This is the amount of energy that the precursor
ions receive as they are accelerated into the Q2 collision cell, where they collide with gas
molecules and fragment.

Ion Energy 1(IE1): The IE1 parameter controls the potential difference between Q0 and
RO1. Although this parameter does affect the sensitivity, it has a greater impact on the
resolution of the peaks, that is, peak shape, and is considered a resolution parameter.
IE1 is used in Q1and MS/MS-type scans. In Q3 scans, the potential applied to Q1 is
called RO1 (Q1 Rod Offset) and helps to transmit ions.

Ion Energy 3 (IE3): The IE3 parameter controls the potential difference between RO2
and RO3. Although this parameter does affect sensitivity, it has a greater impact on the
resolution of the peaks, that is, peak shape, and is considered a resolution parameter.

29
IE3 is used in Q3 and MS/MS-type scans. In Q1 scans, the potential applied to Q3 is
called RO3 (Q3 Rod Offset) and helps to transmit ions.

Compound-Dependent Parameters for LIT-Mode Scans Only

In addition to the compound-dependent parameters that are available on the
compound tab, several parameters are available on the advanced MS tab for LIT-mode
scans that will affect the sensitivity for your sample of interest. The best method of
sample introduction for optimizing these parameters are infusion since you cannot
change them in real time. The acquisition must be stopped between each parameter
change.

Collision Energy Spread (CES): The CES parameter controls the spread of collision
energies used when filling the LIT and applies when you are using AutoFrag. For
example, if you have a CE of 30 and a CES of 5, collision energies of 25, 30, and 35 will
be used.

Fixed LIT Fill Time: The Fixed LIT Fill Time parameter controls amount of time that
you are filling the trap with ions. In general, the default time is appropriate. You want
to fill the linear ion trap so that you have the greatest peak intensity without saturation.

Dynamic Fill Time (DFT): The DFT parameter controls whether the LIT fill time is
dynamic. If DFT is turned on, the software will dynamically calculate the length of time
that ions are collected in the LIT. If you are using DFT, Q0 trapping is turned off by
default.

Q0 Trapping: The Q0 trapping parameter controls the storage of ions in the Q0 region,
which can increase sensitivity. This parameter has only two values: on or off. When Q0
trapping is on, ions are stored in the Q0 region, while ions are being scanned out of the
trap. If the sample is diluted, you will want to turn on Q0 trapping to increase the duty

30
cycle and get better sensitivity. If the sample is concentrated, you will want to turn off
Q0 trapping to prevent saturating the peaks, which results in poor peak resolution. If
you are using DFT, Q0 trapping is turned off by default.

Time Delayed Fragmentation Collision Energy (TDF CE): Available only for TDF
scans, the TDF CE parameter controls the collision energy that is used to fragment the
precursor ions.

Q3 Entry Barrier: The Q3 Entry Barrier parameter controls the potential difference
between RO2 and RO3. It is used to transfer the ions from Q2 into the LIT. If the
compound is fragile, you may want to decrease the value from the default to prevent
fragmentation.

Q3 Empty Time: Available only for EMC scans, the Q3 Empty Time parameter controls
the amount of time that you are removing the singly charged ions from the trap: the
greater the time, the more ions that leave the trap. If the time is too long, the multiply
charged ions will also exit the trap without detection, resulting in decreased sensitivity.

Q3 Cool Time: Available only in TDF scans, the Q3 Cool Time parameter controls the
amount of time that the precursor ions are allowed to cool prior to collecting all of their
fragment ions. As you increase this time, the number and intensity of fragment ions
decreases. In general, the default Q3 Cool Time value is sufficient.

Multi-Charge Separation (MCS) Barrier: The MCS Barrier parameter controls the
voltage used to remove singly charged ions from the LIT and is only available for EMC
scans. This parameter applies only to the 4000 QTRAP system

31
Detector Parameters:
In a quadrupole mode scan, the following parameters are available for optimization.

Channel Electron Multiplier (CEM): The detector degrades with time and the CEM
value should periodically be readjusted using the standard positive PPG calibrant. Do
not change the voltage unless the detector has been replaced or there has been a
reduction of sensitivity. A typical initial value is 2000V. A typical end-of-life value is
3000V.

Deflector (DF): The deflector shows no mass or energy dependence and optimizes over
at least a 100 V plateau. Each detector has its own optimum value. On some systems,
there may be a different optimization value for MS and MS/MS scans.
Note: Over time the detector begins to wear and require a greater voltage for the same
performance. Therefore, adjusting the detector voltage is an important part of ensuring
maximum sensitivity.

TurboIonSpray probe position

The position of the TurboIonSpray Probe relative to the orifice and to the heater
probe is an important factor in optimizing the TurboIonSpray performance. The probe
should point between 5 and 10 mm off axis with respect to the center of the orifice. The
distance of the Heater Probe from the orifice plane is fixed, but the TurboIonSpray can
be adjusted using the scale on the side of the sample inlet arm. Changing from low
solvent flow rates (40 µl/min) to high solvent flow rates (2 mL/min) requires that the
TurboIonSpray be repositioned further away from the orifice to prevent solvation
penetration through the orifice into the mass spectrometer.

TurboIonSpray Positioning Across the Orifice

32
 Also as the aqueous composition of the carrier solvent increases at high flow
rates (2 mL/ min), the more visible the spray becomes and the further away from the
orifice it should be directed. Refer to the fig-1. TurboIonSpray positioning across the
Orifice, where the areas indicated in the figure for the different flow rates are the
optimum target areas for the TurboIonSpray liquid spray. The circle immediately
around the orifice (for example the part of the orifice plate which is visible when
viewing the front of the interface) should remain clear of solvent or solvent drops at all
times. The best position is usually a few millimeters off axis to the left of the curtain
plate aperture. Multiply charged proteins and peptides introduced at a few micro liters
per minute usually require the sprayer to be less than 1 cm from the Curtain Plate.

TurboIonSpray Voltage (IS)

Positive mode, singly charged compounds usually require a high probe voltage
between 4000 to 5500 V. Negative mode compounds usually require a lower voltage -
3000 to -4500 V.
Nebulizer Gas (Gas 1)
It is optimized for signal stability and sensitivity. Typically a value of 5 to 90 is
used as applied by the Applications Computer.

Curtain Gas Flow

The Curtain Gas ensures a stable clean environment for the sample ions entering
the mass spectrometer. The gas curtain prevents air or solvent from entering the
analyzer region of the instrument while permitting the sample ions to be drawn into the
vacuum chamber by the electrical fields generated between the Vacuum Interface and
the TurboIonSpray needle. The presence of the solvent vapor or moisture in the
analyzer region of the mass spectrometer contaminates the Q0 rod set causing a
reduction in resolution, stability, sensitivity, and an increase in chemical background
noise. As a general rule, the Curtain Gas flow should be set as high as possible without
reducing the signal significantly (for example start at a lower value and increase the

33
flow until the signal starts to decrease). In order to prevent instrument contamination
the Curtain Gas flow should be optimized at the highest possible setting but never
below six that does not result in a significant reduction in signal intensity. Refer to the
System Reference Manual for further details of Vacuum Interface operation.

Heater Gas (Gas2) Flow

The Heater Gas (Gas 2) aids in the evaporation of solvent which aids in
increasing the ionization of the sample. The higher the liquid flow or the higher the
aqueous composition of the solvent, the higher the heater gas temperature and gas flow
required. However, too high a temperature can cause premature vaporization of the
solvent, and result in a high chemical background noise, while too high a heater gas
flow can produce a noisy, or unstable signal. For each flow rate, the Curtain Gas flow
rate (from setting 6 to 90 at the application’s computer) should be as high as possible.
The solvent composition used for optimization was 1/1 water/acetonitrile. These
conditions represent a starting point from which to optimize TurboIonSpray. By an
iterative process, the various settings can be optimized using Flow Injection Analysis to
obtain maximum signal-to-noise for the compound of interest.

Turbo Temperature
The quantity and type of sample affects the optimal TurboIonSpray temperature.
At higher flow rates the optimal temperature increases. As the organic content of the
solvent increases the optimal probe temperature should decrease with solvents
consisting of 100 percent methanol or acetonitrile the probe performance may optimize
as low as 300°C. Aqueous solvents consisting of 100 per cent water at flows
approximately 1mL/min require a minimum probe temperature of 425°C. Normal
optimization is usually performed in increments of 25°C. The TurboIonSpray is
normally used with sample flow rates of 5 µL/min to 2000 µL/ min. The heat is used to
increase the rate of evaporation and this improves ionization efficiency resulting in
increased sensitivity.

34
De clustering Potential (DP) and Focusing Potential (FP) Voltages
Optimal de clustering potential and focusing potential operating conditions with
the TurboIonSpray source should be set high enough to reduce the chemical noise but
low enough to avoid fragmentation. Start with the de clustering Potential DP) at 300V
and the Focusing Potential (FP) at 30V.

Solvent Composition
Commonly used solvents and modifiers are acetonitrile, methanol, propanol,
water, acetic acid, formic acid, ammonium formate and ammonium acetate. The
modifiers such as TEA, sodium phosphate, TFA and dodecyl sodium sulfate are not
commonly used because they omplicate the spectrum with their ion mixtures and
cluster combinations. They may also uppress the strength of the target compound ion
signal. The standard concentration of ammonium formate or ammonium acetate is from
2 to 10 mmol per liter for positive ions and 2 to 50 mmol per liter for negative ions. The
concentration of the organic acids is 0.01% to 0.5% by volume.

Source Exhaust Pump

The Source Exhaust system is required for TurboIonSpray operation. The
exhaust pump draws the solvent vapors from the enclosed source chamber and delivers
them to a trap at the rear of the instrument chassis where they can be collected. The
source exhaust system is interlocked to the system electronics, such that if the source
exhaust pump is not operating to specification the instrument electronics are disabled.
The exhaust system lowers the pressure in the source slightly below atmospheric. If the
pressure in the source rises beyond a trip point, the instrument high voltage power
supply is disabled. The adjustment of the source exhaust can affect the TurboIonSpray
operation. The sample pump should be optimized at the flow rate to be used for a
particular sample by adjusting the exhaust flow control regulator located on the Ion
Source Panel.

35
Polarity selection in multiple reaction monitoring mode (LC-MS/MS)
In the case of method development for the known compound it is necessary to
consider the chemical structure of the molecule. Based on the functional groups
presented in the molecule the polarity of the compound is decided for the mass
spectrometer study. For example, if the compound is having basic functional groups
such as primary and secondary amines amides etc. it will accept a proton from the
solution and its molecular weight increases. Thus, for compounds with basic functional
groups positive polarity is selected in the multiple reaction monitoring (MRM).

Similarly if the compound contains acidic functional groups such as phenols,

carboxylic etc. it liberates proton in solution and consequently the molecular weight of
the compound will decreases. Thus, the acidic [H] + compounds which show M-1 peaks
in their mass spectra. Hence, for compounds with acidic functional groups negative
polarity is chosen in the MRM mode. If the compound is having both the acidic and
basic functional groups in its molecule, both the polarities are tested and the one
showing the better and reproducible sensitivity is selected.

36
METHOD DEVELOPMENT AND PLASMA EXTRACTION PROCEDURE

Choice of extraction technique and mobile phase selection

Based on the solubility of the compound the composition of the mobile phase is
judged. From the pKa of the compound, the pH of the buffer used in the mobile phase
can be adjusted (pH = ± 2 of pKa value). Based on the log of partition coefficient (Log P)
value obtained from the literature, if the value is more than 1.0, we can optimization for
liquid – liquid extraction (LLE) technique for extracting the drug from plasma. Even if
the pKa value is below 1.0 we can check with LLE by adjusting the pH of the drug
containing plasma sample. If the drug is bound to plasma proteins (more than 40
percent), the drug containing plasma is treated with 0.1N acetic acid / 0.1N ortho
phosphoric acid to extract the drug. If this technique is unable to extract the drug from
plasma it’s better to optimization for protein precipitation followed by extraction.

Preparation of biological samples

The aim of sample preparation is to enable instrumental analysis or improve the
instrumental analyte signals in comparison to those obtained from non-treated samples.
The sample preparation steps may consist of extraction of the analyte from the sample
matrix, a clean-up step and/or a pre concentration step. Sometime/ the analytes are
chemically modified or derivatised to give them more suitable properties prior to
separation and/or detection. The sample preparation if laborious may become a major
source of error in the overall analytical process. For these reasons, this part of the
analytical chain should ideally be minimized (or avoided if possible). However, in
many cases extensive sample pretreatment is necessary to obtain acceptable analytical
results. This is often the case for bioanalytical methods where biological samples are
processed. Biological samples, such as urine, blood serum or blood plasma, contain
large amounts (and numbers) of endogenous components and are generally referred to
as complex matrices. The components of the matrix if not removed efficiently may often
interfere and adversely affect the subsequent separation and detection. This is especially

37
important if very low amounts of the analytes are present in the samples. Extracting
hydrophilic compounds from these aqueous matrices is an analytical challenge. Blood
contains many components, including a variety of proteins, fats, salts and suspended
cells. The red blood cells can be removed from the plasma by centrifugation after
addition of an anti-coagulant. The simplest form of sample preparation for this kind of
samples involves dilution, centrifugation, filtration and/or evaporation. Some
commonly used techniques for sample preparation, especially for biological fluid clean-
up are briefly described below.

Techniques for sample preparation

In order to determine compounds such as drugs or drug metabolites in biological
fluids, the proteins generally have to be removed prior to the final analysis. Proteins
may get denatured in the solvents or at high temperatures used for GC and cause
clogging of the analytical column. Some common methods employed for removing
proteins are:

Protein precipitation
When a drug strongly binds to the plasma proteins (in case of plasma samples) it
is often difficult to extract the drug from plasma by any means. Then protein
precipitation followed by extraction is only the process to extract the drug from plasma
samples. This separation technique removes proteins from the samples by denaturating
them directly. The protein precipitation is usually done by the addition of a water
miscible organic solvent (e.g. methanol, ethanol, acetonitrile or acetone) or a strong acid
such as trichloroacetic acid. The denatured proteins are then removed from the sample
by centrifugation. Efficient centrifugation will give clear and safe samples for injection.

Liquid-liquid extraction (LLE)

LLE is a classical technique involving the partitioning of solutes between two
immiscible liquids. It is important to select appropriate solvents for this purpose, the

38
solvent should match the analytes polarity while still being immiscible with water and
it should preferably be compatible with the following detection method. A larger
volume of the extraction solvent should be higher compared to the sample. However,
the sample extract can easily be evaporated if a volatile solvent is used to increase the
analyte concentration. Other factors, such as pH and ionic additives may affect the
extraction efficiency.

Solid-phase extraction (SPE)

SPE is a very common type of clean-up technique for bioanalytical purposes, due
to its simplicity and versatility. Many different types of SPE sorbents are commercially
available, for diverse applications. SPE with tailored MIP sorbents (MISPE) is currently
a rapidly growing field. Other examples of extraction techniques are solid-phase micro
extraction (SPME), supercritical fluid extraction (SFE), membrane extraction and affinity
sorbent extraction. SPE involves passing a liquid sample through a solid sorbent bed,
usually consisting of modified silica particles. The aim is to retain the analytes in the
sorbent bed, wash away interferences and finally elute the analytes as a clean extract in
a small volume. The collected extract can then be analyzed by a suitable method, for
instance LC/MS. A wide range of different formats and sorbents for SPE applications is
available.

39
METHOD VALIDATION PARAMETERS AND THEIR ACCEPTANCE CRITERIA

Introduction
Method validation is the process of proving that an analytical method is
acceptable for its intended purpose. The methods are followed by the guidelines of
International Conference on Hormonization (ICH), and Food and Drug Administration
2- 4 (FDA). This guideline introduces the validation terms as defined by the ICH and the
purpose of these guidelines is to details the validation data necessary for High
Performance Liquid Chromatographic (HPLC), and Liquid Chromatography connected
with Mass spectrometer (LC-MS/MS).The validation parameters to be given below.

Selectivity
At least 6 different blank plasma lots were screened for the interference at the
retention times of analyte(s) and internal standard using specified extraction procedure
and chromatography. Spiked one LLOQ level from each plasma lot and extracted the
LLOQ sample as per the extraction procedure. The percent interference was calculated
for endogenous components present in plasma at the retention times of the analyte and
the IS.

Interference of peak response (analyte and ISTD)

Percent Interference = ------------------------------------------------------------- X 100
Average area of six LLOQ samples

Acceptance criteria
The interfering peaks at the retention time of the analyte must be < 20% of the
respective plasma blanks extracted mean LLOQ peak area. Response of interfering
peaks at the retention time of internal standard must be < 5% of the respective mean
response of internal standard in LLOQ sample. At least 80% of the blank screened
matrix lots should be meets the above acceptance criteria.

40
Precision and accuracy (within batch and global)
A minimum of 4 P&A batches have to be analyzed.
The Precision & Accuracy batches are organized in the following manner:
Reconstitution solution / Mobile Phase x 1
Standard blank (without Analyte, Internal Standard) x 1
Standard zero (with internal standard) x 1
6 – 8 non-zero CC standards (LLOQ and ULOQ)
LLOQ QC, LQC, MQC, HQC
The above mentioned set of QC samples (LLOQ QC, Low QC, Middle QC and
High QC) was injected for 6 times. The calibration curve and back calculated the
concentrations of quality control samples are generated. The precision and accuracy at
each concentration level of QC samples are then determined (both within batch and
global).

Acceptance criteria
1. Lower Limit of Quantification (LLOQ)
The lowest standard on the calibration curve should be accepted as the limit of
quantification if the following conditions are met:

The analyte response at the LLOQ should be at least 5 times the response compared to
the blank response. Analyte peak response should be identifiable, discrete, and
reproducible with a precision of 20% and an accuracy of 80-120%.
With respect to the calibration curve 75% or a minimum of 6 out of 8 non-zero
standards should be used to construct the calibration curve including the LLOQ and the
ULOQ. The back calculated concentration should fall within ±15% of the nominal value
for all the calibration standards except LLOQ where it can be ±20%.

Precision:

41
 The within and between batch %CVs for low, medium and high concentrations
should be within 15% except LLOQ QC for which %CV should not exceed by more than
20%.

Accuracy:
The within and between batch mean value should not deviate by more than 15%
of the nominal value at low, medium and high QC concentrations except LLOQ QC
where it should not be more than 20%.

Recovery:
To evaluate the recovery of the analyte (s) and the internal standard (IS) the
respective peak areas are used. In case of combination of drugs, all the analytes must be
present in extracted samples as well as in unextracted samples. Six low, medium, and
high quality control samples from the freezer are retrieved and processed as per
extraction method and then injected. Unextracted quality control samples (post spiked,
spiked with internal standard) were prepared from the stock solutions having a
concentration equivalent to that of extracted samples and were injected.
The recovery was calculated from the mean peak response of extracted samples
and the unextracted samples. The percent recovery at each concentration of LQC, MQC
and HQC levels and the overall mean recovery were computed. Similarly, the percent
recovery of the IS was estimated at MQC level only. The percent recovery of analyte(s)
was determined by using the formula

Mean Analyte peak response in extracted samples

Percent recovery = ------------------------------------------------------------- X 100
Mean Analyte peak response in unextracted (post spiked
samples) samples

42
Acceptance criteria:
The percent recovery of the analyte and the internal standard should not be more than
115%. The CV for the % recovery of analyte across LQC, MQC and HQC levels should
be ≤ 15%.

Stabilities
The stock solutions of the analyte and the IS for stability evaluation were prepared in an
appropriate solvent.
The following stability experiments were conducted.
1. Stock solution stability (short term and long term)
2. Auto injector stability or in-injector stability
3. Dry extract stability
4. Bench top stability
5. Freeze thaw stability
6. Long-term stability of Analyte (s) in matrix

Stock solution stability

Fresh stock solution of the analyte(s) and the internal standard are prepared as
per the procedure. Approximately one mL aliquots are transferred into pre labeled
tubes and stored in a refrigerator for long-term stock solution stability.

Short-term stability
From the fresh stock solution approximately one mL aliquot is taken into a pre
labeled tube and kept on bench for short term stock stability. After a minimum of 6
hours dilutions from this solution are prepared and injected in 6 replicates. Dilutions
from the original stock solution, which is kept in the refrigerator are made and injected
(n=6). The stability is assessed by comparing the mean response of the stability samples
against the mean response of the comparison samples.

43
Acceptance criteria:
The mean peak response of the freshly prepared stock solution of analyte or internal
standard versus the comparable stored solution should be within the range of 90-110%.
Mean peak response of stability stock
% Stability of stock solution = ---------------------------------------------- X 100
Mean peak response of fresh stock

The short-term stability time is computed by subtracting the time when the stability
stock was kept on the work bench from the time when the fresh (comparison) stock is
retrieved from refrigerator.

Long-term stock solution stability

The long-term stock solution stability study is conducted as per the requirement
(depending on the duration of the entire validation process). A fresh stock is prepared
on the day of experimentation and appropriate dilutions are made from the fresh stock
solution as well as from stability stock (Long term stock stability sample) stored in the
refrigerator. Inject 6 replicates of each dilution.

Acceptance criteria:
The mean peak response of the freshly prepared stock solution of analyte or internal
standard versus the comparable stored solution should be within the range of 90-110%.
Mean peak response of stability stock
% Stability of stock solution = ---------------------------------------------- X 100
Mean peak response of fresh stock

The Long-term stock solution stability time is computed by subtracting the time when
the stability stock was kept in the refrigerator from the time when the stock is retrieved
from refrigerator.

In-Injector stability (auto sampler stability)

44
 Six replicates of low and high QC samples are processed and loaded into the
auto injector for a minimum period of 24 hours. After the auto injector storage period,
the stability samples are analyzed against freshly spiked calibration curve standards
and 6 replicates of LQC, HQC samples (comparison samples).
The time interval specified is only an example. The time intervals are selected on
the basis of anticipated analytical batch run time. The Mean, SD, %CV and %nominal
are calculated for the stability samples as well as the comparison samples, %CV and
%nominal should be within 15%. The percent in-injector stability is assessed by using
the formula

Mean concentration of Stability samples

% Stability = ------------------------------------------------------- x100
Mean concentration of comparison samples

Acceptance criteria
The percent stability of stability samples should be within 85-115%. The in-injector
stability time is calculated by subtracting the time when the stability QC samples are
loaded in to the auto sampler from the time when the fresh CC and QC samples are
loaded in to the auto sampler for the assay.

Dry-extract stability (Not applicable for precipitation technique)

The dry extract stability is assessed with a minimum of six QC samples each at
HQC and LQC levels. The stability samples are processed and stored in the refrigerator
without reconstitution after drying. After the intended storage period, the dry extract
samples are reconstituted and analyzed against the freshly spiked and processed
calibration curve standards along with six replicates of LQC, HQC samples
(comparison samples).
The Mean, SD, %CV and %nominal are calculated for the stability samples as
well as the comparison samples, %CV and %nominal should be within 15%. The
percent dry extract stability is assessed by using the formula

45
 Mean concentration of stability samples
% Stability = ---------------------------------------------------------- x100
Mean concentration of comparison samples

Acceptance criteria
The percent stability of stability samples should be within 85-115%. The dry
extract stability time is calculated by subtracting the time when the stability samples are
loaded into the refrigerator from the time when they are retrieved from the refrigerator
for the assay.

Bench top stability

Six replicates of QC samples corresponding to LQC and HQC are retrieved from
the deep freezer (stability samples). These QC samples are kept unprocessed on a bench
at room temperature for a period of about six to 24hrs (generally based on the expected
duration for extraction process). The freshly prepared calibration curve standards and 6
replicates each of LQC and HQC (Comparison samples) are analyzed along with
stability samples. The relevant calibration curve is generated and the concentrations are
back calculated.
The Mean, SD, %CV and %nominal are calculated for the stability samples as
well as the comparison samples, %CV and %nominal should be within 15%. The
percent bench top stability is assessed by using the formula
Mean concentration of stability samples
% Stability = ---------------------------------------------------------- x 100
Mean concentration of comparison samples

Acceptance criteria

46
The percent stability of stability samples should be within 85-115%. The bench top
stability time is calculated by subtracting the time when the stability samples were kept
on bench for thawing from the time when the processing of these samples was started.

Freeze-thaw stability
In Freeze- thaw stability, minimum three cycles are to be conducted. All the six
replicates of LQC and HQC samples after three freeze-thaw cycles are processed along
with freshly prepared calibration curve standards and comparison LQC and HQC
samples. If the third freeze thaw cycle is not stable, then the first and the second freeze
thaw samples are processed. If an analyte is unstable at the storage temperature of –
200C, then stability sample should be frozen at -700C during the three freeze and thaw
cycles.
Three sets of samples (containing six each at LQC and HQC levels) are chosen for
the freeze-thaw stability studies. The samples are initially stored in a deep freezer at -
700C. After a minimum storage of 24 hrs these samples are taken out and allowed to
thaw at room temperature. After the thawing is complete (around 45 min) the samples
are kept back in the freezer.
After a minimum of 12 hours freezing, retrieve two sets of the samples from the
deep freezer and allowed to thaw again (second freeze thaw cycle) and the samples are
again stored in the deep freezer. After a minimum of 12 hours freezing, retrieve one set
of the samples and allowed to thaw. This completes three freeze thaw cycles. Now the
samples are processed along with freshly spiked CC standards and freshly spiked LQC
and HQC samples (comparison samples). The freshly prepared calibration standards,
comparison samples and stability samples are analyzed. The calibration curve is
generated and the concentrations are back calculated.
The Mean, SD, %CV and %nominal are calculated for the stability samples as
well as the comparison samples, %CV and %Nominal should be within 15%. The
percent freeze thaw stability is assessed by using the formula

47
 Mean concentration of stability samples
% Stability = ---------------------------------------------------------- x 100
Mean concentration of comparison samples

Acceptance criteria
The percent stability of stability samples should be within 85-115%.

Long-term stability of the analyte in matrix

Sufficient number of low and high QC samples at LQC and HQC levels is stored
in the deep freezer at the desired temperature (till the end of the validation study). On
the last day of the study six replicates of LQC, HQC samples are retrieved from the
freezer, processed and analyzed along with the freshly spiked calibration standards and
the freshly spiked QC samples (comparison samples). A new stock solution is used for
the preparation of fresh calibration standards and comparison samples. The calibration
curve is generated and the percentage nominal for QC samples is calculated.
The mean of the back-calculated values for the stability samples (long-term
stability samples) is compared against the mean of back-calculated values of
comparison stability samples. The application of a correction factor is necessary (since
there will be a change in the drug concentration in the freshly spiked samples). The
long-term stability is performed on the samples stored at–700C. The Mean, SD, %CV
and %nominal are calculated for the stability samples as well as the comparison
samples, %CV and %nominal should be within 15%. The percent long term stability is
assessed by using the formula

Mean concentration of stability samples

% Stability = ------------------------------------------------------------ x100
Mean concentration of comparison samples
Acceptance criteria
The percent stability of stability samples should be within 85-115%. The long-
term stability time in biological matrix is calculated by subtracting the date and time

48
when the stability samples were logged into the freezer/cold room from the date and
time when they were retrieved from the freezer/cold room for processing.

Dilution integrity
The dilution integrity is assessed by assaying six diluted quality control (DQC)
samples (spiked in screened blank matrix having less than twice the concentration of
ULOQ) diluted by a factor of 1/2 and six DQC samples diluted by a factor of 1/4 with
screened blank matrix prior to extraction, against calibration curve standards.

Acceptance criteria
The mean concentration obtained for dilution integrity QCs should be within
±15% of their nominal concentration and the %CV should not exceed 15%.

Matrix effect (in case of MS/MS procedures)

Two sets of extracted blank plasma samples each containing six tubes (plasma
taken from six different lots) are taken. One set of tubes are reconstituted with
equivalent aqueous concentration of LQC and the other set of tubes are reconstituted
with equivalent aqueous concentration of HQC. These samples are known as post
spiked samples. These samples are analyzed along with equivalent aqueous LQC and
HQC samples. The matrix effect is evaluated by determining the % response ratio using
the formula.

Mean area ratio of post spiked samples

% Response ratio = ----------------------------------------------------------------- X 100
Mean area ratio of equivalent aqueous samples

Acceptance criteria
The percent response ratio at LQC and HQC level should be within 85-115%.

49
THE VALIDATION PARAMETERS AND THEIR ACCEPTANCE CRITERIA
Parameters Acceptance Criteria
Response of interfering peaks at the retention time of analyte (s) must
be ≤ 20% of the mean response of LLOQ standard. Response of
Screening of plasma
interfering peaks at the retention time of IS must be ≤ 5% of the mean
lots and specificity
response of Internal standard. At least 80% matrix lots should meet the
above criteria
A minimum of 6 out of 8 standards, including LLOQ and ULOQ shall
Calibration
fall within ± 15% except LLOQ for which it shall be within ± 20% when
Curve
back calculated. Regression factor should be > 0.98 (r).
Percent recovery for analyte or IS should be ≤ 115%. The %CV of the %
Recovery
Recovery for the analyte at L, M and H QC levels shall be ≤ 15%.
A minimum of 4 P & A batches shall be evaluated. The precision shall
Precision (%CV) not exceed 15% for all the QC samples except for the LLOQ QC where
it shall not exceed 20%.
A minimum of 4 P & A batches shall be evaluated. The mean value
Accuracy
shall be within ± 15% for all QC samples except for the LLOQ QC,
(% Nominal Conc.)
where it shall not deviate by more than ± 20%.
Short term stock
Short term stability against comparison samples for analyte and
solution stability
internal standard shall be within the range of 90-110%.

Long term stock

Long term stability against comparison samples for analyte and
solution stability
internal standard shall be within the range of 90-110%.

The mean concentration obtained for LQC & HQC samples should be
In injector stability within ± 15% of nominal concentration and the %CV shall not exceed
15%. The % Stability when compared with comparison samples shall be
within ± 15%.

The mean concentration obtained for LQC & HQC samples should be
Dry extract
within ± 15% of nominal concentration and the %CV shall not exceed
stability
15%. The % Stability when compared with comparison samples shall be
within ± 15%.

The mean concentration obtained for LQC & HQC samples should be
Bench top stability within ± 15% of nominal concentration and the %CV shall not exceed
15%. The % Stability when compared with comparison samples shall be
within ± 15%.

The mean concentration obtained for LQC & HQC samples should be
Freeze-thaw within ± 15% of nominal concentration and the %CV shall not exceed
stability 15%. The % Stability when compared with comparison samples shall be
within ± 15%.

50
THE VALIDATION PARAMETERS AND THEIR ACCEPTANCE CRITERIA
(contd.)

The mean concentration obtained for LQC & HQC samples should be
Long term stability within ± 15% of nominal concentration and the %CV shall not exceed
in plasma 15%. The % Stability when compared with comparison samples shall
be within ± 15%.

The mean concentration obtained for LQC & HQC samples should be
within ± 15% of nominal concentration and the %CV shall not exceed
Dilution Integrity
15%. The % Stability when compared with comparison samples shall
be within ± 15%.
Accuracy should be within ±15% for all QC samples except for the
LLOQ QC, where it should not deviate by more than ± 20%.
Matrix effect
Precision should not exceed 15% for all the QC samples except for the
LLOQ QC where it shall not exceed 20%.

References
1. Lloyd R. Snyder, Joseph J. Kirkland and Joseph L. Glajah Practical HPLC method
development 2nd Edition, New York, 1997.
2. http://bebac.at/Guidelines.htm.
3. http://www.fda.gov/Drugs/GuidanceComplianceRegulatoryInformation/Gui
dances/default.htm.
4. http://www.emea.europa.eu/docs/en_GB/document_library/Scientific_guidel
ine/2009/12/WC500018062.pdf.

5. API 4000 LC/MS/MS Hardware manual, May 2002.

51