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

13

CHAPTER I

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

14

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.

15

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.

16

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

17

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.

18

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

19

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.

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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

21

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

22

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

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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.

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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

25

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.)

26

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.

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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.

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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

29

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.

30

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

31

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

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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

33

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

34

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.

35

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.

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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.

37

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

38

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

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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.

40

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.

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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:

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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

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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.

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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.


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)

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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

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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.


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

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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.


well as the comparison samples, %CV and %Nominal should be within 15%. The

percent freeze thaw stability is assessed by using the formula

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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

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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%.

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THE VALIDATION PARAMETERS AND THEIR ACCEPTANCE CRITERIA

Parameters Acceptance Criteria

Screening of plasma lots and specificity

Response of interfering peaks at the retention time of analyte (s) must be ≤ 20% of the mean response of LLOQ standard. Response of interfering peaks at the retention time of IS must be ≤ 5% of the mean response of Internal standard. At least 80% matrix lots should meet the above criteria

Calibration Curve

A minimum of 6 out of 8 standards, including LLOQ and ULOQ shall fall within ± 15% except LLOQ for which it shall be within ± 20% when back calculated. Regression factor should be > 0.98 (r).

Recovery Percent recovery for analyte or IS should be ≤ 115%. The %CV of the % Recovery for the analyte at L, M and H QC levels shall be ≤ 15%.

Precision (%CV) A minimum of 4 P & A batches shall be evaluated. The precision shall not exceed 15% for all the QC samples except for the LLOQ QC where it shall not exceed 20%.

Accuracy (% Nominal Conc.)

A minimum of 4 P & A batches shall be evaluated. The mean value shall be within ± 15% for all QC samples except for the LLOQ QC, where it shall not deviate by more than ± 20%.

Short term stock solution stability

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

Long term stock solution stability

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

In injector stability

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

Dry extract stability


Bench top stability


Freeze-thaw stability


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THE VALIDATION PARAMETERS AND THEIR ACCEPTANCE CRITERIA (contd.)

Long term stability

in plasma


Dilution Integrity


Matrix effect

Accuracy should be within ±15% for all QC samples except for the LLOQ QC, where it should not deviate by more than ± 20%. 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_guideline/2009/12/WC500018062.pdf.

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

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

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