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

Transcription

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

2 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

3 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

4 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 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

6 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 instrumentation 1. 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

7 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

8 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 bondedphase 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

9 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 (>30 0 C). Higher temperature operation also gives lower operating pressures and higher plate numbers, because of decrease in mobile phase viscosity. A temperature of C is usually a 21

10 good starting point. However, ambient temperature is required if the method will be used in laboratories that lack column thermostating. Sample size Initially, a 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

11 A mass spectrometer is an instrument that measures the masses of electrically charged molecules, or ions. Mass spectrometer 5 (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

12 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 ( 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

13 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

14 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

15 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 compounddependent 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 quadrupolemode 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

16 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 compounddependent 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

17 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

18 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

19 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

20 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

21 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 to 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

22 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

23 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

24 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

25 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 p Ka of the compound, the ph of the buffer used in the mobile phase can be adjusted (ph = ± 2 of p Ka 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 p Ka 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

26 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 cleanup 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

27 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

28 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

29 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 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 %. 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

30 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

31 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