Introduction 1. INTRODUCTION 1.1. HPLC METHOD DEVELOPMENT AND OPTIMIZATION

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1 1. INTRODUCTION 1.1. HPLC METHOD DEVELOPMENT AND OPTIMIZATION HPLC provides reliable quantitative precision and accuracy for the determination of the active pharmaceutical ingredients and related substances in the same run using a variety of columns, solvents and detectors and can be performed on fully automated instrumentation. HPLC provides excellent reproducibility and is applicable to a wide range of compound types by judicious choice of HPLC column chemistry. Separation of chiral molecules into their respective enantiomers is also possible by HPLC. HPLC, particularly reversed phase HPLC is currently the most suitable method for meeting most of the criteria for quantitative analysis of a number of drugs. It is basically a liquid chromatographic technique involving separation of complex mixtures and quantifications of the resolved components. The separation may be by adsorption, partition, exclusion or ion-exchange depending on the type of the stationary phase packed in the column. METHOD DEVELOPMENT Chromatography includes those methods having goal for the separation of different types of components from a mixture based upon their distribution capacity between a stationary and mobile phase. There are several valid reasons for developing new methods of analysis for determination of a compound or drug. There may not be a suitable method available for a particular analyte in the specific sample matrix. Existing methods may be too error-, artifact-, and /or contamination prone, or they may be unreliable (having poor accuracy or precision). Existing methods may not provide adequate sensitivity or analyte selectivity in samples of interest. Newer instrumentation and techniques may have evolved that provide opportunities for improved analysis identification or detection limits, greater accuracy or precision, or better return on investment. 1

2 Modes of chromatography Chromatographic techniques are categorized as per the interactions between the analyte and the stationary phase, which is raised from different types of forces such as hydrogen bonding, Vander walls forces, electrostatic forces or hydrophobic forces. Different types of liquid chromatographic techniques are available such as: Affinity chromatography Size exclusion Chromatography Ion chromatography Ion- exchange chromatography Reverse phase Chromatography Normal phase Chromatography Reverse Phase-ion pair Chromatography Reverse phase Chromatography In 1960s the Chromatographic operators started modifying the column material silanol polar groups by silica with organic silica or with organic silanes chemically. The aim was to make a non polar or less polar column material so that water soluble polar compounds can be separated using polar solvents. Since the ionic nature of the modified silica is now reversed chemically i.e. the nature of the phase is non polar. The separation carried out with such silica is called as reverse phase chromatography. Chemically bonded stationary phases based on silica are commercially available in large number. In reverse phase chromatography,chemically modified silica based stationary phases are popular. Other adsorbents based on polymer (styrene- divinyl benzene co- polymer) are slowly becoming popular. Less water-soluble Compounds are retained on the modified stationary phase. The retention decreases in the following order: aliphatic > induced dipoles (CCL 4 ) > permanent dipoles (CHC 13 ) > weak Lewis acids (ethers, aldehydes, ketones) > strong Lewis Acids (carboxylic acids). 2

3 With the increase in contact of solute molecule and stationary phase the retention can be increased i.e. by increasing number of water molecules, released during the adsorption phenomenon. Separation of branch chained compounds is more rapid than that of normal isomers. The strong attractive forces between water molecules in reverse phase systems are from the 3 dimentional inter molecular hydrogen bonded network. Only those can interact with the water which are having more polarity. The systems are chemically bonded with Octadecyl silane (ODS), an alkane having eighteen Carbons atoms. it is widely used stationary phase in pharmaceutical field. Strength of the solvent in reversed phase chromatography is changed from that of adsorption chromatography (e.g. silica gel). The interaction of silanol groups with Water is strong, so adsorption of solute molecules becomes strongly prohibited and is rapidly eluted. In reverse phase system exactly opposite thing is implemented i.e. alkyl groups such as non-polar (hydrophobic) C 18 of ODS phase cannot be wet by water and therefore does not interact with bonded phase. Therefore water is regarded as the weakest solvent and gives very slow separation rate. Increase amount of water in the mobile phase leads increase in the separation time (retention time) in reverse phase chromatography 1, 2. Normal phase chromatography Chromatography palys an important role in pharmaceutical analysis for exact differentiation, selective identification and quantitative determination of close structure related compounds. Another important application of chromatographic methods is the testing of the purity of final products and intermediates (detection of decomposition products and byproduces).considering the above points methods of chromatography is occupying an important role in the latest editions of the pharmacopoeias and other testing standards. The systems used will follow one of the four mechanistic types, adsorption, partition, ion exchange and size exclusion. Adsorption chromatography is the interaction between solutes on the surface of the solid stationary phase. Partition chromatography involves a liquid stationary phase, which is immiscible with the eluent and coated on an inert support. Adsorption and partition systems can be normal phase (stationary phase more polar than eluent) or reverse phase 3

4 (stationary phase less polar than eluent). Ion- exchange chromatography involves an anionic or cationic groups on the surface of the solid stationary phase on which solute molecules of opposite charge are attracted. Size exclusion chromatography involves a controlled pore size stationary phase. Depending on the molecular size the solutes are separated; the large molecules, enable to enter the pores eluting first. High performance, high pressure, and high resolution and high speed liquid chromatography are the modern names of the column chromatography. High performance Liquid Chromatography (HPLC) is a special branch of column chromatography in which at high speed the mobile phase is forced through the column. As a result the analysis time is reduced by 1-2 orders of magnitude relative to classical column chromatography and the column efficiency can be increased by the usage of much smaller particles of the adsorbent. The equipment consists of an eluent, reservoir, a high pressure pump, and a injector to introduce the sample, a column containing the stationary phase, a detector and recorder. The development of highly efficient micro particulate bonded phases has increased the versatility of the technique and has greatly improved the analysis of multi component mixtures. The stationary phase is polar and the mobile phase is nonpolar in nature in normal phase mode. In this method non polar compounds travel faster and are eluted first because of the lower affinity between the stationary phase and the non polar compounds. Polar compounds that have higher affinity with the stationary phase are retained for longer time and therefore take more time to separate. Normal phase mode of separation is not generally used for pharmaceutical application because most of the drug molecules are polar in nature and hence take longer time to separate. COMPONENTS OF HPLC SYSTEM Solvent delivery system From one or more reservoirs the mobile phase is pumped under pressure and flows through the column at a constant rate. There is a high-pressure drop across a chromatography column due to micro particle packing. Polarity of the mobile phase, the polarity of the stationary phase and the nature of the sample components determines the eluting power of the mobile phase. By the 4

5 use of combination of two solvents,optimum separating conditions can be obtained. Some parameters which are important for the successful elution are boiling point, viscosity, detector compatibility, flammability and toxicity. In HPLC the pump is the most important part in solvent delivery system as its performance directly related to the retention time, reproducibility and detector sensitivity. Due to its less baseline noise, good flow rate and reproducibility etc a reciprocating pump with twin or triple pistons is widely used among the several solved delivery systems (direct gas pressure, pneumatic intensifier, reciprocating etc.) available. Solvent degassing system The mobile phase should be filtered and degassed before use. The dissolved gases in the mobile phase can be removed using a lot of methods. These methods mainly include vacuum degassing, heating and stirring filtration through a membrane filter, helium purging, ultra sonication or a combination of these methods. Dissolved gases are continuously removed from the mobile phase y online degassing systems provided in HPLC. Gradient elution devices HPLC system either can be isocratic mode i.e. comprising of an eluent of a definite proportion or in the gradient mode having varying mobile phase composition during the run. Sample introduction systems Injection in to a flowing stream and a stop flow injection are the two means of introduction of an analyte on to the column. A syringe or an injection valve is employed for the purpose. Automatic injector is one of the manual universal injector used of microprocessor controlled version. In the auto injector up to 100 samples can be loaded at a time. The flow rate, run time and volume of sample to be injected are selected, stored in memory and sequentially implemented for successive injections. Columns and column packing materials: The columns most commonly used are made with 316-grade stainless steel (Cr-Ni-Mo steel, relatively inert to chemical corrosion). The inside of the stainless steel tube should be as 5

6 smooth as possible, so the tubes are precision drilled or electro-polished after manufacture. Common dimensions are 6.35 mm external diameter, 4.6 mm internal diameter and up to 25 cm long. The columns can be packed with 10, 5, 4 or 3 µm diameter particles. Silica (SiO 2, H 2 O) is the most widely used packing materials. It consists of a network a siloxane linkages (Si-O-Si ) in a rigid three dimensional unit. Thus a wide range of commercial products is available with surface areas ranging from 100 to 800 M 2 /g and particle sizes from 3 to 50 µm. The useful ph range for columns is 2 to 8,since siloxane linkages are cleaved below ph-2 while at ph above eight, silica may be dissolved. Normal phase columns and reverse phase columns are the two types of columns used In HPLC. Ranges of stationary phases (C18, C8, - NH2,- CN, -Phenyl etc.) are available which can be useful for selective separations. Table 1.1: Bonded Phases for HPLC and their Abbreviations Nature of phase Si Elaboration Silica Polar non ionic organic compounds are separated suitably. C 1 Tetra methyl Silane, SAS, Trimethyl Silane Material which is of reversed phase nature. Least retentive of all alkyl group bonded phases for non- polar solvents. C 2 Reversed phase-2, Dimethyl Material is of reversed phase nature and somewhat having less retention power in comparison to C 4, C 8, or C 18 but having more retention power than that of C 1. C 3 Propyl Material is of reversed phase nature, generally having use in hydrophobic interaction chromatography [HIC] for peptides and proteins. 6

7 C 4 C 6 C 8 C 18 Butyl Material is of reversed phase nature, generally having use in ion pairing chromatography offering very low retention than C 8 and C 18 phases for non- polar solutes. It serves as an ideal phase for analysis of large proteins and hydrophobic peptides when bonded to 300A Silica. Hexyl Material is of reversed phase nature, generally having use in ion pairing chromatography offering very low retention than C 8 and C 18 phases. MOS, RP-8, LC8, Octyl Material is of reversed phase nature, with same selectivity as C 18 but retention power is less. Widely applicable (e.g. pharmaceuticals, steroids, nucleosides). It serves as an ideal phase for analysis of small hydrophilic proteins, peptide mapping and peptides when bonded to 300A Silica. ODS, RP- 18, Octadecyl, LC 18 Classic reversed phase material is most retentive for non- polar solutes and is excellent for ion-pairing chromatography. It is having wide applicability for the assay of nucleosides, nucleotides, steroids, pharmaceuticals, vitamins, fatty acids and environmental compounds when bonded to 300 A silica, this phase is perfect for separating small hydrophilic peptides. Detectors Detection in liquid chromatography has long been considered one of the weakest aspects of the technique. Low concentrations of a solute dissolved in a liquid modify the properties of the liquid to a much smaller extent than low concentrations of a solute in a gas. For this reason there is no sensitive universal, or quasiuniversal, detector such as the flame ionization or thermal conductivity detectors for GC. A comprehensive review of detectors has been published by Fielden, as well as two recent books by Scott and Patonay. The Fundamental Review issue of Analytical Chemistry, published in even-numbered years, contains a comprehensive review of developments in instrumentation for LC, including detection techniques. Most common HPLC Detectors: UV visible absorbance detector (UV- VIS) Photo diode array detector (PDA) Fluorescence Detector Electrochemical (ECD) 7

8 Refractive index (RI) Conductometric detector Mass detectors (MS) Chiral detector (Polarimetric & circular dichrosim) Evaporative Light scattering detector (ELSD) Radiochemical detection. Gradient elution or solvent programming is the change of solvent composition during a separation in which the solvent strength increases from the beginning to the end of the separation. It is well suited to the analysis of samples of unknown complexity since good resolution is automatically provided for a wide range of sample polarities. There are two types of gradient systems: Low-pressure gradient mixtures and high pressure gradient mixtures. In the former the solvents are mixed at atmosphere pressure and then pumped to the column, where as in the later, solvents are pumped in to a mixing chamber at high pressure before going in to the column 3, 4. Performance calculations The following values are calculated (which can be included in a custom report) and used to access overall system performance. Relative retention Theoretical plates Capacity factor Resolution Peak asymmetry Plates per meter The parameters used to calculate these system performance values for the separation of two chromatographic components, where the terms W and T both appear in the equation must be expressed in the same units. Relative retention (Selectivity) = (t 2 - t a ) / (t 1 - t a ) 8

9 Theoretical plates n = 16 (t / W) 2 Capacity factor K' = (t 2 / t a ) - 1 Resolution R = 2 (t 2 - t 1 ) / (W 2 + W 1 ) Peak asymmetry Plates per meter Height Equivalent to Theoretical Plates T = W 0.05 / 2f N = n/l L/n Where, α = Relative retention t 2 = Retention Time of the second peak measured from point of injection t 1 = Retention time of the first peak measured from point of injection. t a = Retention time of an inert peak not retained by the column, measured from point of injection, n = Theoretical plates t = Retention time of the component W = Width of the base of the component peak using tangent method. K' = Capacity factor. R = Resolution between a peak of interest and the peak preceding it W 2 = Width of the base of component peak 2. W 1 = Width of the base of component peak 1. T = Peak asymmetry, or tailing factor. W 0.05 = Distance from the leading edge of the peak measured at a point W 0.05 = Distance from the leading edge to the tailing edge of the peak measured at a point 5 % of the peak height from the baseline. f = Distance from the peak maximum to the leading edge of the peak. N = Plates per meter and L = Length of column, in meters. 9

10 1.2. METHOD OPTIMIZATION The initial sets of conditions initiated from the very beginning of first stages of development during the optimization stage are improvised in terms of separation and peak shape, capacity, elution time, detection limits, limit of quantitation and overall ability to quantify the specific analyte of interest. General approaches for Optimization of a method can be studied under two subparts such as: 1. Manual approach 2. Computer driven approach The first approach involves varying one experimental variable at a time, while keeping all others constant. The variables may include flow rates, stationary or mobile Phase composition, detection wavelength and P H. This univariate approach to system optimization is tedious and very expensive. In the computer driven automated method development, efficiency is optimized while experimental input is minimized. This type of approach has many applications. The various method development parameters to be considered are as follows: Separation mode Nature of stationary phase Mobile phase Type of detector Separation mode The mobile phase is more polar than the stationary phase in reversed phase chromatography. The nature of the analyte is the first and the nature of the matrix is the second factor in choosing mode of separation. 10

11 Nature of stationary phase The most important factor for effective method development is to select suitable column. Some of the important parameters considered are as follows: Size of the column Packing material. Particle shape Particle size Carbon loading (%) Pore volume Surface area End capping The column is selected based on the nature and the information of the solute analyte. Reversed phase mode of chromatography facilitates a wide range of column like dimethyl silane (C2), Butyl silane (C1), octyl silane (C8), octadecyl silane(c18),bds phenyl,cyano propyl, nitro amino etc. C 18 was chosen for this study since it is the most retentive one. Generally longer columns having higher theoretical plate numbers provide better resolution. Surface area available for coating increases with decrease in particle size. The best result for efficiency, reproducibility and reliability is obtained with Column of 5µ particle size. Shape of the peak is also playing vital role in method development. To prohibit inaccurate plate number, poor resolution, imprecise quantitation and undetected minor bands in the peak tail suitable column is always preferable. Mobile phase selection The primary separation focus at maximum resolution between all the impurities or degraded products among themselves and from the analyte. The determination of the analyte depends on the nature of mobile phase and composition of it. Solvent polarity is the important factor as the rate of resolution of solute depends upon it. Polar mobile phase is having low retention in case of normal phase chromatography and more in case of reversed phase chromatography. Less water content is preferable in reversed phase chromatography. 11

12 Selection and optimization requires the following parameters to be considered such as: Buffer ph of the buffer Composition of mobile phase Buffer Buffer and its concentration play very important role for getting symmetric peaks and good separation. Followings are the list of commonly used buffers: Phosphoric acid buffers (containing H 3 PO 4 ) Phosphate buffers (containing salts like KH 2 PO 4, k2hpo4, NaH 2 PO 4, Na 2 HPO 4 etc.) Acetic acid buffers (containing CH 3 COOH) Acetate buffers (Ammonium acetate, Sodium acetate etc.) The molar strengths of the buffer are related to the retention times of analytes. Increase in molar strengths leads to increase in retention times. The strength of buffers can be varied depending upon the requirement. P H range of the buffer P H plays a major role in achieving good separation by suppressing the ionization. Mobile phase P H ranging between is always preferable for good separation as most of the columns cannot withstand out of the above range. The reason is that Siloxane linkage is cleaved below ph 2.0; while above 8.0 ph may be dissolved. Mobile phase composition Choosing of appropriate mobile phase composition is a key factor in method developement. Methanol and acetonitrile are most widely used solvents in reversed phase chromatography. The selection of mobile phase is done by checking the ruggedness result should be differed by % at variable composition and P H. 12

13 Selection of detector Selection of appropriate detector is done depending upon some property of analyte such as UV absorbance, fluorescence, conductance, oxidation, reduction etc. Following are some of the factors to be considered when one detector is to be selected such as: Lower detection Low dead volume Non destructive to sample Inexpensive to purchase and operate For good separation to be achieved max should be determined and all measurements should be done at that wavelength. It is better to avoid measurement below 200 nm as noise is observed at this range. Measurements at higher wave length give good separation with high selectivity 5. Table 1.2. Preferred experimental conditions for the initial HPLC separation Separation variable Preferred initial choice Column dimensions (length, ID) 15 x 0.46 cm Particle size 5 µm Stationary phase C 8 or C 18 Mobile phase: solvent A and B Buffer acetonitrile % B % Buffer 25 mm potassium phosphate ph Flow rate Column temperature C Sample volume < 25 µl 13

14 Table 1.3. Separation goals in HPLC method development GOAL Resolution (Rs) Separation time Quantitation Pressure Peak height Solvent consumption COMMENT Precise and rugged quantitative analysis requires that Rs be greater than 1.5. < 5-10 min is desirable for routine procedures % (1SD) for assay; % 5 for less demanding analysis; 15% for trace analysis. < 150 bar is desirable Narrow peaks are desirable for large signal/noise ratios Minimum mobile phase use per run is desirable 1.3 HPLC METHOD VALIDATION Method validation can be defined as Establishing documented evidence, which provides a high degree of assurance that a specific activity will consistently produce a desired result or product meeting its predetermined specifications and quality characteristics according to ICH guidelines. It is an integral part of the method development. It is the process of evaluation of suitability of analytical procedures for the required purpose to support the quality, identity, potency and purity of the active pharmaceutical ingredients. Simply, method validation is the process of evaluating suitability of an analytical method for its suitability of an analytical method for its predetermined objectives or not. For successful chromatographic separation the key parameters to be validated are sensitivity, selectivity, precision, recovery, limit of detection, limit of quantitation, ruggedness, robustness stability and system suitability. Recovery The recovery of the method, determined by adding a previously analyzed test solution with additional drug standard solution at three levels of concentration, and should be well within the range of 90 to 110%. 14

15 It is mathematically expressed as the ratio of response obtained of an analyte spiked in to matrix to that of pure standard in terms of percentage. It is best practiced by comparing the responses of spiked samples at three different levels in replicates of at least 6 with those of non spiked standards, which confirm 100% recovery. The formula for absolute recovery is as follows: Response of an analyte spike into matrix (processed) Absolute recovery = Response of analyte of pure standard (unprocessed) If an internal standard is used its recovery should be determined independently at the concentration levels used in the method. Sensitivity The Method is said to be sensitive if small changes in concentration cause large changes in response to function. The sensitivity of an analytical method is determined from the slope of the calibration line. The limits of quantification (LOQ) or working dynamic range of bio analytical methods are defined as the highest and lowest concentrations, which can be determined with acceptable accuracy. It is suggested that, this should be set at ± 15% for both the upper and lower limit of quantitation respectively. Any sample concentration that falls outside the calibration range cannot be interpolated from the calibration line and extrapolation of the calibration curve is discouraged. If the concentration is over the range, the sample should be diluted in drug-free matrix and re-assayed. Precision It can be defined as the reproducibility of the results. When one considers the criteria according to which an analytical procedure is selected, precision and accuracy are usually the first time to come to mind. Precision and accuracy together determine the error of an individual determination. They are the most important parameters for evaluating analytical procedures by their appreciable results. 15

16 Precision is determined as both repeatability and intermediate precision, in accordance with ICH guidelines. Repeatability of sample injection is determined as intraday variation and intermediate variation. For these determinations, single concentration (μg/ml) at different time intervals and different days, of the solution of API is used. Precision refers to the reproducibility of measurement within a set or the scatter of dispersion about its central value. The term set is defined as referring to a number (n) of independent replicate measurements of some property. One of the most common statistical terms employed is the standard deviation of a population of observation. Standard deviation is the square root of the sum of squares of deviations of individual results for the mean, divided by one less than the number of results in the set. The standard deviation (S) is calculated as following equation: % Relative standard deviation=s x 100/ X Accuracy Accuracy is best determined by the standard addition method. Previously analyzed samples of API are added with standard drug solutions and are analyzed by the proposed method. Recovery (%), RSD (%) and bias (%) are calculated for each concentration. Accuracy is reported as percentage bias, which is calculated from the expression.percentage bias is calculated as follows: (Measured value true value) % Bias = X 100 True value Calibration curve (Linearity) To be useful for quantitative purposes, the detector response should be linear with amount of solute over a reasonable range of solute concentrations. Most often the logarithm of the detector response is plotted versus the logarithm of the amount injected, and the linear range is taken as that concentration range over which the slope of the corresponding line is 1.00 ±

17 However, as Colin et al. have pointed out, such a plot dampens fluctuations and may be misleading. They have recommended that a plot of the ratio of detector signal to amount injected versus the logarithm of amount injected is much more instructive. Methodology: Analyte solutions from limit of quantification level to 120% (or as per protocol) of the target concentration by analysing the sample as per test method should be prepared. Linearity shall be established across the range. Minimum five concentrations across the range are recommended. Coefficient of correlation, Y-intercept and slope of regression line should be calculated. If linearity is not meeting the acceptance criteria, should have to establish the range of concentration in which it is linear. The equation of linearity is given as: Standard Deviation of slope (S b ) Y = mx + c The standard deviation of sloe is proportional to standard error of estimate and inversely proportional to the range and square root of the number of data points. S b = sqrt [Σ(y i - ŷ i ) 2 / (n - 2) ] / sqrt [ Σ(x i - x) 2 ] Where, y i = Dependent variable for observation i, x i= Independent variable for observation i, n = number of observations. Standard deviation of intercept (S a) Intercept values of least squares fits of data are often to evaluate additive errors between or among different methods. Where Xi denotes the arithmetic mean of xi values. Acceptance criteria: Coefficient of correlation should be NLT 0.98; Peak purity should be not less than 990.0; Purity angle should be less than threshold. 17

18 Linearity and sensitivity of the method Knowledge of the sensitivity of the color is important and the following terms and commonly employed for expressing sensitivity. According to Beer Lambert s law: Intensity of incident light (I o ) A = Log = ε ct Intensity of transmitted light (I t ) The absorbance (A) is proportional to the concentration (c) of the absorbing species, if molar extinction co-efficient (ε) and thickness of the medium (t) are constant. When C is in moles per liter and the constant is called molar absorptivity constant.beer s law limits and E max values µg / cm 2 of the drug are expressed as which in a column solution of cross section 1cm2 shows an absorbance of (expressed as µg cm- 2 ). Limit of detection (LOD) The limit of detection of an analytical method development is defined as the minimum concentration at which the instrumental signal is obtained and is different from that of blank. LOD is obtained by analyzing the minimum concentration of the linearity range. The limit of detection (LOD) of an analytical method may be defined as the concentration, which gives rise to an instrument signal that is significantly different from the blank. For spectroscopic techniques or other methods that rely upon a calibration curve for quantitative measurements, the IUPAC approach employs the standard deviation of the intercept (Sa), which may be related to LOD and the slope of the calibration curve, b, by LOD=3 Sa / b Limit of quantification (LOQ) The LOQ is the concentration that can be quantitated reliably with a specified level of accuracy and precision. The LOQ represent the concentration of analyte that would yield a signal-to-noise ratio of 10. LOQ = 10 Sa/b 18

19 Where, Sa is the standard deviation of the peak area ratio of analyte to IS (6 injections) of the drugs and b is slope of the corresponding calibration curve. Ruggedness Method Ruggedness is defined as the reproducibility of results when the method is performed under actual use conditions. This is obtained by changing analysts, place of experiment, instruments, columns, chemicals, solvents etc. Robustness Robustness Influence of small changes in chromatographic conditions such as change in flow rate (± 0.1 ml/min), Wavelength of detection (±2 nm) & acetonitrile content in mobile phase (±2%) studied to determine the robustness of the method are also in favor of (Table 5, % RSD < 2%) the developed RP-HPLC method for the analysis of API. According to ICH guidelines it is defined as a measure of its capacity to remain unaffected by small but deliberate variations in method parameters. It is evaluated by changing the mobile phase composition and its ph, temperature of the column and the buffer content. Methodology: Mobile phase variation: Mobile phases should be prepared by changing organic phase to +/-5% of the mobile phase composition. Flow rate: Flow rate should be changed by +/ 0.1 ml/minute of the target flow rate mentioned in test method. Change in Wavelength: wavelength should be changed by +/ 2nm of the target wavelength mentioned in test method. Change in Column: Different column should be used instead of column used in the test method. Stability To have reproducible and sensitive results the drug products and reagents should be stable for specific time duration (i.e. one day, one week, and one month depending upon the need). 19

20 System suitability The criteria selected will be based on the actual performance of the method as determined during its validation. For example, if sample retention times form a part of the system suitability criteria, their variation (SD) during validation can determine system suitability, which may fall within a =3 SD range INTRODUCTION TO LC-MS/MS TECHINQUE 7 A mass spectrometer 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 volatile samples. However, many thermolabile 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 / 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. Creation of Gas phase ions is the role of the ionization method. Ionization methods available on the instruments within the MS facility are described below. Ionization techniques A variety of ionization techiniques are used for MS. Most ionization techiniques excite the neutral analyte molecule which then ejects electron to form a radical cation (M*). Other ionization techniques involve icn molecule reactions that produce adduct ions (MH*). The most important considerations are the physical state of the analyte and the ionization energy. Various Ionization sources in mass spectrometry Atmospheric pressure photo ionization(appi) Electrospray ionization (ESI) 20

21 Atmospheric pressure chemical ionization (APCI) Fast atom bombardment(fab) Fast desorption/field ionization(fd/fi) Electron impact ionization (EI) Matrix assisted laser desorption ionization (MALDI) Out of available all mass spectrometric configuration atmospheric pressure ionization (ESI or APCI) ionization source with triple quadrupole mass analyser has become world widely accepted because of ease of attachment to liquid chromatography. Fig.1.1 Electrospray Ionisation (ESI) Technique Electrospray lonisation (ESI) It is one of the atmospheric pressure ionization (API), which is well- suited for the analysis of polar molecules ranging from less than 100 Da to more than 1,000,000 Da in molecular mass. Standard electro spray ionization source In standard electro spray ionization a polar, volatile solvent is taken and the sample is dissolved and pumped through a narrow, stainless steel capillary ( micrometers i.d.) at a flow rate of between 1µl./min and 1 ml./min. To the tip of the capillary a high voltage of 3 or 4 kv is applied within the ionization source of the mass spectrometer, and as a result of this strong 21

22 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 neubulising 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. By solvent evaporation the charged droplets diminish in size, folowed by a warm flow of nitrogen known as the drying gas which passes across the front of the ionization source. Eventually charged sample ions, free from solvent, are released from the droplets, some of which pass through a sampling cone or orifice into an intermediate 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). Source-Dependent Parameters Optimum conditions of source dependent parameter values are dependent upon liquid chromatographic conditions. These parameters are optimized using split infusion or FIA. The sensitivity depends upon the positioning of the probe in the source. GS1 The nebulizer gas is controlled by GS1. It affects the stability and sensitivity by forming small droplets of sample flow. It is also called nebulizer gas for API 3000 and API 150EX systems. GS2 The auxiliary or turbo gas is controlled by GS2 parameter. It helps in prevention of entering small droplets in to the instrument by evaporating the sprayed droplets. This parameter is called auxiliary or turbo gas for API 3000 and API 150EX systems. 22

23 Auxiliary Gas (Aux) The Aux parameter commands the auxiliary or turbo gas. It evapourates the sprayed droplets and prevents their entry in to the instrument. This parameter is called Gas 2 for Q TRAP, 4000 Q TRAP, API 2000, and API 4000 systems. Temperature (TEM) In the Turbo Ion Spray source or the temperature of the probe in the heated nebulizer or APCI source, the temperature of the turbo gas is controlled by TEM parameter. Sample ions are converted to gas phase by it. Curtain Gas (CUR) The Curtain Gas is controlled by CUR parameter, which flows between the curtain plate and the orifice. Curtain Gas also meant for preventing solvent droplets to the ion optics. It should be kept at as high as possible without losing sensitivity. Ion spray voltage (IS) The voltage applied to the needle is controlled by the IS parameter which ionizes the sample in the ion source. The stability of the spray and the sensitivity are affected due its polarity. Photo Spray source on an API 2000, API 4000, API 5000, Q TRAP, or 4000 Q TRAP instrument is called Ion Transfer Voltage. Interface Heater (IHE) The on and off of the Interface heater is done by this parameter. Contamination of the ion optics is prevented by heating the interface because of increase of ion signal. For API 4000 and 4000 Q TRAP 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. 23

24 Interface Heater Temperature (IHT) It controls the temperature of the Nano Spray interface heater and is only available if the Nano Spray source and interface are installed. Upto 250 C,the temperature can be adjusted. This parameter applies to the API 4000 and 4000 Q TRAP instruments. Nebulizer, or Needle Current (NC) The current applied to the corona discharge needle in the APCI (atmospheric pressure chemical ionization) probe,used in the Turbo V source can be conrrolled by the NC. The solvent molecules are ionized by the discharge, which in turn ionize the sample molecules. Multi-Source Selector: This parameter controls the selection of the Duo Spray source probe: Turbo Ion Spray or APCI. In the parameter settings file, the options are 1 for Turbo Ion Spray and 2 for APCI. Compound-Dependent Parameters The available compound-dependent parameters vary with instrument type. They mostly consists of lens elements in the ion path. For compound-dependent parameters, the optimal values 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. Compound-Dependent Parameters for Both Quadrupole and LIT-mode Scans For running a quadrupole-mode scan or an LIT-mode scan the following parameters are available for optimization. Collisionally Activated Dissociation Gas (CAD) The pressure of collision gas in the collision cell during Q3 MS, MS/MS, and LIT scans is controlled by the CAD parameter. For Q3 MS scans, the collision gas helps to focus the ions as they pass through the collision cell. The collision gas acts as a target to fragment the precursor ions for MS/MS scans. With the collision gas when the parent ions collide, they can dissociate to 24

25 fragment ions. Although this parameter is on the Source/Gas tab, this parameter is compounddependent and not dependent on the sample flow. De clustering Potential (DP) The DP parameter controls the potential difference between Q0 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. The de clustering potential uses the access ID DP. Focusing Potential (FP) The voltage applied to the focusing ring lens are controlled by FP. Through the skimmer region of the mass spectrometer interface the ions are focused with the help of FP. Fragmentation can be induced in the interface area by FP, similar to the de clustering potential. This parameter does not apply to the API 4000, Q TRAP, or 4000 Q TRAP systems. Entrance Potential (EP) The EP parameter controls the entrance potential, which guides and focuses the ions thorough the high-pressure Q0 region. The entrance optential uses the access ID EP. It is also displayed by its parameter ID Q0. Collision Cell Entrance Potential (CEP) The CEP parameter controls the collision cell entrance potential, which 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 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, API 4000, and 4000 Q TRAP systems, CXP is the potential difference between RO2 and ST3 (the stubby lens 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 25

26 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. IE3 is used in Q3 and MS/MS-type 26

27 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 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 sample of interest. The best method of sample introduction for optimizing these parameters is infusion. The acquisition must be stopped between change of parameters. Collision Energy Spread (CES) The CES parameter controls the spread of collision energies used, using Auto Frag. For example, if there is CE of 30 and a CES of 5, collision energies of 25, 30, and 35 are used. Fixed LIT Fill Time The Fixed LIT Fill Time parameter controls amount of time required to fill the trap with ions. In general, the default time is appropriate. It needs to fill the linear ion trap so that there will be greatest peak intensity without saturation. Dynamic Fill Time (DFT) The DFT parameter controls whether the LIT fill time is dynamic or not. If DFT is turned on, the software will dynamically calculate the length of time that ions are collected in the LIT. If DFT is used, 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, it needs to turn on Q0 trapping to increase the duty cycle and get better sensitivity. If the sample is concentrated, it requires to turn off Q0 trapping to prevent saturating the peaks, which results in poor peak resolution. If DFT is used, Q0 trapping is turned off by default. 27

28 Time Delayed Fragmentation Collision Energy (TDF CE): It is 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 transfers the ions from Q2 into the LIT. It needs to decrease the value from the default to prevent fragmentation if the compound is fragile. Q3 Empty Time It is available only for EMC scans. The Q3 Empty Time parameter controls the amount of time that requires to remove 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. MS/MS/MS Fragmentation Time The MS/MS/MS Fragmentation Time parameter controls the amount of time that the excitation frequency is applied to the second precursor ion. In general, the default time is acceptable. Q3 Cool Time It is found 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 time is increased, the number and intensity of fragment ions decreases. In general, the default Q3 Cool Time value is sufficient. 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 calibrate. The voltage should not be changed unless the detector 28

29 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. Turbo Ion spray Probe Position In optimizing the Turbo Ion Spray performance the position of the Turbo Ion Spray Probe relative to the Orifice and to the Heater Probe is an important factor. With respect to the center of the orifice the Probe should point between 5 and 10 mm off axis. The distance of the Heater Probe from the orifice plane is fixed, but the Turbo Ion Spray 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 (1 ml /min) requires that the Turbo Ion Spray be repositioned further away from the orifice to prevent salvation penetration through the orifice into the mass spectrometer. Turbo Ion Spray Positioning Across the Orifice As the aqueous composition of the carrier solvent increase at high flow rates (1 ml/min), the more visible the spray becomes and the further away from the orifice. 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. Multiple charged proteins is and peptides introduced at a few micro liters per minute usually require the sprayer to be less than 1 cm from the Curtain Plate. Turbo Ion Spray Voltage (IS) Positive mode, a high probe voltage is required for the singly charged compounds between 4000 to 5500 V. Negative mode compounds usually require a lower voltage 3000 to V. 29

30 Nebulizer Gas (Gas 1) It is optimized for signal stability and sensitivity. Typically a value of 5 to 15 is used as applied by the applications computer. Curtain Gas Flow Aa stable clean environment for the sample ions is ensured by the curtain gas entering the mass spectrometer. Air or solvent is prevented by the gas curtain 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 Turbo Ion Spray needle. The QO Rod Set is contaminated by the presence of the solvent vapor or moisture in the analyzer region of the mass spectrometer 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 flow until the signal starts to decreases). In order to prevent instrument contamination the Curtain Gas flow should be optimized at the highest possible setting but never below 6 that does not result in a significant reduction in signal intensity. Heater Gas (Gas2) Flow The Heater Gas (Gas 2) aids in the evaporation of solvent which aids the ionization of the sample to increase. 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, premature vaporization of the solvent can be caused by too high temperature, and result in a high chemical background noise, while a noisy or unstable signal can be caused by too high a Heater Gas flow. For each flow rate, there should be as high as possible Curtain Gas flow rate (from setting 6 to 15 at the application s computer). The solvent composition used for optimization was 1/1 water/acetonitrile. These conditions represent a starting point from which to optimize Turbo Ion Spray. 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. 30