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Development of Instrumental Methods for Simultaneous Estimation of Lansoprazole and DomperidoneA Dissertation Submitted to

Pacheri Bari, Dist.-Jhunjhunu

Rajasthan-333515

In partial fulfillment of the requirements for theMaster of PharmacyinPharmaceutical ChemistryUnder the Supervision ofDr. Mandev B. Patel Mr. Subhajit GhoshAsst. Professor Asst. Professor

Submitted ByTaraknath ChatterjeeEnrollment No. 918501375

CERTIFICATE BY THE GUIDEThis is to certify that the dissertation entitled Development of Instrumental Methods for Simultaneous Estimation of Lansoprazole and Domperidone is a bonafide research work done by Mr. Taraknath Chattetrjee in partial fulfillment of the requirements for the degree of Master of Pharmacy in Pharmaceutical Chemistry. Such materials as obtained from other sources have been duly acknowledged in this thesis. This report is now ready for examination.

Date: Dr. Mandev B. Patel Place: Assistant Professor Dept. of Pharmaceutical Chemistry SHIVDAN SINGH INSTITUTE OF TECHNOLOGY & MANAGEMENT

10th Km. STONE, ALIGARH-MATHURA ROAD, ALIGARH (U.P.)

Contact No. : (0522) 257260, 257280, Fax : (05722) 257337

Website:www.seglko.org

CERTIFICATE BY THE GUIDEThis is to certify that the dissertation entitled Development of Instrumental Methods for Simultaneous Estimation of Lansoprazole and Domperidone is a bonafide research work done by Mr. Taraknath Chatterjee under the guidance of Mr. Subhajit Ghosh, Asst. Professor, Department of Pharmaceutical Sciences, SSITM, Aligarh.

Date: Mr. Subhajit GhoshPlace: Asst. Professor Dept. of Pharmaceutical Chemistry SSITM, Aligarh. SHIVDAN SINGH INSTITUTE OF TECHNOLOGY & MANAGEMENT

10th Km. STONE, ALIGARH-MATHURA ROAD, ALIGARH (U.P.)

Contact No. : (0522) 257260, 257280, Fax : (05722) 257337

Website:www.seglko.org

ENDORSEMENT BY THE HOD OF THE INSTITUTIONThis is to certify that the dissertation entitled Development of Instrumental Methods for Simultaneous Estimation of Lansoprazole and Domperidone is a bonafide research work done by Mr. Taraknath Chatterjee under the guidance of Mr. Subhajit Ghosh, Asst. Professor, Department of Pharmaceutical Sciences, SSITM, Aligarh.

Date:

Mr. Md. Hedaytullah

Place:

HOD, Dept. of Pharmacy

SSITM, Aligarh

DECLARATION BY THE RESEARCH SCHOLARI hereby declare that this dissertation entitled Development of Instrumental Methods for Simultaneous Estimation of Lansoprazole and Domperidone is a bonafide and genuine research work carried out by me under the guidance of Mr. Subhajit Ghosh, Asst. Professor, Department of Pharmaceutical Chemistry, S.S.I.T.M,Aligarh and Dr. Mandev B. Patel, Assistant Professor, Dept. of Pharmaceutical Sciences, Singhania University.

Date:

Place: Aligarh (Taraknath Chatterjee)

I would like to take the golden opportunity to express my humble gratitude on the successful completion of my thesis work. Firstly I am very thankful to Mr. Subhajit Ghosh. Asst. Professor, Department of Pharmaceutical Chemistry, SSITM, Aligarh. And Dr. Mandev B. Patel, Asst. Professor, Department of Pharmaceutical Sciences, Singhania University. I am honored to work under his guidance and have his precious guidelines, constant encouragement and support throughout my research work. I am very obliged for the patience with which he guided me at every step of my research work.

My sincere thanks to Mr. Md. Hedaytullah Professor & Head, Dept. of Pharmaceutical Science, SSITM, Aligarh, for his valuable guidelines and timely procurement apparatus and chemical required.I am very grateful to all the teaching and non teaching staff of SSITM, Especially Mr. Yogesh, Mr. Deepak, Mr. Santosh and Librarians whose valuable assistance had helped me a lot in the experiments and literature survey.The discipline, cooperation, and perseverance which I had learned being in SSITM, will be cherished in all the walks of my life.I will not forget to thank my friends Anupam, Kishor, Sayantan and Subha for their love and affection.I would like to remember my juniors, Sandeep, Roop and Santosh for their support through the course and I thank them all for their suggestions, care and friendship.My heartily thanks to my loving family, my mother Mrs. Suchitra Chatterjee, my, father Dr. Biswanath Chatterjee, Especially to my wife Priyanka who has been my strength, my inspiration, and who had always been there, to make my dreams come true. I am deeply indebted to my family for their love and affection.Lastly, I sincerely thank god for all his blessings and all those who had helped me directly or indirectly during my research work.

Date:

Place: C O N T E N T SChapter No. TitlePage No.

1.INTRODUCTION1-35

1.1Introduction to Analytical Chemistry1-3

1.2Steps for Analytical Development3-6

1.3Introduction to Spectroscopy6-8

1.4 Introduction to HPLC8-12

1.5 Analytical Method Validation13-21

1.5.1. Objective of Validation13

1.5.2. Benefits14

1.5.3. Analytical validation characteristics14-21

1.6Multicomponent analysis21-25

1.6.1. Spectrophotometric multicomponent analysis21-22

1.6.2.Various spectrophotometric techniques22-26

1.7 Chemometrics

1.7.1. Application of Chemometrics on spectral data

1.7.2. Statistical parameters to evaluate the efficiency of calibrations mode26-29

27-29

30-30

1.8Drug Profile 31-35

2.OBJECTIVE 36

3.REVIEW OF LITERATURE 38-41

4.METHODOLOGY42-111

Part A

UV Spectrophotometric methods for Simultaneous Estimation Of Domperidone and Lansoprazole.42-92

4.1. Introduction 42-46

4.2. Simultaneous equation method47-55

4.3. First derivative zero crossing method 56-64

4.4. Ratio derivative Spectrophotometric method65-76

4.5. Chemometric method (PLS, PCR and CLS)77-92

Part B

4.6. RP - HPLC Method for Simultaneous Estimation ofDomperidone and Lansoprazole.93-111

5.RESULTS AND DISCUSSION112-114

6.CONCLUSION115

7.SUMMARY116-117

8.BIBLIOGRAPHY118-125

9.ANNEXURE

9.1. List of Tables

9.2. List of Figures126-129

LIST OF TABLESTable No.TitlePage No.

4.1.1Composition of DOMP and LANS in preparation of Binary mixture 46

4.2.1(a)Intra day precision (repeatability)52

4.2.1(b)Inter day precision (intermediate precision)52

4.2.2Application of standard addition technique to the analysis of DOMP and LANS in Lanspro-D Capsules 53

4.2.3Reproducibility of the method by three different analysts54

4.2.4LOD and LOQ of DOMP and LANS54

4.2.5Analysis of commercial formulation55

4.3.1Optimized method parameters59

4.3.2(a)Intra day precision (repeatability)61

4.3.2(b)Inter day precision (intermediate precision)61

4.3.3Application of standard addition technique to the analysis of DOMP and LANS in Lanspro-D Capsules62

4.3.4LOD and LOQ of DOMP and LANS62

4.3.5Reproducibility of the method by three different analysts63


4.4.1(a)Analytical data of the calibration graphs for the determination of DOMP and LANS by the ratio spectra derivative method 67

4.4.1(b)Application of the proposed procedure for the simultaneous determination of the two drugs in laboratory prepared mixtures69

4.4.2Calibration data (Linearity data) for standard dilutions of DOMP and LANS73

4.4.3Intra day precision for determination of DOMP and LANS 74

4.4.4Inter day precision for determination of DOMP and LANS74

4.4.5Recovery study from synthetic mixture75

4.4.6LOD and LOQ75


4.5.1Composition of the concentration (calibration) set 80

4.5.2Statistical parameters of chemometric methods in calibration step of Zero-order spectra85

4.5.3Statistical parameters of chemometric methods in prediction step of Zero-order spectra90

4.5.4Application of standard addition technique to the analysis of DOMP and LANS in Lanspro-D Capsules90

4.5.5Recovery results in prediction from Zero- order spectra for DOMP and LANS in synthetic mixtures by proposed chemometric techniques91

4.5.6Results obtained for the pharmaceutical samples by using chemometric calibrations91

4.5.7Precision study results of prepared binary mixture92

4.6.1Composition of the concentrations and recoveries results100

4.6.2Linearity data for DOMP and LANS 101

4.6.3Characteristic parameters of calibration equation for the proposed HPLC method for simultaneous determination of DOMP and LANS. 103

4.6.4Precision study results of prepared binary mixture105

4.6.5Application of standard addition technique to the analysis of DOMP and LANS in Lanspro-D Capsules. 106

4.6.6Ruggedness report of DOMP and LANS107

4.6.7Robustness of chromatographic method108

4.6.8Results of system suitability parameters109

4.6.9Results obtained for the pharmaceutical samples by using formulation110

LIST OF FIGURESFigure No.TitlePageNo.

1.3(a)UV Visible Double Beam Spectrophotometer.8

1.4.(a)The Nomenlclature of Chromatogram9

4.2.1(a)Zero-order absorption spectra for DOMP and LANS a represents DOMP showing max at 287nm and b represents LANS showing max at 294nm. 48

4.2.1(b)Overlay Zero-order absorption spectra for standard dilution of DOMP and LANS48

4.2.2(a)Calibration curve for determination of DOMP at 289 nm50

4.2.2(b)Calibration curve for determination of LANS at 294 nm51

4.3.1Overlay first derivative absorption spectra of standard dilutions of DOMP and LANS, arepresents the zero crossing of DOMP at 287nm and brepresents the zero crossing of LANS at 294nm.57

4.3.2Overlay first derivative absorption spectra of binary mixture of DOMP and LANS57

4.3.3Calibration curve for determination of DOMP59

4.3.4Calibration curve for determination of LANS60

4.4.1Ratio spectra (A) and first derivative of the ratio spectra (B): a) 4, b) 8, c) 12, d) 16, e) 20, f) 24, g) 28, h)32, i)36 g ml-1 solution of DOMP when 12 g ml-1 of LANS used as divisor ( = 4 nm); scaling factor, 100. 70

4.4.2Ratio spectra (A) and first derivative of the ratio spectra (B): a) 2, b) 4, c) 6, d) 8, e) 10, f) 12, g) 14, h) 16, i)18 g ml-1 solutions of LANS when 20 g ml-1 of DOMP used as divisor ( = 4 nm); scaling factor, 100. 71

4.4.3Calibration curve for determination of DOMP at 273 nm (12g ml-1 of LANS as divisor).72

4.4.4Calibration curve determination of LANS at 289nm (20 g ml-1 of DOMP as divisor). 72

4.5.1Zero order overlay absorption spectra of standard dilution of (a) DOMP (4 -36 g ml-1) and (b) LANS (2 -18 g ml-1) 86

4.5.2Zero order overlay absorption spectra of 25 laboratory prepared binary mixtures of DOMP and LANS for chemometric calibration86

4.5.3MSEP plots of a calibration set obtained using leave-one-out (LOO) cross validation of PLS-model for A) DOMP and B) LANS in zero-order absorption data 87

4.5.4MSEP plots of a calibration set obtained using leave-one-out (LOO) cross validation of PCR-model for A) DOMP and B) LANS in zero order absorption data.88

4.5.5Plot of concentrations residuals (residuals.pcrP) against the predicted concentrations (Composition. new) of A) DOMP and B) LANS in prediction set89

4.6.1Zero-order overlay absorption spectra: a) 20 g ml-1 of DOMP, b) 12 g ml-1 of LANS and c) their binary mixture96

4.6.2Chromatogram showing Retention Time (Rt) of (a) 16 g ml1 of DOMP (4.380 min) and (b) 12 g ml1 of LANS (5.827 min) in laboratory-prepared mixture96

4.6.3HPLC 3-Dimensional Chromatograms set of seven standard dilutions of DOMP (in triplicate) using 10 g ml1 of LANS as internal standard98

4.6.4HPLC 3-Dimensional Chromatograms set of seven standard dilutions of LANS (in triplicate) using 20 g ml1 of DOMP as internal standard98

4.6.5HPLC 3-Dimensional Chromatograms set of 25 binary mixtures of DOMP and LANS prepared by four factorial designs for prediction99

4.6.6Linearity curve of DOMP102

4.6.7Linearity curve of LANS 102

4.6.8HPLC 3-Dimensional Chromatograms of DOMP and LANS binary mixtures used for Precision (repeatability) analysis104

4.6.9(a)HPLC 3-Dimensional Chromatograms of DOMP and LANS binary mixtures in Lanspro-D capsules used for Accuracy studies106

INTRODUCTION1. INTRODUCTION1.1 ANALYTICAL CHEMISTRY28 Analytical chemistry is the study of the chemical composition of natural and artificial materials. Unlike other major sub disciplines of chemistry such as inorganic chemistry and organic chemistry, analytical chemistry is not restricted to any particular type of chemical compound or reaction. Properties studied in analytical chemistry include geometric features such as molecular morphologies and distributions of species, as well as features such as composition and species identity. To be effective and efficient, analyzing samples requires expertise in:

1. The chemistry that can occur in a sample

2. Analysis and sample handling methods for a wide variety of problems (the tools-of-the-trade) 3.Proper data analysis and record

Types22 Traditionally, analytical chemistry has been split into two main types qualitative and quantitative:

1. Qualitative Qualitative analysis seeks to establish the presence of a given element or compound in a sample.

2. Quantitative Quantitative analysis seeks to establish the amount of a given element or compound in a sample. Specific Technologies and Instrumentation23A) Spectrometric techniques Ultraviolet and visible Spectrophotometry

Fluorescence and phosphorescence Spectrophotometry

Atomic Spectrometry (emission and absorption)

Infrared Spectrophotometry

Raman Spectroscopy

X-Ray Spectroscopy

Radiochemical Techniques including activation analysis

Nuclear Magnetic Resonance Spectroscopy

Electron Spin Resonance Spectroscopy

B) Electrochemical Techniques Potentiometry

Voltametry

Voltametric Techniques

Amperometric Techniques

Colorimetry

Electrogravimetry

Conductance Techniques

C) Chromatographic Techniques Gas Chromatography

High performance Liquid Chromatography

Thin Layer Chromatography

D) Miscellaneous Techniques Thermal Analysis

Mass Spectrometry

Kinetic Techniques

E) Hyphenated Techniques GC-MS (Gas Chromatography Mass Spectrometry)

ICP-MS (Inductivity Coupled Plasma Mass Spectrometry)

GC-IR (Gas Chromatography Infrared Spectroscopy)

MS-MS (Mass Spectrometry Mass Spectrometry)

1.2 STEPS FOR ANALYTICAL DEVELOPMENT24 Methods are developed for new products when no official methods are available. Alternate methods for existing (non-pharmacopoeial) products are developed to reduce the cost and time for better precision and ruggedness. Trial runs are conducted, method is optimized and validated.1. Analyte standard characterization:a. All information about the analyte i.e., physical and chemical properties, toxicity, purity, hygroscopic nature, solubility and stability.

b. The standard analyte (100% purity) is obtained. Made an arrangement for the proper storage (refrigerator, desiccators and freezer).

c. When multiple components are to be analyzed in the sample matrix, the number of components is noted, data is assembled and the availability of standards for each one is determined.

d. Only those methods (MS, GC, HPLC etc.) that are compatible with sample stability are considered.

2. Method requirements:The goals of the analytical method that need to be developed are considered. The detection limits, selectivity, linearity, range, accuracy and precision are defined. 3. Literature search and prior methodology:The information related to the analyte is surveyed. For synthesis, physical and chemical properties, solubility and relevant analytical methods. Books, periodicals and USP / NF and publications are reviewed. Chemical Abstracts Service (CAS) automated computerized literature searches are convenient.4. Choosing a method:a. Using the information in the literatures, methodology is adapted. The methods are modified wherever necessary. Sometimes it is necessary to acquire additional instrumentation to reproduce, modify, improve or validate existing methods for in-house analytes and samples.

b. If there are no prior methods for the analyte in the literature, from analogy, the compounds that are similar in structure and chemical properties are investigated and are worked out. There is usually one compound for which analytical method already exist that is similar to the analyte of interest.

5. Instrumental setup and initial studies:The required instrumentation is setup. Installation, operational and performance qualifications of instrumentation using laboratory standard operating procedures (SOPs) are verified.

Always new solvents, filters are used, for example, method development is never started on a HPLC column that has been used earlier.

The analyte standard in a suitable injection / introduction solution and in known concentrations and solvents are prepared. It is important to start with an authentic, known standard rather than with a complex sample matrix. If the sample is extremely close to the standard (e.g. bulk drug) then it is possible to start work with the actual sample. Analysis is done using analytical conditions described in the existing literature.6. Optimization:During optimization one parameter is changed at a time, and set of conditions are isolated, rather than using a trial and error approach. Work has been done from an organized methodical plan, and every step is documented (in a lab notebook) in case of dead ends.7. Documentation of analytical figures of merit:The originally determined analytical figures of merit limit of quantitation (LOQ), Limit of detection (LOD), linearity, time per analysis, cost, sample preparation etc. are documented.8. Evaluation of method development with actual samples:The sample solution should lead to unequivocal, absolute identification of the analyte peak of interest apart from all other matrix components.9. Determination of percent recovery of actual sample and demonstration of quantitative sample analysis:Percent recovery of spiked, authentic standard analyte into a sample matrix that is shown to contain no analyte is determined. Reproducibility of recovery (average +/- standard deviation) from sample to sample and whether recovery has been optimized and shown. It is not necessary to obtain 100% recovery as long as the results are reproducible and known with a high degree of certainty.

The validity of analytical method can be verified only by laboratory studies. Therefore documentation of the successful completion of such studies is a basic requirement for determining wheather a method is suitable for its intended applications.1.3 SPECTROSCOPY25Spectroscopy is the measurement and interpretation of Electro Magnetic Radiation (EMR) absorbed or emitted when the molecule or atoms or ions of a sample move from one energy state to another energy state. This change may be from Ground state to excited state or excited state to Ground state. At ground state, the energy of a molecule is the sum of rotational, vibrational and electronic energy. In other words, spectroscopy measures the changes in rotational, vibrational and /or electronic energies.ULTRAVIOLET SPECTROSCOPY:Ultraviolet-visible spectroscopy or ultraviolet-visible spectrophotometry (UV-Vis or UV/Vis) refers to absorption spectroscopy or reflectance spectroscopy in the ultraviolet-visible spectral region. This means it uses light in the visible and adjacent (near-UV and near-infrared (NIR)) ranges. The absorption or reflectance in the visible range directly affects the perceived color of the chemicals involved. In this region of the electromagnetic spectrum, molecules undergo electronic transitions. This technique is complementary to fluorescence spectroscopy, in that fluorescence deals with transitions from the excited state to the ground state, while absorption measures transitions from the ground state to the excited state.

Any molecule has either n, or or a combination of these electrons. These bonding ( and ) and non bonding (n) electrons absorb the characteristic radiation and undergoes transition from ground state to excited state. By the characteristic absorption peaks, the natures of the electrons present and hence the molecule structure can be elucidated. LAW GOVERNING ABSORPTION OF RADIATION:Beers law (related to concentration of absorbing species)

The intensity of a beam of monochromatic light decreases exponentially with increase in the concentration of absorbing species arithmetically.Lamberts law (related to thickness/path length of absorbing species)

The rate of decrease of intensity (monochromatic light) with the thickness of the medium is directly proportional to the intensity of incident light.The method is most often used in a quantitative way to determine concentrations of an absorbing species in solution, using the Beer-Lambert law:

A= log10(IO/I) =e.c.l

where A is the measured absorbance, I0 is the intensity of the incident light at a given wavelength, I is the transmitted intensity, L the pathlength through the sample, and c the concentration of the absorbing species. For each species and wavelength, is a constant known as the molar absorptivity or extinction coefficient. This constant is a fundamental molecular property in a given solvent, at a particular temperature and pressure, and has units of 1 / M * cm or often AU / M * cm.

The absorbance and extinction are sometimes defined in terms of the natural logarithm instead of the base-10 logarithm.

The Beer-Lambert Law is useful for characterizing many compounds but does not hold as a universal relationship for the concentration and absorption of all substances. A 2nd order polynomial relationship between absorption and concentration is sometimes encountered for very large, complex molecules such as organic dyes (Xylenol Orange or Neutral Red).

Ultraviolet and visible radiation interacts with matter which causes electronic transitions (promotion of electrons from the ground state to a high energy state). The ultraviolet region falls in the range between 190-380 nm, the visible region fall between 380-750 nm.

The following electronic transitions are possible:

( -( * (pi to pi* transition)

n -( * (n to pi* transition)

s - s* (sigma to sigma* transition)

n - s* (n to sigma*transition)

The lowest energy transition is between the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) in the ground state. The absorption of the Electromagnetic radiation excites an electron to the LUMO and create an excited state. The more highly conjugated the system, the smaller the HOMO-LUMO gap, DE, and therefore the lower the frequency and longer the wavelength, (.7

ENERGY LEVELS IN UV SPECTROSCOPY

UV INSTRUMENTATION:It consists of the followings:

A light source (tungsten lamp, sod. Lamp etc.)

A sample cell ( quartz cuvette cell )

A detector (phototube, goley cell etc)

A beam splitter ( diffraction grating prism )

A sensitive recorder

Figure No. 1.3(a): UV Visible double beam spectrophotometer1.4 Introduction to High Performance Liquid Chromatography 27:Chromatography was discovered by M. S. Tswett in 1903. Chromatography encompasses a diverse but related group of methods that permit the separation, isolation, identification and quantification of components in a mixture. HPLC is one mode of chromatography, the most widely used analytical technique. The process occurs as a result of many sorptions-desorption steps during the movement of sample components through the stationary phase. Separation is due to differences in the distribution coefficients of the sample components.

HPLC utilizes a liquid mobile phase to separate the components of a mixture. These components (or analytes) are first dissolved in a solvent, and then forced to flow through a chromatographic column under a high pressure. In the column, the mixture is resolved into its components. The interaction of the solute with mobile and stationary phases can be manipulated through different choices of both solvents and stationary phases. As a result, HPLC acquires a high degree of versatility not found in other chromatographic systems and it has the ability to easily separate a wide variety of chemical mixtures.High-performance liquid chromatography (HPLC) is a form of liquid chromatography to separate compounds that are dissolved in solution. HPLC instruments consist of a reservoir of mobile phase, a pump, an injector, a separation column, and a detector. Compounds are separated by injecting a plug of the sample mixture onto the column. The different components in the mixture pass through the column at different rates due to differences in their partitioning behavior between the mobile liquid phase and the stationary phase.

Solvents must be degassed to eliminate formation of bubbles. The pumps provide a steady high pressure with no pulsating, and can be programmed to vary the composition of the solvent during the course of the separation. Detectors rely on a change in refractive index, UV-VIS absorption, or fluorescence after excitation with a suitable wavelength

.

INSTRUMENTATION:

A solvent reservoir

A degasser ( ultrasonicator etc. )

A high pressure pump (pneumatic pump)

A pre or guard column

An analytical column

A sample injection port

A detector (UV detector)

A sensitive recorder

A SCHEMATIC DIAGRAM OF HPLC

Modes of HPLC:1. Reverse phase or normal phase- analysis of small (< 2000 Da) organic molecule

2. Ion chromatography- analysis of ions

3. Size exclusion chromatography for separation of polymers

4. Chiral HPLC determination of enentiomeric purity

1. Normal phase chromatography:It is based upon highly polar stationary phases such as triethylene glycol or water and a relatively non-polar mobile phase such as hexane or tetrahydrofuran.

2. Reverse phase chromatography: In the reverse phase Partition HPLC the relative polarities of the stationary and mobile phases are opposite to those in normal HPLC.

Choice of mode of separation:

To select the most appropriate column type for the analyte requires some knowledge of physical characteristics of the sample. The following flow chart gives general outlines for the selection of the chromatographic method.Sample

Water-soluble Soluble in organic solvent

Ionic Non-ionic Polar

Non-polar

IEC IPC LSC BPC RPC BPC RPC LSC

IEC Ion exchange chromatography IPC Ion pair chromatography Ionic

LSC Liquid solid chromatography BPC Bonded pair chromatography

RPC Reversed phase chromatography

Flow chart showing Mode of separation in chromatography

STRATEGY FOR METHOD DEVELOPMENT IN HPLC

Selection of suitable chromatography method for Organic compounds:

First reverse phase should be tried,

If not successful, Normal-phase should be taken into consideration.

Before making experimentation with ion exchange or ion-Pair chromatography, ion suppression by pH controls and Reverse - Phase chromatography should be tried. For ion-forming organic compounds Ion-Pair chromatography should be preferred to Ion-Exchange chromatography.

Reverse phase HPLC:

Reverse phase chromatography is usually a method of first choice because of convenience, wide applicability, and good understanding of operating principles.36 In the reverse - phase partition HPLC the relative polarities of the stationary and mobile phases are opposite to those in normal HPLC, i.e. the stationary phase is less polar than the mobile phase and consequently the solutes are eluted in order of their decreasing polarities. These phases are prepared by treating surface silanol groups of silica with orthogonochlorosilane reagent.Mobile phase selection:In reverse - phase chromatography, the mobile phase is more polar than the stationary phase. Mobile phase in these systems is usually mixture of two or more individual solvents with or without additives or organic solvent modifiers. The usual approach is to choose what appears to be the most appropriate column, and then to design a mobile phase that will optimize the retention and selectivity of the system. Separations in these systems are considered to be due to different degrees of hydrophobicity of the solutes. The polarity of organic modifier and its proportion control the rate of elution of the components in the mobile phase. The rate of elution is increased by reducing the polarity. The simple alteration of composition of the mobile phase or of the flow rate allows the rate of the elution of the solutes to be adjusted to an optimum value and permits the separation of a wide range of the chemical types.

HPLC measurements are susceptible to variations in analytical conditions, these should be suitably controlled or a precautionary statement should be included in the method. One consequence of the evaluation of robustness and ruggedness should be that a series of system suitability parameters is established to ensure that the validity of the analytical method is maintained wherever used. Typical variations are the stability of analytical solutions, different equipment, and different analysts.

Acceptance criteria: The RSD variation of the results 2.5%.System suitability specifications and tests for HPLC26: The accuracy and precision of HPLC data collected begin with a well behaved chromatographic system. The system suitability specifications and tests are parameters that provide assistance in achieving this purpose.

1. Capacity factor (k')k' = (t R- t0 ) / t 0

The capacity factor is a measure of the degree of retention of an analyte relative to an unretained peak, where tR is the retention time for the sample peak and to is the retention time for an unretained peak.

Recommendations:The peak should be well-resolved from other peaks and the void volume. Generally the value of k' is > 2.

2. Resolution (Rs) Ability of a column to separate chromatographic peaks. Resolution can be improved by increasing column length, decreasing particle size, increasing temperature, changing the eluent or stationary phase. It can also be expressed in terms of the separation of the apex of two peaks divided by the tangential width average of the peaks.

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Rs = tR / 0.5 (W1 + W2)

Where tR = t2 t1For reliable quantitation, well-separated peaks are essential.

Recommendations:Rs of > 2 between the peak of interest and the closest potential interfering peak (impurity, excipient, degradation product, internal standard, etc) are desirable.

3. Tailing factor (T)A measure of the symmetry of a peak given by the following equation where W0.05 is the peak width at 5% height and f is the distance from peak front to apex point at 5% height. Ideally, peaks should be Gaussian in shape or totally symmetrical.

T = W0.05 / 2f

The accuracy of quantitation decreases with increase in peak tailing because of the difficulties encountered by the integrator in determining where/when the peak ends and hence the calculation of the area under the peak. Integrator variables are preset by the analyst for optimum calculation of the area for the peak of interest.

Recommendations:T of 2

4. Theoretical plate number / Efficiency (N)A measure of peak band spreading determined by various methods, some of which are sensitive to peak asymmetry. The most common are shown here, with the ones most sensitive to peak shape shown first:

4-sigma / tangential

N = 16 (tR / W )2 = L / H

Half height

N = 5.54 (tR / W)2 = L / H

Theoretical plate number is a measure of column efficiency that is, how many peaks can be located per unit run-time of the chromatogram, where tR is the retention time for the sample peak and W is the peak width.

N is fairly constant for each peak on a chromatogram with a fixed set of operating conditions. H or HETP, the height equivalent of a theoretical plate, measures the column efficiency per unit length (L) of the column. Parameters which can affect N or H include peak position, particle size in column, flow-rate of mobile phase, column temperature, viscosity of mobile phase and molecular weight of the analyte.

Recommendations:The theoretical plate number depends on elution time but in general should be > 2000.

General Recommendation:System suitability testing is essential for the assurance of the quality performance of the chromatographic system. The amount of testing required will depend on the purpose of the test method. For dissolution or release profile test methods using an external standard method, k', T and RSD are minimum recommended system suitability tests. For acceptance, release, stability or impurities/degradation methods using external or internal standards, k', T, Rs and RSD are recommended as minimum system suitability testing parameters. In practice, each method submitted for validation should include an appropriate number of system suitability tests defining the necessary characteristics of that system.

Additional tests may be selected at the discretion of the reviewer.1.5 ANALYTICAL METHOD VALIDATIONValidation is defined as follows by different agencies:

Food and Drug administration (FDA) Establishing documentation evidence, which provides a high degree of assurance that specific process, will consistently produce a product meeting its predetermined specification and quality attributes.

World Health Organization (WHO)Action of providing that any procedure, process, equipment, material, activity or system, actually leads to the expected results.

European Committee (EC)31Action of providing in accordance with the principles of good manufacturing practice, that any procedure, process, equipment material, activity or system actually lead to the expected results. In brief validation is a key process for effective Quality Assurance.1.5.1 Objective of Validation32The primary objective of validation is to form a basis for written procedures for production and process control which are designed to assure that the drug products have the identity, strength, quality and purity they purport are represented to process.

Quality, safety and efficacy must be designed and built into the products.

Each step of the manufacturing process must be controlled to maximize the probability that the finished product meets all quality and design specifications.

Steps followed for validation procedures 1. Proposed protocols or parameters for validations are established.

2. Experimental studies are conducted.

3. Analytical results are evaluated

4. Statistical evaluation is carried out.

5. Report is prepared documenting all the results.

1.5.2 Benefits of Method Validation29A fully validated process may require less in-process control and end product testing. It deepens the understanding of processes, decrease the risks of processing problems, and thus assure the smooth running of the process.1.5.3 Typical validation characteristics which should be considered are listed below 30 Accuracy

Precision

-Repeatability

-Reproducibility

Specificity

Linearity

Range

Detection limit

Quantitation limit

Ruggedness

Robustness etc.

AccuracyThe accuracy of an analytical method is defined as the degree to which the determined value of analyte in a sample corresponds to the true value. Accuracy may be measured in different ways and the method should be appropriate to the matrix. The accuracy of an analytical method may be determined by any of the following ways:

Analysing a sample of known concentration and comparing the measured value to the true value. However, a well characterised sample (e.g. reference standard) must be used.

Spiked placebo (product matrix) recovery method. In the spiked placebo recovery method, a known amount of pure active constituent is added to formulation blank [sample that contains all other ingredients except the active(s)], the resulting mixture is assayed, and the results obtained are compared with the expected result.

Standard addition method. In the standard addition method, a sample is assayed, a known amount of pure active constituent is added, and the sample is again assayed. The difference between the results of the two assays are compared with the expected answer. In both methods (spiked placebo recovery and standard addition method), recovery is defined as the ratio of the observed result to the expected result expressed as a percentage.The accuracy of a method may vary across the range of possible assay values and therefore must be determined at several different fortification levels. The accuracy should cover at least 3 concentrations (80, 100 and 120%) in the expected range.

Accuracy may also be determined by comparing test results with those obtained using another validated test method. Acceptance criteria:

The expected recovery depends on the sample matrix, the sample processing procedure and on the analyte concentration. The mean % recovery should be within the following ranges:% Active/impurity content Acceptable mean recovery

10

1

0.1 1

< 0.1 98 102%

90 110%

80 120%

75 125%

PrecisionThe precision of an analytical procedure expresses the closeness of agreement (degree of scatter) between a series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions. Precision may be considered at three levels: repeatability, intermediate precision and reproducibility. For these guidelines, a simple assessment of repeatability will be acceptable. The precision of an analytical procedure is usually expressed as the variance, standard deviation or coefficient of variation of a series of measurements. A minimum of 5 replicate sample determinations should be made together with a simple statistical assessment of the results, including the percent relative standard deviation. If considered appropriate, a suitable test for outliers (Dixons or Grubbs Test) may be applied to the results, where outliers have been discarded, that fact must be clearly indicated. An explanation as to the reason for the occurrence of individual outliers must be attempted.

The following levels of precision are recommended.

Component measured in sample Precision > 10.0% 2%

1.0 up to 10.0% 5%

0.1 up to 1.0% 10%