Rajiv Gandhi University of Health Sciences s53

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, BANGALORE, KARNATAKA, ANNEXURE II PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION

1. Name of the Candidate C.JAYA HANISHA and Address a. Permanent Address D/O C.Vijay Kumar, Thangavel nagar, Alagapuram Salem-636 016 Tamil Nadu. b. Postal Address Krupanidhi College of Pharmacy Chikka Bellandur, Carmelaram Post, Varthur Hobli, Bangalore-560 035 Karnataka. 2. Name of the Institute and Address Krupanidhi College of Pharmacy Chikka Bellandur, Carmelaram Post, Varthur Hobli, Bangalore – 560035. Karnataka. 3. Course of Study and Subject MASTERS OF PHARMACY Pharmaceutical analysis 4. Date of Admission to Course 22/06/2011

Title of the Topic: 5. “ANALYTICAL METHOD VALIDATION OF GLATIRAMER ACETATE INJECTION BY HPLC”

1 6. BRIEF RESUME OF THE INTENDED WORK: 6.1 Need of study: METHOD VALIDATION "Validation of an analytical method is the process which is established by laboratory studies, that the performance characteristics of the method meet the requirements for the intended analytical application ".

Validation is required for any new or amended method to ensure that it is capable of giving reproducible and reliable results, when used by different operators employing the same equipment in the same or different laboratories.

Immunosuppressant: An agent that can suppress or prevents the immune response. Immunosuppressants are used to prevent rejection of a transplanted organ and to treat autoimmune diseases such as psoriasis, rheumatoid arthritis and crohn’s disease. Some treatments for cancer act as immunosuppressants1.

Various guidelines like, ICH, WHO etc suggest that analytical method used for the analysis of drug must be validated. The drug under consideration for validation is glatiramer acetate. Drug information1:

Class: Immunosuppressive Chemical Name: l-Glutamic acid polymer with l-alanine, l-lysine, and l-tyrosine, acetate

Molecular Formula: (C5H9NO4 • C3H7-5H9NO4 • C3H7NO2 • C6H14N2O2 • C9H11NO3)x • x(C2H4O2) Brands: Copaxone

2 Mechanism of action:

The mechanism of action for glatiramer is unknown, although several have been proposed. Administration of glatiramer shifts the population of T cells from pro-inflammatory Th1 cells to regulatory Th2 cells that suppress the inflammatory response. Glatiramer may also act as a sort of decoy, diverting an autoimmune response against myelin1.

6.2 Review of Literature:

A combined method of liquid chromatography (LC) with tandem mass spectrometry (MS/MS) was developed to quantify tacrolimus in whole blood. Ascomycin was used as an internal standard. The standard curve was composed of seven points ranging from 1 to 50 µg/l (average r2 = 0.9999). Limits of detection and quantitation were 0.25 and 0.75 µg/l, respectively. Imprecision was <5% across the therapeutic range. Tacrolimus recovery averaged 62%. The most abundant metabolites detected in clinical samples were 13-O- and 15-O- demethyl tacrolimus. This method was used in a comparison study with a microparticle enzyme immunoassay (MEIA): MEIA = 1.03 LC/MS/MS - 0.084 (µg/l), (Sy/x = 1.43), r2 = 0.933. With its high sensitivity and specificity, this LC/MS/MS method presents a good method for immunoassay evaluation2.

A HPLC method was developed and validated for the estimation of mycophenolate mofetil in bulk and tablet dosage form. In this method RP-C18 column (250 mmx4.6 mm I.D., 5μm particle size) with mobile phase consisting of acetonitrile and 0.03 M phosphate buffer in the ratio of 60:40 v/v in isocratic mode was used. The detection wavelength is 254 nm and the flow rate is 0.8 ml/min. In the range of 20-120 μg/ml, the linearity of mycophenolate mofetil showed a correlation coefficient of 0.99953.

Two specific high performance liquid chromatography (HPLC) methods for determination of azathioprine metabolites, both employing a mercurial cellulose resin for extraction, are presented. One method using a strong anion exchange column allows detection of 6-thioguanosine di- and triphosphate (thioguanine nucleotides) in red blood cells (RBC) with a sensitivity of 30 pmol/100 microliters RBC. 6-mercaptopurine (MP) and 6-thiouric acid (TUA) in plasma and urine were analyzed simultaneously by reversed-phase HPLC with a sensitivity of 5 ng/ml. The average (median values are given) steady state concentrations of thioguanine nucleotides in erythrocytes came to 267 pmol/100 microliters RBC (range 53-613)

3 with and to 246 pmol/100 microliters RBC (range 39-629) without concomitant enalapril medication. Mean plasma concentrations of MP and TUA 3 hours after drug intake came to 14.8 +/- 9.9 ng/ml and 398 +/- 262 ng/ml, respectively, during enalapril comedication. Withdrawal of enalapril did not influence these metabolite levels coming to 15.3 +/- 9.1 and 451 +/- 253 after stopping enalapril treatment. Thioguanine nucleotides in RBCs were neither related to the dose of azathioprine given (r = -0.113, p > 0.05) nor to hemoglobin levels (r = 0.278, p > 0.05). However, azathioprine dose/kg body weight seemed to be related to hemoglobin concentration, with and without enalapril co-medication4.

A high-performance liquid chromatographic method was developed for the determination of methotrexate in plasma involving on-line pre-column oxidative cleavage and fluorimetric detection. Plasma samples were subjected to protein precipitation followed by solvent purification and then injection into the chromatographic system. Cerium (IV) trihydroxyhydroperoxide (CTH) was used as a packed oxidant. The oxidative cleavage of methotrexate occured during the flow of 0.04M phosphate buffer (PH3.5) containing the drug through CTH column with a flow-rate of 0.2 mL/min at 40°C. The separation was performed on a reversed-phase column (ODS/TM) using a mobile phase consisting of phosphate buffer (0.05M, PH6.6) and acetonitrile (90:10v/v). The fluorescent products were monitored fluorimetrically at emission and excitation wavelengths of 463 and 367 nm respectively5.

A RP-HPLC method was developed and validated for rapid assay of Sirolimus in tablet dosage form. Isocratic elution at a flow rate of 1.0 ml/min was employed on a symmetry C18 (250x4.6 mm, 5 µm in particle size) column at ambient temperature. The mobile phase consisted of acetonitrile: methanol 20:80 (V/V). The UV detection wavelength was 272 nm and 20µl sample was injected. The retention time for sirolimus was 2.740 min. The percentage RSD for precision and accuracy of the method was found to be less than 2%. The method was validated as per the ICH guidelines6.

A RP-HPLC method was developed for the determination of cyclosporine in capsule dosage form. XTerra C18 column was used as stationary phase with mobile phase acetonitrile in combination with 0.1% trifluoro acetic acid buffer and pH is adjusted to 1.4. Method was developed in an isocratic run of 20% trifluoro acetic acid with 80% acetonitrile for 10 min, at flow rate of 1 ml/min. Effluents were monitored at 210 nm. Retention time of

4 cyclosporine was 3.855 min. The method was validated for specificity, linearity, accuracy, precision, and limit of quantification, limit of detection, robustness and solution stability. Limit of quantification and limit of detection of cyclosporine was found to be 100 ng/ml and 200 ng/ml. Recovery was found to be in the range of 98.08-101.55% 7.

Multivariate calibration methods, partial least squares (PLS-1) and principle component regression (PCR) have been developed and validated for the simultaneous determination of 6-MP (Mercaptopurine) and its oxidative metabolites (6TUA, 8-hydroxo-6- mercaptopurine (8OH6MP) and 6TX) without analyte separation in spiked human plasma. Mixtures of 6MP, 8-8OH6MP, 6TX and 6TUA have been resolved by PLS-1 and PCR to their UV spectra. Recoveries (%) obtained for 6MP, 8-8OH6MP, 6TX and 6TUA were 94.5-97.5, 96.6-103.3, 95.1-96.9 and 93.4-95.8, respectively, using PLS-1 and 96.7-101.3, 96.2-98.8, 95.8- 103.3 and 94.3-106.1, respectively, using PCR. The NAS (Net analyte signal) concept was used to calculate multivariate analytical figures of merit such as limit of detection (LOD), selectivity and sensitivity. The limit of detections for 6MP, 8-8OH6MP, 6TX and 6TUA were calculated to be 0.734, 0.439, 0.797 and 0.482 μ mol L-1, respectively, using PLS and 0.724, 0.418, 0783 and 0.535 μ mol L-1, respectively, using PCR. HPLC was also applied and validated8.

A RP-HPLC method was developed for estimation of cyclophosphamide in whole blood and plasma. The pre-chromatography isolation of the drug involves salting-out of acetonitrile with simultaneous extraction of cyclophosphamide from whole blood and plasma. A short column packed with 5-micron reversed-phase octadecylsilane (ODS) spherical particles was used with an isocratic elution of 5 mM potassium phosphate (pH 6.80)- acetonitrile (80:20, v/v). The cyclophosphamide was monitored at 190 nm and 0.40-0.002 a.u.f.s. At a flow-rate of 1.0 ml/min, the retention time of cyclophosphamide was 9 min. The completion time for the assay was less than 20 min and the assay had a detection limit of 0.30 microgram/ml9.

6.3 Objective of study: The objective of present study is to validate a simple, precise and rapid high-performance liquid chromatography (HPLC) method for glatiramer acetate. The type of validation programme required depends entirely on the particular method and its proposed applications. Validation is necessary for the following reasons:

5 1. To determine whether the process is under control. 2. To set up the appropriate in-process controls.

Typical analytical parameters used in assay validation include:

ACCURACY Accuracy is a measure of the closeness of test results obtained by a method to the true value. Accuracy indicates the deviation between the mean value found and the true value.

PRECISION

The 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.

Repeatability Repeatability expresses the precision under the same operating conditions over a short interval of time. Repeatability is also termed intra-assay precision.

Intermediate precision Intermediate precision expresses within-laboratories variations: different days, different analysts, different equipment, etc.

Reproducibility

Reproducibility expresses the precision between laboratories (collaborative studies, usually applied to standardization of methodology).

RANGE The range of the method is the interval between the upper and lower levels of an analyte that have been determined with acceptable precision, accuracy and linearity.

LINEARITY The linearity of an analytical procedure is its ability (within a given range) to obtain test results which are directly proportional to the concentration (amount) of analyte in the sample during

6 normal usage.

ROBUSTNESS The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small, but deliberate variations in method parameters and provides an indication of its reliability during normal usage.

LIMIT OF DETECTION The lowest concentration in a sample that can be detected, but not necessarily quantitated, under the stated experimental conditions.

LIMIT OF QUANTITATION The quantitation limit of an analytical procedure is the lowest concentration of analyte in a sample that can be quantitatively determined with acceptable precision and accuracy.

SELECTIVITY Selectivity is the ability to measure accurately and specifically the analyte in the presence of components that may be expected to be present in the sample matrix.

SPECIFICITY Specificity for an assay ensures that the signal measured comes from the substance of interest, and that there is no interference from excipient and/or degradation products and/or impurities10.

SYSTEM SUITABILITY TESTING It is an integral part of many analytical procedures .The tests are based on concept that the equipment, electronics, analytical operations and samples to be analysed constitute an integral system that can be evaluated as such. The simplest form of an HPLC system suitability test involves a comparison of the chromatogram trace with a standard trace (as shown below). This allows a comparison of  the peak shape  peak width  baseline resolution

7 So below parameters are calculated experimentally to provide a quantitative system suitability test report: a. Number of theoretical plates (efficiency) b. Capacity factor c. Separation (relative retention) d. Resolution e. Tailing factor

f. Relative Standard Deviation (Precision)11

7. MATERIALS AND METHODS: 7.1 Source of Data: Data will be obtained from Internet facilities, Literatures and related articles from libraries of Krupanidhi College of Pharmacy, other Research Publications and Journals.

7.2 Method of Collection of Data: Data will be collected for analytical method validation for glatiramer acetate by HPLC technique by carrying the following steps: 1. Selection of drug in dosage form 2. Selection of chromatographic conditions 3. Obtaining & preparing standard & reference samples 4. Preparation of sample solutions 5. Validation of developed method. 7.3 Does the study require any investigation or interventions to be conducted on patients or the human or animals? If so please describe briefly: No 7.4 Has ethical clearance been obtained from your institute Not applicable

8 8. List of References:

1. Johnson KP, Brooks BR, Cohen JA, et al. "Extended use of glatiramer acetate (Copaxone) is well tolerated and maintains its clinical effect on multiple sclerosis relapse rate and degree of disability. Copolymer 1 multiple sclerosis study group". Neurology 1998; (3): 701–8.

2. Zhang Q, Simpson J, Aboleneen HI. A specific method for the measurement of tacrolimus in human whole blood by liquid chromatography/tandem mass spectrometry. Ther Drug Monit. 1997; 19(4):470-6.

3. Lakshmana Rao A, Vijay Srinivas P, Rao JVLNS. A new validated RP-HPLC method for the estimation of mycophenolate mofetil in pure and tablet dosage form. J Pharm Res Health Care.2001; 2(3):266-9.

4. Weller S, Thurman P, Rietbrock N, Grossmann J, Scheuermann EH. HPLC analysis of azathioprine metabolites in red blood cells, plasma and urine in renal transplant recipients. Int J Clin Pharmacol Ther.1995; 33(12):639-45.

5. Samy Emara, Saied Razee, Alaa Khedr, Tsutomu Masujima, Samy Emara. On-line precolumn derivatization for HPLC determination of methotrexate using a column packed oxidant. Biomed Chromatogr.1997 ;( 2):42–6.

6. Basaveswara Rao MV, Nagendrakumar AVD. Development and validation of RP – HPLC method for the estimation of sirolimus in formulation and serum. Int J Chem. 2011 ;( 1):47-54.

7. Aziz F, Gupta A, and Khan MF. Development and Validation a RP-HPLC Method for Determination of Cyclosporine in Capsule. Indian J Pharm Sci. 2010; 72(2): 252–5.

8. Hossein Sorouraddin, Yaser Khani M, Kaveh Amini, Abdolhossein Naseri, Davoud Asgari, Reza Rashidi M. Simultaneous determination of 6-mercaptopurine and its oxidative metabolites in synthetic solutions and human plasma using spectrophotometric multivariate calibration methods. Bio impacts.2011; 1(1)53-62.

9. Rustum AM, Hoffman NE. Determination of cyclophosphamide in whole blood and plasma by reversed-phase high-performance liquid chromatography. J Chromatogr. 1987; 422:125-34.

10. Quality Assurance of Pharmaceuticals. A compendium of guidelines and related materials. WHO, Geneva. 2nd ed :131-2.

11. ICH harmonized tripartite guideline. Validation of Analytical Procedures: text and methodology Q2(R1).Available at www.ich.org.com (Retrieved on 2011 , December 11).

9. SIGNATURE OF THE CANDIDATE:

(C.JAYA HANISHA) 10 . REMARKS OF THE GUIDE: Analytical method validation by HPLC technique is to be carried out has been discussed under the supervision of the official guide. Immunosupressive agents are gaining importance for the treatment of multiple sclerosis and to prevent the rejection of a transplanted organ. Hence the proposed work is recommended for persual.

11. Name and Designation of

11.1 Guide Mrs. NAIRA NAYEEM Department of Pharmaceutical Analysis, Krupanidhi College of Pharmacy, Bangalore-560 035.

11.2 Signature

(Mrs.NAIRA NAYEEM)

11.3 Head of the Department Mrs. NAIRA NAYEEM ASST.PROFESSOR, Department of Pharmaceutical Analysis, Krupanidhi College of Pharmacy, Bangalore-560 035.

11.3 Signature

(Mrs.NAIRA NAYEEM)

10 12 . 12.1 Remarks of the Dean: The program and research work that is undertaken by C.JAYA HANISHA has potential implication in the field of Pharmaceutical Analysis; hence the project is recommended and requested for clearance and approval.

12.2 Signature

Mr. N. PREM KUMAR DEAN Krupanidhi College of Pharmacy, Chikka Bellandur, Carmelaram Post, Varthur Hobli, Bangalore – 560035.