RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES,

BANGALORE, KARNATAKA,

ANNEXURE II

PROFORMA FOR REGISTRATION OF SUBJECTS FOR

DISSERTATION

1. / Name of the Candidate
and Address / P. PRIYANKA
a. Permanent Address
D/O P.Madhavareddy
QC NO NC-20 8’incline colony
Godhavarikhani
Karimnagar
Andhrapradesh
b. Postal Address
Krupanidhi College of Pharmacy
Chikka Bellandur,
Carmelaram Post, Varthur Hobli,
Bangalore-35
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

/ Master of Pharmacy
(Pharmaceutical Analysis)
4. /

Date of Admission to Course

/ NOV 2010
5. / Title of the Topic:
“METHOD DEVELOPMENT AND VALIDATION FOR SIMULTANEOUS ESTIMATION OF TENOFOVIR-LAMIVUDINE BY HPLC.’’
6. / BRIEF RESUME OF THE INTENDED WORK:
6.1 Need of study:
Various guidelines like, ICH, WHO etc. suggest that analytical method used for the analysis of drug must be stability indicating and validated.
Generally, chromatographic method (HPLC, HPTLC, and GC) is used because it fulfills requirement of various guidelines by effectively separating degraded product from the drug substance.
So the interest of work emphasizes on HPLC, because in the modern pharmaceutical industry, high-performance liquid chromatography (HPLC) is the major and integral analytical tool applied in all stages of drug discovery, development, and production. It is ideal for the analysis of many drugs in dosage forms due to its simplicity, high specificity and wide range of sensitivity.
HPLC currently accounts for 35% of all instrument usage across the pharmaceutical and cosmetic industries and remains the fastest growing technique in both industries.
HPLC provides reliable quantitative precision and accuracy, along with a linear dynamic range (LDR) sufficient to allow for the determination of the API and related substances in same run using a variety of detectors, and can be performed on fully automated instrumentation. HPLC provides excellent reproducibility and is applicable to wide array of compound types by judicious choice of HPLC column chemistry
Best column, best mobile phase, best detection wavelength, efforts in their selection can make a world of difference while developing HPLC method for routine analysis determining the ideal combination of these factors assures faster delivery of desired results –a validated method for separation
Acquired immune deficiency syndrome (AIDS) is a disease of the human immune system caused by the human immunodeficiency virus (HIV) .In recent times the introduction of combination therapy has reduced the morbidity and mortality of the human immune deficiency (HIV) infected individuals. Several drugs from various classes are combined to form fixed dose combinations (FDCs).
HPLC methods are useful in the determination of drugs in pharmaceutical formulations especially those containing more than one active component. Therefore, the aim of this work was to develop a relatively simple HPLC method for simultaneous quantification of tenofovir-lamivudine.
6.2 Review of Literature:
A simple, rapid reversed-phase high performance liquid chromatographic method had been developed and validated for estimation of emtricitabine and tenofovir disoproxil fumarate in tablet dosage form. Estimation was carried out on Luna C18 (25cm x 4.60 mm, particle size 5μm) column with a mixture of acetonitrile: potassium dihydrogen phosphate buffer (pH 3.0 ± 0.05 adjusted with orthophosphoric acid): triethylamine in the ratio of 70:30:0.5(v/v) as mobile phase. UV detection was performed at 260 nm. The method was validated for linearity, accuracy, precision, specificity and sensitivity as per ICH norms1.
A simple and rapid high-performance liquid chromatographic method for the simultaneous quantification of efavirenz and nevirapine in human plasma suitable for therapeutic drug monitoring. Sample pre-treatment consisted of protein precipitation with acetonitrile and subsequently dilution with distilled water. The drugs were separated from endogenous compounds by isocratic reversed-phase high-performance liquid chromatography with ultraviolet detection at 275 nm. The method was validated over the therapeutically relevant concentration range of 0.05–15.0 mg and 0.25–15.0 mg for efavirenz and nevirapine, respectively, using a volume of 100 ml of plasma. The calibration curves were linear over this concentration range. Internal standard used was Carbamazepine. The assay proved to be accurate and precise. This validated assay is suited for use in pharmacokinetic studies with efavirenz and nevirapine2.
An accurate, sensitive and specific reversed phase high performance liquid chromatographic method (RP-HPLC) for the simultaneous quantitative determination of lamivudine, stavudine , nevirapine in pharmaceutical fixed dose combinations is described. Chromatography was carried on C-18 column by gradient elution with two mobile phase components: mobile phase (A) comprising of 80% of 10mM acetate buffer adjusted to pH 3.5 with glacial acetic acid and 20% methanol and mobile phase (B) comprising of 50% acetonitrile with 50% isopropyl alcohol. Mobile phase was pumped at a flow rate of 0.6 ml min−1 and UV detection was employed at 270 nm. The average retention times for lamivudine, stavudine and nevirapine were 5.9, 8.8 and 14.2 min, respectively. The calibration curves were linear in the range for each analyte. The method is accurate and precise. No spectral or chromatographic interferences from the tablet excipients were found. This method which is rapid, simple and does not require any separation process has been successfully applied to the assay of commercial fixed dose formulation3.
Two methods are described for the simultaneous determination of lamivudine (3TC) and stavudine (d4T) in combined pharmaceutical tablets. The first method depends on first derivative UV-spectrophotometry with zero-crossing measurement technique. The first derivative absorbances at 280 and 300 nm were selected for the determination of stavudine and lamivudine, respectively. The second method is based on the separation of both drugs by high performance liquid chromatography using methanol:water (20:80)as the mobile phase at 0.6 ml/min on a reverse phase column with detection at 270 nm. The methods showed good linearity, reproducibility and precision. No spectral or chromatographic interferences from the tablet excipients were found. The proposed methods were suitably applied to the assay of commercial formulations. The procedure was rapid, simple and suitable for routine quality control application4.
An accurate, sensitive and simple reverse-phase (RP) high-performance liquid chromatography (HPLC) assay for the simultaneous quantitative determination of emtricitabine and tenofovir in human blood plasma is described. Using 200 mL of plasma and BOND ELUT-C18 Varian columns, the solid phase extraction (SPE) method results in a clean baseline and high extraction efficiencies (100% for emtricitabine and 98.6% for tenofovir). An AtlantisTM dC-18 analytical column is used along with an 18 min linear gradient elution of phosphate buffer (pH 5.7) and methanol to provide sharp peaks for emtricitabine at 280 nm, tenofovir at 259 nm, and the internal standard 2 ,3 didoxyuridine (DDU) at 262 nm. The method was validated over the range of 10–10,000 ng/mL for both analytes, and is accurate and precise. This method is suitable for use in clinical pharmacokinetic studies5.
A sensitive high-performance liquid chromatographic assay has been developed to determine the concentrations of indinavir in human plasma. The sample pretreatment involved a protein precipitation procedure using 100 ml of human plasma and 400 ml of acetonitrile. Chromatography was carried out on an Octadecyl column using a mobile phase of acetonitrile–water (40:60, v/v). The water phase contained 50 mM phosphate buffer pH 6 and 4 g/ l tetramethylammoniumchloride. Ultraviolet detection at 210 nm was used. The method has been validated with regard to specificity, detection limit, lower and upper limit of quantitation, recovery, accuracy, and inter- and intra-assay precision. Stability tests under various conditions were performed. The bioanalytical assay is now in use for the determination of Indinavir in several clinical pharmacokinetic studies in HIV-infected patients6.
A simple and rapid high-performance liquid chromatographic method for the quantification of abacavir in human plasma suitable for pharmacokinetic research purposes is described. Sample pretreatment consists of protein precipitation with perchloric acid. The supernatant is injected directly into the chromatographic system after centrifugation. The drug is separated from endogenous compounds by isocratic reversed-phase high-performance liquid chromatography with ultraviolet detection at 285 nm. The method has been validated over the range of 20–2000 ng/ml using a volume of 300 ml of plasma. The assay is linear and precise. The lower limit of quantitation is 20 ng/ml and the recovery of abacavir is 88.1% (61.3%). Frequently coadministered drugs did not interfere with the described methodology. This validated assay is suited for use in pharmacokinetic studies with abacavir in human plasma7.
A reversed phase high performance liquid chromatography method was developed for the simultaneous quantitative determination of the lamivudine, didanosine, stavudine, zidovudine and abacavir in plasma. The method involved solid-phase extraction with Oasis MAX cartridges from plasma, followed by high performance liquid chromatography with a Symmetry Shield RP 18 column and ultraviolet detection set at a wavelength of 260 nm. The assay was validated over the concentration range of 0.015–5 mg/l for all five NRTIs. This method proved to be simple, accurate and precise, and is currently in use in our laboratory for the quantitative analysis of NRTIs in plasma8.
A simple reversed-phase high-performance liquid chromatography assay for the simultaneous quantitative determination of nevirapine, delavirdine, and efavirenz in human blood plasma is described. The method was validated over the range of 10 ng/ml to 50 mg/ml for nevirapine, 25 ng/ml to 25 mg/ml for delavirdine, and 10 ng/ml to 10 mg/ml for efavirenz. The method is accurate and precise. All three non-nucleoside reverse transcriptase inhibitors proved to be stable under various conditions. Due to its simplicity, this assay can readily be used for investigational or clinical monitoring of plasma concentrations9.
A fast, simple isocratic reversed-phase high-performance liquid chromatography method with a 30-mm long column for assaying nevirapine in human serum. After deproteinization of 200 ml serum samples with 50% trichloroacetic acid, the supernatant was injected into a reversed-phase C column, using 10 mM phosphate buffer (pH 5)–acetonitrile (82:18, v/v) 18 as the mobile phase. Peak detection was performed at 240 nm. Nevirapine retention time was 2 min. The method was 2 validated over 0.1–10 mg/ml and the assay was linear and precise. The lower limit of quantification was 0.1 mg/ ml10.
A simple and rapid high-performance liquid chromatographic method for the quantification of delavirdine in human plasma suitable for drug monitoring in patients is described. Sample pretreatment consists of protein precipitation with acetonitrile and subsequent evaporation of the extract to concentrate the analyte. The drug is separated from endogenous compounds by isocratic reversed-phase, high-performance liquid chromatography coupled with fluorescence detection. The optimal excitation and emission wavelengths are 300 and 425 nm, respectively. The method has been validated over the range of 50–50 000 ng/ml using only 200 ml of plasma samples. The lower limit of quantitation is 50 ng/ ml. Recovery of delavirdine from human plasma is 93.8%. vThe method is found to be linear and precise. This validated assay is suited for use in pharmacokinetic studies with delavirdine11.
An isocratic reversed-phase high-performance liquid chromatographic method with ultraviolet detection at 205 nm has been validated for the determination of indinavir, ritonavir and lopinavir (ABT 378) in human plasma. The ritonavir analogue A-86093.0 was used as internal standard. Good chromatographic separation was achieved using a stainless steel column packed with 5 mm Phenomenex phenyl hexyl material operated at 40 8C, and a mobile phase consisting of acetonitrile–10 mM potassium phosphate buffer (50:50, v/v). The calibration curve for indinavir was linear over the range of 50 to1000 mg/l while the ritonavir and lopinavir calibration curves were linear over the range of 100 to 15000 mg/l. The lower limit of quantitations for indinavir, ritonavir and lopinavir were 50, 100 and 100 mg/l, respectively, using 500 ml of human plasma. The validation data showed that the assay is sensitive, specific and reproducible for determination of indinavir, ritonavir and lopinavir12.
A simple, high-performance liquid chromatographic method has been developed and validated for the quantitative determination of efavirenz in human plasma. The method involved solid-phase extraction of the drug and the internal standard (L-737,354) from 300 ml of human plasma. The analysis was via UV detection at 250 nm using a reversed-phase C analytical column 8 and a isocratic mobile phase consisting of phosphate buffer (pH 5.75)–acetonitrile that resolved the drug and internal standard from endogenous matrix components and potential co-administered drugs. This method is found to be precise. The lower limit of quantification was 0.1 mg/ ml. Recovery of efavirenz from human plasma was greater than 83%. This validated assay is being used in pharmacokinetic studies with efavirenz13.
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6.3 Objective of study:
The objective of present study is to develop and validate a simple, precise and rapid high-performance liquid chromatography (HPLC) method for simultaneous estimation of nevirapine-lamivudine
SPECIFIC OBJECTIVES:
·  Step 1 - Selection of the HPLC method and initial system
·  Step 2 - Selection of initial conditions
·  Step 3 - Selectivity optimization
·  Step 4 - System optimization
·  Step 5 - Method validation.
Method development involves a series of sample steps; based on what is known about the sample, a column and detector are chosen; the sample is dissolved, extracted, purified and filtered as required; an eluent survey (isocratic or gradient) is run; the type of final separation (isocratic or gradient) is determined from the survey; preliminary conditions are determined for the final separation; retention efficiency and selectivity are optimized as required for the purpose of the separation (quantitative, qualitative or preparation); the method is validated using ICH guidelines. The validated method and data can then be documented.
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 method development and validation of tenofovir-lamivudine by carrying the following steps:
·  Selection of raw materials.
·  Preparation of raw materials.
·  Selection of instrument.
·  Chromatographic conditions.
·  Preparation of standard solutions.
·  Preparation of sample solutions.
·  HPLC method development and optimization.
·  Validation of developed method.