ANALYTICAL METHOD DEVELOPMENT AND VALIDATION FOR

SIMULTANEOUS ESTIMATION OF ANTI-INFLUENZA VIRAL DRUGS BY

RP- HPLC BY BIOANALYSIS

A. BRIEF RESUME OF THE INTENDED WORK:

Need of study:

Studies to measure bioavailability and/or establish bioequivalence of a product are important elements in support of orally administered drug products in investigational new drug applications (INDs)1, new drug applications (NDAs)2, abbreviated new drug applications (ANDAs)1, and their supplements. The systemic exposure profile determined during clinical trials in the IND period can serve as a benchmark for subsequent BE studies.

Until recently, bioavailability (rate and extent of absorption of medicaments from drug delivery systems) of drugs was not emphasized. It was more or less assumed that if the physical and chemical integrities of a drug product were assured pharmacological performance would be observed. It is now recognized that formulation factors can influence the biological availability of a medicament from a dosage unit in mammalian systems. Consequently, it has become common practice to establish bioavailability3 by measurement of blood levels of drugs following administration of dosage forms.

However, it should be noted that neither bioavailability nor bioequivalence4 data could not be generated without analytical methodology to accurately measure drugs in biological fluids.

Currently there is a need in the pharmaceutical environment to develop analytical methods for the determination of Anti-influenza drugs by Bioanalysis. The developed method could then be applied to clinical trials to obtain accurate pharmacokinetic parameters.

The need to validate an analytical or bio analytical method is encountered by analysts in the pharmaceutical industry on an almost daily basis, because adequately validated methods are a necessity for approvable regulatory filings. What constitutes a validated method, however, is subject to analyst interpretation because there is no universally accepted industry practice for assay validation.

Several methods were developed for quantitative estimation of Anti-influenza drugs such as voltametric, capillary electrophoresis, spectroflurometer, spectrophotometer, and liquid chromatography (LC)5. Moreover, voltametric, capillary electrophoresis spectrophotometry; spectroflurometry involves tedious procedure and too many steps which do not satisfy the determination of the samples. Quantification of Anti-influenza drugs using HPLC was developed by few researchers,which involved longer run time. Out of thesesome methods do not have good recovery, and some methods have not covered the wide linearity range and some are more expensive.

The study will be aimed to develop & validate a suitable, highly specific, and sensitive analytical method for the quantification of Anti-influenza drugs using RP-HPLC6 technique which could have involved less manual work, have a wide range of linearity, good recovery & a short run time . The study will also aim at applying the method for pharmacokinetic study.

Review of Literature:

● The determination of rivaridine by spectrophotometric method in methanol has been developed for the determination of anti-influenza in bulk drug and its pharmaceutical formulation. The results of analysis have been validated statistically and also by recovery studies. The method was found to be simple economical accurate and reproducible and can be adopted in routine analysis of anti-influenza drugs in bulk drug and Pharmaceutical dosage form7.

●In the determination of lamivudine, nevirapine and rimantidine in human plasma by HPLC with Ultraviolet detection, chromatographic separation was achieved on 10 μm columns having 250 × 4.6 mm ID with a mobile phase containing 15 mM aqueous phosphate buffer: acetonitrile (65:35 % v/v) in isocratic mode. The flow rate was 1.0 ml / min and effluents were monitored at 283 nm.The linearity of the method was good (r2 0.9995), as also were intra-day and inter-day precision. The method was validated for accuracy, specificity, limit of quantification, limit of detection, robustness and stability8.

● A simple, accurate, precise, economical and reproducible UV spectrophotometric method was developed in pure bulk drug and in tablet dosage form of zanamavir and rimantidine. The stock solutions were prepared in 0.5M HCl followed by the further required dilutions with distilled water. The λ max was found to be 280.2nm and 312nm respectively. Linearity in concentration range of 5-25 mg/ mL, 5-50mg/ mL was reported for the two drugs. In the marketed tablets the proposed method has estimated zanamivir -97.56% and rimantidine- 98.03%9 .

● A rapid and sensitive liquid chromatography random mass spectrometry (LC–MS/MS) method has been developed and validated for simultaneous quantification of Tenofovir (TEN) and Emtricitabine (EMT) in human plasma using Chromolith Speed Rod RP18. The mass transition ion-pair has been followed as m/z 288.10→176.10 for TEN, m/z 248.20→130.20 for EMT Lamivudine was used as the internal standard. The proposed method has been validated with a linear range of 10–600 ng/ml for TEN and 25–2500 ng/ml for EMT. The intra-run and inter-run precision values are within 12.0% for TEN and 15.6% or EMT at their respective LOQ levels. The overall recoveries for TEN and EMT were 84.3% and 68.5%, respectively. Total elution time was as low as 2 min10.

● A UPLC–ESI-MS/MS method is developed and validated for the selective determination of protease inhibitors – lopinavir (LPV) and ritonavir (RTV) in human plasma. Plasma samples were prepared by solid phase extraction of the analytes. The chromatographic separation was achieved in a run time of 1.2 min on Waters Acquity UPLC BEH C18 column (50mm×2.1mm, 1.7mm) under isocratic conditions. A linear dynamic range of 2.9–1452 ng/mL and 29.6–14379 ng/mL was established for ritonavir and lopinavir respectively using 0.1mL human plasma. The mean relative recovery of lopinavir (96.6%), ritonavir (97.5%), d8-lopinavir (85.5%) and d6-ritonavir (86.3%) from spiked plasma samples was consistent and reproducible11.

●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 has been reported using 200 mL of plasma and BOND ELUT-C18 variant columns, the solid phase extraction (SPE) method resulted in a clean baseline and high extraction efficiencies (100% for emtricitabine and 98.6% for tenofovir). An Atlantis TM 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 precise12.

●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. 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 lopinavir13.

● A simple, precise, accurate and rapid high performance thin layer chromatographic method has been developed and validated for the estimation of emtricitabine and tenofovir simultaneously in combined dosage form. The stationary phase used was precoated silica gel 60F 254. The mobile phase used was a mixture of chloroform: methanol (9:1 v/v). The detection of spots was carried out at 265 nm. The method was validated in terms of linearity, accuracy, precision and specificity. The calibration curve was found to be linear between 200 to 1000 ng with regression coefficient of 0.9995. The proposed method can be successfully used to determine the drug content of marketed tablet formulation14.

Objective of study:

The objective of present study is to develop and validate an accurate, precise and rapid RP-HPLC method for estimation of Anti-influenza drugs.

SPECIFIC OBJECTIVES:

Method development is a complex process that involves a number of steps, which are as follows:

Method selection and information of sample

  • Selection of initial method conditions
  • Checking the analytical method in appropriate solvents
  • Development and optimization of sample processing method
  • Checking the analytical method in biological matrix
  • Pre-validation

B. MATERIALS AND METHODS:

Source of Data:

Data will be obtained from Internet facilities (Helinet, science direct, scirus, PubMed) Literature and related articles from libraries of Krupanidhi College of Pharmacy, other Research Publications and Journals.

Method of Collection of Data:

Data will be collected for analytical method development and validation of Anti-influenza drugs by carrying the following steps:

  • Selection of raw materials
  • Preparation of raw materials
  • Selection of instrument
  • Chromatographic conditions
  • Preparation of standard stock solution
  • Preparation of internal standard stock dilution
  • Preparation of standard solutions
  • Preparation of sample solutions
  • Method development and optimization
  • Validation of developed method

Does the study require any investigation or interventions to be conducted on patients or the human or animals? If so please describe briefly:

NO

Has ethical clearance been obtained from your institute

Not applicable

C. List of References

1.Available from: en.wikipedia.org/wiki/Abbreviated_New_Drug_Applicationhtml Retrived on: 10/12/12.

2. Available from: en.wikipedia.org/wiki/New_Drug_Applicationhtml Retrived on: 10/12/12.

3. British pharmacopoeia. London, medical and health care products regulatory agency (MHRA) 2005; 1:171-2:943.

4.United States Pharmacopoeia Convention, INC. Washington D.C; United States pharmacopoeia 27, NF 22, Asian Ed; 2004:183-4:917-8.

5.Available from: en.wikipedia.org/wiki/Analytical_technique Retrived on: 10/12/12

6.Naveen K, Nishant V, Omveer S, Naveen J, Kanwar GS. RP-HPLC. E- J Chem. 2010; 7(3):962-6.

7.Amudhavalli V and Lakshmi KS. Derivative spectrophotometric estimation of Rivaridine in pharmaceutical dosage form. J Chem Pharm Res 2010; 2(5):502-5.

8.Vaishali PN, Kishore PB. A validated UV spectrophotometric method for the simultaneous estimation of Lamivudine, Nevirapine and Zidovudine in combined tablet dosage form.J Pharm Res 2009; 2(4):666-9.

9.Purnima H, Mitesh P, Priti P and Nitul S. Determination of Zanamavir and Rimantidine in human plasma by High performance liquid chromatography with Ultraviolet detection. Int J Pharm Technol Res 2010; 2(2):1316-24.

10.Noel AG, Vikas VV, Ashutosh P, Santosh SJ, Sagar AP. Liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for simultaneous determination of Tenofovir and Emtricitabine in human plasma and its application to a bioequivalence study. J Pharm Bio Anal 2008; (48):918–26.

11.Manish Y, Rajasekhar R, Hemal K, Puran S, Sailendra G, Pranav SS. Application of a rapid and selective method for the simultaneous determination of protease inhibitors, Lopinavir and Ritonavir in human plasma by UPLC–ESI-MS/MS for bioequivalence study in Indian subjects. J Pharm Bio Anal 2009; 49:1115–22.

12.Naser LR, Rustin DC, Angela DM. 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. J Chrom B 2005; 822:201–8.

13.John R, Edna P, Dianne C. 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. J Chrom B 2002; 775:225–30.

14.Maithilee J, Nikalje AP, Shahed M, Dehghan M. HPTLC Method for the Simultaneous Estimation of Emtricitabine and Tenofovir in Tablet Dosage Form. Ind J Pharm Sci 2009; 71(1):95–97.

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