New Analytical HPLC Method Development and Validation for the Simultaneous Quantification of Paritaprevir Ombitasvir and Ritonavirin Spiked Human Plasma .

A rapid HPLC bio-analytical method has been developed and validated for the simultaneous quantification of Ombitasvir (OMBTSR), Ritonavir (RTNVR), Paritaprevir (PTVR) in human plasma. OMBTSR and PTVR are used to control Hepatitis-C infection. RTNVR is used in the treatment of HIV/AIDS. The method was developed with Column (Intersil ODSC 18, 250 mm × 4.6 mm × 5μ) at 230 nm wave length and at flow rate of 1.0 mL/min. The mobile phase consisted of 20% Acetonitrile, 20% Methanol, 60% 1mM NH4H2PO4Buffer(P H 6.5 v/v). The retention times of RTNVR, OMBTSR, PTVR are 5.7 min. 7.8 min. and 12.8 min. respectively. The method was developed and validated in terms of linearity, interday precision, intraday precision, accuracy, LOQ, LOD and stability study. The proposed method is useful in pharmacokinetic studies using HPLC or LC-MS.


INTRODUCTION
Ritonavir (RTNVR) is an antiretroviral drug used along with other medications to treat HIV/AIDS and hepatitis C. [1][2][3] .Ombitasvir (OMBTSR) is an antiviral drug for the treatment of hepatitis C virus.Mostly it is used in the combinations with in combination with Paritaprevir, Ritonavir and Dasabuvir 4 .Paritaprevir (PTVR) is best drug in the treatment of hepatitis C. The usual side effects of this combination are Diarrhea, Vomiting, loss of appetite and numbness of the hands and feet.The serious side effects are liver problems, pancreatitis, allergic reactions, and Arrhythmias 5 .From last few decades, Pharmacokinetics playing major important role in drug development.At the time of identification of drug's biological properties, metabolic feature of the drug and understanding of pharmacokinetic the bioanalytical methods playing key role.The bioanalytical method used to determine the drug and its metabolites in serum, plasma, urine [6][7][8][9][10][11][12] .
Literature survey revealed that Nourah Zoman et al 12 submitted a method for the estimation of RTNVR, OMBTSR, PTVR in pharmaceutical formulations.Other methods are reported for analysis of individual and combination of two drugs.From the literature survey there is no bio analytical method reported.Andrew J Ocque et al 13 developed a method to determination of Paritaprevir and Ritonavir in rat liver tissue samples.In this method accuracy range is 6.68 to 10.1%.Jyoti.M. Salunke et al 14 developed a method to estimate and RTNVR and Lopinavir in combined dosage form with C18 column (250 x 4.6mm id,5µm) at 1.5 ml/min.flow rate.Methanol, Acetonitrile and Potassium Dihydrogen Phosphate as a mobile phase.Rajasekhar et al 15 developed a method for LCMS method to determination of Lopinavir and RTNVR in Human Plasma by Protein Precipitation method.The method was developed with mobile phase consisting of CH 3 CN and 5mM CH 3 COONH 4 buffer.The linearity range of the method is 50.67-10,008.82ng/mL for Lopinavir and 5.066-1,000.693ng/ml for Ritonavir.K Vinod Kumar et al 16 reported a HPLC method developed for the simultaneous quantification of RTNVR and Lopinavir in tablet dosage form and in plasma.The concentration range of Ritonavir and Lopinavir are 5-50 µg/ml, 20-200 µg/ ml respectively.The method was carried out on RP C18 column and methanol: water (85:15v/v) as a mobile phase.M Jagadees waran et al 17 submitted a method developed a method for quantitative estimation of Lopinavir and RTNVR.The method was reported with reversed-phase C18 Column, mixture of Buffer: Acetonitrile (45:55 v/v) as mobile phase at p H 4.5.The recovery of the method was between 102.1% and 100.1% for Lopinavir and Ritonavir respectively.Suneetha.A et al 18 developed a method for estimation of Lopinavir and RTNVR in combined dosage.The method was developed with Potassium di hydrogen phosphate buffer, CH 3 CN and CH 3 OH in the ratio of 50:35:15v/v at pH 6.0 linear in the ranges are 400-600µg/ml for Lopinavir and 100-150 µg/ml for RTNVR.

Objective
The present study is concerned with the development and validation of RTNVR, OMBTSR and PTVR in spiked human plasma by HPLC.

Apparatus
The new method was developed and validated with Peak LC P7000 HPLC (Isocratic) system rheodyne injector with 20 µL and UV/Vis detector UV7000 and PEAK Chromatographic version 1.06.The Ombitasvir, Ritonavir Paritaprevir were scanned with UV-Visible spectrophotometer (Tech comp-UV 2301, make Japan) with Hitachi software.OMBTSR, RTNVR and PTVR were obtained from Manus Aktteva Biopharma LLP (India).HPLC grade solvents Water, Acetonitrile, Methanol and Tri Ethyl Amine (TEA) were procured from Merck, Mumbai.Method was developed at 230 nm with Intersil ODS C18 column (250 mm × 4.6 mm×5µ).

Chromatographic conditions
The HPLC method conditions were optimized by using different columns, different mobile phases and different buffers.The finalized mobile phase is 20% Acetonitrile, 20% Methanol, 60% 0.001 M NH 4 H 2 PO 4 Buffer v/v.The mobile phase P H was adjusted to 6.5 with TEA for perfect separation.Analysis was performed with flow rate 1.0 ml/min.at ambient temperature.
Standard stock solution preparation 19 The plasma samples was extracted by liquid-liquid extraction process.The fixed dosages (10mg) are spiked in to 10ml plasma and stored in freezer for 24 hours.For processing, the stored spiked samples were taken out from freezer and allowed to thaw at room temperature.An aliquot of 500µL was transferred to prelabeled 10.0ml polypropylene centrifuge tubes.Extraction solvent, 5.0ml of ethyl acetate, was then added to extract the drug.The samples were then kept on a vibramax unit and vortexed for 15 minute.Samples were centrifuged at 5000 rpm for 5 min.in a refrigerated centrifuge (4°C).Supernatant solution, 1 ml was then transferred into pre-labeled polypropylene tubes and was allowed to evaporate to dryness under N 2 at 40°C.The dried residue dissolved in 200µL of mobile phase and transferred into shell vials containing vial inserts for analysis.Samples, 20µL by volume, were then injected into the column and analyzed by HPLC on the same day to avoid any degradation.The column temperature was maintained at ambient temperature.

Validation Linearity Test
The Linearity test was conducted in the range of 25% to 200%.For RTNVR and PTVR the concentrations ranges are 0.15 to 1.2µg/ml.For OMBTR the linearity range is 0.25 µg/ml to 2.0 µg/ ml .The results are showed in below Table .1.The target concentration solutions are prepared by

Precision
Intraday precision and Interday precision tests were conducted at standard concentrations of RTNVR, OMBTSR and PTVR i.e 0.6 µg/ml, 1.0µg/ ml and 0.6µg/ml respectively.The Precision was expressed as the percentage of RSD(Relative Standard Deviation).The calculated percentage RSD of intraday and inter day precision test of each drug is below 2.0.

Recovery
The recovery test was conducted at three different concertinos levels 50%, 100% and 150%.Recovery of RTNVR, OMBTSR and PTVR was evaluated by comparing of observed peak area with    .2.

Sensitivity
The sensitivity of method was calculated by signal to noise ratio.The plasma sample solution was diluted serially and injected at developed conditions.Similarly, blank plasma samples were also processed and injected.The results were showed in Table .3.

Stability
The stability experiments were aimed at testing all possible conditions that the samples might experience after collecting and prior the analysis.Short term BT (Bench-Top) stability test was evaluated after 24 h.at room temperature.Autosampler stability test was evaluated on QCs extracts maintained in the auto sampler at 10 o C for 24 h, by comparing their concentrations with freshextracts.Freeze and Thaw stability test evaluated after three cycles at -20 0 C to room temperature, by comparison with freshly prepared.Stability of RTNVR, OMBTSR and PTVR solutions was observed at room and temperature and in refrigerated conditions for period of 48 hours.Acceptable stability has been considered as percent difference in concentration lower than 5%.The stability study results were showed in Table