Simultaneous determination of six HIV nucleoside analogue reverse transcriptase inhibitors and nevirapine by liquid chromatography with ultraviolet absorbance detection

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Abstract

An accurate, sensitive and specific reversed-phase high-performance liquid chromatography assay for the simultaneous quantitative determination of the nucleoside reverse transcriptase inhibitors zalcitabine, lamivudine, didanosine, stavudine, zidovudine, and abacavir with the non-nucleoside reverse transcriptase inhibitor nevirapine in human blood plasma is described. The new Polarity dC C18 silica column used in this method provides better resolution and peak shape than all other columns tested. Also, four different ultraviolet wavelengths were used for accurate and specific quantitation of the analytes. The method was validated over the range of 10–10 000 ng/ml for all analytes except zalcitabine (10–5000 ng/ml). This method is accurate (average accuracies of three different concentrations ranged from 97.2 to 105%), and precise (within- and between-day precision measures ranged from 0.5 to 5.1% and 0.5 to 5.6%, respectively), and is currently being used for determination of plasma drug concentrations in our laboratory.

Introduction

Combination antiretroviral therapy is the most effective approach to managing HIV infection [1], [2], [3]. Current treatment guidelines state that antiretroviral regimens should contain at least two nucleoside reverse transcriptase inhibitors (NRTIs), and either a non-nucleoside reverse transcriptase inhibitor (NNRTI) such as nevirapine, or a protease inhibitor [4]. To date, six nucleoside reverse transcriptase inhibitors (NRTIs) have been approved for the treatment of HIV infection: zidovudine (ZDV), didanosine (ddI), zalcitabine (ddC), stavudine (d4T), lamivudine (3TC) and abacavir (ABC). Since multiple drugs are used in a single patient, sensitive and specific analytical methods are needed for simultaneously determining plasma concentrations for as many HIV medications as possible [5]. A clinician may use such methods to provide valuable information about patient treatment in several regards: malabsorption, drug interactions, adherence, and individual drug pharmacokinetics [6].

To date, most published analytical methods for the NRTIs and nevirapine use reversed-phase high-performance liquid chromatography (RP-HPLC) with ultraviolet (UV) detection or mass spectrometry (MS). UV detection is widely used for relatively simple bioanalytical assays. The majority of these methods are for single drugs: didanosine [7], [8], zidovudine [9], [10], [11], [12], lamivudine [13], [14], [15], [16], stavudine [17], [18], [19], abacavir and its two major metabolites [20] and nevirapine [21], [28]. Several other methods have been published for two or more NRTIs [6], [22], [23], [24], [25].

This paper describes the development and validation of the first RP-HPLC method with UV detection for the simultaneous determination of all six NRTIs and nevirapine in human blood plasma after an optimized solid-phase extraction procedure.

Section snippets

Chemicals

Zidovudine, didanosine, stavudine, and hexobarbital were purchased from Sigma (St. Louis, MO, USA). Nevirapine, lamivudine, abacavir, and zalcitabine were obtained from the NIH AIDS Research & Reference Reagent Program (McKesson HBOC BioServices, Rockville, MD, USA). HPLC grade chemicals were purchased from Fisher Scientific (Norcross, GA, USA). Purified compressed nitrogen gas used was obtained from National Welders Supply (Charlotte, NC, USA).

Equipment

A high-performance liquid chromatography (HPLC)

Linearity

The peak area ratio values of the calibration standards were proportional to the concentration of each drug in plasma over the range tested. The calibration curves were fitted by unweighted least-squares linear regression. The mean±SD of three standard curve slopes for zalcitabine, lamivudine, didanosine, stavudine, zidovudine, abacavir and nevirapine were 0.7817±0.078, 1.0879±0.044, 1.6533±0.022, 1.3960±0.025, 1.2281±0.033, 1.8719±0.035and 2.1452±0.011, respectively. The concentration range

Discussion

At present, two HPLC–UV methods have been published for the simultaneous determination of multiple NRTIs [6], [24]. However, the method of Aymard et al. [24] does not include zalcitabine or internal standard, and the method recently published by Simon et al. [6] does not include nevirapine or internal standard. A capillary electrochromatographic method for NRTIs was published by Mesplet et al. [27], but did not include abacavir or nevirapine.

Our optimized HPLC method provides a sensitive and

Conclusion

The HPLC assay here described represents an accurate, precise, specific, and highly reproducible HPLC method for the direct measurement of seven ARV drugs in plasma. The solid-phase extraction method was optimized to concentrate the samples and provided excellent clean up. This method yields high recoveries, shows good linearity, precision and accuracy within a wide concentration range for each drug. The method is currently being used to analyze samples of patients treated with combination

Acknowledgments

This research was supported by the The University of North Carolina at Chapel Hill Center for AIDS Research, #9 P30 AI50410, and the University of North Carolina BIRCWH Career Development Program HD01441.

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