Validation of a rapid and sensitive high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) assay for the simultaneous determination of existing and new antiretroviral compounds

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Abstract

Clinical pharmacokinetic studies of antiretrovirals require accurate and precise measurement of plasma drug concentrations. Here we describe a simple, fast and sensitive HPLC–MS/MS method for determination of the commonly used protease inhibitors (PI) amprenavir, atazanavir, darunavir, lopinavir, ritonavir, saquinavir and the non-nucleoside reverse transcriptase inhibitor (NNRTI) nevirapine, as well as the more recent antiretrovirals, the CCR5 antagonist maraviroc and the “second generation” NNRTI etravirine and rilpivirine. An internal standard (quinoxalone; QX) was added to plasma aliquots (100 μl) prior to protein precipitation with acetonitrile (500 μl) followed by centrifugation and addition of 0.05% formic acid (200 μl) to the supernatant. Chromatographic separation was achieved using a gradient (acetonitrile and 0.05% formic acid) mobile phase on a reverse-phase C18 column. Detection was via selective reaction monitoring (SRM) operating in positive ionization mode on a triple-quadrupole mass spectrometer. All compounds eluted within a 5 min run time. Calibration curves were validated over concentration ranges reflecting therapeutic concentrations observed in HIV-infected patients from pharmacokinetic data reported in the literature. Correlation coefficients (r2) exceeded 0.998. Inter- and intra-assay variation ranged between 1% and 10% and % recovery exceeded 90% for all analytes. The method described is being successfully applied to measure plasma antiretroviral concentrations from samples obtained from clinical pharmacokinetic studies.

Introduction

Highly active antiretroviral therapy (HAART) has dramatically reduced HIV-1-associated mortality and morbidity [1] and currently comprises 25 drugs from five different classes; the nucleoside and non-nucleoside reverse transcriptase inhibitors (NRTI and NNRTI), protease inhibitors (PI), and most recently entry inhibitors and integrase inhibitors. Current 2008 British HIV Association guidelines recommend for treatment naive patients, a combination of three or more antiretroviral agents; an NNRTI or a ritonavir boosted PI in combination with a dual NRTI backbone [2]. However, despite the long-term benefits of HAART, an estimated 8% of treatment naive and 33% of experienced patients do not achieve viral suppression or experience viral rebound within 12 months of initiating HAART [3].

For patients harbouring resistant virus and those failing multiple regimens, antiretroviral drug combinations have become increasingly complex and in recent years new and more potent agents have been introduced which possess activity against both wild-type and resistant viral strains. The chemokine receptor (CCR5) antagonist maraviroc was approved by the FDA and EMEA in 2007. Maraviroc acts specifically against CCR5-trophic (R5) HIV-1 and prevents R5 virus engaging with the CCR5 co-receptor located on the host CD4 cell membrane, but is not effective against CXCR4-trophic (X4) and dual/mixed trophic strains which become increasingly dominant in the later stages of HIV-1 infection [4]. The integrase inhibitor raltegravir (also licensed in 2007) inhibits the integration of pro-viral DNA into the host genome and has demonstrated potent antiviral activity in multi-drug experienced patients [5], [6]. Also, in view of increasing resistance, new NNRTI were also urgently needed. The second generation NNRTI, etravirine (TMC125) and rilpivirine (TMC278) are diarylpyrimidine compounds which possess favorable binding interactions toward reverse transcriptase of both mutant HIV-1 strains as well as wild-type virus, including the common K103N mutation [7]. In 2008, the FDA and EMEA granted accelerated approval of etravirine based on data from the phase III DUET_1/2 studies [8], [9]. Rilpivirine, shown in initial phase IIb studies to be equivalent to the standard-of-care efavirenz [10], is not yet licensed and is currently undergoing non-inferiority phase III trials (ECHO and THRIVE) for use in treatment naive and experienced patients.

The PI and NNRTI undergo cytochrome P450 mediated metabolism via CYP3A4 and to a lesser extent by CYP2B6, CYP2D6 and CYP2C19 which renders them prone to variable pharmacokinetics and extensive drug–drug interactions when given in combination or with other concomitant medications [11]. Moreover, they can variably affect their own metabolism through the induction and inhibition of these enzymes. All PI inhibit CYP3A4, with ritonavir being the most potent and is used exclusively at sub-therapeutic doses to “boost” other PI [12], [13]. In addition, ritonavir, lopinavir and amprenavir have CYP enzyme inducing properties [14], [15], [16]. The first generation NNRTI nevirapine and efavirenz are substrates and inducers of CYP3A4 and CYP2B6 (the major enzyme involved in the metabolism of efavirenz) [17]. The second generation NNRTI rilpivirine is metabolized primarily by CYP3A4, and etravirine by CYP3A4, CYP2C9, CYP2C19 [18]. Maraviroc is a substrate for both CYP3A4 and the efflux transporter P-gycloprotein, and has shown clinically significant interactions with both PI and NNRTI, rendering mandatory maraviroc dosage adjustments with some associations [19], [20].

The quantification of antiretrovirals from plasma is a valuable pharmacological tool since PI and NNRTI demonstrate pharmacokinetic/pharmacodynamic (PK/PD) [21], [22], [23], [24] and pharmacokinetic/toxicity relationships [25], [26], [27]. Thus characterisation of the relationship between antiretroviral pharmacokinetics (systemic exposure or a single concentration) and drug response (beneficial or adverse) is key to the selection of an optimal dose for a drug, understanding inter- and intra-subject variability, and to design strategies to optimize response and tolerability while avoiding unwanted toxicity. For this reason, comprehensive pharmacokinetic studies investigating drug interactions, as well as those assessing new dosing strategies require accurate and precise measurement of drug concentrations to ensure that correct and meaningful data are fed back into clinical care. Indeed, routine therapeutic drug monitoring (TDM) and pharmacokinetic drug interaction studies between existing and new antiretrovirals, and with concomitant medications are essential for the optimization and management of antiretroviral therapy, in order to maintain efficacy and prevent drug toxicity and resistance. Also important are clinical studies investigating pharmacokinetics in specific patient groups including pregnant women and children, who are in need of tailored antiretroviral dosage regimens.

Several methodologies have been reported in the literature which simultaneously determine PI and NNRTI plasma concentrations using high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) [28], [29], [30], [31], [32], [33]. However, some methods are limited by relatively long analytical run times (10–25 min), which reduce turnover when analysing multiple samples and others have only moderate sensitivity. As new drugs emerge it is important that assay methodology incorporates both new (maraviroc, raltegravir, etravirine and rilpivirine) and existing antiretroviral classes.

A number of previously published HPLC–UV and LC–MS/MS assays have quantified darunavir from human plasma either alone [34] or with other antiretroviral drugs [30], [35], [36], [37]. Raltegravir has been quantified separately using HPLC with fluorescent detection [38] and LC–MS/MS methods [39], [40]. Also, LC–MS/MS assays for maraviroc [19], [41], [42] and etravirine [18], [43], [44] have been briefly described as part of in vivo pharmacokinetic and drug interaction studies but not in the form of a comprehensive method development and validation. A more recent LC–MS/MS method quantified raltegravir, maraviroc, darunavir and etravirine together in a separate assay, but existing agents (with exception of ritonavir) were not included [45], thereby, limiting the assay's utility in a routine setting. Only one bio-analytical method, using solid phase extraction and HPLC coupled to photodiode array detection, has simultaneously quantified raltegravir and etravirine alongside existing PI and NNRTI, however, the chromatographic separation did not allow for the simultaneous quantification of amprenavir and darunavir, which due to similarities in their chemical structure, co-eluted as a single peak [36]. LC–MS/MS methods have been described for the quantification of rilpivirine for pre-clinical studies in dog and rat plasma [46], [47]; however, to date, no bio-analytical assay has measured rilpivirine alongside current antiretrovirals.

Here we describe a simple, fast and sensitive HPLC–MS/MS method for the determination of the commonly used PI [amprenavir (APV), atazanavir (ATV), darunavir (DRV), lopinavir (LPV), ritonavir (RTV), saquinavir (SQV)] and NNRTI [nevirapine (NVP)], as well as recently licensed CCR5 antagonist maraviroc (MVC) and the second generation NNRTI etravirine (ETV) and rilpivirine (RPV). The method was adapted from a previous assay used within our laboratory to measure 7 PI [48]. Validation was conducted based on modified Westgard regulations and FDA international guidelines for bio-analytical assay validation [49], [50]. In particular, we emphasise that the novelty of this analytical methodology is that it includes RPV, which has not been incorporated into past LC–MS/MS methods. As RPV is not yet licensed, determination of RPV plasma concentrations as part of intensive pharmacokinetic and drug interaction studies will be crucial for the future safe and effective use of this agent in the wider HIV-infected population.

Section snippets

Chemicals

APV was kindly donated by Glaxo Wellcome Research and Development (Middlesex, UK), ATV (atazanavir sulphate) by Bristol-Myers Squibb (Hounslow, UK), SQV by Roche Discovery (Welwyn, UK), LPV and RTV by Abbott Laboratories (Chicago, IL, USA) and NVP by Boehringer Ingelheim Pharmaceuticals, Inc. (Berkshire, UK). DRV (darunavir ethanolate), ETV and RPV (rilpivirine hydrochloride) were kindly contributed by Tibotec (Mechelen, Belgium) and MVC was donated by Pfizer (Sandwich, Kent, UK). The internal

Detection and chromatography

Compound specific parameters including the tube lens (V) and the relative collision energy (V) were optimized for a maximum of six transitions per analyte using TSQ Tune Software (Thermo Electron Corporation, Hemel Hempstead, UK) and the two fragment ions with the highest signal-to-noise ratio were selected for quantification. The parent-to-fragment [m/z] transitions, tube lens and relative collision energies used are summarized in Table 2. APV, ATV, DRV, LPV, RTV, SQV, NVP, MVC, ETV and RPV

Discussion

The development and validation of an ultra-sensitive assay to simultaneously quantify 10 antiretroviral compounds, including both existing and new classes, with accuracy and precision, has been described. This bio-analytical method is now being successfully applied to measure antiretroviral plasma concentrations obtained from clinical pharmacokinetic studies.

The calibration curves for all compounds were constructed to reflect therapeutic concentrations observed in HIV-infected patients from

Funding

The authors thank the National Institute of Health Research (NIHR–Department of Health) for infrastructural and project support.

Conflict of interest

S.K. and D.B. have received research grants and travel bursaries from Merck, Bristol-Myers Squibb, GlaxoSmithKline, Pfizer, Abbott, Boehringer Ingelheim and Tibotec.

L.E., V.W., J.T., A.H. and M.S.: none to declare.

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