Journal of Pharmaceutical and Biomedical Analysis 94 (2014) 65–70

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A validated HPLC-MS method for quantification of the CCR5 inhibitor maraviroc in HIV+ human plasma Marco Simiele, Lorena Baietto, Alessio Audino, Mauro Sciandra, Stefano Bonora, Giovanni Di Perri, Antonio D’Avolio ∗ Unit of Infectious Diseases, University of Turin, Department of Medical Sciences, Amedeo di Savoia Hospital, Turin, Italy

a r t i c l e

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Article history: Received 3 August 2013 Received in revised form 20 January 2014 Accepted 22 January 2014 Available online 31 January 2014 Keywords: Maraviroc HPLC-MS HIV Therapeutic drug monitoring Plasma

a b s t r a c t Maraviroc is a CCR5 inhibitor approved in 2007 for treatment of therapy experienced adult patients infected with CCR5-tropic HIV-1 virus. International guidelines for HIV therapy indicate a plasma concentration cutoff of maraviroc for response. We developed and validated a new HPLC-MS method to quantify maraviroc concentrations in human plasma. 6,7-Dimethyl-2,3-di(2-pyridyl)quinoxaline was used as internal standard and added to 100 ␮L of plasma. Samples were then treated with 500 ␮L of acetonitrile for the protein precipitation procedure. An analytical T3 Atlantis column (150 mm × 4.6 mm i.d.) with a particle size of 5 ␮m was used to separate the compounds and ions were detected at m/z 257.5 and 313.3 for maraviroc and quinoxaline respectively. The calibration curve was linear up to 2500 ng/mL. The mean recovery of maraviroc was 89.1%. All validation data results were in accordance to Food and Drug Administration and European Medicines Agency requirements. The HPLC-MS method reported here could be used routinely to monitor plasma concentrations of maraviroc in HIV-infected patients. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Since Highly Active Antiretroviral Therapy (HAART) became widely available in 1996, the number of AIDS-related deaths deeply decreased, and it led to improvement of quality of life to the people living with HIV. Combination therapy includes at least one protease inhibitor (PI) or a non nucleoside transcriptase inhibitor (NNRTI) and/or one or more nucleoside or nucleotide transcriptase inhibitors (NRTIs-NtRTI) and/or a fusion inhibitor (FI). HIV virions might acquire mutations conferring cross resistance to different compounds of each class, especially when the patient does not intake the therapy correctly. Therefore, new therapeutic compounds are needed that able to overcome the extensive class-resistances observed in multi-drug treated patients [1]. New compounds as raltegravir (RGV), the first integrase inhibitor, and rilpivirine (TMC278) the last NNRTI approved, have shown to be active against multidrug-resistant viral strains [2–6]. In contrast to other classes of drugs maraviroc has a target direct of the cell host rather than viral components. Maraviroc (MVC; Celsentri® , Selzentry® ) is a chemokine CCR5 co-receptor antagonist

∗ Corresponding author at: Laboratory of Clinical Pharmacology and Pharmacogenetics, Unit of Infectious Diseases, University of Turin, Department of Medical Sciences, Amedeo di Savoia Hospital, CorsoSvizzera 164, 10149 Turin, Italy. Tel.: +39 011 4393979; fax: +39 011 4393996. E-mail address: [email protected] (A. D’Avolio). 0731-7085/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jpba.2014.01.031

[7–15] that is used at present in experienced R5-tropic HIV-infected patients, for whom previous antiretroviral regimens have failed. MVC is administered orally at a usual dose of 300 mg twice daily, but can be taken at different doses (150 mg or 600 mg) mainly depending on other concomitant drugs [16]. MVC is a substrate for CYP3A4, so potentially can interact with several other antiretrovirals. Therefore, it is administered at 150 mg bid if co-administered with a boosted protease inhibitor, at 300 mg bid if co-administered with nevirapine (NVP) or tipranavir/ritonavir (TPV/r), and at 600 mg bid if co-administered with etravirine(ETV) or efavirenz (EFV) [16,17]. MVC is also a substrate of P-glycoprotein [18]. Ritonavir, which is commonly administered as a booster for protease inhibitors, is a strong inhibitor of both CYP3A4 and P-glycoprotein, and can consequently cause an increase of MVC plasma concentrations. On the basis of the exposure-response analysis from the MOTIVATE studies, an approximate maximal efficacy is achieved at a maraviroc trough concentration (Ctrough ) above 50 ng/ml determined in a population predominantly receiving MVC (150 mg) with boosted protease inhibitors [19]. Although average plasma concentrations (Cavg ) have a stronger correlation with virological efficacy, the measurement of average plasma concentrations in the clinical setting is difficult because of the requirement for multiple samples and the associated cost. Therefore, Ctrough is more commonly used. Accurate measurement of antiretroviral plasma concentrations is crucial for pharmacokinetic/pharmacodynamic analyses, drug–drug interaction studies, and therapeutic drug monitoring (TDM) [20]. The latter is currently considered a useful tool for the

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Table 1 Chromatographic conditions for gradient HPLC analysis of maraviroc: Mobile phase: Solvent A (HPLC grade water + 0.05% formic acid) and Solvent B (HPLC grade acetonitrile + 0.05% formic acid). Temperature of the column was set at 35 ◦ C. Time (min)

Flow (ml/min)

Solvent A %

Solvent B %

0 4 7.5 8.1 15 15.1 20

1 1 1 1 1 1 1

80 60 30 5 5 80 80

20 40 70 95 95 20 20

optimization of antiretroviral therapy in international guidelines [20–23]. An understanding of the pharmacokinetics (PK) of MVC in the clinical setting, pharmacokinetic/pharmacodynamic properties and drug interaction profiles, is still limited due to the recent availability of this compound. Moreover, recent data suggest the involvement of pharmacogenetic factors in the PK of MVC [24]. Therefore, PK studies of MVC are requested in order to define the possible role of TDM in the clinical context. Few methods for MVC quantification in human plasma have been published to date [25–31]. The aim of our study was to develop and validate a cheap, fast and reliable HPLC-MS analytical method for the quantification of MVC in human plasma.

voltage of 35 V for MVC and 312.3 → m/z 313.3 with a cone voltage of 50 V for QX (IS). 2.3. Stock solutions, standards (STD) and quality controls (QC) MVC and QX stock solutions were made in a solution of methanol and HPLC grade water (90:10, v/v) to obtain a final concentration of 1 mg/mL; all stock solutions were then refrigerated at 4 ◦ C until use, within 1 month. A working solution of Internal Standard (IS) was made for every validation analyses with QX (3 ␮g/mL) in methanol and HPLC grade water (50:50, v/v). The highest calibration standard (STD 10) and four quality controls (QCs) were prepared adding a stock solution to blank plasma; the others STDs were prepared by serial dilution from STD 10 (2500 ng/mL) to STD 1 (4.9 ng/mL) with blank plasma, to obtain 10 different spiked concentrations plus a blank sample (STD 0). QC concentrations were 2000 ng/mL, 500 ng/mL, 50 ng/mL and 12.5 ng/mL for QC-High, QCMedium, QC-Low and QC-Low-Low, respectively. The range of MVC concentrations was established in accord with relevant clinical studies [8–12,14–16] in order to include all described concentrations. As for patient samples, STDs and QCs were inactivated for 35 min at 58 ◦ C to provide HIV-free plasma, prior to storing them at −20 ◦ C for no more than three months. They were thawed in the beginning of analysis avoiding more then two freeze–thaw cycles. 2.4. STD, QC and samples preparation

2. Experimental 2.1. Chemicals Maraviroc was purchased from Pfizer Inc. (Groton, CT, USA). Acetonitrile HPLC grade and methanol HPLC grade were purchased from J.T. Baker (Deventer, Holland). HPLC grade water was produced with Milli-DI system coupled with a Synergy 185 system by Millipore (Milan, Italy). Quinoxaline [6,7-dimethyl-2,3-di(2pyridyl)quinoxaline] (QX) and formic acid were obtained from Sigma–Aldrich (Milan, Italy). Blank plasma from healthy donors was kindly supplied by the Blood Bank of Maria Vittoria Hospital (Turin, Italy). 2.2. Chromatographic and MS conditions The HPLC-MS instrument used was a Waters system (Milan, Italy), with binary pump model 1525, AF degasser, 717-plus autosampler, and Micromass ZQ mass detector. LC–MS Empower 2 Pro software (version year 2007, Waters; Milan, Italy) was used. Chromatographic separation was performed at 35 ◦ C using a column oven, and an Atlantis T3 5 ␮m column (150 mm × 4.6 mm i.d.) (Waters, Milan, Italy), protected by a SecurityGuard with C18 (4.0 mm × 3.0 mm i.d.) pre-column (Phenomenex, CA). Runs were performed with a gradient (Table 1), and the mobile phase was composed of Solvent A (HPLC grade water + 0.05% formic acid) and Solvent B (HPLC grade acetonitrile + 0.05% formic acid). A “T” switch tube was applied post-column to introduce only 200 ␮L/min of total flow (1 mL/min) into the MS detector. Optimization of the MS conditions has been performed by direct infusion of reference standards (1 ␮g/mL) in mobile phase (solvent A:solvent B (50:50)) at 10 ␮L/min; at the same time a flow of 1 mL/min of mobile phase (solvent A:solvent B (50:50)) was introduced in the column to perform the infusion in combined mode. MS parameters were been optimized to maximize sensitivity as follows: ESI, positive polarity ionization; capillary voltage, 3.5 kV; source temperature, 110 ◦ C; desolvation temperature, 350 ◦ C; nitrogen desolvation flow, 400 l/h; nitrogen cone flow, 50 L/h. The ion transitions and cone voltages were: 513.3 → m/z 257.5 with a cone

Patients receiving standard dosing of MVC (150 mg, 300 mg or 600 mg) underwent blood sampling for the measurement of plasma drug concentrations. The study was conducted in compliance with the Declaration of Helsinki and with the local Review Board regulations; all patients gave written informed consent according to the local ethic committee standards. Blood samples were collected in lithium heparin tubes (7 mL), and plasma was obtained after centrifugation at 1400 × g for 10 min at +4 ◦ C (Jouan Centrifuge, Model BR4i; Saint-Herblain, France) and then underwent heat inactivation as described above. A protein precipitation procedure was performed for extraction of maraviroc from plasma. Fifty ␮L of IS working solution was added to 100 ␮L of sample (STDs, QCs or patient samples) in a PTFE microfuge tube, then 500 ␮L of protein precipitation solution (acetonitrile 100%) was added. The tube was vortexed for 10 s and then centrifuged at 13,000 × g for 10 min at 4 ◦ C. Supernatant was transferred into glass tubes and treated by vortex vacuum evaporation to dryness at 60 ◦ C and then reconstituted with 125 ␮L of HPLC grade water and acetonitrile solution (70:30, v/v). Fifty ␮L were injected into the HPLC column. All QCs samples were performed in duplicate during validation sessions, and all procedure steps were carried out at room temperature. During every routine analysis session, the patients samples have been processed together with STDs from 0 to 10 (STD0 was a blank plasma) and QCs (High, Medium, Low, Low-Low). Before the injection of these samples a series of blank (water and acetonitrile solution (70:30, v/v)) was injected for the conditioning of the HPLC-MS system. A fresh mix with the analytes (10 ␮g/mL in water:acetonitrile solution (70:30, v/v)) was also injected to verify the retention times. Concentrations data of patients samples were considered reliable if standard deviation of all QCs from nominal values were below 15%. 2.5. Specificity and selectivity Interference from endogenous compounds was investigated by analysis of six different blank plasma samples. Potential interference by antiretroviral drugs concomitantly administered

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to the patients was also evaluated by spiking blank plasma. These included zidovudine (AZT), didanosine (ddI), stavudine (d4T), lamivudine (3TC), abacavir (ABV), tenofovir (TDF), emtricitabine (FTC), and enfuvirtide (T-20). Other possible concomitant drugs were also investigated, including amodiaquine, desacetylamodiaquine, amoxicillin, caspofungin, ceftazidime, ciprofloxacin, clavulanic acid, ethambutol, furosemide, insulin, isoniazid, levofloxacin, nimesulide, omeprazole, pravastatin, and ribavirin. An “interfering drug” was considered to be a molecule that exhibits a retention time within 0.3 min of the analyte, and with the potential capability to cause ion suppression. 2.6. Matrix effect The “matrix effect” was investigated on six lots of blank plasma from individual donors, as requested by guidelines [32,33]. Peak areas from blank extracts spiked with all analytes at three QC concentrations were compared with peak areas from standard solutions (water and acetonitrile, 70:30, v/v) spiked with analytes in the some way, as described by Taylor [34]. The possible “matrix effect” was calculated, as deviation %, by comparing the peak area obtained from the plasma extract with the peak area obtained from the standard solution. 2.7. Accuracy, precision, limit of quantification and limit of detection

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Table 2 Accuracy and precision data for validation of maraviroc LC–MS method. Accuracy (%), intraday, and interday precision of the four quality control (QC), expressed as percent relative standard deviation (RSD%). MVC

QC H QC M QC L QC LL

Accuracy %

0.37% 1.03% 6.55% 8.47%

Precision (RSD%) Intra-day

Inter-day

2.37% 3.48% 2.01% 6.68%

3.55% 7.51% 7.35% 9.24%

standing for 24 h at room temperature, by comparing the peak areas of drugs in the two sample injections. The stability of IS was not performed because it was extensively evaluated and used in similar condition in many previous articles [26,36–47]. 2.10. Carry over Carry-over was investigated in triplicate by injecting extracted blank plasma samples immediately after samples containing target analytes at ten-fold concentration of STD 10. A value ≤20% of the lower limit of quantification (LLOQ) and a value ≤5% for IS were considered as absence of carry over. 3. Results

Intra-day and inter-day accuracy and precision were determined by assaying ten spiked plasma samples at four different concentrations (QCs). Accuracy was calculated as the percent deviation from the nominal concentration. Inter-day and intra-day precision was expressed as the standard deviation at each QC concentration. Each calibration curve was obtained using ten calibration points, ranging from 4.9 to 2500 ng/mL. Calibration curves were created by plotting the peak area ratios of each drugs relative to the IS against the various drugs concentrations in the spiked plasma standards. A 1/X weighted quadratic regression was used for all curves in order to obtain the best fit for all calibration points and particularly for low concentration points, close to theoretical range and cut-off of activity (50 ng/mL) [35] of Ctrough concentration of maraviroc. The limit of detection (LOD) in plasma was defined as the lowest concentration that yields a signal-to-noise ratio of at least 3/1. The lowest concentration levels that could be determined with a percent deviation from the nominal concentration and relative standard deviation T), and its MVC Ctrough has been described [24]. The clinical value of MVC TDM has yet to be fully evaluated, and a method for the quantification of MVC will be an essential tool for PK studies and management of special populations. For this purpose, a novel analytical method for quantification of MVC in plasma of HIV-infected patients was developed using protein precipitation extraction, and liquid chromatography with mass detection. Other methods, for measuring MVC using liquid chromatography coupled with UV or mass spectrometry, have been reported previously [25–31,37]. Our assay extraction is simpler than the solid phase extraction (SPE) described by Notari et al. [30] and the isocratic run described, without a wash step, appears to be unsuitable in clinical routine practice. Moreover mass spectrometry detection is more sensitive than UV-detection, and facilitates the measurement of analytes at very low concentrations. Other authors used very expensive instrumentation like HPLC-MS/MS, and other previously described methods are limited to a narrow concentration range for MVC that appears inadequate for quantifying the Cmax of this drug [25,27–29]. Only one known method uses our same type of instrumentation (HPLC coupled with mass spectrometry detection) to quantify MVC in human plasma [31]. But this other method, with respect our procedure, has a longer chromatographic run time (40 min vs 20 min) and requires a five-fold higher volume of plasma (500 vs 100 ␮L). While the run time is only 20 min, it still ensures a good separation of drugs and avoids potential matrix effects. Our method has been fully validated and has been proven to be precise and accurate. Calibration curves cover a wide range of MVC concentrations that correspond to the lowest and highest values reported in clinical settings and PK studies [8–16]. A quadratic through zero regression was chosen respect to a linear regression due to better signal-response, because the highest STDs of the curves were slightly in saturation and the response was not a linear function of the concentration of the analyte. The method was optimized to achieve low levels of quantification (see Section 3.2), as requested from HIV guidelines [19,35]. The evaporation step to concentrate the analytes, after protein precipitation with acetonitrile, allows to obtain a high signal also for concentration of maraviroc very low. All analytes were adequately retained; ensuring a good capacity factor.

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Reliability, cost, simply to perform, and reproducibility are key points for the measurement of drug plasma concentrations. Our assay extraction is cheaper than SPE extraction. Likewise, our instrumentation is very inexpensive with regard to liquid chromatography coupled with tandem-mass spectrometry; even though it is more expensive than HPLC-UV instruments [26,30]. The use of QX as IS, an inexpensive and easy to purchase substance, improved reliability and reproducibility of our assay. QX has been used by our laboratory in many other works [26,36–47,49–51] and it was fully used for maraviroc in a previous UV method [26], moreover it showed a chemical and physical behavior similar to the different class of antiretroviral drugs. Relative error, in intraday and interday precision (Table 2) demonstrate acceptable the accuracy and precision of our procedure. Absence of matrix effects at the retention times of the analytes of interest allowed an accurate measurement of MVC plasma concentrations, even in samples from patients administered with several concomitant drugs. 5. Conclusion This method has been developed and validated following FDA and EMA guidelines [32,33] and according to EN UNI ISO 9001:2008 certification planning rules of our laboratory [52,53]. TDM of MVC could be a useful tool for clinicians to verify correct plasma exposure in drug-drug interactions or special populations. This method, based on a simple precipitation extraction and HPLC-MS instrumentation, for quantify plasma concentration of MVC is accurate and reproducible and can be used in a wide range of PK, clinical studies and routinely in HIV infected patients. Conflicts of interest The authors disclose no conflicts. Funding This study was not supported. References [1] B.O. Taiwo, C.B. Hicks, Darunavir: an overview of an HIV protease inhibitor developed to overcome drug resistance, AIDS Read 17 (2007) 151–156, 159–161. [2] C.J. Cohen, J.M. Molina, I. Cassetti, P. Chetchotisakd, A. Lazzarin, C. Orkin, F. Rhame, H.J. Stellbrink, T. Li, H. Crauwels, L. Rimsky, S. Vanveggel, P. Williams, K. Boven, Week 96 efficacy and safety of rilpivirine in treatment-naive HIV-1 patients in two Phase III randomized trials, AIDS 27 (2012) 939–950. [3] C.A. Hughes, L. Robinson, A. Tseng, R.D. MacArthur., New antiretroviral drugs: a review of the efficacy, safety, pharmacokinetics, and resistance profile of tipranavir, darunavir, etravirine, rilpivirine, maraviroc, and raltegravir, Expert Opin. Pharmacother. 10 (2009) 2445–2466. [4] A. Wilkin, A.L. Pozniak, J. Morales-Ramirez, S.H. Lupo, M. Santoscoy, B. Grinsztejn, K. Ruxrungtham, L.T. Rimsky, S. Vanveggel, K. Boven, Long-term efficacy, safety, and tolerability of rilpivirine (RPV, TMC278) in HIV type 1-infected antiretroviral-naive patients: week 192 results from a phase IIb randomized trial, AIDS Res. Hum. Retroviruses 28 (2011) 437–446. [5] T. Correll, O.M. Klibanov, Integrase inhibitors: a new treatment option for patients with human immunodeficiency virus infection, Pharmacotherapy 28 (2008) 90–101. [6] M. Markowitz, B.Y. Nguyen, E. Gotuzzo, F. Mendo, W. Ratanasuwan, C. Kovacs, G. Prada, J.O. Morales-Ramirez, C.S. Crumpacker, R.D. Isaacs, L.R. Gilde, H. Wan, M.D. Miller, L.A. Wenning, H. Teppler, Rapid and durable antiretroviral effect of the HIV-1 integrase inhibitor raltegravir as part of combination therapy in treatment-naive patients with HIV-1 infection: results of a 48-week controlled study, J. Acquir. Immune Defic. Syndr. 46 (2007) 125–133. [7] U.F. Bredeek, M.J. Harbour, CCR5 antagonists in the treatment of treatmentnaive patients infected with CCR5 tropic HIV-1, Eur. J. Med. Res. 12 (2007) 427–434. [8] H. Dhami, C.E. Fritz, B. Gankin, S.H. Pak, W. Yi, M.J. Seya, R.B. Raffa, S. Nagar, The chemokine system and CCR5 antagonists: potential in HIV treatment and other novel therapies, J. Clin. Pharm. Ther. 34 (2009) 147–160. [9] J.A. Este, A. Telenti, HIV entry inhibitors, Lancet 370 (2007) 81–88.

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