Accepted Manuscript Title: A liquid chromatography–tandem mass spectrometry assay for quantification of rilpivirine and dolutegravir in human plasma Author: M. Gr´egoire G. Deslandes C. Renaud R. Bouqui´e C. Allavena F. Raffi P. Jolliet E. Dailly PII: DOI: Reference:
S1570-0232(14)00573-X http://dx.doi.org/doi:10.1016/j.jchromb.2014.09.006 CHROMB 19111
To appear in:
Journal of Chromatography B
Received date: Revised date: Accepted date:
1-4-2014 27-8-2014 7-9-2014
Please cite this article as: M. Gr´egoire, G. Deslandes, C. Renaud, R. Bouqui´e, C. Allavena, F. Raffi, P. Jolliet, E. Dailly, A liquid chromatographyndashtandem mass spectrometry assay for quantification of rilpivirine and dolutegravir in human plasma, Journal of Chromatography B (2014), http://dx.doi.org/10.1016/j.jchromb.2014.09.006 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Highlights
A method is proposed to quantify novel drugs(rilpivirine, dolutegravir) in plasma
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This assay is based on liquid chromatography–tandem mass spectrometry
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This method is sensitive, accurate, precise
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This method can be used for therapeutic drug monitoring in HIV infected patients
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A liquid chromatography–tandem mass spectrometry assay for
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quantification of rilpivirine and dolutegravir in human plasma.
Clinical Pharmacology Department, CHU de Nantes, Nantes, France
an
(2)
, F Raffi(3), P
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Jolliet(1,2) , E Dailly(1,4)§
(1)
(3)
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M Grégoire(1), G Deslandes(1), C Renaud(1), R Bouquié(1,2), C Allavena
EA 4275 Biostatistique, Recherche Clinique et Mesures Subjectives en Santé, Faculté de
M
Médecine–Pharmacie, Université de Nantes, France
Infectious Diseases Department, CHU de Nantes, Nantes, France
(4)
EA 3826 Thérapeutiques Cliniques et Expérimentales des Infections, Faculté de Médecine–
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(3)
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Pharmacie, Université de Nantes, France
§ corresponding author : Eric Dailly, Laboratoire de Pharmacologie clinique, Hôtel Dieu, 9 Quai Moncousu, 44093 Nantes Cedex, France email:
[email protected] , Tel. : 33 2 40 08 40 95, Fax.: 33 2 40 08 39 96
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Abstract A liquid chromatography–tandem mass spectrometry assay requiring a 100 µL aliquot of human plasma for simultaneous determination of rilpivirine, a second generation non-
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nucleoside reverse transcriptase inhibitors of HIV and dolutegravir, a novel integrase stand transfer inhibitors of HIV concentrations has been developed. Sample pre-treatment is limited
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to protein precipitation with a mixture of methanol and zinc sulfate. After centrifugation the supernatant is injected in the chromatographic system, which consists of on-line solid phase
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extraction followed by separation on a phenyl-hexyl column. This 2.5 minutes method, with
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its simple sample preparation provides sensitive (the limit of quantitation is 25 ng/mL for each compound), accurate and precise (the intra-day and inter-day imprecision and inaccuracy
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are lower than 15%) quantification of the plasma concentration of these drugs and can be used
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for therapeutic drug monitoring in patients infected with HIV.
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Keywords: mass spectrometry, dolutegravir, rilpivirine
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1. Introduction Several assays were proposed to simultaneously measure plasma concentrations of antiretroviral drugs in HIV infected patients by liquid chromatography–tandem mass
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spectrometry assay [1-6]. These methods allow to determine plasma concentrations of antiretroviral agents including protease inhibitors of HIV and the non-nucleoside reverse
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transcriptase inhibitors of HIV (NNRTIs). Most of these methods do not include rilpivirine, a second generation NNRTI. Four assays based on the chromatography coupled with mass
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spectrometry [1, 7-9] have been reported in the literature to measure plasma concentration of
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rilpivirine, the last licensed NNRTI. Dolutegravir is a promising antiretroviral drug belonging to the integrase stand transfer inhibitor of HIV (INSTI) class [10]. Two assays were reported
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to determine plasma concentration of dolutegravir [11,12]. The assay employed by Castellino et al was not enough precisely described to be operated by a laboratory in order to measure
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dolutegravir concentrations [12]. Currently, no assay allows the simultaneous measurement of
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the plasma concentration of dolutegravir and rilpivirine. This work presents a liquid chromatography–tandem mass spectrometry method with a simple sample pre-treatment
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which can be used for the simultaneous quantification of the novel antiretroviral agents, rilpivirine and dolutegravir, in the plasma of HIV infected patients.
2. Material and methods
2.1. Chemicals:
Methanol, zinc sulfate, acetic acid and ammonium acetate were purchased from VWR International (Fontenay-sous-bois, France). The following antiretroviral agents are listed in Table 1, each molecule is measured using a deuterated internal standard: rilpivirine and
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dolutegravir (Alsachim, Strasbourg, France), dolutegravir-13C d5 (Alsachim, Strasbourg, France) and rilpivirine-d6 (Toronto research chemicals inc., North York, Canada). The
2.2. Preparation of standards:
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chemical structures of these compounds are presented in fig. 1.
Stock solutions were prepared as follows: rilpivirine, rilpivirine-d6, dolutegravir, and
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dolutegravir-13C d5 at a concentration of 1000 mg/L (methanol)
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For the calibration samples, a working solution was prepared by diluting the stock solution in methanol to a final concentration of 20 µg/mL for each agent. Seven-point calibration curves
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and blanks were prepared for the calibration of each analyte by diluting known volumes of the working solution in methanol to obtain a final volume of 1000 µL. Fifty microliters of these
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solutions were mixed with 450 µL of drug-free human plasma (collected with K3EDTA as
described in section 2.3.
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anticoagulant) to prepare the calibration samples. The calibrators were further treated as
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For the quality controls, 3 different levels solutions (high, medium and low) were prepared by diluting the working solution in methanol to respective-final concentrations of 8, 1.5 and 0.3 µg/mL for each drug. Fifty microliters of these solutions were mixed with 450 µL of drugfree human plasma to prepare quality controls for high, medium and low levels. The quality controls were further treated as described in section 2.3. The final plasma concentrations of the low, medium and high controls were 30, 150 and 800 ng/mL each drug.
2.3. Sample preparation:
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The blood samples with K3EDTA as anticoagulant were centrifuged at 1800 g for 10 min at + 4°C. One hundred microliters of plasma were treated with 200 µL of precipitation reagent [methanol/0.2M zinc sulfate (80/20 v/v)] including 0.4 mg/L of rilpivirine-d6 and
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dolutegravir-13C d5, used as internal standards, in a 1.5 mL conical plastic Eppendorf test tube. Samples were immediately vortexed and then centrifuged at 13 000 g for 15 min at
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+4°C. Then, 150 µL of supernatant were transferred into a sample vial with a 200 µL volume
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micro-insert and 25 µL were injected into the chromatographic system.
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2.4. Calibration curves:
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Seven-point calibration curves (25, 50, 100, 200, 400, 1000, 2000 ng/mL for each agent) were calculated (area ratio using the internal standard versus nominal concentration) and fitted
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either by a linear regression. The concentrations were back-calculated and the model with the
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2.5. Instruments:
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lowest deviation between the calculated and nominal concentrations was retained.
The instrument set up is shown in fig.2. The chromatographic system consisted of Agilent (Palo Alto, CA, USA) 1200 Series components including a binary pump, isocratic pump, column oven, 2 ten-port switching valves and an autosampler. The hardware configuration included an Applied Biosystems(Foster City, CA, USA) API 3200 equipped with a TurboIonspray source. On-line extraction was performed using a perfusion column (POROS R1/20, 2.1 mm x 30 mm, Applied Biosystems, Foster City, CA, USA). The HPLC column was a short phenyl-
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hexyl column (Phenomenex Luna 5µm Phenyl-Hexyl, 2 mm x 50 mm, Aschaffenburg, Germany). Data analysis was performed using the Analyst 1.4.2 software package (Applied Biosystems,
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2.6. On-line solid phase extraction (SPE) and chromatographic separation:
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Foster City, CA, USA).
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The chromatographic conditions are presented in fig.2 and the pumps and valves
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configurations are summarized in Table 2. During the first-dimension chromatography the binary pump supplied 90% of eluent A (100% water, 10 mM ammonium acetate, 0.1% acetic
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acid) and 10% of eluent B (100% acetonitrile) for dolutegravir and rilpivirine binding on a Poros column and to elute the most hydrophilic molecules at a flow rate of 3000 µL/min for
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0.45 min (0.05-0.5 min) (Position A of the valve 1). At zero time, a 25 µL aliquot of
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supernatant from each prepared sample was injected. Simultaneously eluent C (66% acetonitrile, 34% water, 10 mM ammonium acetate, 0.1% acetic acid) was introduced to the
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HPLC- tandem mass spectrometry detection at a flow rate of 700 µL/min, supplied by the isocratic pump. The SPE elution and analytes transfer to the HPLC column were performed by switching valve 1 after 0.5 min (Position B of the valve 1). The configuration developed offered complete SPE elution in flush mode and analytes transfer to the HPLC column and tandem mass spectrometer by means of eluent C at 700 µL/min, which was well compatible with the TurboIonspray source. Eluent C was not able to separate the analytes completely using the phenyl-hexyl HPLC column but fortunately the highly selective tandem mass spectrometry detector in multiple reaction monitoring (MRM, precursor/product ion) detection mode was suitable for simultaneous detection of more than one analyte without retention time differences. The retention times are shown in Table 1. After 1.5 min, valve 1
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was switched and the binary pump supplied eluent B to wash the Poros column at a flow rate of 3000 µL/min (Position A of the valve 1). After 1.7 min, the binary pump stopped supplying 100% of eluent B for 90% of eluent A and 10% of eluent B for 0.5 min at a flow rate of 3000
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µL/min to re-equilibrate the Poros column. A total of analysis time of 2.5 min was obtained
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for all molecules including the internal standard.
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2.7 Tandem mass spectrometry analysis:
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One positive ion mode MRM transition was used for each antiretroviral drug and their deuterated internal standard. All transitions are listed in Table 1 including declustering
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potential (DP), entrance potential (EP), cell entrance potential (CEP), collision energy (CE) and cell exit potential (CXP). The dwell time was set to 50 ms for each MRM transition. The
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TurboIonspray interface settings and collision gas pressure were manually optimized (Ion
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spray voltage: 5500 V, temperature: 400°C, collision gas: 5.0 psi, curtain gas: 25.0 psi). Tandem- mass spectrometry was performed with nitrogen as collision gas. The procedure was
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completely automatic and performed using the Analyst 1.4.2 software package.
2.8. Analytical method validation
2.8.1 Inaccuracy, imprecision and limits of quantitation and detection Inaccuracy and imprecision were evaluated by analyzing quality control samples at low, medium and high concentrations on 5 different days. For intra-day validation, 5 samples of each quality control were analyzed on the same day. For inter-day validation, concentrations of the quality control samples were determined on 5 separate days [13]. Inaccuracy is defined as the percentage of deviation from the nominal level and imprecision as the coefficient of
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variation (%CV) within a single run (intra-assay) and between different assays (inter-assay). The imprecision and the inaccuracy should not exceed 15% except for the lower limit of quantitation for which 20% deviation was allowed. Signal/noise ratio should be greater than
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than 3 for the limit of detection.
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2.8.2 Matrix effects
The matrix effects were investigated according to the European Medicines Agency guidelines
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[14]. In the case of on-line sample preparation, the variability of the response from lot to lot
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should be assessed by analysing at least 6 lots of plasma matrix, spiked at a low and at a high level of concentration (30 and 800 ng/mL). The overall %CV calculated for the concentration
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should not be greater than 15 %.
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2.8.3 Carry-over effects
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Carry-over effects were assessed by testing successively 3 high levels of concentration plasmas (H1, H2 and H3) and 3 low levels of concentration plasmas (L1, L2 and L3)
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(respectively 800 and 30 ng/mL) according to the French committee of accreditation recommendations [15]. This step was reproduced 3 times. There should not be found any statistical difference between L1 and L3 averages using a Student test.
2.8.4 Recovery
Recovery was assessed by testing 3 spiked samples (30, 150 and 800 ng/mL) directly injected on the column of chromatography without protein precipitation and any solid phase extraction and compared by 6 different sources of plasma with 3 spiked samples for each plasma (30, 150 and 800 ng/mL) injected after protein precipitation followed or not by one line extraction. The recovery is the mean value of the ratio done for each of the 18 samples tested.
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2.8.5 Stability
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Stability of the analytes (aliquots of each level of quality control samples) was investigated in plasma stored at –20°C, after 3 freeze and thaw cycles, and thawed at room temperature and
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kept at this temperature for 4 hours, in plasma stored at -20°C for 3 months and in plasma stored at + 60°C for one hour (viral inactivation).Otherwise, stability was assessed in plasma
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stored at +4°C for 3 days. The mean concentration at each level should be within ±15% of the
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nominal concentration [14]. The stability of the stock solutions of drugs and internal standard
2.8.6 Glucuronide metabolite separation
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stored at –20°C was also evaluated.
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The presence of the glucuronide conjugate was investigated in the plasma of 8 patients treated
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with dolutegravir. To ensure that glucuronide metabolite of dolutegravir is well eliminated during the first-dimension chromatography, we assessed the dolutegravir transition (420.0-
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277.1) and the glucuronide transition (596.0-420.0) in standard conditions (10% of acetonitrile and 90% of water) and in more hydrophilic conditions (100% of water). Moreover, we assessed these two transitions before and after beta-glucuronidase sample pretreatment in standard conditions.
3. Results: The calibration curves were satisfactorily fitted by linear regression (1/x weighting). Deviations of the back calculated concentrations were within 85% and 115 % of the nominal
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concentrations (80% and 120 % for the lower level which is the limit of quantitation) and the correlation coefficients for all calibration curves were above 0.990. The intra-day (iad) and inter-day (ied) imprecision and inaccuracy were less than 15% for quality control samples.
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These results are presented in Table 3. The limits of quantitation and detection were respectively 25 and 6.25 ng/mL for each one of these components. Fig. 3 shows the
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chromatograms for the lowest calibration sample. Carry-over effects proved to be moderate and acceptable and any statistical difference between L1 and L3 could not be found using a
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Student test. The exact carry-over values were respectively -0.049% and +0.087% for
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rilpivirine and dolutegravir. No critical matrix effects have been observed, the overall CV calculated for the concentration were not greater than 15 %. The stability of the frozen plasma
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samples and stock solutions for a period of at least 3 months under our storage conditions and the stability of the analytes in plasma after 3 freeze and thaw cycles and thawed at room
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temperature and kept at this temperature 4 hours were checked as the variation for each drug
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was within the ±15% of the nominal concentrations. Moreover, stability (Table E1 in Online Supplemental Material) was guaranteed in plasma stored at +4°C for 3 days. Besides, stability
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at viral-inactivation temperature of 60°C for one hour was demonstrated. As shown in Fig. 4, (A) when the solid phase extraction was realized with 0% of acetonitrile and 100% of water, glucuronide metabolite was retained by the Poros column and chromatographed just before the dolutegravir. A metabolite-ion source dissociation was observed in the 8 patients tested and logically revealed by a little peak at the glucuronide retention time for the dolutegravir transition. (B) No glucuronide metabolite was observed after beta-glucuronidase sample pretreatment or (C) in standard conditions (10% of acetonitrile, 90% of water) and no difference was observed between peak area before and after beta-glucuronidase sample pretreatment in standard conditions. The respective total recoveries for dolutegravir and rilpivirine were 14% and 83%. These results are presented in Table 4. Fig. 5 shows the
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chromatogram of an HIV-infected subject who was treated with rilpivirine at a dosage of 25 mg once-daily in combination tenofovir/emtracitabine 245mg/200mg once-daily (A) and the chromatogram of an HIV-infected subject who was treated with dolutegravir at a dosage of
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Respective drug concentrations were 165 ng/mL and 1330 ng/mL.
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50mg once-daily in combination abacavir/lamivudine 600mg/300mg once-daily (B).
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4. Discussion
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This method allows accurate and precise determination of plasma concentrations of two recently licensed antiretroviral agents including the novel INSTI dolutegravir. The limits of
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quantification are consistent with trough plasma concentrations of antiretroviral agents [16]. The MRM transitions (m/z) used (dolutegravir 420.1-277.1; rilpivirine 367.1-195.2) are
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consistent with the transitions retained by other authors (dolutegravir 420-277 [12]; rilpivirine
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367.2-194.9 [1]; rilpivirine 367.2-195.1 [8]). Contrary to other assays, our method included the one-line solid phase extraction before liquid chromatography–tandem mass spectrometry
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to reduce the matrix effect interferences. Compared with a direct liquid chromatography– tandem mass spectrometry assay, the one-line solid phase extraction before liquid chromatography–tandem mass spectrometry prevents the introduction of endogenous compounds into the mass spectrometer to limit the matrix effects as previously demonstrated [17]. Moreover, the use of deuterated-internal standards contrary to other assays developed to quantify rilpivirine level plasma [1,8] reduces also matrix effect interferences. Studies investigating the metabolism of dolutegravir indicate that the primary route of metabolism is glucuronidation via UDP-glucuronosyl transferase 1A1 (UGT1A1) [12]. Especially in case of severe renal impairment, elevated dolutegravir glucuronide concentrations have been described, so, it was important ascertain that dolutegravir and its glucuronide conjugate were
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not analysed together after ion-source dissociation [19]. To be sure to not be disturbed by these metabolites after their ion-source dissociation from glucuronide to parent drug, as observed with other INSTIs [7,18], samples of patients treated with dolutegravir were
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assessed in different conditions with glucuronide transition and after or not betaglucuronidase sample pretreatment. According to other methods [11,12], the analysis in
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patients samples did not reveal the presence of dolutegravir glucuronide. In fact dolutegravir glucuronide is not retained by the Poros column in the solid-phase extraction conditions.
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Conversely, in more hydrophylic conditions, dolutegravir glucuronide is retained and found
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with a retention time slightly shorter than dolutegravir. These last results allow us to maintain that dolutegravir glucuronide conjugate do not interfere with its parent drug measurement in
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this method. However, these chromatographic conditions which avoid the interference between dolutegravir and its glucuronide affect the recovery. A poor recovery was observed
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for dolutegravir which is attributed to the less effective trapping of dolutegravir by Poros
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column under the conditions used. Nevertheless, dolutegravir can be detected at low plasma concentration and the limit of quantitation of dolutegravir is clinically relevant with plasma
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trough level . According to the FDA guidelines, the recovery of the analyte need not to be 100%, but the extent of recovery of an analyte should be consistent, precise, and reproducible [13]. The coefficient of variation accessed on 6 different sources of plasma was enough low (7%) to estimate that the recovery was reproductible.
5. Conclusions
To our knowledge, this assay is the only one developed to measure simultaneously dolutegravir and rilpivirine concentrations in plasma using on line extraction LC/MS-MS and deuterated-internal standard to prevent matrix effect. The results of our validation indicate
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that this method is sensitive, accurate, precise and can be used for therapeutic drug monitoring
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of these novel antiretroviral agents in patients infected with HIV.
References
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[1] L Else, V Watson, J Tjia, A Hughes, M Siccardi, S Khoo, et al. Validation of a rapid and sensitive high-performance liquid chromatography-tandem mass spectrometry (HPLC-
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[3] B.H. Jung, N.L. Rezk, A.S. Bridges, A.H. Corbett, A.D. Kashuba. Simultaneous determination of 17 antiretroviral drugs in human plasma for quantitative analysis with liquid chromatography-tandem mass spectrometry. Biomed. Chromatogr.21 (2007) 10951104.
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Figure captions
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fig. 1 : Chemical structures of antiretroviral drugs and internal standards
fig. 2 Column switching system. (I), system configuration for loading, analysis, rinsing and
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re-equilibration. (II), system configuration for transfer
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fig. 3 Chromatograms of the lowest calibration sample (25 ng/mL for rilpivirine and
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dolutegravir)
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fig. 4 Chomatograms of dolutegravir and its glucuronide conjugate in hydrophilic on-line solid phase extraction conditions (100% of water) (A), after beta-glucuronidase sample
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pretreatment in hydrophilic on-line solid phase extraction conditions (B) and in standard on-
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line solid phase extraction conditions (10% of acetonitrile and 90% of water (C).
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fig. 5 Chromatograms of rilpivirine (A) (obtained from a man infected with HIV who was receiving rilpivirine 25 mg once-daily in combination tenofovir/emtracitabine 245mg/200mg once-daily, the plasma concentration of rilpivirine was 165 ng/mL) and dolutegravir (B) (obtained from a man infected with HIV who was receiving dolutegravir 50 mg once a day and abacavir/lamivudine 600mg/300mg once a day., the plasma concentration of dolutegravir was 1330 ng/mL)
Table captions
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Table 1: Antiretroviral drugs and MRM transitions used for detection, declustering potential (DP), entrance potential (EP), cell entrance potential (CEP), collision energy (CE) and cell
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exit potential (CXP) for API 3200, and retention times for the phenyl-hexyl HPLC column
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Table 2: Configuration for the pumps and valve
Table 3: Inter-day (ied; n =5) and intra-day (iad; n =5) precision and accuracy for
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antiretroviral drugs : Inaccuracy is defined as the percentage deviation from the nominal level
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and the imprecision as the coefficient of variation. The units for plasma concentration are
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ng/mL
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Table 4: Recoveries after protein precipitation followed or not by one line extraction.
19
Page 19 of 30
Table 1
retention time
Transition
(min)
DP
EP
10.5
367.1-195.2
1.07
96
rilpivirine-d6
373.1-201.2
1.07
96
dolutegravir
420.1-277.1
0.97
dolutegravir-13C d5
426.1-277.1
0.97
dolutegravir-glucuronide
596.0-420.0
CE
CXP
18
51
6
18
51
6
12
18
37
8
61
12
18
37
8
12
18
20
8
an
61
30
Ac ce p
te
d
M
-
10.5
us
rilpivirine
cr
(m/z)
CEP
ip t
MRM
20
Page 20 of 30
Table 2 Time
Binary pump (eluents A and B)
Isocratic pump (eluent C)
10-port switching
B (%)
Flow rate eluent (µL/min)
0.0
500
90
10
700
0.05
3000
90
10
700
0.5
3000
90
10
700
B
0.55
500
90
10
700
B
1.10
500
90
10
700
B
1.2
3000
90
10
700
B
1.3
3000
0
100
700
B
1.5
3000
0
100
700
A
1.8
3000
90
10
700
A
2.2
3000
90
10
700
A
2.5
500
10
700
A
cr
us
A
A
Ac ce p
te
90
Position
ip t
A (%)
M
Flow rate eluent (µL/min)
an
valve 1
d
(min)
21
Page 21 of 30
Table 3 High level (800 ng/mL)
average measured plasma level
average measured plasma level
average measured plasma level
(imprecision %-inaccuracy%)
(imprecision %-inaccuracy%)
(imprecision %-inaccuracy%)
ied
iad
ied
31.24
27.59
154.00
154.50
(8.35-4.13)
(13.27-8.04)
(3.41-2.67)
30.96
25.53
(2.68-3.20)
(14.93-14.89)
iad
ied
ip t
iad
845.75
(7.17-3.00)
(1.67-8.12)
(6.17-5.72)
157.00
156.75
897.80
833.95
(3.37-4.67)
(2.82-4.50)
(1.65-12.22)
(6.34-4.24)
d
M
an
us
cr
865.00
te
dolutegravir
Medium level (150 ng/mL)
Ac ce p
rilpivirine
Low level (30 ng/mL)
22
Page 22 of 30
Table 4
Protein precipitation followed Protein precipitation recovery by on-line extraction recovery
ip t
(%)
(%) CV (%)
cr
CV (%) 93
14
us
dolutegravir 10
7
103
83
an
rilpivirine
8
Ac ce p
te
d
M
11
23
Page 23 of 30
Figure
ip t
13
dolutegravir
rilpivirine-d6
Ac
ce pt
rilpivirine
ed
M
an
us
cr
dolutegravir-13C d5
Page 24 of 30
Figure
fig.2 Binary pump
ip t
Valve 2
Valve 1 (position A)
Waste
Poros
us
cr
Waste
Waste
an
Isocratic pump
MS/MS detector
M
I
HPLC column
ed
Binary pump
Valve 2
Waste
ce pt
Valve 1 (position B)
Poros
Ac
Waste
HPLC column
Isocratic pump
II
MS/MS detector
Page 25 of 30
Figure
cr
ce pt
ed
M
an
us
rilpivirine MRM transition (m/z) : 367.1-195.2
ip t
Fig 3
Ac
dolutegravir MRM transition (m/z) : 420.0-277.1
Page 26 of 30
cr
ip t
Figure
Fig. 4
dolutegravir glucuronide MRM transition (m/z) : 596.0-420.0
ep te
d
M
an
us
dolutegravir MRM transition (m/z) : 420.1-277.1
A
Ac c
0.92
Fig. 5b
Page 27 of 30
ip t cr us
dolutegravir glucuronide MRM transition (m/z) : 596.0-420.0
ep te
d
M
an
dolutegravir MRM transition (m/z) : 420.1-277.1
Ac c
B
Page 28 of 30
ip t cr us an
dolutegravir glucuronide MRM transition (m/z) : 596.0-420.0
ep te
d
M
dolutegravir MRM transition (m/z) : 420.1-277.1
Ac c
C
Page 29 of 30
Figure
Fig. 5
A
M
an
us
cr
ip t
rilpivirine MRM transition (m/z) : 367.1-195.2
ce pt
ed
B
Ac
dolutegravir MRM transition (m/z) : 420.1-277.1
Page 30 of 30