Simultaneous quantification of emtricitabine and tenofovir nucleotides in peripheral blood mononuclear cells using weak anion-exchange liquid chromatography coupled with tandem mass spectrometry

doi:10.1016/j.jchromb.2010.01.002

Journal of Chromatography B

Volume 878, Issues 7–8, 1 March 2010, Pages 621-627

https://doi.org/10.1016/j.jchromb.2010.01.002 Get rights and content

Abstract

Emtricitabine (FTC) and tenofovir (TFV) are widely used antiviral agents that require intracellular phosphorylation to become active. This article describes the development and validation of an assay for the simultaneous quantification of FTC mono-, di- and triphosphate (FTC-MP, -DP and -TP), TFV and TFV mono- and diphosphate (TFV-MP and -DP) in peripheral blood mononuclear cells. Reference compounds and internal standards were obtained by thermal degradation of FTC-TP, TFV-DP, stable isotope-labeled TFV-DP and stable isotope-labeled cytosine triphosphate. Cells were lysed in methanol:water (70:30, v/v) and the extracted nucleotides were analyzed using weak anion-exchange chromatography coupled with tandem mass spectrometry. Calibration ranges in PBMC lysate from 0.727 to 36.4, 1.33 to 66.4 and 1.29 to 64.6 nM for FTC-MP, FTC-DP and FTC-TP and from 1.51 to 75.6, 1.54 to 77.2 and 2.54 to 127 nM for TFV, TFV-MP and TFV-DP, respectively, were validated. Accuracies were within −10.3 and 16.7% deviation at the lower limit of quantification at which the coefficients of variation were less than 18.2%. At the other tested levels accuracies were within −14.3 and 9.81% deviation and the coefficients of variation lower than 14.7%. The stability of the compounds was assessed under various analytically relevant conditions. The method was successfully applied to clinical samples.

Introduction

Emtricitabine (2′,3′-dideoxy-5-fluoro-3′-thiacytidine; FTC; Fig. 1) and tenofovir (TFV; Fig. 1) are a nucleoside and a nucleotide reverse transcriptase inhibitor (NRTI and NtRTI), respectively. NRTIs and NtRTIs form the backbone of highly active antiretroviral therapy (HAART), which is used against human immunodeficiency virus (HIV) infections. A combination of both agents with the non-nucleoside reverse transcriptase inhibitor efavirenz has recently been approved as the first once-daily, single pill combination therapy against HIV.

Both analogs require intracellular phosphorylation to become active. In cells, the nucleoside analog FTC is first phosphorylated to its monophosphate (FTC-MP) by deoxycytidine kinase after which it is converted further to its di- and triphosphate (FTC-DP and FTC-TP; Fig. 1).

The acyclic nucleotide analog TFV, on the other hand, already contains a phosphonate moiety. To increase the bioavailability and cell penetration, this polar molecule is administered as a disoproxil fumarate prodrug (TDF) that first requires hydrolization to TFV. Subsequently, TFV is phosphorylated intracellularly to its mono- and diphosphate (TFV-MP and TFV-DP; Fig. 1) containing a phosphonate and one or two phosphate groups in total, respectively. FTC-TP and TFV-DP are the actual active metabolites that inhibit viral reverse transcriptase, thereby inhibiting viral replication.

The intracellular pharmacokinetics of the phosphorylated metabolites are different from the plasma pharmacokinetics of the parent compounds. The plasma half-lifes of FTC and TFV are, for example, 8–10 [1] and 14 [2] h, whereas intracellular half-lifes of 39 [1] and 150 [3] h have been observed for FTC-TP and TFV-DP, respectively. Moreover, metabolite levels are subject to inter-individual variation and intracellular drug-drug interactions [4]. Finally, low triphosphate levels can cause viral resistance, whereas high levels have been associated with treatment toxicity [5]. Thus, monitoring of intracellular FTC and TFV nucleotide levels will give more insight in the pharmacology of FTC and TFV.

FTC-TP and TFV-DP have indirectly been determined in peripheral blood mononuclear cells (PBMCs) after dephosphorylation, using high performance liquid chromatography with ultraviolet (HPLC–UV) [6] and mass spectrometric (LC–MS) detection [7].

Direct LC–MS determination of FTC-TP and TFV-DP has been performed using the ion-pairing agents tetrabutylammonium [1], [3], [4], [8], [9], [10], [11] and dimethylhexylamine [12], [13], [14], [15], [16] and weak anion-exchange liquid chromatography (WAXLC–MS/MS) [17]. Although some of these methods also include TFV and/or TFV-MP [4], [10], [11], [12], none include FTC-MP or FTC-DP.

In this paper we describe the development and validation of a method for the quantification of FTC-MP, -DP and -TP in combination with TFV, TFV-MP and TFV-DP in human PBMCs.

Section snippets

Chemicals

FTC and TFV were purchased from Sequoia Research products (Pangbourne, UK). ¹³C₉,¹⁵N₃(U)-labeled cytidine triphosphate (^*CTP; Fig. 1) was purchased from Buchem BV (Apeldoorn, The Netherlands), whereas ¹³C₅-labeled TFV-DP (^*TFV-DP, Fig. 1) was obtained from Moravek (Brea, CA, USA). Unlabeled FTC-TP and TFV-DP were kindly provided by Gilead Sciences Inc. (Foster city, CA, USA).

Methanol and acetonitrile were obtained from Biosolve Ltd. (Amsterdam, The Netherlands) and distilled water was obtained

Reference standards

Because some analytes were not commercially available we thermally degraded FTC-TP, ^*CTP, TFV-DP and ^*TFV-DP into mixtures of their lower phosphates. Since the chromophores of a nucleoside and its nucleotides are identical we could quantify the obtained mixtures on the isocratic HPLC–UV systems, using reference FTC and TFV material. The thus obtained solutions contained 32.7, 59.8 and 58.2 μM (FTC-MP, -DP and -TP, respectively), 68.1, 69.4 and 115 μM (TFV, TFV-MP and -DP, respectively). The ^*TFV

Conclusions

A sensitive method for the simultaneous quantification of FTC-MP, -DP and -TP, and TFV, TFV-MP and -DP has been developed and validated. Assuming a mean of 10 million PBMCs per sample the LLOQ of the method is 14.5, 26.6 and 25.9 fmol/10⁶ PBMCs for FTC-MP, FTC-DP and FTC-TP and 30.3, 30.9 and 50.9 fmol/10⁶ PBMCs for TFV, TFV-MP and TFV-DP, respectively. The method requires minimal sample pretreatment and is the first for the simultaneous quantitation all 6 analytes.

References (24)

T. King et al.
J. Chromatogr. B
(2006)
J.E. Vela et al.
J. Chromatogr. B
(2007)
L.H. Wang et al.
AIDS Res. Hum. Retroviruses
(2004)
P. Barditch-Crovo et al.
Antimicrob. Agents Chemother.
(2001)
T. Hawkins et al.
J. Acquir. Immune. Defic. Syndr.
(2005)
K. Borroto-Esoda et al.
Antivir. Ther.
(2006)
D.J. Back et al.
J. Acquir. Immune. Defic. Syndr.
(2005)
A. Darque et al.
Antimicrob. Agents Chemother.
(1999)
R.L. Claire
Rapid Commun. Mass Spectrom.
(2000)
S. Bartley et al.

W.E. Delaney et al.

Antimicrob. Agents Chemother.

(2006)

A.S. Ray et al.

Antivir. Ther.

(2005)

Cited by (21)

Detection and quantification of Covid-19 antiviral drugs in biological fluids and tissues
2021, Talanta
Since coronavirus disease 2019 (COVID-19) started as a fast-spreading pandemic, causing a huge number of deaths worldwide, several therapeutic options have been tested to counteract or reduce the clinical symptoms of patients infected with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Currently, no specific drugs for COVID-19 are available, but many antiviral agents have been authorised by several national agencies. Most of them are under investigation in both preclinical and clinical trials; however, pharmacokinetic and metabolism studies are needed to identify the most suitable dose to achieve the desired effect on SARS-CoV-2. Therefore, the efforts of the scientific community have focused on the screening of therapies able to counteract the most severe effects of the infection, as well as on the search of sensitive and selective analytical methods for drug detection in biological matrices, both fluids and tissues. In the last decade, many analytical methods have been proposed for the detection and quantification of antiviral compounds currently being tested for COVID-19 treatment. In this review, a critical discussion on the overall analytical procedure is provided, i.e (a) sample pre-treatment and extraction methods such as protein precipitation (PP), solid-phase extraction (SPE), liquid–liquid extraction (LLE), ultrasound-assisted extraction (UAE) and QuEChERS (quick, easy, cheap, effective, rugged and safe), (b) detection and quantification methods such as potentiometry, spectrofluorimetry and mass spectrometry (MS) as well as (c) methods including a preliminary separation step, such as high performance liquid chromatography (HPLC) and capillary electrophoresis (CE) coupled to UV–Vis or MS detection. Further current trends, advantages and disadvantages and prospects of these methods have been discussed, to help the analytical advances in reducing the harm caused by the SARS-CoV-2 virus.
The determination of human peripheral blood mononuclear cell counts using a genomic DNA standard and application in tenofovir diphosphate quantitation
2019, Analytical Biochemistry
A fluorescent quantitation method to determine PBMC-derived DNA amounts using purified human genomic DNA (gDNA) as the reference standard was developed and validated. gDNA was measured in a fluorescence-based assay using a DNA intercalant, SYBR green. The fluorescence signal was proportional to the amount (mass) of DNA in the sample.
The results confirmed a linear fit from 0.0665 to 1.17 μg/μL for gDNA, corresponding to 2.0 × 10⁶ to 35.0 × 10⁶ cells/PBMC sample. Intra-batch and inter-batch accuracy (%RE) was within ±15%, and precision (%CV) was <15%. Benchtop stability, freeze/thaw stability and long term storage stability of gDNA in QC sample matrix, PBMC pellets samples, and pellet debris samples, respectively, as well as dilution linearity had been established. Consistency between hemocytometry cell counting method and gDNA-based counting method was established. 6 out of 6 evaluated PBMC lots had hemocytometry cell counts that were within ±20% of the cell counts determined by the gDNA method.
This method was used in conjunction with a validated LC-MS/MS method to determine the level of tenofovir diphosphate (TFV-DP), the active intracellular metabolite of the prodrugs tenofovir alafenamide (TAF) and tenofovir disoproxil fumarate (TDF), measured in PBMCs in clinical trials of TAF or TDF-containing fixed dose combinations.
Electrochemical and spectroscopic studies of the interaction of antiviral drug Tenofovir with single and double stranded DNA
2018, Bioelectrochemistry
Citation Excerpt :
tnf restrains the activity of viral reverse transcriptase by competing with deoxyadenosine 5′-triphosphate, terminating the elongation of DNA [17]. Several chromatographic [18–22], spectroscopic [23, 24], and electrochemical [25–27] methods have been developed for tenofovir determination. However, thus far, tenofovir interaction with double-strand DNA (dsDNA) and single-strand DNA (ssDNA) has not been investigated.
In the present study, the electrochemical behavior of the antiviral drug tenofovir (tnf) on a boron-doped diamond electrode (BDDE) has been studied using square-wave voltammetry (SWV). The experimental conditions such as the supporting electrolyte composition and the SWV parameters were optimized. Under the optimized conditions, a simple and sensitive SWV procedure for tnf determination was developed in the concentration range of 5.0 × 10⁻⁶ to 1.0 × 10⁻⁴ mol L⁻¹. The calculated limit of detection and limit of quantification were equal to 5.6 × 10⁻⁷ and 1.9 × 10⁻⁶ mol L⁻¹, respectively. The biological significance of the developed method was demonstrated by a quantitative analysis of the pharmaceutical formulations Viread and Tenofovir disoproxil Teva. Moreover, both voltammetric and spectroscopic techniques were used to study the interaction between tnf and DNA. Changes in the electrochemical and spectroscopic behavior of tnf in the presence of DNA were used to calculate the binding constants of the formed complexes. The estimated values of Gibbs free energy revealed that the formation of the drug–DNA complexes was a spontaneous and favorable process. Moreover, for free and bound tenofovir, kinetic parameters such as heterogeneous rate constant, electron transfer coefficient, and diffusion coefficient were determined.
Simultaneous determination of tenofovir alafenamide and its active metabolites tenofovir and tenofovir diphosphate in HBV-infected hepatocyte with a sensitive LC–MS/MS method
2017, Journal of Pharmaceutical and Biomedical Analysis
Tenofovir (TFV), a first-line anti-viral agent, has been prepared as various forms of prodrugs for better bioavailability, lower systemic exposure and higher target cells loading of TFV to enhance efficacy and reduce toxicity. TFV undergoes intracellular phosphorylation to form TFV diphosphate (TFV-DP) in target cell to inhibit viral DNA replication. Hence, TFV-DP is the key active metabolite that exhibits anti-virus activity, its intracellular exposure and half-life determine the final activity. Therefore, simultaneous monitoring prodrug, TFV and TFV-DP in target cells will comprehensively evaluate TFV prodrugs, both considering the stability of ester prodrug, and the intracellular exposure of TFV-DP. Thus we intended to develop a convenient general analytical method, taking tenofovir alafenamide (TAF) as a representative of TFV prodrugs. A sensitive LC–MS/MS method was developed, and TAF, TFV and TFV-DP were separated on a XSelect HSS T3 column (4.6 mm × 150 mm, 3.5 μm, Waters) with gradient elution after protein precipitation. The method provided good linearity for all the compounds (2–500 nM for TFV and TAF; 20–5000 nM for TFV-DP) with the correlation coefficients (r) greater than 0.999. Intra- and inter-day accuracies (in terms of relative error, RE < 10.4%) and precisions (in terms of coefficient of variation, CV < 14.1%) satisfied the standard of validation. The matrix effect, recovery and stability were also within acceptable criteria. Finally, we investigated the intracellular pharmacokinetics of TAF and its active metabolites in HepG2.2.15 cells with this method.
The search for nucleoside/nucleotide analog inhibitors of dengue virus
2015, Antiviral Research
Nucleoside analogs represent the largest class of antiviral agents and have been actively pursued for potential therapy of dengue virus (DENV) infection. Early success in the treatment of human immunodeficiency virus (HIV) infection and the recent approval of sofosbuvir for chronic hepatitis C have provided proof of concept for this class of compounds in clinics. Here we review (i) nucleoside analogs with known anti-DENV activity; (ii) challenges of the nucleoside antiviral approach for dengue; and (iii) potential strategies to overcome these challenges. This article forms part of a symposium in Antiviral Research on flavivirus drug discovery.
Development of an LC-MS/MS assay for the quantitative determination of the intracellular 5-fluorouracil nucleotides responsible for the anticancer effect of 5-fluorouracil
2015, Journal of Pharmaceutical and Biomedical Analysis
Citation Excerpt :
In this paper the suffix -XP is used to refer to the obtained mixtures of nucleoside monophosphates, diphosphates and triphosphates. Thermal degradation of the nucleoside triphosphates into lower phosphates was monitored using the HPLC-UV system previously described for the analysis of emtricitabine and tenofovir with some minor adjustments [19]. The mobile phase consisted of 10 mM tetrabutylammonium dihydrogen phosphate with 70 mM potassium dihydrogen phosphate in water adjusted to pH 7 and 7% methanol (v/v), and it was delivered isocratically to a Synergi hydro-RP column (150 mm × 2.0 mm ID, 4 μm particles; Phenomenex, Torrance, CA, USA) with a flow rate of 0.25 mL/min.
5-Fluorouracil (5-FU) and its oral prodrug capecitabine are among the most widely used chemotherapeutics. For cytotoxic activity, 5-FU requires cellular uptake and intracellular metabolic activation. Three intracellular formed metabolites are responsible for the antineoplastic effect of 5-FU: 5-fluorouridine 5′-triphosphate (FUTP), 5-fluoro-2′-deoxyuridine 5′-triphosphate (FdUTP) and 5-fluoro-2′-deoxyuridine 5′-monophosphate (FdUMP).
In this paper, we describe the development of an LC–MS/MS assay for quantification of these active 5-FU nucleotides in peripheral blood mononuclear cells (PBMCs). Because the intracellular 5-FU nucleotide concentrations were very low, maximization of the release from the cell matrix and minimization of interference were critical factors. Therefore, a series of experiments was performed to select the best method for cell lysis and nucleotide extraction. Chromatography was optimized to obtain separation from endogenous nucleotides, and the effect of different cell numbers was examined.
The assay was validated for the following concentration ranges in PBMC lysate: 0.488–19.9 nM for FUTP, 1.66–67.7 nM for FdUTP and 0.748–30.7 nM for FdUMP. Accuracies were between −2.2 and 7.0% deviation for all analytes, and the coefficient of variation values were ≤4.9%.
The assay was successfully applied to quantify 5-FU nucleotides in PBMC samples from patients treated with capecitabine and patients receiving 5-FU intravenously. FUTP amounts up to 3054 fmol/10⁶ PBMCs and FdUMP levels up to 169 fmol/10⁶ PBMCs were measured. The FdUTP concentrations were below the lower limit of quantification. To our knowledge, this is the first time that 5-FU nucleotides were quantified in cells from patients treated with 5-FU or capecitabine without using a radiolabel.

View all citing articles on Scopus

View full text

Simultaneous quantification of emtricitabine and tenofovir nucleotides in peripheral blood mononuclear cells using weak anion-exchange liquid chromatography coupled with tandem mass spectrometry

Abstract

Introduction

Section snippets

Chemicals

Reference standards

Conclusions

J. Chromatogr. B

J. Chromatogr. B

AIDS Res. Hum. Retroviruses

Antimicrob. Agents Chemother.

J. Acquir. Immune. Defic. Syndr.

Antivir. Ther.

J. Acquir. Immune. Defic. Syndr.

Antimicrob. Agents Chemother.

Rapid Commun. Mass Spectrom.

Antimicrob. Agents Chemother.

Antivir. Ther.