Analysis of Anti-Hiv Nucleoside Inhibitors by Capillary Electrophoresis-Electrospray Ionization Mass Spectrometry

Agrofoglio, Luigi A.; Cahours, Xavier; Tran, Thu Thi; Dessans, Hélène; Kiéda, Claudine; Morin, Pascal

doi:10.1081/ncn-100002309

Cited by 16 publications

(21 citation statements)

References 11 publications

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“…The sensitivity of our test is comparable to the liquid chromatography MS/MS analytical method recently described, involving the detection of ddATP by an indirect method with a limit of ddA quantification of 0.1 ng/ml (4 x 10 Ϫ10 M) (1,11). It is important to note that the ddATP content found in our PBMC model (picomole) is much higher than that reported in the literature (femtomole) (18,23,27).…”

Section: Discussioncontrasting

confidence: 37%

Determination of ddATP Levels in Human Immunodeficiency Virus-Infected Patients Treated with Dideoxyinosine

Saint

Terreux

Duval

et al. 2004

Antimicrob Agents Chemother

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Clinical failures of the highly active antiretroviral therapy could result from inefficient intracellular concentrations of antiviral drugs. The determination of drug contents in target cells of each patient would be useful in clinical investigations and trials. The purpose of this work was to quantify the intracellular concentration of ddATP, the active metabolite of dideoxyinosine (ddI), in peripheral blood mononuclear cells (PBMCs) of human immunodeficiency virus (HIV)-infected patients treated with ddI. We have raised antibodies against ddA-citrate, a stable isostere of ddATP selected on the basis of its structural and electronic analogies with ddATP. The anti-ddA-citrate antibodies recognized ddATP and ddA with nanomolar affinities and cross-reacted neither with any of the nucleotide reverse transcriptase inhibitors used in HIV therapy nor with their phosphorylated metabolites. The three phosphorylated metabolites of ddI (ddAMP, ddADP, and ddATP) were purified by anion exchange chromatography and the amount of each metabolite was determined by radioimmunoassay with or without prior phosphatase treatment. The intracellular levels of the three ddI metabolites were measured both in an in vitro model and in PBMCs of HIV-infected patients under ddI treatment. The possibility to measure intracellular levels of ddATP from small blood samples of HIV-infected patients treated with ddI could be exploited to develop individual therapeutic monitoring.Highly active antiretroviral therapy has been used successfully for treatment of human immunodeficiency virus (HIV) disease. The most common highly active antiretroviral therapy regimens consist of a combination of at least one protease inhibitor and two nucleoside reverse transcriptase inhibitors. Contrary to protease inhibitors, the expression of nucleoside reverse transcriptase inhibitor activity requires intracellular metabolism of the nucleoside precursor into its corresponding 5Ј-triphosphate nucleotide by the host cell kinases. The active metabolite (nucleoside reverse transcriptase inhibitor-triphosphate) competitively inhibits the HIV reverse transcriptase and acts as a chain terminator of the proviral DNA.The presence and activity of the intracellular kinases are highly dependent on the type and activation state of the target cell (37). Studies conducted in HIV-infected patients failed to establish a clear relationship between the plasma nucleoside reverse transcriptase inhibitor concentration and the antiviral efficiency of these drugs (3, 4, 18, 39). However, a clinical study showed a significant and linear relationship between the intracellular nucleoside reverse transcriptase inhibitor-triphosphate (zidovudine-triphosphate and lamivudine-triphosphate) concentrations, the percent change in CD4 ϩ cells and the rate of decline of HIV RNA in plasma (17). Thus, intracellular contents of active drugs in target cells seem to give a much better indication of therapeutic efficiency than plasma concentrations of drug precursors.The intracellular metabolism of ddI leads...

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Section: Discussioncontrasting

confidence: 37%

Determination of ddATP Levels in Human Immunodeficiency Virus-Infected Patients Treated with Dideoxyinosine

Saint

Terreux

Duval

et al. 2004

Antimicrob Agents Chemother

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“…Recently, Benech and co-workers [17][18][19][20][21] used LC-MS/ MS with selected reaction monitoring (SRM) to directly quantitate the intracellular triphosphorylated metabolites of anti-HIV nucleosides, alone or in combination, in human PBMCs of HIV-positive patients. As part of our on going program to develop new bioanalytical methods, our group has previously reported that CE coupled with ESI-MS/MS was an excellent technique for the separation and quantitation of various anti-HIV nucleosides [22,23]. Thus, we turned our attention to the on-line combination of CE with electrospray (ES) MS (CE-MS/MS) for the quantitation of their phosphorylated metabolites.…”

Section: Introductionmentioning

confidence: 99%

Analysis and validation of the phosphorylated metabolites of two anti‐human immunodeficiency virus nucleotides (stavudine and didanosine) by pressure‐assisted CE‐ESI‐MS/MS in cell extracts: Sensitivity enhancement by the use of perfluorinated acids and alcohols as coaxial sheath‐liquid make‐up constituents

et al. 2006

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A CE method utilizing triple quadrupole electrospray (ES) MS (MS/MS) detection was developed and validated for the simultaneous measurement of nucleoside 5'-triphosphate and 5'-monophosphate anabolites of the anti-HIV (human immunodeficiency virus) didanosine (ddAMP, ddATP) and stavudine (d4TMP, d4TTP), among a pool of 14 endogenous 5'-mono-, di-, and triphosphate nucleosides. These compounds were spiked and extracted from peripheral blood mononuclear cells (PBMCs) which are the sites of HIV replication and drug action. An acetic acid/ammonia buffer (pH 10, ionic strength of 40 mM) was selected as running electrolyte, and the separation was performed by the simultaneous application of a CE voltage of +30 kV and an overimposed pressure of 28 mbar (0.4 psi). The application of pressure assistance was needed to provide stable ES conditions for successful coupling. The coupling was carried out with a modified sheath-flow interface, with one uninterrupted capillary (80 cmx 50 microm id; 192 microm od) in a dimension that fits into the ESI needle to get a stable ion spray. Some CE-MS parameters such as overimposed pressure, sheath-liquid composition, sheath-liquid and sheath-gas flow rates, ES voltage, and the CE capillary position were optimized in order to obtain an optimal sensitivity. The use of perfluorinated alcohols and acids in the coaxial sheath-liquid make-up (2,2,2-trifluoroethanol + 0.2 mM tridecafluoroheptanoic acid) appeared to provide the best MS sensitivity and improve the stability of spray. The linearity of the CE-MS and CE-MS/MS methods was checked under these conditions. Validation parameters such as accuracy, intraday and interday precision, and LOQs were determined in CE-MS/MS mode. Finally, the quantitation of d4T-TP and ddA-TP was validated in this CE-MS/MS system.

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“…Again, applications in the field of CE-MS dealing with real samples are scarce. Cahours and co-workers [37][38][39], for example, published several papers about the development of a CZE-ESI-MS/MS method for the analysis of the active triphosphate derivates of several NRTIs as their corresponding nucleoside, among a pool of natural nucleosides, however, always using standard solutions. An example does exist, albeit based on spiked samples.…”

Section: Nucleoside Analoguesmentioning

confidence: 99%

“…The traditional approach involves an initial separation of the triphosphate from the other metabolites using ion-exchange cartridges or HPLC, followed by nucleotide dephosphorylation and further quantification of the corresponding nucleoside, leading in return to the concentration of the triphosphate. Quantitation can be accomplished by HPLC-UV [42], HPLC-radioimmunoassay (RIA) [43], RIA [44], enzymatic assay [45], LC-MS/ MS [46,47], and CE-MS/MS [37][38][39]. In the last years, efforts have been made in developing direct methods for the analysis of the active triphosphate metabolites using LC-MS/MS [48][49][50][51], CE-MS/MS [40], and MALDI-time of flight (TOF)-MS [52].…”

Section: Nucleoside Analoguesmentioning

confidence: 99%

Analysis of nucleic acid constituents by on‐line capillary electrophoresis‐mass spectrometry

et al. 2005

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This review is focused on the capillary electrophoresis-mass spectrometric (CE-MS) analysis of nucleic acid constituents in the broadest sense, going from nucleotides and adducted nucleotides over nucleoside analogues to oligonucleotides. These nucleic acid constituents play an important role in a variety of biochemical processes. Hence, their isolation, identification, and quantification will undoubtedly help reveal the process of life and disease mechanisms, such as carcinogenesis, and can also be useful for antitumor and antiviral drug research to provide valuable information about mechanism of action, pharmacokinetics, pharmacodynamics, toxicity, therapeutic drug level monitoring, and quality control related to this substance class. Fundamental investigations into their structure, the search for modifications, the occurrence and biochemical impact of structural variation amongst others, are therefore of great value. In view of the related bioanalytical procedures, the coupling of CE to MS has emerged as a powerful tool for the analysis of the complex mixtures of nucleic acid constituents: CE confers rapid analysis and efficient resolution, while MS provides high selectivity and sensitivity with structural characterization of minute amounts of compound. After an introduction about the biochemical and analytical perspectives on the nucleic acid constituents, the different modes of CE used in this field of research as well as the relevant CE-MS interfaces and the difficulties associated with quantitative CE-MS are briefly discussed. A large section is finally devoted to field-oriented applications.

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Analysis of Anti-Hiv Nucleoside Inhibitors by Capillary Electrophoresis-Electrospray Ionization Mass Spectrometry

Cited by 16 publications

References 11 publications

Determination of ddATP Levels in Human Immunodeficiency Virus-Infected Patients Treated with Dideoxyinosine

Determination of ddATP Levels in Human Immunodeficiency Virus-Infected Patients Treated with Dideoxyinosine

Analysis of nucleic acid constituents by on‐line capillary electrophoresis‐mass spectrometry

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