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Development and bioanalytical validation of a liquid chromatographic-tandem mass spectrometric (LC-MS/MS) method for the quantification of the CCR5 antagonist maraviroc in human plasma.

ABSTRACT: Maraviroc is a CCR5 antagonist that has been utilized as a viral entry inhibitor in the management of HIV-1. Current clinical trials are pursuing maraviroc drug efficacy in both oral and topical formulations. Therefore, in order to fully understand drug pharmacokinetics, a sensitive method is required to quantify plasma drug concentrations.Maraviroc-spiked plasma was combined with acetonitrile containing an isotopically-labeled internal standard, and following protein precipitation, samples were evaporated to dryness and reconstituted for liquid chromatographic-tandem mass spectrometric (LC-MS/MS) analysis. Chromatographic separation was achieved on a Waters BEH C8, 50×2.1 mm UPLC column, with a 1.7 ?m particle size and the eluent was analyzed using an API 4000 mass analyzer in selected reaction monitoring mode. The method was validated as per FDA Bioanalytical Method Validation guidelines.The analytical measuring range of the LC-MS/MS method is 0.5-1000 ng/ml. Calibration curves were generated using weighted 1/x(2) quadratic regression. Inter-and intra-assay precision was ?5.38% and ?5.98%, respectively; inter-and intra-assay accuracy (%DEV) was ?10.2% and ?8.44%, respectively. Additional studies illustrated similar matrix effects between maraviroc and its internal standard, and that maraviroc is stable under a variety of conditions. Method comparison studies with a reference LC-MS/MS method show a slope of 0.948 with a Spearman coefficient of 0.98.Based on the validation metrics, we have generated a sensitive and automated LC-MS/MS method for maraviroc quantification in human plasma.

SUBMITTER: Emory JF

PROVIDER: S-EPMC4029414 | biostudies-literature | 2014 Apr

REPOSITORIES: biostudies-literature

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Development and bioanalytical validation of a liquid chromatographic-tandem mass spectrometric (LC-MS/MS) method for the quantification of the CCR5 antagonist maraviroc in human plasma.

Emory Joshua F JF Seserko Lauren A LA Marzinke Mark A MA

Clinica chimica acta; international journal of clinical chemistry 20140219

<h4>Background</h4>Maraviroc is a CCR5 antagonist that has been utilized as a viral entry inhibitor in the management of HIV-1. Current clinical trials are pursuing maraviroc drug efficacy in both oral and topical formulations. Therefore, in order to fully understand drug pharmacokinetics, a sensitive method is required to quantify plasma drug concentrations.<h4>Methods</h4>Maraviroc-spiked plasma was combined with acetonitrile containing an isotopically-labeled internal standard, and following ...[more]

PMID: 24561264

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Project description:BackgroundTopical microbicidal agents are being actively pursued as a modality to prevent HIV viral transmission during sexual intercourse. Quantification of antiretroviral agents in specimen sources where antiviral activity is elicited is critical, and drug measurements in cervicovaginal fluid can provide key information on local drug concentrations. Two antiretroviral drugs, dapivirine and maraviroc, have gained interest as vaginal microbicidal agents, and rugged methods are required for their quantification in cervicovaginal secretions.MethodsCervicovaginal fluid spiked with dapivirine and maraviroc were applied to ophthalmic tear strips or polyester-based swabs to mimic collection procedures used in clinical studies. Following sample extraction and the addition of isotopically labeled internal standards, samples were subjected to liquid chromatographic-tandem mass spectrometric (LC-MS/MS) analysis using a Waters BEH C8, 50mm×2.1mm, 1.7μm particle size column, on an API 4000 mass analyzer operated in selective reaction monitoring mode. The method was validated according to FDA Bioanalytical Method Validation guidelines.ResultsDue to the disparate saturation capacity of the tested collection devices, the analytical measuring ranges for dapivirine and maravirocin cervicovaginal fluid on the ophthalmic tear strip were 0.05-25ng/tear strip, and 0.025-25ng/tear strip, respectively. As for the polyester-based swab, the analytical measuring ranges were 0.25-125ng/swab for dapivirine and 0.125-125ng/swab for maraviroc. Dilutional studies were performed for both analytes to extended ranges of 25,000ng/tear strip and 11,250ng/swab. Standard curves were generated via weighted (1/x(2)) linear or quadratic regression of calibrators. Precision, accuracy, stability and matrix effects studies were all performed and deemed acceptable according to the recommendations of the FDA Bioanalytical Method Validation guidelines.ConclusionsA rugged LC-MS/MS method for the dual quantification of dapivirine and maraviroc in cervicovaginal fluid using two unique collection devices has been developed and validated. The described method meets the criteria to support large research trials.

Project description:BACKGROUND:The nucleotide reverse transcriptase inhibitor tenofovir (TFV) is widely administered in a disoproxil prodrug form (tenofovir disoproxil fumarate, TDF) for HIV management and prevention. Recently, novel prodrugs tenofovir alafenamide fumarate (TAF) and hexadecyloxypropyl tenofovir (CMX157) have been pursued for HIV treatment while minimizing adverse effects associated with systemic TFV exposure. Dynamic and sensitive bioanalytical tools are required to characterize the pharmacokinetics of these prodrugs in systemic circulation. Two parallel methods have been developed, one to combinatorially quantify TAF and TFV, and a second method for CMX157 quantification, in plasma. METHODS:K2EDTA plasma was spiked with TAF and TFV, or CMX157. Following the addition of isotopically labeled internal standards and sample extraction via solid phase extraction (TAF and TFV) or protein precipitation (CMX157), samples were subjected to liquid chromatographic-tandem mass spectrometric (LC-MS/MS) analysis. For TAF and TFV, separation occurred using a Zorbax Eclipse Plus C18 Narrow Bore RR, 2.1?×?50?mm, 3.5??m column and analytes were detected on an API5000 mass analyzer; CMX157 was separated using a Kinetex C8, 2.1?×?50?mm, 2.6??m column and quantified using an API4500 mass spectrometer. Methods were validated according to FDA Bioanalytical Method Validation guidelines. RESULTS:Analytical methods: were optimized for the multiplexed monitoring of TAF and TFV, and CMX157 in plasma. The lower limits of quantification (LLOQs) for TAF, TFV, and CMX157 were 0.03, 1.0, and 0.25?ng/mL, respectively. Calibration curves were generated via weighted linear regression of standards. Intra- and inter-assay precision and accuracy studies demonstrated %CVs???14.4% and %DEVs ??±?7.95%, respectively. Stability and matrix effects studies were also performed. All results were acceptable and in accordance with the recommended guidelines for bioanalytical methods. Assays were also applied to quantify in vivo concentrations of prodrugs and TFV in a preclinical study post-rectal administration. CONCLUSIONS:Sensitive, specific, and dynamic LC-MS/MS assays have been developed and validated for the multiplexed quantification TAF and TFV, as well as an independent assay for CMX157 quantification, in plasma. The described methods meet sufficient throughput criteria to support large research trials.

Project description:BackgroundThe non-nucleoside reverse transcriptase inhibitor dapivirine has been evaluated as a topical microbicidal agent to prevent HIV-1 acquisition. Several clinical trials have evaluated the pharmacokinetics of dapivirine when administered as a 25-mg intravaginal ring. Recent studies have focused on the distribution of dapivirine into breast milk. Drug distribution during lactation and breastfeeding can have implications in terms of infant drug exposure. Thus, sensitive bioanalytical tools are required to characterize the pharmacokinetics of dapivirine in breast milk.MethodsWhole breast milk was spiked with dapivirine and internal standard. Lipid content was disrupted via pre-treatment with n-hexane, and supernatants were subjected to solid phase extraction. Extracted materials were subjected to liquid chromatographic-tandem mass spectrometric (LC-MS/MS) analysis. Separation occurred using a Waters BEH C8, 50 × 2.1 mm UPLC column with a 1.7 µm particle size and dapivirine was detected on an API 5000 mass analyzer. Methods were validated in accordance with FDA Bioanalytical Method Validation recommendations.ResultsThe analytical method was optimized for dapivirine extraction from breast milk. The analytical measuring range of the assay was 10-1000 pg/mL. Calibration curves were generated via weighted linear regression of standards. Intra- and inter-assay precision and accuracy studies demonstrated %CVs ≤ 14.6% and %DEVs ≤ ±12.7%. Stability and matrix effects studies were also conducted and deemed acceptable. The method was applied to a previously reported phase 1 clinical trial and demonstrated appropriate performance in the quantitation of dapivirine in breast milk samples from lactating women.ConclusionsAn ultrasensitive LC-MS/MS assay has been developed and validated for the quantitation of dapivirine in breast milk. The described method meets validation acceptance criteria and has been applied to a phase 1 clinical trial.

Project description:BackgroundValproic acid (VPA) is a clinically used antiepileptic drug, but it is associated with a significant risk of a low verbal intelligence quotient (IQ) score, attention-deficit hyperactivity disorder and autism spectrum disorder in children when it is administered during pregnancy. Prenatal VPA exposure has been reported to affect neurogenesis and neuronal migration and differentiation. In addition, growing evidence has shown that microglia and brain immune cells are activated by VPA treatment. However, the role of VPA-activated microglia remains unclear.MethodsPregnant female mice received sodium valproate on E11.5. A microglial activation inhibitor, minocycline or a CCR5 antagonist, maraviroc was dissolved in drinking water and administered to dams from P1 to P21. Measurement of microglial activity, evaluation of neural circuit function and expression analysis were performed on P10. Behavioral tests were performed in the order of open field test, Y-maze test, social affiliation test and marble burying test from the age of 6 weeks.ResultsPrenatal exposure of mice to VPA induced microglial activation and neural circuit dysfunction in the CA1 region of the hippocampus during the early postnatal periods and post-developmental defects in working memory and social interaction and repetitive behaviors. Minocycline, a microglial activation inhibitor, clearly suppressed the above effects, suggesting that microglia elicit neural dysfunction and behavioral disorders. Next-generation sequencing analysis revealed that the expression of a chemokine, C-C motif chemokine ligand 3 (CCL3), was upregulated in the hippocampi of VPA-treated mice. CCL3 expression increased in microglia during the early postnatal periods via an epigenetic mechanism. The CCR5 antagonist maraviroc significantly suppressed neural circuit dysfunction and post-developmental behavioral disorders induced by prenatal VPA exposure.ConclusionThese findings suggest that microglial CCL3 might act during development to contribute to VPA-induced post-developmental behavioral abnormalities. CCR5-targeting compounds such as maraviroc might alleviate behavioral disorders when administered early.

Dataset Information

Development and bioanalytical validation of a liquid chromatographic-tandem mass spectrometric (LC-MS/MS) method for the quantification of the CCR5 antagonist maraviroc in human plasma.

Publications

Development and bioanalytical validation of a liquid chromatographic-tandem mass spectrometric (LC-MS/MS) method for the quantification of the CCR5 antagonist maraviroc in human plasma.

Similar Datasets

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