Simultaneous quantitation of favipiravir and its hydroxide metabolite in human plasma and hamster matrices using a UPLC-MS/MS method.

Favipiravir, a broad-spectrum RNA-dependent RNA polymerase inhibitor, is currently being evaluated in preclinical and clinical studies for the treatment of various infectious diseases including COVID-19. We developed an ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) assay for the quantification of favipiravir and its hydroxide metabolite (M1), in human and hamster biological matrices. Analytes were separated on an Acquity UPLC HSS T3 column (2.1 × 100 mm, 1.8 µm) after a simple protein precipitation with acetonitrile. The mobile phase consisted of water and methanol, each containing 0.05% formic acid. Experiments were performed using electrospray ionization in the positive and negative ion mode, with protonated molecules used as the precursor ion and a total run time of 6 min. The MS/MS response was linear over the concentration ranges from 0.5-100 µg/ml for favipiravir and 0.25-30 µg/ml for M1. Intra- and inter-day accuracy and precision were within the recommended limits of the European Medicines Agency guidelines. No significant matrix effect was observed, and the method was successfully applied to inform favipiravir dose adjustments in six immunocompromised children with severe RNA viral infections. In conclusion, the UPLC-MS/MS assay is suitable for quantification of favipiravir over a wide range of dosing regimens, and can easily be adapted to other matrices and species.

Determination of 5-azacitidine in human plasma by LC-MS/MS: application to pharmacokinetics pilot study in MDS/AML patients.

PURPOSE: Azacitidine (Vidaza®, AZA) is a mainstay for treating acute myeloid leukemia (AML) in patients unfit for standard induction and other myelodysplastic syndromes (MDS). However, only half of the patients usually respond to this drug and almost all patients will eventually relapse. Predictive markers for response to AZA are yet to be identified. AZA is metabolized in the liver by a single enzyme, cytidine deaminase (CDA). CDA is a ubiquitous enzyme coded by a highly polymorphic gene, with subsequent great variability in resulting activities in the liver. The quantitative determination of AZA in plasma is challenging due the required sensitivity and because of the instability in the biological matrix upon sampling, possibly resulting in erratic values. METHODS: We have developed and validated following EMA standards a simple, rapid, and cost-effective liquid chromatography-tandem mass spectrometry method for the determination of azacitidine in human plasma. RESULTS: After a simple and rapid precipitation step, analytes were successfully separated and quantitated over a 5-500 ng/mL range. The performance and reliability of this method were tested as part of an investigational study in MDS/AML patients treated with standard azacitidine (75 mg/m2 for 7 days a week every 28 days). CONCLUSION: Overall, this new method meets the requirements of current bioanalytical guidelines and could be used to monitor drug levels in MDS/AML patients.

Development and Validation of a Simple and Rapid Ultrahigh-Performance Liquid Chromatography Tandem Spectrometry Method for the Quantification of Hydroxychloroquine in Plasma and Blood Samples in the Emergency Context of SARS-CoV-2 Pandemic.

ABSTRACT: Therapeutic drug monitoring of hydroxychloroquine (HCQ) has been recommended to optimize the treatment of patients with COVID-19. The authors describe an ultrahigh-performance liquid chromatography tandem spectrometry method developed in a context of emergency, to analyze HCQ in both human plasma and blood samples. After adding the labeled internal standard and simple protein precipitation, plasma samples were analyzed using a C18 column. Blood samples required evaporation before analysis. The total chromatographic run time was 4 minutes (including 1.5 minutes of column equilibration). The assay was linear over the calibration range (r2 > 0.99) and up to 1.50 mcg/mL for the plasma samples (5.00 mcg/mL for the blood matrix). The limit of quantification was 0.0150 mcg/mL for plasma samples (0.05 mcg/mL blood matrix) with accuracy and precision ranging from 91.1% to 112% and from 0.750% to 11.1%, respectively. Intraday and interday precision and accuracy values were within 15.0%. No significant matrix effect was observed in the plasma or blood samples. This method was successfully applied to patients treated for COVID-19 infection. A simple and rapid ultrahigh-performance liquid chromatography tandem spectrometry method adapted to HCQ therapeutic drug monitoring in the context of SARS-CoV-2 infection was successfully developed and validated.

Simultaneous determination of cytosine arabinoside and its metabolite uracil arabinoside in human plasma by LC-MS/MS: Application to pharmacokinetics-pharmacogenetics pilot study in AML patients.

Purine analogs like aracytine (AraC) are a mainstay for treating acute myeloid leukemia (AML). There are marked differences in drug dosing and scheduling depending on the protocols when treating AML patients with AraC. Large inter-patient pharmacokinetics variability has been reported, and genetic polymorphisms affecting cytidine deaminase (CDA), the liver enzyme responsible for the conversion of Ara-C to inactive uracil arabinoside (AraU) could be a culprit for either life-threatening toxicities or poor efficacy related to substantial changes in plasma exposure levels among patients. The quantitative determination of Ara-C in plasma is challenging due the required sensitivity because of the short half-life of this drug (i.e., <10 min) and the metabolic instability in biological matrix upon sampling possibly resulting in erratic values. We developed and validated a liquid chromatography tandem mass spectrometry method (UPLC-MS/MS) for the simultaneous determination of Ara-C and Ara-U metabolite in human plasma. After simple and rapid precipitation, analytes were successfully separated and quantitated over a 1-500 ng/ml range for Ara-C and 250-7500 ng/ml range for AraU. The performance and reliability of this method was tested as part of an investigational study in AML patients treated with low dose cytarabine and confirmed marked differences in drug exposure levels and metabolic ratio, depending on the CDA status of the patients. Overall, this new method meets the requirements of current bioanalytical guidelines and could be used to monitor drug levels in AML patients with respect to their CDA phenotypes.

Validation of an electrospray ionization LC/MS/MS method for quantitative analysis of vincristine in human plasma samples.

Vincristine is a natural vinca alkaloid widely used in paediatric cancer treatment. Vincristine pharmacokinetics has been already studied, but few data are available in paediatric populations. A sensitive and specific liquid chromatography-tandem mass spectrometry (LC/MS/MS) method was developed for the quantification of vincristine in plasma in order to investigate pharmacokinetics in a paediatric population. Two hundred microliters of plasma was added to vinblastine, used as internal standard. Chromatographic separation was achieved on a C8 HPLC column (Phenomenex Luna 50 mm x 2.0 mm, 3.0 microm) with a mobile phase gradient at a flow rate of 0.2 ml/min. Quantification was performed using the transition of 825.4-->765.4 (m/z) for vincristine and 811.4-->751.4 (m/z) for vinblastine. Chromatographic separation was achieved in 8 min. The limit of quantification was 0.25 ng/ml with a precision of 10.2% and an accuracy of 99.6%. The calibration curve was linear up to 50.0 ng/ml. Intra-day precision and accuracy ranged from 6.3% to 10% and from 91.9% to 100.8%, respectively. Inter-assay precision and accuracy ranged from 3.8% to 9.7% and from 93.5% to 100.5%, respectively. No significant matrix effect was observed for vincristine. A rapid, specific and sensitive LC/MS/MS method for quantification of vincristine in human plasma was developed and is now successfully applied for pharmacokinetic studies in paediatric patients.

Validation of an electrospray ionization LC-MS/MS method for quantitative analysis of raltegravir, etravirine, and 9 other antiretroviral agents in human plasma samples.

BACKGROUND: Raltegravir is the first human immunodeficiency virus-1 (HIV-1) integrase inhibitor used in treatment-experienced patients who have evidence of viral replication and HIV-1 strains resistance to multiple antiretroviral regimens. Etravirine is a novel NNRTI, active against HIV-1 strains harboring multiple NNRTI mutations. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the quantification of raltegravir, etravirine, and 9 other antiretroviral agents (amprenavir, atazanavir, darunavir, efavirenz, indinavir, lopinavir, ritonavir, saquinavir, and tipranavir) in plasma at the concentrations associated with therapy. MATERIALS AND METHODS: The ritonavir analog, methyl indinavir, and lopinavir-d8 were used as internal standards, added to 100 microL of plasma sample prior to a protein precipitation using methanol. Chromatographic separation was achieved on a C18 HPLC column (Waters Sunfire 100 x 2.1 mm, 3.5 microm) with a mobile phase gradient at a flow rate of 0.3 mL/min. Five microL of sample were injected into the LC-MS/MS system (Waters Quattro Premier XE) to determine concentrations of raltegravir, etravirine, and other antiretroviral agents. RESULTS AND DISCUSSION: This method showed an excellent linearity for all calibration curves (r2 > 0.998). The lower limit of quantification was established at 5 ng/mL for raltegravir and 40 ng/mL for etravirine, with precision and accuracy within +/-20% and 80% to 120% for all analytes. Intraassay and interassay precision and inaccuracy ranged from -9.2% to 6.9% for raltegravir and from -14.3% to 12.3% for etravirine and were less than 15% for all other compounds. No matrix effect was observed for any of the antiretrovirals studied. CONCLUSION: A rapid, specific, and sensitive LC-MS/MS method for quantification of raltegravir, etravirine, and 9 other antiretrovirals in human plasma was developed and was successfully applied for routine therapeutic drug monitoring.