Multiplexed quantification of venlafaxine and metabolites in human plasma by liquid chromatography-tandem mass spectrometry.

BACKGROUND: Venlafaxine (VEN) and its O-demethylated metabolite, O-desmethylvenlafaxine (ODV), are commonly prescribed serotonin-norepinephrine reuptake inhibitors, approved for the treatment of depression and anxiety. Both are metabolized to inactive metabolites via cytochrome P450 enzymes. While previous studies have focused on quantifying VEN and ODV, bioanalytical methods for the simultaneous measurement of all metabolites are needed to fully characterize the pharmacology of VEN and ODV. METHODS: K2EDTA plasma was spiked with VEN, ODV, N-desmethylvenlafaxine (NDV), N,O-didesmethylvenlafaxine (NODDV), and N,N-didesmethylvenlafaxine (NNDDV). Drugs and metabolites were extracted via protein precipitation and quantified using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The multiplexed assay was validated in accordance with regulatory recommendations, and evaluated in remnant plasma samples from persons prescribed venlafaxine. RESULTS: The analytical measuring range for venlafaxine and all four metabolites was 5-800 ng/mL. Standard curves were generated via weighted quadratic (NNDDV) or linear (VEN, ODV, NDV, NODDV) regression of calibrators. Inter-assay imprecision was between 1.9-9.3% for all levels of all analytes. Minor matrix effects were observed, and both recovery efficiency and process efficiency were >96% for all analytes. All other assay validation assessments met acceptance criteria. Drug concentrations measured from remnant plasma specimens obtained from patients with current venlafaxine prescriptions (37.5-450 mg/day) yielded NDDV, NDV, and NODDV metabolite concentrations in 6/21, 14/21, and 20/21 samples, respectively. The ratio of active to inactive analytes ranged from 0.74 to 14.5, with a median of 6.39. CONCLUSIONS: An efficient and accurate LC-MS/MS method was developed and validated for the quantification of VEN, ODV, and all three inactive metabolites in plasma. The assay met all acceptance criteria, and may be used in future studies of the pharmacokinetics of these drugs.

Development and validation of a liquid chromatographic-tandem mass spectrometric assay for the quantification of the direct acting antivirals glecaprevir and pibrentasvir in plasma.

BACKGROUND: Direct acting antiviral (DAA) therapies are effective in the treatment and management of chronic HCV infections. Glecaprevir (GLE) and pibrentasvir (PIB) are pangenotypic DAAs that are delivered alone or as a fixed-dose oral formulation to treat chronic HCV infections with or without cirrhosis. Sensitive and dynamic bioanalytical assays are needed to understand the pharmacology of GLE and PIB. METHODS: Drug free K2EDTA plasma was spiked with GLE, PIB, and their internal standards. Drugs were extracted from plasma via protein precipitation, and subsequently quantified using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The method was validated according to regulatory recommendations, and evaluated in remnant plasma samples from individuals prescribed GLE and PIB. RESULTS: The analytical measuring ranges for GLE and PIB were 0.25-2000 ng/mL and 0.25-1000 ng/mL, respectively. The method showed acceptable accuracy and precision for both DAAs. GLE and PIB in plasma were stable following six freeze thaw cycles and at room temperature for up to 67 h. All analytes were stable in whole blood incubated at room temperature for 24 h, and at 40 °C and 100% humidity for 2 h. Negligible percent matrix effects were observed for PIB and PIB-IS across the measuring range of the assay. Significant ion suppression was observed for GLE, with an average matrix effects of 27.9%. However, relative matrix effects were < 6.3% between drug and internal standard, and deemed acceptable. Assay validation assessments in alternative matrices also met acceptance criteria. Both DAAs were successfully measured in remnant plasma samples from individuals administered GLE and PIB. CONCLUSIONS: An LC-MS/MS method for GLE and PIB quantification in plasma has been developed and validated. The assay met acceptable performance criteria and may be used in downstream applications to characterize DAA pharmacology for HCV treatment.

Development and validation of a multiplexed assay for the measurement of long-acting hormonal contraceptives in plasma via liquid chromatography-tandem mass spectrometry.

BACKGROUND: Exogenous progestins are an effective tool for hormonal contraception and family planning. Progestins may be delivered as oral pills, intramuscular or subcutaneous injections, vaginal rings, or intrauterine devices. Drug concentrations may vary based on the route and duration of delivery. Measurement of synthetic steroids in blood plasma can aid in determination of product adherence, evaluation of drug-drug interactions, and investigation of unintended pregnancies. METHODS: Drug-free K2EDTA plasma was spiked with the synthetic steroids etonogestrel (ETO), levonorgestrel (LNG), medroxyprogesterone acetate (MPA), and norethisterone (NET). Plasma was combined with isotopically labeled internal standards, and drugs were extracted via liquid-liquid extraction. Samples were then subjected to liquid chromatographic-tandem mass spectrometric (LC-MS/MS) analysis. The method was validated in accordance with regulatory recommendations. The assay was evaluated in a cohort of remnant plasma samples in individuals using one of the aforementioned progestins. RESULTS: The analytical measuring range for ETO, MPA, and NET was 20-10,000 pg/mL; the primary linearity for LNG was 20-20,000 pg/mL. The method showed acceptable precision and accuracy for all progestins. Stability was established for 72 h with room temperature storage and through 3 freeze-thaw cycles. All analytes were stable in whole blood incubated at room temperature for 25 h, and at 40°C and 100% humidity for 2 h. Ion suppression was observed for all analytes spiked in plasma; average ion suppression was 31.6%, 66.6%, 32.1% and 41.2% for ETO, LNG, MPA, and NET, respectively. However, internal standards showed comparable ion suppression, and relative matrix effects were minimal. ETO, LNG, MPA, and NET could also be quantified accurately in K3EDTA plasma and serum. Progestins were successfully measured in remnant samples from individuals using hormonal contraceptives. CONCLUSIONS: A multiplexed LC-MS/MS assay for the quantification of ETO, LNG, MPA, and NET has been developed and validated. The assay met acceptable performance characteristics and may be used in downstream studies to evaluate progestin pharmacology.