A bioanalytical method was developed and validated to estimate donepezil, 6-desmethyl donepezil and 5-desmethyl donepezil simultaneously in human plasma using galantamine as an internal standard (IS). The chromatographic separation was achieved on a reverse-phase XTerra RP (150 × 4.6 mm, 5 µm) column without affecting recovery (mean recovery > 60% with CV < 10%) for all analytes. ESI-MS/MS multiple reaction monitoring in positive polarity was used to detect mass pairs for donepezil (m/z 380.3 → 91.3), 6-desmethyl donepezil (m/z 366.4 → 91.3), 5-desmethyl donepezil (m/z 366.4 → 91.3) and galantamine m/z (288.1 → 213.0). The linearity was established over a dynamic range of 0.339-51.870, 0.100-15.380 and 0.103-15.763 ng/mL for donepezil, 6-desmethyl donepezil and 5-desmethyl donepezil, respectively. The current method shows that minimal conversion of labile metabolites to parent donepezil in plasma as stability was successfully achieved for 211 days at -15 °C storage temperature. The method was successfully applied to a clinical study after administration of 10 mg donepezil tablets to healthy male Indian volunteers.
A reliable, selective and sensitive liquid chromatography tandem mass spectrometry method was developed and validated for the quantification of lamotrigine in human plasma using lamotrigine-13C3, d3 as an internal standard. Analyte and internal standard were extracted from human plasma by solid-phase extraction and detected in positive ion mode by tandem mass spectrometry with electrospray ionization (ESI) interface. Chromatographic separation was performed on a Chromolith® SpeedROD; RP-18e column (50−4.6 mm i.d.) using acetonitrile: 5±0.1 mM ammonium formate solution (90:10, v/v) as the mobile phase at a flow rate of 0.500 mL/min. The calibration curves were linear over the range of 5.02–1226.47 ng/mL with the lower limit of quantitation validated at 5.02 ng/mL. The analytes were found stable in human plasma through three freeze (−20 °C)-thaw (ice-cold water bath) cycles and under storage on bench-top in ice-cold water bath for at least 6.8 h, and also in the mobile phase at 10 °C for at least 57 h. The method has shown good reproducibility, as the intra- and inter-day precisions were within 3.0%, while the accuracies were within ±6.0% of nominal values. The validated LC–MS/MS method was applied for the evaluation of pharmacokinetic and bioequivalence parameters of lamotrigine after an oral administration of 50 mg lamotrigine tablet to thirty-two healthy adult male volunteers.
Terbinafine, a widely used antifungal drug, is a challenging molecule for quantitative bioanalysis due to certain factors contributing assay variability. Despite previous attempts at human plasma determination of terbinafine, exhaustive stability of the drug or an internal standard was lacking. Internal standard stability with negligible variation throughout the analysis is an indicator of a reliable bioanalytical method as the majority of LC-MS/MS assays are based on analyte/IS response ratios for quantitation. A newly developed high-throughput simple LC-MS/MS method is described for human plasma determination of terbinafine using naftifine internal standard and eluting all compounds within 2 min. A solid-phase extraction of terbinafine achieving mean recovery of 84.3% (CV < 4%) without compromising sensitivity (limit of quantitation 5.11 ng/mL) or linearity (5.11-3014.19 ng/mL) is delineated in this paper. A heated nebulizer in positive multiple reaction monitoring mode was employed with transitions m/z 292.2 →141.1 and 288.2 →117.0 for terbinafine and naftifine, respectively, resulting in excellent chromatographic separation on a Hypurity Advance (50 x 4.6 mm, 5 µm) column. The developed method was successfully applied to clinical samples and for the first time demonstrated marked improved extraction efficiency and reliable long-term plasma stability results without any internal standard response variation during the entire course of study.
A LC-MS/MS method was developed for simultaneous estimation of valacyclovir and acyclovir in human plasma. Plasma sample was extracted with solid phase extraction technique and chromatographic condition was set with Inertsil CN-3 (5 µm) column and mobile phase (1 mM ammonium acetate buffer -methanol, 50:50 v/v). Valacyclovir, acyclovir, Valacyclovir D4 and acyclovir D4 were detected in positive polarity in multiple reactions monitoring mode at mass transitions (m/z) 325.2→152.1, 226.2→152.1, 329.3→152.1 and 230.2→152.1, respectively. The validated calibration curve range for valacyclovir is 4.09 to 725.63 ng/mL and for acyclovir is 50.35 to 10017.29 ng/mL. During method development, stability of acyclovir in whole blood could not be established over the period for 2 hr as the K WB / P ratio for acyclovir is greater than 1 and although for valacyclovir it is less than 1. Therefore, the drug distribution of acyclovir was investigated in whole blood and plasma. Experimental data showed that an initial drop of acyclovir level in plasma due to the cellular uptake of acyclovir by erythrocytes. Hence, the spiked comparison samples were allowed to reach equilibrium (between RBC and plasma). After reaching the equilibration time (30.0 min), plasma was harvested from the spiked whole blood and processed as per the proposed protocol. From the blood stability data, we concluded that valacyclovir and acyclovir both are stable in blood for 2 hrs. The developed method was validated as per current regulatory guidelines and applied for valacyclovir and acyclovir bio-equivalence study.
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