Controlled ex-vivo plasma hydrolysis of valaciclovir to acyclovir demonstration using tandem mass spectrometry.

Plasma estimation of valaciclovir, an antiviral drug, is challenging due to both in-vivo and ex-vivo hydrolysis to active metabolite acyclovir. A simultaneous method is described involving the solid-phase ion-exchange extraction procedure requiring 100 µL of plasma volume, a reverse-phase Lichrosphere RP Select B (125 × 6 mm, 5 µm) column and isocratic mobile phase to achieve the desired chromatographic separation. ESI-MS/MS multiple reaction monitoring in positive polarity, detected mass pairs for valaciclovir (m/z 325.5 → 152.2), acyclovir (m/z 226.3 → 152.2) and respective internal standards valganciclovir (m/z 307.1 → 220.3) and acyclovir-d4 (m/z 230.2 → 152.0). Fully fledged method validation was evaluated as per current regulatory requirements and results were deemed acceptable. The plasma samples showed extensive hydrolysis of valaciclovir when collected or processed at room temperature, without buffer stabilization prior to storage at -15°C. Our results showed that using prechilled K3 EDTA vacutainers immersed in an iced-water bath during blood sample collection, and addition of 50% orthophosphoric acid solution to plasma samples prior to storage at -50°C for at least 120 days controlled the hydrolysis of valaciclovir to acyclovir. While monitoring drug absorption into systematic circulation, the valaciclovir to acyclovir formation ratio was improved to 1:20 in healthy volunteers for the first time.

Simultaneous determination of propranolol and 4-hydroxy propranolol in human plasma by solid phase extraction and liquid chromatography/electrospray tandem mass spectrometry.

A very sensitive, reliable, reproducible and highly selective assay for the simultaneous determination of free and total (conjugated and unconjugated) propranolol and its equipotent hydroxyl metabolite, 4-hydroxy propranolol, in human plasma was developed and validated. The analytes were simultaneously extracted from 0.300 mL of human plasma using solid phase extraction and detected in positive ion mode by tandem mass spectrometry with a turbo ionspray interface. Deuterium-labeled propranolol and 4-hydroxy propranolol, propranolol-d7 and 4-hydroxy propranolol-d7, were used as internal standards. The method has a lower limit of quantitation (LOQ) of 0.20 ng/mL for both analytes with the limits of detection (LOD) 50 and 100 pg/mL for propranolol and 4-hydroxy propranolol, respectively, based on a signal-to-noise ratio of 5. The assay was linear over a range 0.20-135.00 ng/mL for free propranolol and 0.20-25.00 ng/mL for free 4-hydroxy propranolol and linear over range 1.00-500.00 ng/mL for total propranolol and 1.00-360.00 ng/mL for total 4-hydroxy propranolol, with coefficient of determination greater than 0.99 for both analytes. The extraction recoveries were >96 and >64% on an average for propranolol and 4-hydroxy propranolol, respectively. The analytes were found stable in human plasma through five freeze (-15 degrees C)-thaw (room temperature) cycles and under storage on bench-top for at least 6.5 h, and also in mobile phase at 10 degrees C for at least 48 h. The method has shown tremendous reproducibility, with intra- and inter-day precision <11.3% (RSD), and intra- and inter-day accuracy <11% of nominal values, for both analytes, and has proved to be highly reliable for the analysis of clinical samples.