A sensitive liquid chromatography-tandem mass spectrometry method for the quantification of valacyclovir and its...

It is challenging to conduct a pharmacokinetic (PK) study on mice due to the limited amount of plasma one can obtain, which is also true for some clinical studies. Here, we developed and validated a simple, sensitive and robust LC-MS/MS method for measuring the prodrug valacyclovir (VACV) and its metabolite acyclovir (ACV) in mouse and human plasma. This assay utilized an acetonitrile protein precipitation method with isotope-labeled internal standards (IS) and enabled precise and accurate quantification of VACV and ACV in 10 μL plasma samples with a nine-min gradient. The analytes were separated on a Waters Atlantis T3 C18 column. The precursor-product ion transitions for VACV (m/z 325.2 > 152.1), ACV (m/z 226.2 > 152.1), VACV-D4 (m/z 329.2 > 152.1, IS) and ACV-D4 (m/z 230.2 > 152.1, IS) were detected in a multiple reaction monitoring (MRM) positive ion mode using an API4000 LC-MS/MS system. The lower limit of quantification (LLOQ) was 2 nM for both VACV and ACV. The linear range was validated over the concentration ranges of 2-200 nM and 200-5000 nM for both compounds. The matrix effect and stability of VACV and ACV were also evaluated. This assay was successfully applied to a PK study in mice.