Simultaneous determination of a potassium channel opener and its metabolite in rat plasma with column-switching liquid chromatography using atmospheric pressure chemical ionisation

A specific LC-MS assay was developed for simultaneous determination of Ro 31-7837 (I) and its metabolite Ro 31-6930 (II) in rat plasma, using on-line SPE by column-switching reversed-phase HPLC combined with atmospheric pressure chemical ionization (APCI) tandem mass spectrometry for detection in the selected reaction monitoring mode. The method involved precipitation of plasma proteins with ethanol and automatic injection of a 1-ml aliquot of the supernatant onto a standard bore trapping column (LC-ABZ, 20x4.6 mm) for compound retention. Using the backflush mode, the analytes were transferred onto the analytical column (Kromasil C18, 125x4.0 mm) for chromatographic separation and mass spectrometric detection. The mean precision and accuracy for I and II in the concentration range 0.25-100 ng/ml were found to be 3.7% and 101%, and 3.5% and 106%, respectively. The data were assessed from QC samples during the validation phase of the assay. The lower limit of quantification for both I and II was 0.25 ng/ml, using a 0.5-ml plasma aliquot. This LC-MS method provided the requisite specificity, sensitivity, accuracy and precision to assess the pharmacokinetics of the compounds in the rat.