SIMULTANEOUS DETERMINATION OF A POTASSIUM CHANNEL OPENER AND ITS METABOLITE IN RAT PLASMA WITH COLUMN-SWITCHING LIQUID-CHROMATOGRAPHY USINGATMOSPHERIC-PRESSURE CHEMICAL-IONIZATION

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, usin g on-line SPE by column-switching reversed-phase HPLC combined with at mospheric pressure chemical ionization (APCI) tandem mass spectrometry for detection in the selected reaction monitoring mode. The method in volved precipitation of plasma proteins with ethanol and automatic inj ection of a I-mi aliquot of the supernatant onto a standard bore trapp ing column (LC-ABZ, 20x4.6 mm) for compound retention. Using the backf lush mode, the analytes were transferred onto the analytical column (K romasil C-18, 125x4.0 mm) for chromatographic separation and mass spec trometric 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 sampl es during the validation phase of the assay. The lower limit of quanti fication for both I and II was 0.25 ng/ml, using a 0.5-ml plasma aliqu ot. This LC-MS method provided the requisite specificity, sensitivity, accuracy and precision to assess the pharmacokinetics of the compound s in the rat.