Simultaneous determination of three dipeptides (JBP485, Gly-Sar and JBP923) in the cell lysates by liquid chromatography-tandem mass spectrometry: application to identify the function of the PEPT1 transfected cell.

A simple and rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous determination of JBP485, Gly-Sar and JBP923 in the cell lysates using methanol as a deproteinization solvent was developed and validated. Detection was performed by turbo ionspray ionization in multiple reaction monitoring mode using the transitions of m/z 147.1 → m/z 90.1 for Gly-Sar, m/z 201.1 → m/z 86.1 for JBP485, m/z 219.1 → m/z 86.1 for JBP923 and m/z 152.0 → m/z 110.0 for paracetamol (internal standard). The analytes were separated on a Hypersil ODS C18 HPLC column using isocratic elution mode with a mobile phase containing 0.1% formic acid in water-methanol (97:3, v/v) at a flow rate of 0.2 mL/min. The calibration curves were demonstrated to be linear over the concentration range of 5.00-5000 nm with coefficient of 0.9968 for Gly-Sar, 0.9975 for JBP485 and 0.9952 for JBP923. The intra- and inter-day precisions were <10.2% for each quality contro; level, and the accuracy was within ±5.6% for each analyte. The matrix effect, the extraction recovery and stabilities of LC-MS/MS analysis were also investigated. This validated method was successfully applied to the simultaneous determination of JBP485, Gly-Sar and JBP923 in the cell lysates for identification of stably transfected HeLa cells with human PEPT1.