Small molecule adduct formation with the components of the mobile phase as a way to analyse valproic acid in human serum with liquid chromatography-tandem mass spectrometry.

Abstract

A valproic acid (VPA) LC-MS/MS analytical method using analyte adduct formation was developed and validated in human serum. The fragmentation of the sodium acetate adduct (mass transition: 225/143) and acetic acid adduct (mass transition: 203/143) were used as the target and qualifier mass transition, respectively. A Luna 5 μm C18 (2) 100 A, 150 mm×2 mm analytical column and a mobile phase consisting of A (H2O/methanol=95/5, v/v) and B (H2O/methanol=3/97, v/v), both with 10mM ammonium acetate and 0.1% acetic acid (pH=3.2) were used. A binary flow pumping mode with a total flow rate of 0.4 mL/min was applied. Protein precipitation with 1 mL of the mobile phase B was used as sample preparation. The calculated limit of detection/quantification was 0.45/1.0 μg/mL and the inter-/intra-day precision was <6%. The application of a deuterated internal standard resulted in a good adduct formation reproducibility. The strategy applied made the VPA LC-MS/MS analysis in human serum on the basis of two mass transitions possible. Therefore, it is an interesting alternative for the VPA pseudo multiple reaction monitoring methods (mass transition 143/143) and a proof that the developed strategy is also useful for the analysis of compounds which do not produce any stable ion fragments detectable by tandem mass spectrometry.