Determination of the major metabolite of betahistine (2-pyridyl acetic acid) in human plasma by high performance liquid chromatography-tandem mass spectrometry

A specific HPLC−MS/MS assay was developed for the determination of the major metabolite of betahistine (2-pyridyl acetic acid) in plasma. The analyte was extracted from plasma by solid phase extraction technique. 3-Pyridyl acetic acid-d6 was used as the internal standard. Samples were applied to a Waters 515 HPLC system to deliver a mobile phase composed of a mixture of deionized water and acetonitrile in a ratio of 90: 10, (v/v) plus 0.2% formic acid. 2-Pyridyl acetic acid was monitored at the molecular ion m/z 137.818 and MS/MS (daughter) at m/z 91.998. 3-Pyridyl acetic acid-d6 was monitored at the molecular ion m/z 144.320 and MS/MS (daughter) at m/z 99.100. The separation was performed at 40°C. The analytical method was validated according to the Guidance for industry, Bioanalytical Method Validation, Food and Drug Administration (FDA). The results met the acceptance criteria as stated in the aforementioned guidance. The method was proved to be sensitive and specific. Linearity was established for the range of concentrations 2.5–600 ng/mL with a correlation coefficient (R) not less than 0.994. The lower limit of quantification (LLOQ) was identifiable and reproducible at 2.5 ng/mL with a precision of 3.6% of the coefficient of variation (CV). Long-term stability of 2-pyridyl acetic acid in plasma samples was investigated for a period beyond that needed for finalizing authentic (subject) samples analysis. Stability in the autosampler and at bench top was also established.