Determination of valproic acid concentration in human serum by LC-MS/MS and its application in therapeutic drug monitoring in patients with epilepsy

Background Valproic acid (VPA), a first-line broad-spectrum antiepileptic drug, is widely used in the treatment of various types of epilepsy. It has a narrow effective therapeutic concentration range (50–100 μg·mL -1 ) and significant individual variability in pharmacokinetics. Therefore, the implementation of therapeutic drug monitoring (TDM) for VPA is crucial for achieving individualized and precise medication in epilepsy patients. Objective The objective of this study was to develop and validate an accurate and rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for determining VPA concentrations in human serum, and to apply it to routine TDM in epilepsy patients. Methods Human serum samples were pretreated using a one-step precipitation method, with VPA-D6 as the internal standard (IS). Chromatographic separation was performed on a Kinetex® C18 column (50 × 2.1 mm, 2.6 μm) at a column temperature of 40 o C, using gradient elution at a flow rate of 0.4 mL·min -1 , with an injection volume of 1 μL. Mass spectrometry was conducted using an electrospray ionization (ESI) source in negative ion mode with multiple reaction monitoring (MRM) for quantitative analysis. The quantitative detection ion pairs for VPA and the IS were m/z 143.0→143.0 and m/z 149.0 → 149.0, respectively. The method's specificity, linearity, lower limit of quantification (LLOQ), accuracy, precision, and stability were comprehensively validated according to bioanalytical guidelines, and it was applied to TDM analysis of serum samples from 337 epilepsy patients. Results The concentration of VPA showed excellent linearity in the range of 5–200 μg·mL -1 (r = 0.9998), with an LLOQ was 5 μg·mL -1 . The relative standard deviation ( RSD ) of intra-day and inter-day precision was less than 5%. The accuracy ranged from 90.6% to 103.9%. Clinical application results indicated that among 337 TDM samples from epilepsy patients, 215 cases were within the effective therapeutic concentration, with a compliance rate of 63.80%. Conclusion The LC-MS/MS method established in this study is simple, rapid, and reliable. It satisfies the clinical requirements for routine TDM of VPA in epilepsy patients and provides technical support for optimizing individualized and precise medication management.