Development of a Simple and Rapid Method to Measure Free Fraction of Valproic Acid in Plasma Using Ultrafiltration and Ultra High Performance Liquid Chromatography–Mass Spectroscopy: Application to Therapeutic Drug Monitoring

Abstract

Valproic acid (VPA) is widely used as an antiepileptic drug in children, and it is usually coadministered with other antiepileptic drugs. Because of its narrow therapeutic range and large variations in pharmacokinetic/pharmacodynamic behavior in different individuals, therapeutic drug monitoring of the trough total VPA concentration is commonly used to guide dosing. However, as only the free fraction of VPA that exerts pharmacological and toxic effects, it may be more meaningful to determine the unbound VPA concentration in plasma. Free fraction of VPA in plasma was extracted by ultrafiltration. Ultra high performance liquid chromatography coupled with mass spectroscopy was used to measure VPA in the negative ionization mode. This method was validated by studies of its selectivity, linearity, lower limit of quantification, accuracy, precision, recovery, matrix effect, and stability. The method was validated over a linear range of 0.2-25 mcg/mL, and its lower limit of quantification was 0.2 mcg/mL. The method's relative standard deviations for intra- and inter-day precision were <15%, and its accuracy (relative error) was ±3.22%. The recoveries and matrix effect of unbound VPA at 3 different concentrations satisfied our requirements for the analysis of biological samples, and no significant degradation of VPA was observed under different storage conditions. Simple ultra high performance liquid chromatography coupled with mass spectroscopy showed good performance when used to measure unbound VPA concentration, this method may be used to study the relationship between unbound VPA concentrations and its effectiveness by the use of therapeutic drug monitoring.