New assay for measuring valproic acid in plasma by conjugation to hydrazides

Abstract

Valproic acid (VA) is an anticonvulsant used to treat mania, schizophrenia, bipolar disorders, panic states and post‐traumatic stress disorder. Recently, VA has been shown to improve survival to hemorrhagic and septic shock in rats and pigs. However, the measurement of VA in plasma is difficult by standard chromatography and spectroscopy. We have developed a method that extracts VA and an internal standard (2‐phenyl butyric acid) from 10μl plasma after addition of 20μl 1M NaCl, 100μl ethyl acetate and 400μl acetonitrile. Sample is vortexed, centrifuged, and the supernatant removed and dried, and conjugated to 7‐diethylaminocoumarin‐3‐carboxylic acid, hydrazide (DCCH, Invitrogen). The conjugate is measured by Fluorometry (2020 Jasco) with λex=415nm, λem=465nm after separation by Reverse Phase High Pressure Liquid Chromatography (Beckman Coulter). A 2‐parameter exponential non‐ linear regression, y‐a(1‐bx), was fitted (r2=0.991) to the standard curve (0.033 to 10mg/ml VA, or 12.5μg to 41ng/injection). Coefficient of Variation was 3.9 and 1.8% for high and low pools. This method can be used to measure the reported dosage range of VA in human or animal plasma. Supported by the ARMY MRMC.