HPLC determination of valproic acid in human plasma by derivatization with O-p-nitrobenzyl-N,N′-diisopropylisourea

A sensitive and specific method for the determination of valproic acid in plasma has been developed. After the proteins in the plasma have been precipitated with a saturated solution of ammonium sulfate in 1N HCl, the valproic acid, together with the internal standard, is extracted from the plasma with dichloromethane. An aliquot of the organic solution is taken for derivatization of the valproic acid and the internal standard with O‐p‐nitrobenzyl‐N,N′‐diisopropylisourea. Separation is carried out by HPLC using two chromatographic systems: an adsorption system with a μ Porasil column, hexane‐chloroform (94:6) as mobile phase, and caproic acid as internal standard and a partition reverse phase system comprising a μ Bondapak TM/C18 column, acetonitrile/methanol/0.0035 M phosphate buffer (60:10:30), and caprylic acid as internal standard. UV detection is at 254 nm. This method, developed in both systems, permits the determination of plasma levels of valproic acid in the reported range of 50‐100 μg/mL. With adequate sensitivity, specificity, precision, and accuracy. The plasma levels of valproic acid may be determined by this method without interference from the commonest antiepileptic drugs. Good correlation is obtained with the enzymatic immune analytic method: EMIT.