GLC determination of plasma acenocoumarol levels

A method for the quantitative estimation of acenocoumarol in plasma is described. Plasma containing acenocoumarol, to which a known amount of gamma-oxo derivative of phenylbutazone is added as an internal standard, is acidified and extracted with ethylene dichloride. The drug and the internal standard are then back-extracted into alkali, which, in turn, is acidified and reextracted with ethylene dichloride. The organic extract is evaporated and treated with an ethereal solution of diazomethane (100 microliter). The reacted mixture is evaporated, and the residue is dissolved in 25 microliter of carbon disulfide. Aliquots (2-3 microliter) are injected into a gas chromatograph equipped with a flame-ionization detector. The methyl derivatives of acenocoumarol and the internal standard give sharp, well-separated, symmetrical peaks. The method is of sufficient sensitivity to determine 0.25 microgram/ml of the drug in plasma with a relative standard deviation of 4%.