A method is described for a one step acetonitrile precipitation of serum or plasma and subsequent analysis of pentobarbital by reverse phase HPLC. The results of using two internal standards, N,N-Diethyl-m-toluamide and 5-(p-Methylphenyl)-5-phenylhydantoin are compared. Internal standard is added to serum (as little as 25 μL) and vortex-mixed with acetonitrile followed by centrifugation. An aliquote of the supernatant is analyzed on a C18 reverse phase column eluted with metanol/0.05 M (NH4)2HPO4, pH 8/water (55/20/25, v/v/v). The effluent is monitored at 220 nm.
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Ferrell et al. (1983) studied this question.
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