Supercritical Fluid Chromatography for Therapeutic Drug Monitoring and Toxicology: Methodological Considerations for Open Capillary Tubular Column for the Analysis of Phenobarbital in Serum

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Abstract
Methodological considerations are presented here for the application of SFC for clinical drug analysis using an open-tubular capillary column with polymethylsiloxane as the stationary phase. Preliminary studies of analysis of phenobarbital in serum showed that the use of liquid-liquid extractions, and recently introduced microfilters did not prevent rapid deterioration of column performance. Through systematic studies with solid-phase, C-18 extraction columns, a retention gap and a non-polar mixture of n-pentane/methylene chloride(25:75) for reconstituting the extracts, the feasibility was established. As a result of the lack of chromatographic interferences as shown by analysis of extract of drug-free serum, the procedure was used to estimate a patient's phenobarbital concentration of about 20 mg/L, comparable to a clinically established determination by fluorescence polarization immunoassay. Precision studies showed comparable mean concentrations for the measurement of quality control samples, but with marginally acceptable coefficients of variation. Preliminary extraction studies showed that other antiepileptics - phenytoin, secobarbital and pentobarbital were identifiable in SFC chromatograms.