An enzymic digestion and solid-phase extraction procedure for the screening for acidic, neutral, and basic drugs in liver using gas chromatography for analysis.

Analysis of liver specimens is an important issue in forensic toxicology, but suitable workup and extraction methods for general screening purposes have been lacking until now. A workup and extraction scheme based on a recently developed procedure for the screening of biological fluids was developed that can be used for the screening of acidic, neutral, and basic drugs in liver. This method uses a single solid-phase extraction (SPE) column and gas chromatography-flame ionization detection (GC-FID) for the final analysis. First, the homogenized liver sample is sonicated and centrifuged; the resulting supernatant is applied to the SPE column. Elution of acidic, neutral, and some weakly basic drugs is then performed with acetone-chloroform and analyzed by GC-FID. Next, the pellet of tissue material obtained from the centrifugation is enzymically digested by subtilisin Carlsberg. This frees the drugs bound to the liver tissue. The resulting clear liquid is brought to the reconditioned SPE column. A wash step is introduced to remove acidic and neutral interferences and the basic drugs can then be eluted with ammoniated ethyl acetate. Using 100-mg wet liver samples spiked with 2 micrograms of amounts of various drugs, recoveries were 70-102% with relative standard deviations less than 9%. The resulting GC-FID chromatograms were virtually free of endogenous interferences. GC-nitrogen-phosphorous detection detected smaller amounts of nitrogen-containing drugs, again without endogenous interferences. With the SPE columns currently used, which contain a bed mass of 130 mg, the liver samples should be smaller than 200 mg because the endogenous compounds obtained after the digestion of the tissue will overload the column, which results in a lower recovery of the drugs of interest. Drugs that decompose under the digestion conditions (pH 10.5 at 60 degrees C for 1 h) may be lost in the present procedure. This phenomenon is being investigated further.