Use of enzymatic solubilization of tissues and direct injection on pre-columns of large volumes for analysing biological samples by high-performance liquid chromatography.
A pre-column technique, which can be used to introduce all types of biological samples into high-performance liquid chromatographic systems, is described. Fluids are injected directly, whereas faeces are homogenized and centrifuged and tissue samples are solubilized by the enzyme subtilisin A. Acetonitrile is added to all samples (no precipitation of proteins is seen), fluids (10%), solubilized tissue (25%) and faeces (20%), to obtain better wettability of the packing and to counteract binding. Samples of up to 50 ml are injected onto a 6 X 4 mm I.D. pre-column from which the compounds are backflushed onto the analytical column. Different packing materials for the pre-column have been used; LiChroprep RP-18 gave the best results. This system has been used for a series of different drugs, using different analytical columns and different detectors. Both gradient and isocratic elution have been used. High recoveries, good reproducibility and low detection limits are seen for routine analysis. For metabolic work, a very large enrichment factor is obtained.
D001711:Biotransformation; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods*; D004798:Enzymes; D005243:Feces / Q000032:analysis; D006801:Humans; D004364:Pharmaceutical Preparations / Q000032:analysis / Q000097:blood; D012997:Solvents
D004798:Enzymes; D004364:Pharmaceutical Preparations; D012997:Solvents