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A sensitive and specific GLC method is described to determine therapeutic levels of tilidine and its two main metabolites, nortilidine and bisnortilidine, in plasma and urine. The method involves the extraction of the compounds and an internal standard with cyclohexane from alkalinized samples, followed by back-extraction into 1NHCl. The hydrochloric acid solution is evaporated to dryness. After liberation of the free bases with ammonia, the residue is subjected to GLC analysis with a nitrogen phosphorus detector and a 1.8-m (6-ft) glass column packed with 1% CRS 101 and 1.5% LAC-4-R-886 on Gas Chrom Q. Sensitivity in plasma and urine is ~1ng/ml for a 5-ml sample.