A validated method for simultaneous LCMSMS quantification of nicotine, cocaine, 6-acetylmorphine (6AM), codeine, and metabolites in 100 mg fetal human brain was developed and validated. After homogenization and solid-phase extraction, analytes were resolved on a Hydro-RP analytical column with gradient elution. Empirically determined linearity was from 5-5,000 pg/mg for cocaine and benzoylecgonine (BE), 25-5,000 pg/mg for cotinine, ecgonine methyl ester (EME) and 6AM, 50-5000 pg/mg for trans-3-hydroxycotinine (OH-cotinine) and codeine, and 250-5,000 pg/mg for nicotine. Potential endogenous and exogenous interferences were resolved. Intra- and inter-assay analytical recoveries were > or = 92%, intra- and inter-day and total assay imprecision were < or = 14% RSD and extraction efficiencies were > or = 67.2% with < or = 83% matrix effect. Method applicability was demonstrated with a postmortem fetal brain containing 40 pg/mg cotinine, 65 pg/mg OH-cotinine, 13 pg/mg cocaine, 34 pg/mg EME, and 525 pg/mg BE. This validated method is useful for determination of nicotine, opioid, and cocaine biomarkers in brain.