SummaryA high-performance liquid chromatography (HPLC) method was developed for the simultaneous analysis of trimethoprim (TMP), sulphamethoxazole (SMX), and acetylsulphamethoxazole (AcSMX) in small amounts of blood. The method involved precipitation with 50 &mgr;L trichloracetic acid (1M) to 125 &mgr;L plasma or serum sample. 60 &mgr;L supernatant was added to 60 &mgr;L mobile phase, modified with 50 &mgr;L 1 M sodium hydroxide/mL. The mobile phase consisted of 20% acetonitrile and 80% phosphate buffer adjusted to pH 6.15. Using 125 &mgr;L of the sample, limits of quantitation were 0.1 &mgr;g/mL for TMP, 1.0 &mgr;g/mL for SMX, and 1.0 &mgr;g/mL for AcSMX. The precision of the method was 2% to 11% over the range of concentrations tested, 0.5–30 &mgr;g/mL for TMP, 5–300 &mgr;g/mL for SMX, and 2.5–150 &mgr;g/mL for AcSMX, respectively. No interference with other commonly used drugs was observed. The method is rapid, simple, specific, and sensitive enough for pharmacokinetic studies. The small amount of blood required makes it suitable for pediatric patients. The method was used to analyze samples from Tanzanian children aged 6–59 months participating in a cotrimoxazole (TMP/SMX)/chloroquine randomized trial for the treatment of uncomplicated malaria. Venous blood samples from 68 children were collected 2 hours after the first dose of TMP/SMX (4 mg/kg TMP/20 mg/kg SMX at two divided doses for 5 days) and again at treatment day 4. Individual variations in plasma concentrations of TMP, SMX, and AcSMX were considerable. The mean and SEM plasma concentrations (g/mL) of TMP, SMX, and AcSMX 2 hours after the first treatment dose were 2.0 ± 1.0 (range 0.5–6), 53 ± 22 (range 24–146), and 13.5 ± 12 (range 0–65), respectively. On the fourth day the attained plasma concentrations were not significantly different from samples collected after the first dose.