A reversed-phase high-performance liquid chromatography method for the determination of cotrimoxazole (trimethoprim/sulphamethoxazole) in children treated for malaria

A high-performance liquid chromatography (HPLC) method was developed for th e simultaneous analysis of trimethoprim (TMP), sulphamethoxazole (SMX), and acetylsulphamethoxazole (AcSMX) in small amounts of blood. The method invo lved precipitation with 50 mu L trichloracetic acid (1M) to 125 mu L plasma or serum sample. 60 mu L supernatant was added to 60 mu L mobile phase, mo dified with 50 mu L 1 M sodium hydroxide/mL. The mobile phase consisted of 20% acetonitrile and 80% phosphate buffer adjusted to pH 6.15. Using 125 mu L of the sample, limits of quantitation were 0.1 mu g/mL for TMP, 1.0 mu g /mL for SMX, and 1.0 mu g/mL for AcSMX. The precision of the method was 2% to 11% over the range of concentrations tested, 0.5-30 mu g/mL for TMP, 5-3 00 mu g/mL for SMX, and 2.5-150 mu g/mL, for AcSMX, respectively. No interf erence with other commonly used drugs was observed. The method is rapid, si mple, specific, and sensitive enough for pharmacokinetic studies. The small amount of blood required makes it suitable for pediatric patients. The met hod was used to analyze samples from Tanzanian children aged 6-59 months pa rticipating in a cotrimoxazole (TMP/SMX)/chloroquine randomized trial for t he treatment of uncomplicated malaria. Venous blood samples from 68 childre n 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. Indi vidual variations in plasma concentrations of TMP, SMX, and AcSMX were cons iderable. The mean and SEM plasma concentrations (g/mL) of TMP, SMX, and Ac SMX 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 th e fourth day the attained plasma concentrations were not significantly diff erent from samples collected after the first dose.