Determination of trimethoprim in low-volume human plasma by liquid chromatography

Trimethoprim is an anti-infective agent used in the treatment of urinary and respiratory tract infections and mild to moderate pneumocystis carinii pneumonia. Trimethoprim is also a selective in vitro inhibitor of cytochrome P450 2C8 and may have utility as an in vivo inhibitor of this enzyme. A simplified high performance liquid chromatography (HPLC) method was developed to determine trimethoprim in human plasma. Samples are processed by protein precipitation with perchloric acid and chromatographic separation is achieved on a Synergi Polar-RP column (4 micron, 150 mm×4.6 mm) using a mobile phase consisting of 50 mM ammonium formate-acetonitrile-methanol (pH=3.0; 90:6:4 (v/v/v)). Detection is monitored at 280 nm. Intra- and inter-day precision ranged from 1.1 to 1.9 and 0.9 to 4.1%, respectively. The assay is simple, economical, precise, and is directly applicable to human studies involving steady state trimethoprim pharmacokinetics.