Determination of methotrexate in serum by high-performance liquid chromatography

A sensitive high-performance liquid chromatographic method with ultraviolet detection was developed to quantitate methotrexate in serum-based calibrators, controls and patient samples. Sample clean-up was achieved with C18 Sep-Pak Classic cartridges. The chromatographic separation was accomplished on a 5-microns Ultrasphere ODS Beckman column. 8-Chlorotheophylline was used as an internal standard. The method was validated by recovery, linearity, accuracy and precision studies. Two standard curves were constructed to cover the high and the low ends of the calibrator range (0.05-1.0 mumol/l). Response was found linear over the whole range of the calibrator set with a correlation coefficient of 0.999 and 1.00 for the low-level and the high-level curves, respectively. Accuracy varied from 12% at the lowest level to 1.2% at the highest level. The precision study showed a C.V. of 14.4% at the lowest level and 3.3% at the highest level.