Abstract: Objective To establish a internal standard method to determine the serum concentration of Methotrexate(MTX) and apply in the low-dose MTX therapy. Methods The serum MTX concentration was determined by HPLC, with metron-idazole as internal standard. The separation was carried out using Shimadzu VP-ODS C18(250mm×4.6mm, 5μm) column with mobile phase of methanol-sodium acetate buffer(pH3.5) (10:90) at a flow rate of 1.0mL/min． The detection wavelength was 306nm and the column temperature was 25℃． And the serum concentration was examined within 1.5~2h after intravenous injection of the patients who received low-dose MTX. Then the patients were followed-up to observe the adverse reaction. Rusults The linear range was 20~1 000μg/L(r=0.9992, n=8)． The intra-day and inter-day precision were both less than 15%. The serum MTX concentration in patients with adverse reactions were significantly higher than those without them. Conclusion The method is sensitive, rapid and accurate. We demonstrated that serum MTX concentration is worthy of detection in low-dose MTX therapy.

Key words: methotrexate, serum concentration, adverse reaction