A reversed-phase high performance liquid chromatography method for quantification of methotrexate in cancer patients serum

• Silver nitrate as a precipitant of protein was developed.
• A rapid and sensitive HPLC method for determination of methotrexate was developed.
• The method was applied to therapeutic drug monitoring of MTX in cancer patients.

A simple, rapid and sensitive reversed-phase high performance liquid chromatography (HPLC) method has been developed for the determination of methotrexate in human serum. After deproteinization of the serum with 40% silver nitrate solution, methotrexate and internal standard (IS) were separated on a reversed-phase column with a mobile phase consisting of 10 mM sodium phosphate buffer (pH6.40)-methanol (78:22%, v/v) and ultraviolet detection at 310 nm. The linearity is evaluated by a calibration curve in the concentration range of 0.05–10.0 μg/mL and presented a correlation coefficient of 0.9995. The absolute recoveries were 97.52 ± 3.9% and 96.87 ± 3.7% for methotrexate and ferulic acid (internal standard), respectively. The intra- and inter-day precision were less 6.19 and 5.89%, respectively (n = 6). The limit of quantitation was 0.02 μg/mL and the limit of detection was 0.006 μg/mL. The complete analysis was achieved less than 10 min with no interference from endogenous components or 22 examined drugs. This method was validated by using serum samples from high-dose methotrexate treated patients with osteosarcoma, breast cancer, acute leukemia and lymphoma. The method was demonstrated to be a simple, rapid and reliable approach in quantification of methotrexate in serum samples from patients with high-dose methotrexate therapy.