B-331 Homogeneous Enzyme Immunoassay for the Quantitative Determination of Methotrexate

Abstract

Abstract Background Methotrexate (MTX), a classical antifolate, can be safely administered over a wide dose range as maintenance chemotherapy for acute lymphoblastic leukemia and treatment of nononcologic diseases including rheumatoid arthritis or psoriasis. When combined with leucovorin (LV) rescue, high-dose MTX (HDMTX; doses of 1000–33 000 mg/m2) is usually administered as a prolonged i.v. infusion for a variety of cancers, including acute lymphoblastic leukemia, lymphoma, osteosarcoma, breast cancer, and head and neck cancer. HDMTX can be safely administered to patients with normal renal function by vigorously hydrating and alkalinizing the patient to enhance the solubility of MTX in urine. Serum levels may reach 1000 μmol/L or more. Pharmacokinetically guided LV rescue by monitoring MTX serum levels is required to prevent potentially lethal MTX toxicity. Ability to measure MTX accurately at 0.050 μmol/L enables clinical determination of non-toxic status Methods The ARK™ Methotrexate II Assay is a homogeneous enzyme immunoassay for the quantitative determination of methotrexate in human serum or plasma on automated clinical chemistry analyzers. The assay was evaluated on the Beckman AU680 analyzer. The assay consists of two reagents, six-level calibrators, and six-level (0.070, 0.400, 0.800, 5.0, 50.0, 500.0 μmol/L) quality controls. Performance of the assay was determined by assessing precision, limit of quantitation, linearity, endogenous substances interference, high sample dilution (1:10 serial dilutions), on-board auto dilution (at 1:10 and 1:50), cross-reactivity, and method comparison. Results Within-lab precision (%CV) for control levels were 3.00% (0.070 µmol/L), 1.40% (0.400 µmol/L), 2.05% (0.800 µmol/L), 1.58% (5.0 µmol/L), 2.30% (50.0 µmol/L), and 1.62% (500.0 µmol/L). Limit of Detection and Quantitation: LoD was 0.004 µmol/L and LoQ was 0.030 µmol/L (4.87% CV, 113.6% analytical recovery). The ARK Methotrexate II Assay showed less than 8% deviation from linearity over the range 0.030 to 1.300 µmol/L. Endogenous substances did not interfere with measurement of MTX at the levels tested. High sample manual dilution (2.0, 20.0, 200.0, 1200.0, 5.0, 50.0, and 500.0 µmol/L) and on-board auto dilution (1:10 and 1:50) results were within 10% of nominal values. Cross-reactivity to compounds (potentially co-administered drugs, folate derivatives, and compounds of similar structure) tested was <10% interference. The major metabolite, 7-hydroxy MTX, at 50 µmol/L produced less than 10% interference for MTX levels of 0.050 and 0.500 µmol/L. Passing Bablok analysis of the results for 90 patient specimens of ARK MTX II Assay compared to liquid chromatography with tandem mass spectrometry (LC-MS/MS) was 1.03 + 0.00 (r2 = 0.98). Conclusion The ARK™ Methotrexate Assay II quantitated methotrexate accurately and precisely in serum and plasma. Performance on the Beckman AU680 automated clinical chemistry analyzer demonstrated excellent precision, recovery, and clinical accuracy vs LC-MS/MS.