Nephrotoxicity, the most important toxicity in high-dose methotrexate (MTX) therapy, is partly caused by the formation of crystal deposits in the kidney due to poor water solubility of MTX and its metabolites 7-hydroxy methotrexate (7-OH MTX), deoxyaminopteroic acid (DAMPA) and 7-hydroxy deoxyaminopteroic acid (7-OH DAMPA). Plasma MTX level-guided urine alkalinization, leucovorin rescue and glucarpidase detoxification are common strategies to overcome MTX-related nephrotoxicity. However, overestimation is a problem for MTX analysis by immunoassays due to the cross-reactivity of MTX metabolites (7-OH MTX and DAMPA). An UHPLC-MS/MS method for the simultaneous determination of MTX, 7-OH MTX, DAMPA and 7-OH DAMPA in human urine was developed, validated and applied in clinical practice. Samples were treated by one-step protein precipitation and analyzed within 3 min. The calibration range was 0.02 to 4 μmol/L for MTX and DAMPA, and 0.1 to 20 μmol/L for 7-OH MTX and 7-OH DAMPA. For all analytes, the intra-day and inter-day bias and imprecision were -8.0 to 7.6 and <9.0%, the internal standard normalized recovery and matrix factor were 92.34 to 109.49 and <20.68%. The plasma MTX and 7-OH MTX levels increased with the urine drug levels, age, serum creatinine and alanine transaminase, but urine could not replace blood for MTX monitoring due to their poor correlation (R2, 0.16 to 0.51). Dose-normalized urine and plasma MTX and 7-OH MTX levels were similar between different patient groups (urine pH <7 or ≥7). Due to the large inter-individual variance of the analytes levels in both plasma and urine, these findings should be treated with caution.
Keywords: 7-hydroxy deoxyaminopteroic acid; 7-hydroxy methotrexate; UHPLC-MS/MS; deoxyaminopteroic acid; method development and validation; methotrexate; urine.