Simultaneous Determination of Urine Methotrexate, 7-Hydroxy Methotrexate, Deoxyaminopteroic Acid, and 7-Hydroxy Deoxyaminopteroic Acid by UHPLC-MS/MS in Patients Receiving High-dose Methotrexate Therapy.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).  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. 
UHPLC-MS/MS
However, overestimation is a problem for MTX analysis by immunoassays due to the cross-reactivity of MTX metabolites (7-OH MTX and DAMPA). 
immunoassays
Samples were treated by one-step protein precipitation and analyzed within 3 min. 
Protein precipitation