Patent ID: 7563590

Claim:
A method for determining a level of methotrexate polyglutamates (MTXPGs) in a cellular lysate, comprising the steps of: (a) converting said MTXPGs to methotrexate (MTX) in said cellular lysate with gamma glutamyl hydrolase under conditions suitable for efficient conversion of MTXPGs to MTX; (b) determining said level of MTXPGs by UV irradiating and chromatographing said MTX under conditions effective to produce a fluorescent MTX photolytic product, having an excitation wavelength in the range of 250 nm to 290 nm, wherein the fluorescence excitation maxima of said photoproduct is 274 nm in water; (c) detecting fluorescence at an emission wavelength in the range of 440 nm to 500 nm, wherein the fluorescence emission maxima of said photoproduct is 464 nm in water; and (d) correlating the level of fluorescent MTX photolytic product with said level of MTXPGs in said cellular lysate by a standard curve.