The use of xanthine oxidase for a specific and sensitive fluorimetric determination of 6-mercaptopurine in serum

Abstract

A specific and highly sensitive fluorimetric method was developed for the determination of 6-mercaptopurine (6MP) in serum. The method is based on the enzymatic oxidation of 6MP with xanthine oxidase to the oxypurine, followed by oxidation with acidic chromate to the corresponding 6-sulfonate. The fluorescent product has excitation and emission maxima at 330 and 400 nm, respectively. The limit of sensitivity was approximately 22 pg/ml for 6MP in water. The sensitivity limit for 6MP in serum containing azathioprine was approximately 2.2 ng/ml. The rate constants for conversion of 6MP into the final product (6-thiouric acid) and the apparent Michaelis constant were also determined by a nonlinear regression analysis based on the integrated Michaelis-Menten equation using the ultraviolet absorbance data and the simplified complementary tristimulus colorimetry.