HPLC DETERMINATION OF 1,2-DIETHYL-3-HYDROXYPYRIDIN-4-ONE (CP94), ITS IRON COMPLEX [FE(III) (CP94)3] AND GLUCURONIDE CONJUGATE [CP94-GLUC] IN SERUM AND URINE OF THALASSEMIC PATIENTS

Sensitive and selective high performance liquid chromatographic (HPLC) methods for the quantification of 1,2-diethyl-3-hydroxypyridin-4-one (CP94), its iron complex [Fe(III) (CP94)3] and glucuronide metabolite (CP94-GLUC) in urine and serum of thalassaemic patients are described. Three separate analyses are involved. The first assay quantifies both CP94 and its iron complex. This procedure requires the conversion of the iron complex to the free ligand and is carried out using diethylenetriaminepentaacetic acid (DTPA). CP94 and the internal standard, 1-propyl-2-ethyl-3-hydroxypyridin-4-one (CP95) present in either serum or urine are then extracted at pH 7.0 with dichloromethane. Extraction efficiency is 96.0 +/- 5.6% and 100 +/- 7.1% for CP94 and CP95, respectively, and 31.2 +/- 2.1% at 30 muM and 53.2 +/- 4.2% at 300 muM for the corresponding iron complex. In the second assay, samples are incubated (16 h) with beta-glucuronidase and processed as before. In this assay, the drug, its iron complex and glucuronide conjugate are measured. In the third assay the iron complex of CP94, [Fe(III) (CP94)3] is quantified. From the three separate analyses it is possible to calculate the individual concentrations of the three separate components present in serum and urine of thalassaemic patients. Calibration for both components, i.e. CP94 (assays 1 and 2) and its iron complex (assay 3) are linear with correlation coefficients >0.99 and are reproducible over the required concentration range of 0-500 muM for the free ligand and 0-100 muM for the iron complex. The minimum quantifiable level is 0.5 muM for the free ligand and 1.0 muM for the iron complex.