RESUMO
Objective: To establish an HPLC-fluorescence detection method for the determination of thioguanosine-monophosphate (TGMP), thioguanosine-diphosphate (TGDP) and thioguanosine-triphosphate (TGTP) in red blood cells (RBC), as well as quantify the individual thioguanine nucleotides metabolites in kidney transplant recipients with azathioprine (AZA) therapy.Methods: The individual thioguanosine phosphates were extracted from RBC by dichloromethane and subsequently oxidized by potassium permanganate.The separation was achieved on a Nucleosil C18 column (150 mm×4.6 mm,5 μm) with an ion pairing reagent and detected by a fluorescence detector (excitation at 315 nm, emission at 390 nm).The mobile phase consisted of 20 mmol·L-1 potassium phosphate buffer (pH was adjusted to 6.8 by 5 mmol·L-1 tetrabutylammonium hydrogensulfate)-acetonitril (80:20) with the flow rate of 1.0 ml·min-1.Results: TGMP, TGDP and TGTP were quantified from RBC within the range of 50-500, 50-1000 and 100-5 000 pmol·ml-1, respectively.The limit of quantification (LOQ) was 50, 50 and 100 pmol·ml-1 RBC for TGMP, TGDP and TGTP, respectively.The intra-and inter-day RSDs were below 7.0% with the method recovery between 95.0% and 103.6%.The mean extraction recovery was above 90%.The assay was applied in the blood samples of 30 kidney transplant recipients with AZA therapy, and the results indicated that TGTP was the predominant phosphate metabolite in RBC.Conclusion: The method is simple, rapid, sensitive and specific, and it can quantitatively determine the individual thioguanosine phosphates in RBC of kidney transplant recipients with AZA therapy.