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Development of a HPLC method for the determination of losartan urinary metabolic ratio to be used for the determination of CYP2C9 hydroxylation phenotypes

Abstract:

Background: Losartan is metabolized to losartan carboxylic acid (E-3174) by the polymorphic cytochrome CYP2C9. The aim of the study was to develop a high-performance liquid chromatographic (HPLC) method with fluorescence detection for simultaneously measuring losartan and its metabolite E-3174 in urine to evaluate the losartan urinary metabolic ratio (MR: losartan/E-3174) for CYP2C9 phenotyping in humans. Methods: The compounds were separated in a reversedphase chromatographic column and detected by fluorescence at a wavelength of 250 nm for excitation and of 370 nm for emission. Results: No analytical interferences with endogenous compounds were found, and the extraction recoveries were over 88%. Limits of quantification of 2 ng mL-1 for losartan and 5 ng mL-1 for E-3174 were achieved, as well as good reproducibility with coefficients of variation of < 9%in all cases. Analyses with the present HPLC method show significant differences (p < 0.05) in losartan MRs between the four CYP2C9 genotype groups in 13 Spanish healthy volunteers. Conclusions: The method developed is simple and affordable, as well as sensitive and reliable to calculate the MR. Therefore, it appears to be useful for CYP2C9 phenotyping using losartan as a drug test in populations, such as Hispanics with different allele combinations.