Comprehensive screening procedure for diuretics in urine by high-performance liquid chromatography

Abstract

A rapid and reliable screening procedure using high-performance liquid chromatography for the detection of 23 diuretics (belonging to five different pharmacological groups) in urine has been developed. Two aliquots of 2-ml urine samples were extracted separately under acidic and basic conditions. The acidic and basic extracts were pooled, evaporated to dryness and reconstituted in methanol. The methanolic extact was injected onto a Hewlett-Packard Hypersil ODS C 18 (5 μm) column (column I) and a Hewlett-Packard LiChrosorb RP-18 (5 μm) column (column II; an alternative column). The same gradient mobile phase was used for both columns. A diode array ultraviolet detector was set to monitor the signal to the integrator (Chem Station) at 230 and 275 nm. Recovery studies of the 23 diuretics were performed under acidic and basic conditions. The overall lower liits for detection on column I using both extraction procedures ranged from 0.5 to 1.5 μg/ml of urine (average 1.0 μg/ml). Amiloride, ethacrynic acid and probenecid could not be detected below 5 μg/ml of urine. No interference from the biological matrix was apparent. Amiloride could be detected in urine 4 h after oral administration of 15 mg of amiloride to a healthy volunteer, when the sample was extracted under alkaline conditions. The suitability of the screening method for the analysis of urine samples was tested by studying the variation with time of chlorthalidone, furosemide, probenecid, acetazolamide, quinethazone, spironolactone, bendroflumethiazide, bumetanide, triamterene and hydrochlorothiazide concentrations in the urine of normal human volunteers after minimum single or multiple (probenecid) doses. The results obtained indicate that the screening method employing either column I or II would be rapid and reliable to be used in doping control and clinical laboratories.