Application of high-performance liquid chromatography–mass spectrometry to detection of diuretics in human urine

A rapid, sensitive and reliable high-performance liquid chromatographic–mass spectrometric method for the detection of 25 diuretics in human urine has been developed. Atmosphere pressure chemical ionization (APCI) and electrospray ionization (ESI) modes were evaluated. A 2-ml volume of urine was extracted under basic conditions and separated on an Agilent Zorbax SB-C18 column (150×2.1 mm, 5 μm). The mobile phase consisted of formic ammonium–formic acid buffer (pH 3.5) and acetonitrile. The effects of capillary temperature, sheath gas pressure and compositions of mobile phase on the sensitivity were studied. The recoveries of most of the diuretics were 75–95%. In the full scan mode, the limits of detection of the 25 diuretics were 0.25–25 ng/ml for APCI and 0.6–250 ng/ml for ESI. Under the optimal conditions, 14 diuretics from authentic urine samples were detected successfully by LC–APCI-MS. To obtain more fragmentation information on the chemical structure for positive confirmation, tandem mass analysis was also investigated.