Bile acids, synthesized from cholesterol by hepatic enzymes, are excreted into the duodenum via the bile duct as their glycine and taurine conjugates. After assisting in the lipolysis and absorption of fats in the intestinal lumen, bile acids are then returned to the liver. According to this enterohepatic circulation, very small quantities of bile acids are present in blood of healthy subjects. However, in hepatobiliary diseases, disturbances in their synthesis and clearance by the liver, as well as absorption by the intestines cause changes in the level of not only bile acids but also their sulfates and glucuronides in the blood and urine. 1 Since the first report of the occurrence of the bile acid glucuronide in human urine, it has been believed that bile acids are conjugated with glucuronic acid through the inherent 3α -hydroxy group on the steroid nucleus to form their ether type glucuronides. 2-6 Recently, we have disclosed the formation of ester-type acyl glucuronides of bile acid conjugated through the 24-carboxy group in an incubation mixture with hepatic microsomal fractions from rats 7 , employing liquid chromatography (LC)/atmospheric pressure chemical ionization (APCI)-mass spectrometry (MS); here the acyl glucuronides were characterized as their acetate-methyl ester derivatives. In addition, we have synthesized the acyl glucuronides of cholic, chenodeoxycholic, deoxycholic, ursodeoxycholic and lithocholic acids as authentic specimens. 8 The present paper deals with the development of the simultaneous determination of the bile acid 24-glucuronides in human urine using LC/electrospray ionization (ESI)-MS without derivatization.
Experimental
LC/ESI-MSThe liquid chromatographic separation was performed with a Nano-space SI-1 (Shiseido, Ltd., Tokyo, Japan) on a Develosil ODS-HG-5 column (5 µm, 150×2.0 mm i.d.) (Nomura Chemical, Aichi, Japan) with a flow rate of 150 µl/min. The mass spectrometric detection was carried out using a JMS-LCmate doublefocusing mass spectrometer (JEOL, Tokyo, Japan) equipped with an ESI system in the negative ion mode. The resolution of the mass spectrometer was set at 750 and 5000 for the low-resolution and high-resolution analyses, respectively. For the rock-mass signal at m/z 602.9125, the standard solution containing sodium trifluoroacetic acid and sodium formic acid (each 50 µg/ml) in MeOH was added to the postcolumn effluent using a "T-type" connector at a flow rate of 50 µl/min.
MaterialsBile acid 3-9 and 24-glucuronides 8 (Fig. 1) A method for the separation and determination of bile acid 24-glucuronides using liquid chromatography (LC)/electrospray ionization (ESI)-mass spectrometry (MS) has been developed. In the ESI mode, 24-glucuronides were characterized by an intense deprotonated molecule [M-H] -with a fragment ion [M-H-176] -formed by elimination of the glucuronic acid moiety. The ratio of these negative ions was significantly influenced by the orifice-1 and the ring lens voltages. Bile acid 24-glucuronides in human urine were extracted with an OASIS HLB car...