Determination of intralumenal individual bile acids by HPLC with charged aerosol detection

Abstract

An isocratic HPLC charged aerosol detector (CAD) method was developed, validated, and applied for the determination of individual bile acids in human gastric and duodenal aspirates. The method requires a low volume of aspirates (50-100 ml) and minimal sample pretreatment. A Hypersil BDS RP-C 18 column (250 3 4.6 mm, 5 mm particle size) was equilibrated with a mobile phase composed of methanol-[ammoniun formate 20 mM, formic acid 0.5%, triethylamine 0.2% (pH 3)] 67:33 v/v. Its flow rate was 1 ml/min. The elution times for taurocholate, glycocholate, taurochenodeoxycholate, ursodeoxycholate, glycochenodeoxycholate, cholate, and glycodeoxycholate were approximately 9.9, 16.2, 18.2, 21.3, 31.6, 34.5, and 38.5 min, respectively. Calibration curves in the mobile phase were constructed in the concentration range of 0.5-500 mM. Limits of detection and quantification were in the range of 0.07-0.60 mM and 0.20-1.80 mM, respectively. This method was applied first, in gastric aspirates collected in the fasted state, in which bile acid presence is minimal and, second, in duodenal aspirates collected in the fed state, in which a large number of potentially interfering compounds exists. Intra-day relative standard deviation in fasted gastric aspirates and in fed duodenal aspirates was less than 2.2% and 6.0%, respectively.-Vertzoni, M., H. Archontaki, and C. Reppas. Determination of intralumenal individual bile acids by HPLC with charged aerosol detection. J. Lipid Res. 2008Res. . 49: 2690Res. -2695

Drug Supersaturation in Simulated Human Intestinal Fluids Representing Different Nutritional States

“…9 The method requires a low volume of aspirates (50-100 mL) and minimal sample pretreatment. A Hypersil BDS RP-C 18 column (250 mm  4.6 mm, 5 mm particle size) was equilibrated with a mobile phase composed of methanol [ammonium formate 20 mM, formic acid 0.5%, triethylamine 0.2% (pH 3)] 67:33 (v/v).…”

Kinetic Evidence for the Presence of Putative Germination Receptors in C lostridium difficile Spores

“…The concentration of taurocholate in intestinal fluids has been reported to be in the millimolar range in the upper intestine (40). In contrast, in the lower intestine, where C. difficile is postulated to germinate (15), taurocholate is deconjugated into taurine and cholate by the intestinal flora (35).…”

“…In the normal upper intralumenal space, chenodeoxycholate is undetectable (40). However, two inactive bile salts, taurochenodeoxycholate (compound X) and glycochenodeoxycholate (compound XI) are deconjugated in the lower intestine, increasing chenodeoxycholate concentration.…”

“…Using estimated metabolite concentration gradients in the digestive tract (18,40) together with our kinetic analysis, we hypothesize that in healthy individuals, C. difficile spores will remain dormant in the lower intestine regardless of the glycine concentration. In contrast, strong antibiotic treatment will deplete the gut flora.…”

Increasing the biorelevance of simulated intestinal fluids for better predictions of drug equilibrium solubility in the fasted upper small intestine

“…Ox bile salt extract was from Fluka Chemie GmbH, Germany (product No 86340, lot # 386645/1 12601). According to our HPLC-CAD analysis [12] the total bile salts content of the crude material used in the presenet study is 71.5 % w/w, and the following bile acids are present: taurocholic 22.2 % w/w, glycocholic 24.7 % w/w, taurochenodeoxycholic 1.7 % w/w, ursodeoxycholic 7.1 % w/w, glycochenodeoxycholic 1.3 % w/w, cholic 7.5 % w/w, glycodeoxycholic 7.1 % w/w. [3].…”