Abstract
A rapid and sensitive method is described for the quantification of ursodiol and its major metabolites glycoursodeoxycholic acid (GUDCA) and tauroursodeoxycholic acid (TUDCA) in human plasma using single internal standard (Ursodeoxycholic Acid d4). Solid phase extraction was performed and chromatographic separation of 5µL injected sample was achieved using Waters Xterra, 5µm column with a mobile phase comprised of methanol and 5 mM ammonium formate with 0.1 % acetic acid ( 70 : 30, v/v ). The mass spectrometer was used in negative ion mode and multiple reactions monitoring using electro spray ionization mode as an interface. The method was fully validated and the calibration curves were linear over the concentration range of 25.9 to 15300.1 ng/mL for ursodiol, 2.7 to 1587.5ng/mLfor tauroursodeoxycholicacid and 25.4 to 15040.9 ng/mL for glycoursodeoxycholic acid. The method was sensitive and specific, with the lower limit of quantification of 25.9, 2.7 and 25.4 ng/ml for ursodiol, tauroursodeoxycholic acid and glycoursodeoxycholic acid respectively. The present method includes a simple and rapid sample preparation with shorter analysis run time and less flow rate compared to previously reported methods. The method was applied successfully for a bioequivalence study in healthy subjects.
Highlights
Ursodiol, 3α,7β-dihydroxy-5β-cholan-24-oic acid is a naturally occurring bile acid found in small quantities in human plasma
The present study describes a highly sensitive, rapid, reliable liquid chromatography/tandem mass spectrometry (LC-MS/MS) method for the simultaneous quantification of ursodiol, tauroursodeoxycholic acid and glycoursodeoxycholic acid in human plasma using single internal standard Ursodeoxycholic Acid D4
The assay was found to be linear for Ursodiol concentrations in the range 25.88 to 15300.05 ng/mL, 2.68 to 1587.48 ng/mL for Tauroursodeoxycholic acid and 25.44 to 15040.93 ng/mL for Glycoursodeoxycholic acid
Summary
3α,7β-dihydroxy-5β-cholan-24-oic acid is a naturally occurring bile acid found in small quantities in human plasma. Several analytical methods [4,5,6,7] have been developed for the determination of bile acids in biological fluids. These methods may not be suitable for the processing of multiple samples in a limited period of time for pharmacokinetic studies.
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