Quantitative bile acid profiling by liquid chromatography quadrupole time-of-flight mass spectrometry: monitoring hepatitis B therapy by a novel Na+-taurocholate cotransporting polypeptide inhibitor

• Published in: Analytical and bioanalytical chemistry Vol. 407; no. 22; pp. 6815 - 6825
• Main Authors: Haag, Mathias, Hofmann, Ute, Mürdter, Thomas E, Heinkele, Georg, Leuthold, Patrick, Blank, Antje, Haefeli, Walter E, Alexandrov, Alexander, Urban, Stephan, Schwab, Matthias
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer Berlin Heidelberg 01.09.2015
• Subjects: Adult; adverse effects; Analytical Chemistry; bile acids; Bile Acids and Salts - blood; Biochemistry; Biomarkers - blood; blood proteins; blood serum; Characterization and Evaluation of Materials; Chemistry; Chemistry and Materials Science; chronic hepatitis B; clinical trials; Drug Monitoring - methods; Female; Food Science; Hepatitis B - blood; Hepatitis B - diagnosis; Hepatitis B - drug therapy; History, Ancient; Humans; Laboratory Medicine; Male; mass spectrometry; methanol; monitoring; Monitoring/Environmental Analysis; polypeptides; Reproducibility of Results; Research Paper; reversed-phase liquid chromatography; Sensitivity and Specificity; Spectrometry, Mass, Electrospray Ionization - methods; therapeutics; Treatment Outcome; viruses; volunteers; Bile acids; Na; Liquid chromatography quadrupole time-of-flight mass spectrometry; Myrcludex B; Hepatitis D; taurocholate cotransporting polypeptide (NTCP); Hepatitis B
• ISSN: 1618-2642; 1618-2650
• DOI: 10.1007/s00216-015-8853-5

A novel analytical approach for the targeted profiling of bile acids (BAs) in human serum/plasma based on liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) is presented. Reversed-phase chromatography enabled the baseline separation of 15 human BA species which could be readily detected by accurate mass analysis in negative ion mode. Blood proteins were removed by methanol precipitation in the presence of deuterium-labeled internal standards which allowed BA quantification in 50 μl plasma/serum. The assay was validated according to FDA guidance achieving quantification limits from 7.8 to 156 nM. Calibration curves prepared in charcoal-stripped serum/plasma showed excellent regression coefficients (R ² > 0.997) and covered quantities from 7.8 to 10,000 nM depending on the analyzed species. Intra- and inter-day accuracy and precision were below 15 % for all analytes. Apparent extraction recoveries were above 97 %, and ion suppression rates were between 4 and 53 %. Mean BA level in serum/plasma from healthy volunteers ranged from 11 ± 4 nM (tauroursodeoxycholic acid) to 1321 ± 1442 nM (glycochenodeoxycholic acid). As a proof of concept, the assay was applied to plasma samples derived from a clinical phase I study of myrcludex B, a novel first-in-class virus entry inhibitor for the treatment of chronic hepatitis B and D. The results demonstrate that myrcludex-induced inhibition of the hepatic BA transporter Na⁺-taurocholate cotransporting polypeptide (NTCP) significantly affects plasma BA level. These observations provide novel insights into drug-induced metabolic responses and will be indispensable for the assessment of side effects and dose-finding processes during future clinical trials.