Bile Acid Profiling and Quantification in Biofluids using. Ultra-Performance Liquid Chromatography Tandem. Mass Spectrometry

Transcription

1 Bile Acid Profiling and Quantification in Biofluids using Ultra-Performance Liquid Chromatography Tandem Mass Spectrometry Magali H. Sarafian *, Matthew R. Lewis *, Alexandros Pechlivanis, Simon Ralphs, Mark J. W. McPhail, Vishal C. Patel#, Marc-Emmanuel Dumas, Elaine Holmes, Jeremy K. Nicholson ** Imperial College London, Division of Computational Systems Medicine, Department of Surgery and Cancer, Sir Alexander Building, Exhibition Road, South Kensington, London SW7 2AZ, UK, Imperial College London, MRC NIHR National Phenome Centre, Department of Surgery and Cancer, IRDB building, Du Cane Road, London W12 0NN, UK, Department of Hepatology, Imperial College London, St Mary's Hospital, Paddington, London, #Institute of Liver Sciences, King's College Hospital NHS Foundation Trust, Division of Transplantation Immunology & Mucosal Biology, MRC Centre for Transplantation, King s College London * These authors contributed equally to the work **Corresponding Author j.nicholson@imperial.ac.uk (J.K.N.) ABSTRACT: Bile acids are important end products of cholesterol metabolism. While they have been identified as key factors in lipid emulsification and absorption due to their detergent properties, bile acids have also been shown to act as signaling molecules and intermediates between the host and the gut microbiota. To further the investigation of bile acid functions in humans, an advanced platform for high throughput analysis is essential. Herein we describe the development and application of a 15 minute UPLC chromatographic procedure for the separation of bile acid species from human biofluid samples requiring minimal sample preparation. High resolution time of flight mass spectrometry was applied for profiling applications, elucidating rich bile acid profiles in both normal and disease state plasma. In parallel, a second mode of detection was developed utilizing tandem mass spectrometry for sensitive and quantitative targeted analysis of 145 bile acid (BA) species including primary, secondary and tertiary bile acids. The latter system was validated by testing the linearity (LLQ nm and ULQ µm), precision ( 6.5%) and accuracy ( %) on inter- and intra-day analysis achieving good recovery of bile acids (serum/plasma 88% and urine 93%). The UPLC-MS/MS targeted method was successfully applied to plasma, serum, and urine samples in order to compare the bile acid pool compositional difference between pre-prandial and post-prandial states, demonstrating the utility of such analysis on human biofluids. S-1

2 S N + CH 3 C H 3 H 3 C H CH 3 H 3 C H 3 C H Sulfur trioxide pyridine complex H Lithocholic acid S H Lithocholic acid sufated Figure S-1. Sulfation scheme with sulfur trioxide-pyridine complex. Example shown for lithocholic acid with sulfation on carbon position 3 in the steroid nucleus. S-2

3 Table S-1. UPLC-MS/MS settings for the 57 bile acid standards, including 36 non-conjugated, 12 conjugated with taurine, 9 conjugated with glycine and 16 deuterated standards. Individual optimization of selected ion monitoring (SIM) and multiple reaction monitoring (MRM) transitions, collision energy (CE) and retention time (RT) was implemented. Abbreviations of BAs observed in Figure 1. Numbers Names and abbreviations SIM (m/z) CE (ev) RT (min) 1 Ursocholanic Acid Lithocholenic Acid Cholenic Acid-3β-ol Ketocholanic Acid Isolithocholic Acid Allolithocholic Acid α,12α, 23-Nordeoxycholic Acid (11), (5β)-Cholenic Acid-3α-ol-12-one α-Cholanic Acid-3, 6-dione ,7-Diketocholanic Acid ,6-Diketocholanic Acid ,12-Diketocholanic Acid (14),(5β)-Cholenic Acid-3α, 12α-diol β-Cholenic Acid-7α-ol-3-one α-Cholanic Acid-3α-ol-6-one α-Hydroxy-7 Ketolithocholic Acid α-Hydroxy-12 Ketolithocholic Acid Lithocholic acid β-Cholanic Acid-3β, 12α-diol Chenodeoxycholic Acid (CDCA) Deoxycholic Acid (DCA) Hyodeoxycholic acid Isodeoxycholic Acid Murocholic Acid (MuroCA) Ursodeoxycholic acid (UDCA) ,7,12 Dehydrocholic acid α-Hydroxy-7,12-DiketoCholanic Acid α-Hydroxy-6,7-DiketoCholanic Acid β-Cholanic Acid-3α, 6α-diol-7-one Dehydrocholic Acid Dehydrocholic Acid α Muricholic Acid β Muricholic Acid ω Muricholic Acid Cholic acid (CA) Hyocholic acid (HCA) Numbers Names and abbreviations MRM transitions and SIM (m/z) CE (ev) RT (min) 37 Glyco-ursocholanic Acid Glycolithocholic Acid (GLCA) Glycoursodeoxycholic Acid (GUDCA) Glycohyodeoxycholic Acid (GHDCA) Glycochenodeoxycholic Acid (GCDCA) Glycodeoxycholic acid (GDCA) ,7,12 Glycodehydrocholic acid Glycocholic Acid (GCA) Glycohyocholic Acid (GHCA) Taurolithocholic Acid (TLCA) Tauro-ursocholanic Acid Tauro-ursodeoxycholic Acid (TUDCA) Taurohyodeoxycholic Acid Taurochenodeoxycholic Acid (TCDCA) Taurodeoxycholic Acid (TDCA) ,7,12 Taurodehydrocholic acid Taurohyocholic Acid (THCA) Tauro-β Muricholic Acid Tauro-α Muricholic Acid Tauro ω-muricholic Acid (TωM) Taurocholic Acid (TCA) Lithocholic Acid-d Deoxycholic Acid-13C Chenodeoxycholic Acid-d Ursodeoxycholic Acid-d Hyodeoxycholic Acid-d Cholic Acid-d Glycolithocholic Acid-d Glycochenodeoxycholic Acid-d Glycodeoxycholic Acid-d Glycoursodeoxycholic Acid-d Glycocholic Acid-d Taurolithocholic Acid-d Taurochenodeoxycholic Acid-d Tauroursodeoxycholic Acid-d Taurocholic Acid-d Taurodeoxycholic Acid-d S-3

4 Table S-2. UPLC-MS/MS settings for mono-sulfated bile acid standards sorted according to sulfation ratio and retention time, including; 44 BAs sulfated in position H-C3, 15 sulfated in position H-C6, 21 sulfated in position H-C7 and 8 sulfated in position H-C12 sulfated. Individual optimization of multiple reaction monitoring (MRM) transitions, collision energy (CE) and retention time (RT) was implemented. Abbreviations of BAs observed in Figure 1. BA number Names an Abbreviations H group MRM transitions CE (m/z) (ev) a b c 74 Lithocholenic acid 3-H Cholenic Acid-3β-ol 3-H Isolithocholic Acid 3-H Allolithocholic Acid 3-H Lithocholic acid (LCA-S) 3-H α,12α, 23-Nordeoxycholic Acid 3-H; 12-H (11), (5β)-Cholenic Acid-3α-ol-12-one 3-H β-Cholenic Acid-7α-ol-3-one 3-H α-Cholanic Acid-3α-ol-6-one 3-H α-Hydroxy-7 Ketolithocholic Acid 3-H α-Hydroxy-12 Ketolithocholic Acid 3-H (14),(5β)-Cholenic Acid-3α, 12α-diol 3-H; 12-H Murocholic Acid 3-H; 6-H Isodeoxycholic Acid 7-H; 12-H β-Cholanic Acid-3β, 12α-diol 3-H; 6-H Chenodeoxycholic Acid 3-H; 7-H Hyodeoxycholic acid 3-H; 6-H Ursodeoxycholic acid 3-H; 7-H Deoxycholic Acid 3-H; 12-H α-Hydroxy-7,12-DiketoCholanic Acid 3-H α-Hydroxy-6,7-DiketoCholanic Acid 3-H β-Cholanic Acid-3α, 6α-diol-7-one 3-H; 6-H Dehydrocholic Acid 7-H; 12-H Dehydrocholic Acid 3-H; 7-H α Muricholic Acid 3-H; 6-H; 7-H ω Muricholic Acid 3-H; 6-H; 7-H β Muricholic Acid 3-H; 6-H; 7-H Cholic acid 3-H; 7-H; 12-H Hyocholic acid 3-H; 6-H; 7-H Glycolithocholic Acid 3-H Glycoursodeoxycholic Acid 3-H; 7-H Glycohyodeoxycholic Acid 3-H; 6-H Glycochenodeoxycholic Acid (GCDCA-S) 3-H; 7-H Glycodeoxycholic acid 3-H; 12-H Glycocholic Acid 3-H; 7-H; 12-H Glycohyocholic Acid 3-H; 6-H; 7-H Taurolithocholic acid 3-H Tauro-ursodeoxycholic Acid 3-H; 7-H Taurohyodeoxycholic Acid 3-H; 6-H Taurochenodeoxycholic Acid 3-H; 7-H Taurodeoxycholic Acid 3-H; 12-H Taurohyocholic Acid 3-H; 6-H; 7-H Tauro-β Muricholic Acid 3-H; 6-H; 7-H Tauro-α Muricholic Acid 3-H; 6-H; 7-H Tauro ω-muricholic Acid 3-H; 6-H; 7-H Taurocholic Acid 3-H; 7-H; 12-H S-4

5 Table S-3. Chromatographic gradient used for BA profiling and targeted analysis. Time(min) Flow Rate %A %B Curve Initial Initial S-5

6 Table S-4. Percent yield of the 16 reactions (3 replicates) of lithocholic acid (LCA) modified into sulfated lithocholic acid (LCA-S). CHCl 3 Sulfation conditions Pyridine 1h 24h 1h 24h 1h 24h 1h 24h 1h 24h 1h 24h 1h 24h 1h 24h RT RT 55 C 55 C RT RT 55 C 55 C RT RT 55 C 55 C RT RT 55 C 55 C Sodium sulfate Sodium sulfate LCA LCA-S standard deviation S-6

7 Table S-5. Intra- and inter-day validation of accuracy and precision over 57 bile acids with (10nM), (50nM), (100nM) and (750nM). Limit of detection (LD), lower limit of quantification (LLQ), upper limit of quantification and R 2 INTRA DAY INTER DAY Accuracy % Precision % Accuracy % Precision % LD (nm) LLQ (nm) ULQ (μm) R S-7

8 LD LLQ ULQ R2 (nm) (nm) (μm) INTRA DAY INTER DAY Accuracy % Precision % Accuracy % Precision % S-8

9 Table S-6. Quantitative matrix effect was evaluated by comparing integrated peak area expressed as a percentage of deuterated standards spiked in solvent and spiked in plasma samples for the (10 nm) and (100 nm) (n=3). Labelled standards S-9

10 Table S-7. Carry-over was evaluated on the integrated area under the peak of each labelled deuterated standards with the following run sequence; Blank 1 was injected first then (10nM), QC5 (5µM) and Blank 2. Labelled standards Blank 2 vs QC5 % Blank 2 vs % Blank 1 vs % S-10

11 % recovery Serum % recovery Plasma % recovery Urine Figure S-2. Recoveries of the 16 deuterated standards evaluated at (10 nm) and (100 nm) in human plasma, serum and urine. The error bars show the standard deviation of the replicates recovery (n=6). S-11

12 Table S-8. Recoveries and standard deviations (SD) of 16 internal labelled standards evaluated at (10 nm) and (100 nm) for human plasma, serum and urine (n=6). Labelled standards Serum Plasma Urine Recoveries Recoveries Recoveries QC SD SD % % % SD S-12