#METABOLOMICS WORKBENCH mcglinchey_aidan_20220518_032304 DATATRACK_ID:3255 STUDY_ID:ST002495 ANALYSIS_ID:AN004093 PROJECT_ID:PR001610 VERSION 1 CREATED_ON February 27, 2023, 9:12 am #PROJECT PR:PROJECT_TITLE Disrupted intestinal microbiota contributes to the pathogenesis of anorexia PR:PROJECT_TITLE nervosa PR:PROJECT_SUMMARY Anorexia nervosa (AN) is an eating disorder with a high mortality affecting PR:PROJECT_SUMMARY about 1% of women, where no evidence-based effective treatment exists. The PR:PROJECT_SUMMARY pathogenesis likely involves genetic and environmental alterations. We PR:PROJECT_SUMMARY hypothesized that a disrupted gut microbiota contributes to AN pathogenesis. In PR:PROJECT_SUMMARY analyses comparing 70 AN with 77 healthy females, we found multiple taxa, PR:PROJECT_SUMMARY functional modules, structural variants and growth rates of bacterial gut PR:PROJECT_SUMMARY microbiota, and viral gut microbiota that were altered in AN with parts of these PR:PROJECT_SUMMARY perturbations linked to estimates of eating behavior and mental health. In PR:PROJECT_SUMMARY silico, causal inference analyses implied serum bacterial metabolites mediated PR:PROJECT_SUMMARY parts of the impact of altered gut microbiota on AN behavior, and in vivo, three PR:PROJECT_SUMMARY independent fecal microbiota transplantation from AN cases to germ-free mice PR:PROJECT_SUMMARY under energy restricted feeding mirroring AN eating behavior consistently PR:PROJECT_SUMMARY induced a lower body weight gain and hypothalamic and adipose tissue gene PR:PROJECT_SUMMARY expressions related to aberrant energy metabolism and eating and mental PR:PROJECT_SUMMARY behavior. PR:INSTITUTE Örebro University PR:LAST_NAME McGlinchey PR:FIRST_NAME Aidan PR:ADDRESS Room 2217, Södra Grev Rosengatan 30, 70362 Örebro PR:EMAIL aidan.mcglinchey@oru.se PR:PHONE +46 0736485638 #STUDY ST:STUDY_TITLE Disrupted intestinal microbiota contributes to the pathogenesis of anorexia ST:STUDY_TITLE nervosa (Part 2) ST:STUDY_SUMMARY Anorexia nervosa (AN) is an eating disorder with a high mortality affecting ST:STUDY_SUMMARY about 0.5% of women, where no evidence-based effective treatment exists. The ST:STUDY_SUMMARY pathogenesis likely involves genetic and environmental alterations. We ST:STUDY_SUMMARY hypothesized that a disrupted gut microbiota contributes to AN pathology. In ST:STUDY_SUMMARY analyses comparing 70 AN with 77 healthy females, we found multiple taxa, ST:STUDY_SUMMARY functional modules, structural variants and growth rates of bacterial gut ST:STUDY_SUMMARY microbiota, and viral gut microbiota that were altered in AN with parts of these ST:STUDY_SUMMARY perturbations linked to estimates of eating behavior and mental health. In ST:STUDY_SUMMARY silico, causal inference analyses implied bacterial metabolites mediated parts ST:STUDY_SUMMARY of the impact of altered gut microbiota on AN behavior, and in vivo, fecal ST:STUDY_SUMMARY microbiota transplantation from AN cases to germ-free mice induced a lower body ST:STUDY_SUMMARY weight and hypothalamic and adipose tissue gene expressions related to aberrant ST:STUDY_SUMMARY energy metabolism and eating and mental behavior. ST:INSTITUTE Örebro University ST:LAST_NAME McGlinchey ST:FIRST_NAME Aidan ST:ADDRESS Room 2217, Södra Grev Rosengatan 30, 70362 Örebro ST:EMAIL aidan.mcglinchey@oru.se ST:PHONE +46 0736485638 #SUBJECT SU:SUBJECT_TYPE Human SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:GENDER Male and female #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS - sample_0001 Category:1 - Healthy RAW_FILE_NAME=OR_LC_20200224_sample_0001.mzdata.xml SUBJECT_SAMPLE_FACTORS - sample_0002 Category:2 - Anorexia RAW_FILE_NAME=OR_LC_20200224_sample_0002.mzdata.xml SUBJECT_SAMPLE_FACTORS - sample_0003 Category:2 - Anorexia RAW_FILE_NAME=OR_LC_20200224_sample_0003.mzdata.xml SUBJECT_SAMPLE_FACTORS - sample_0004 Category:2 - Anorexia RAW_FILE_NAME=OR_LC_20200224_sample_0004.mzdata.xml SUBJECT_SAMPLE_FACTORS - sample_0005 Category:2 - Anorexia RAW_FILE_NAME=OR_LC_20200224_sample_0005.mzdata.xml SUBJECT_SAMPLE_FACTORS - 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sample_0082 Category:2 - Anorexia RAW_FILE_NAME=OR_LC_20200224_sample_0082.mzdata.xml SUBJECT_SAMPLE_FACTORS - sample_0083 Category:1 - Healthy RAW_FILE_NAME=OR_LC_20200224_sample_0083.mzdata.xml SUBJECT_SAMPLE_FACTORS - sample_0084 Category:2 - Anorexia RAW_FILE_NAME=OR_LC_20200224_sample_0084.mzdata.xml SUBJECT_SAMPLE_FACTORS - sample_0085 Category:2 - Anorexia RAW_FILE_NAME=OR_LC_20200224_sample_0085.mzdata.xml SUBJECT_SAMPLE_FACTORS - sample_0086 Category:1 - Healthy RAW_FILE_NAME=OR_LC_20200224_sample_0086.mzdata.xml SUBJECT_SAMPLE_FACTORS - sample_0087 Category:1 - Healthy RAW_FILE_NAME=OR_LC_20200224_sample_0087.mzdata.xml SUBJECT_SAMPLE_FACTORS - sample_0088 Category:1 - Healthy RAW_FILE_NAME=OR_LC_20200224_sample_0088.mzdata.xml SUBJECT_SAMPLE_FACTORS - sample_0089 Category:2 - Anorexia RAW_FILE_NAME=OR_LC_20200224_sample_0089.mzdata.xml SUBJECT_SAMPLE_FACTORS - sample_0090 Category:1 - Healthy RAW_FILE_NAME=OR_LC_20200224_sample_0090.mzdata.xml SUBJECT_SAMPLE_FACTORS - sample_0091 Category:1 - 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Healthy RAW_FILE_NAME=OR_LC_20200224_sample_0110.mzdata.xml SUBJECT_SAMPLE_FACTORS - sample_0111 Category:2 - Anorexia RAW_FILE_NAME=OR_LC_20200224_sample_0111.mzdata.xml SUBJECT_SAMPLE_FACTORS - sample_0112 Category:2 - Anorexia RAW_FILE_NAME=OR_LC_20200224_sample_0112.mzdata.xml SUBJECT_SAMPLE_FACTORS - sample_0113 Category:1 - Healthy RAW_FILE_NAME=OR_LC_20200224_sample_0113.mzdata.xml SUBJECT_SAMPLE_FACTORS - sample_0114 Category:1 - Healthy RAW_FILE_NAME=OR_LC_20200224_sample_0114.mzdata.xml SUBJECT_SAMPLE_FACTORS - sample_0115 Category:1 - Healthy RAW_FILE_NAME=OR_LC_20200224_sample_0115.mzdata.xml SUBJECT_SAMPLE_FACTORS - sample_0116 Category:2 - Anorexia RAW_FILE_NAME=OR_LC_20200224_sample_0116.mzdata.xml SUBJECT_SAMPLE_FACTORS - sample_0117 Category:2 - Anorexia RAW_FILE_NAME=OR_LC_20200224_sample_0117.mzdata.xml SUBJECT_SAMPLE_FACTORS - sample_0118 Category:2 - Anorexia RAW_FILE_NAME=OR_LC_20200224_sample_0118.mzdata.xml SUBJECT_SAMPLE_FACTORS - sample_0119 Category:1 - Healthy RAW_FILE_NAME=OR_LC_20200224_sample_0119.mzdata.xml SUBJECT_SAMPLE_FACTORS - sample_0120 Category:1 - Healthy RAW_FILE_NAME=OR_LC_20200224_sample_0120.mzdata.xml SUBJECT_SAMPLE_FACTORS - sample_0121 Category:2 - Anorexia RAW_FILE_NAME=OR_LC_20200224_sample_0121.mzdata.xml SUBJECT_SAMPLE_FACTORS - sample_0122 Category:1 - Healthy RAW_FILE_NAME=OR_LC_20200224_sample_0122.mzdata.xml SUBJECT_SAMPLE_FACTORS - sample_0123 Category:2 - Anorexia RAW_FILE_NAME=OR_LC_20200224_sample_0123.mzdata.xml SUBJECT_SAMPLE_FACTORS - sample_0124 Category:1 - Healthy RAW_FILE_NAME=OR_LC_20200224_sample_0124.mzdata.xml SUBJECT_SAMPLE_FACTORS - sample_0125 Category:2 - Anorexia RAW_FILE_NAME=OR_LC_20200224_sample_0125.mzdata.xml SUBJECT_SAMPLE_FACTORS - sample_0126 Category:2 - Anorexia RAW_FILE_NAME=OR_LC_20200224_sample_0126.mzdata.xml SUBJECT_SAMPLE_FACTORS - sample_0127 Category:1 - Healthy RAW_FILE_NAME=OR_LC_20200224_sample_0127.mzdata.xml SUBJECT_SAMPLE_FACTORS - sample_0128 Category:1 - Healthy RAW_FILE_NAME=OR_LC_20200224_sample_0128.mzdata.xml SUBJECT_SAMPLE_FACTORS - sample_0129 Category:1 - 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sample_0139 Category:2 - Anorexia RAW_FILE_NAME=OR_LC_20200224_sample_0139.mzdata.xml SUBJECT_SAMPLE_FACTORS - sample_0140 Category:2 - Anorexia RAW_FILE_NAME=OR_LC_20200224_sample_0140.mzdata.xml SUBJECT_SAMPLE_FACTORS - sample_0141 Category:2 - Anorexia RAW_FILE_NAME=OR_LC_20200224_sample_0141.mzdata.xml SUBJECT_SAMPLE_FACTORS - sample_0142 Category:2 - Anorexia RAW_FILE_NAME=OR_LC_20200224_sample_0142.mzdata.xml SUBJECT_SAMPLE_FACTORS - sample_0143 Category:1 - Healthy RAW_FILE_NAME=OR_LC_20200224_sample_0143.mzdata.xml SUBJECT_SAMPLE_FACTORS - sample_0144 Category:1 - Healthy RAW_FILE_NAME=OR_LC_20200224_sample_0144.mzdata.xml SUBJECT_SAMPLE_FACTORS - sample_0145 Category:2 - Anorexia RAW_FILE_NAME=OR_LC_20200224_sample_0145.mzdata.xml SUBJECT_SAMPLE_FACTORS - sample_0146 Category:2 - Anorexia RAW_FILE_NAME=OR_LC_20200224_sample_0146.mzdata.xml SUBJECT_SAMPLE_FACTORS - sample_0147 Category:2 - Anorexia RAW_FILE_NAME=OR_LC_20200224_sample_0147.mzdata.xml SUBJECT_SAMPLE_FACTORS - sample_0148 Category:1 - Healthy RAW_FILE_NAME=OR_LC_20200224_sample_0148.mzdata.xml #COLLECTION CO:COLLECTION_SUMMARY The metabolites listed as gut microbiota-related metabolites is based on CO:COLLECTION_SUMMARY literature mining. Serum samples were randomized, and the sample preparation was CO:COLLECTION_SUMMARY done as described previously. Shortly, 400 μL of MeOH containing internal CO:COLLECTION_SUMMARY standards (heptadecanoic acid, deuterium-labeled DL-valine, deuterium- labeled CO:COLLECTION_SUMMARY succinic acid, and deuterium-labeled glutamic acid, c= 1 μg/ml) was added to 30 CO:COLLECTION_SUMMARY μl of the serum samples which were vortex mixed and incubated on ice for 30 min CO:COLLECTION_SUMMARY after which they were centrifuged (9400 × g, 3 min) and 350 μL of the CO:COLLECTION_SUMMARY supernatant was collected after centrifugation. The solvent was evaporated to CO:COLLECTION_SUMMARY dryness and 25 μL of MOX reagent was added and the sample was incubated for 60 CO:COLLECTION_SUMMARY min at 45 °C. 25 μL of N-Methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) CO:COLLECTION_SUMMARY was added and after 60 min incubation at 45 °C 25 μL of the retention index CO:COLLECTION_SUMMARY standard mixture (n-alkanes, c=10 μg/ml) was added. The analyses were done by CO:COLLECTION_SUMMARY Agilent 7890B gas chromatography (GC) coupled to Agilent 7200 auadrupole CO:COLLECTION_SUMMARY time-of-flight mass spectrometry (Q-TOF MS) (Agilent Technologies, Palo Alto, CO:COLLECTION_SUMMARY CA, USA). Injection volume 1 μL with 100:1 split on PTV at 70 °C, heating to CO:COLLECTION_SUMMARY 300 °C at 120 °C /min. Column: Zebron ZB- SemiVolatiles. Length: 20m, I.D. CO:COLLECTION_SUMMARY 0.18mm, film thickness: 0.18 μm. With initial Helium flow 1.2 mL/min, CO:COLLECTION_SUMMARY increasing to 2.4 mL/min after 16 mins. Oven temperature program: 50 °C (5 CO:COLLECTION_SUMMARY min), then to 270°C at 20 °/min and then to 300 at 40 °/min (5 min). EI CO:COLLECTION_SUMMARY source: 250 °C, 70 eV electron energy, 35μA emission, solvent delay 3 min. CO:COLLECTION_SUMMARY Mass range 55 to 650 amu, acquisition rate 5 spectra/s, acquisition time 200 CO:COLLECTION_SUMMARY ms/spectrum. Quad at 150 °C, 1.5 mL/min N2 collision flow, aux-2 temperature: CO:COLLECTION_SUMMARY 280 °C. Calibration curves were constructed using alanine, citric acid, fumaric CO:COLLECTION_SUMMARY acid, glutamic acid, glycine, lactic acid, malic acid, 2-hydroxybutyric acid, CO:COLLECTION_SUMMARY 3-hydroxybutyric acid, linoleic acid, oleic acid, palmitic acid, stearic acid, CO:COLLECTION_SUMMARY cholesterol, fructose, glutamine, indole-3-propionic acid, isoleucine, leucine, CO:COLLECTION_SUMMARY proline, succinic acid, valine, asparagine, aspartic acid, arachidonic acid, CO:COLLECTION_SUMMARY glycerol-3-phosphate, lysine, methionine, ornithine, phenylalanine, serine and CO:COLLECTION_SUMMARY threonine purchased from Sigma-Aldrich (St. Louis, MO, USA) at concentration CO:COLLECTION_SUMMARY range of 0.1 to 80 μg/ml. An aliquot of each sample was collected and pooled CO:COLLECTION_SUMMARY and used as quality control samples, together with National Institute of CO:COLLECTION_SUMMARY Standards and Technology (NIST) CRM1950 serum sample, an in-house pooled serum CO:COLLECTION_SUMMARY sample. The relative standard deviation of the concentrations was on average 16% CO:COLLECTION_SUMMARY for the pooled QC samples, 10% for the NIST samples. Analysis of serum bile CO:COLLECTION_SUMMARY acids and serum semipolar metabolites The sample preparation procedure was CO:COLLECTION_SUMMARY performed as described previously 80 . The plate was preconditioned with 450 μL CO:COLLECTION_SUMMARY acetonitrile before the addition of 100 μL of sample and 10 μL of PFAS and BA CO:COLLECTION_SUMMARY internal standard mixture (200 ng/mL and 1000 ng/mL respectively). Thereafter, CO:COLLECTION_SUMMARY 450 μL of acetonitrile containing 1% formic acid were added to each well and CO:COLLECTION_SUMMARY the samples extracted using a 10” vacuum manifold. The eluate was evaporated CO:COLLECTION_SUMMARY to dryness under nitrogen gas flow and reconstituted to 80 μL of MeOH/2 mM CO:COLLECTION_SUMMARY aqueous NH4AC. Chromatographic separation was carried out using an Acquity UPLC CO:COLLECTION_SUMMARY BEH C18 column (100 mm × 2.1 mm i.d., 1.7 μm particle size), fitted with a C18 CO:COLLECTION_SUMMARY precolumn (Waters Corporation, Wexford, Ireland). Mobile phase A consisted of CO:COLLECTION_SUMMARY H2O:MeOH (v/v 70:30) and mobile phase B of MeOH with both phases containing 2mM CO:COLLECTION_SUMMARY ammonium acetate as an ionization agent. The flow rate was set at 0.4 mLmin-1 CO:COLLECTION_SUMMARY with the elution gradient as follows: 0-1.5 min, mobile phase B was increased CO:COLLECTION_SUMMARY from 5% to 30%; 1.5-4.5 min, mobile phase B increased to 70%; 4.5-7.5 min, CO:COLLECTION_SUMMARY mobile phase B increased to 100% and held for 5.5 min. A post-time of 5 min was CO:COLLECTION_SUMMARY used to regain the initial conditions for the next analysis. The total run time CO:COLLECTION_SUMMARY per sample was 18 min. The dual ESI ionization source was settings were as CO:COLLECTION_SUMMARY follows: capillary voltage was 4.5 kV, nozzle voltage 1500 V, N2 pressure in the CO:COLLECTION_SUMMARY nebulized was 21 psi and the N2 flow rate and temperature as sheath gas was 11 L CO:COLLECTION_SUMMARY min-1 and 379 °C, respectively. In order to obtain accurate mass spectra in MS CO:COLLECTION_SUMMARY scan, the m/z range was set to 100-1700 in negative ion mode. MassHunter B.06.01 CO:COLLECTION_SUMMARY software (Agilent Technologies, Santa Clara, CA, USA) was used for all data CO:COLLECTION_SUMMARY acquisition. Identification of compounds was done by in-house spectral library CO:COLLECTION_SUMMARY using MS (and retention time), MS/MS information. Quantitation was based on a CO:COLLECTION_SUMMARY matrix matched calibration curve spiked with native compounds. The calibration CO:COLLECTION_SUMMARY curve consisted of concentrations ranging from 0 – 1600 ng mL -1 for BAs. The CO:COLLECTION_SUMMARY RSD for the BAs was on average 17.8 % for the QC samples and 19.4 % for the NIST CO:COLLECTION_SUMMARY samples. CO:SAMPLE_TYPE Blood (serum) CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY No treatment was applied. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Serum samples were randomized, and the sample preparation was done as described SP:SAMPLEPREP_SUMMARY previously 43,79 . Shortly, 400 μL of MeOH containing internal standards SP:SAMPLEPREP_SUMMARY (heptadecanoic acid, deuterium-labeled DL-valine, deuterium- labeled succinic SP:SAMPLEPREP_SUMMARY acid, and deuterium-labeled glutamic acid, c= 1 μg/ml) was added to 30 μl of SP:SAMPLEPREP_SUMMARY the serum samples which were vortex mixed and incubated on ice for 30 min after SP:SAMPLEPREP_SUMMARY which they were centrifuged (9400 × g, 3 min) and 350 μL of the supernatant SP:SAMPLEPREP_SUMMARY was collected after centrifugation. The solvent was evaporated to dryness and 25 SP:SAMPLEPREP_SUMMARY μL of MOX reagent was added and the sample was incubated for 60 min at 45 °C. SP:SAMPLEPREP_SUMMARY 25 μL of N-Methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) was added and SP:SAMPLEPREP_SUMMARY after 60 min incubation at 45 °C 25 μL of the retention index standard mixture SP:SAMPLEPREP_SUMMARY (n-alkanes, c=10 μg/ml) was added. The analyses were done by Agilent 7890B gas SP:SAMPLEPREP_SUMMARY chromatography (GC) coupled to Agilent 7200 auadrupole time-of-flight mass SP:SAMPLEPREP_SUMMARY spectrometry (Q-TOF MS) (Agilent Technologies, Palo Alto, CA, USA). Injection SP:SAMPLEPREP_SUMMARY volume 1 μL with 100:1 split on PTV at 70 °C, heating to 300 °C at 120 °C SP:SAMPLEPREP_SUMMARY /min. Column: Zebron ZB- SemiVolatiles. Length: 20m, I.D. 0.18mm, film SP:SAMPLEPREP_SUMMARY thickness: 0.18 μm. With initial Helium flow 1.2 mL/min, increasing to 2.4 SP:SAMPLEPREP_SUMMARY mL/min after 16 mins. Oven temperature program: 50 °C (5 min), then to 270°C SP:SAMPLEPREP_SUMMARY at 20 °/min and then to 300 at 40 °/min (5 min). EI source: 250 °C, 70 eV SP:SAMPLEPREP_SUMMARY electron energy, 35μA emission, solvent delay 3 min. Mass range 55 to 650 amu, SP:SAMPLEPREP_SUMMARY acquisition rate 5 spectra/s, acquisition time 200 ms/spectrum. Quad at 150 °C, SP:SAMPLEPREP_SUMMARY 1.5 mL/min N2 collision flow, aux-2 temperature: 280 °C. Calibration curves SP:SAMPLEPREP_SUMMARY were constructed using alanine, citric acid, fumaric acid, glutamic acid, SP:SAMPLEPREP_SUMMARY glycine, lactic acid, malic acid, 2-hydroxybutyric acid, 3-hydroxybutyric acid, SP:SAMPLEPREP_SUMMARY linoleic acid, oleic acid, palmitic acid, stearic acid, cholesterol, fructose, SP:SAMPLEPREP_SUMMARY glutamine, indole-3-propionic acid, isoleucine, leucine, proline, succinic acid, SP:SAMPLEPREP_SUMMARY valine, asparagine, aspartic acid, arachidonic acid, glycerol-3-phosphate, SP:SAMPLEPREP_SUMMARY lysine, methionine, ornithine, phenylalanine, serine and threonine purchased SP:SAMPLEPREP_SUMMARY from Sigma-Aldrich (St. Louis, MO, USA) at concentration range of 0.1 to 80 SP:SAMPLEPREP_SUMMARY μg/ml. An aliquot of each sample was collected and pooled and used as quality SP:SAMPLEPREP_SUMMARY control samples, together with National Institute of Standards and Technology SP:SAMPLEPREP_SUMMARY (NIST) CRM1950 serum sample, an in-house pooled serum sample. The relative SP:SAMPLEPREP_SUMMARY standard deviation of the concentrations was on average 16% for the pooled QC SP:SAMPLEPREP_SUMMARY samples, 10% for the NIST samples. Analysis of serum bile acids and serum SP:SAMPLEPREP_SUMMARY semipolar metabolites The sample preparation procedure was performed as SP:SAMPLEPREP_SUMMARY described previously 80 . The plate was preconditioned with 450 μL acetonitrile SP:SAMPLEPREP_SUMMARY before the addition of 100 μL of sample and 10 μL of PFAS and BA internal SP:SAMPLEPREP_SUMMARY standard mixture (200 ng/mL and 1000 ng/mL respectively). Thereafter, 450 μL of SP:SAMPLEPREP_SUMMARY acetonitrile containing 1% formic acid were added to each well and the samples SP:SAMPLEPREP_SUMMARY extracted using a 10” vacuum manifold. The eluate was evaporated to dryness SP:SAMPLEPREP_SUMMARY under nitrogen gas flow and reconstituted to 80 μL of MeOH/2 mM aqueous NH4AC. SP:SAMPLEPREP_SUMMARY Chromatographic separation was carried out using an Acquity UPLC BEH C18 column SP:SAMPLEPREP_SUMMARY (100 mm × 2.1 mm i.d., 1.7 μm particle size), fitted with a C18 precolumn SP:SAMPLEPREP_SUMMARY (Waters Corporation, Wexford, Ireland). Mobile phase A consisted of H2O:MeOH SP:SAMPLEPREP_SUMMARY (v/v 70:30) and mobile phase B of MeOH with both phases containing 2mM ammonium SP:SAMPLEPREP_SUMMARY acetate as an ionization agent. The flow rate was set at 0.4 mLmin-1 with the SP:SAMPLEPREP_SUMMARY elution gradient as follows: 0-1.5 min, mobile phase B was increased from 5% to SP:SAMPLEPREP_SUMMARY 30%; 1.5-4.5 min, mobile phase B increased to 70%; 4.5-7.5 min, mobile phase B SP:SAMPLEPREP_SUMMARY increased to 100% and held for 5.5 min. A post-time of 5 min was used to regain SP:SAMPLEPREP_SUMMARY the initial conditions for the next analysis. The total run time per sample was SP:SAMPLEPREP_SUMMARY 18 min. The dual ESI ionization source was settings were as follows: capillary SP:SAMPLEPREP_SUMMARY voltage was 4.5 kV, nozzle voltage 1500 V, N2 pressure in the nebulized was 21 SP:SAMPLEPREP_SUMMARY psi and the N2 flow rate and temperature as sheath gas was 11 L min-1 and 379 SP:SAMPLEPREP_SUMMARY °C, respectively. In order to obtain accurate mass spectra in MS scan, the m/z SP:SAMPLEPREP_SUMMARY range was set to 100-1700 in negative ion mode. MassHunter B.06.01 software SP:SAMPLEPREP_SUMMARY (Agilent Technologies, Santa Clara, CA, USA) was used for all data acquisition. SP:SAMPLEPREP_SUMMARY Identification of compounds was done by in-house spectral library using MS (and SP:SAMPLEPREP_SUMMARY retention time), MS/MS information. Quantitation was based on a matrix matched SP:SAMPLEPREP_SUMMARY calibration curve spiked with native compounds. The calibration curve consisted SP:SAMPLEPREP_SUMMARY of concentrations ranging from 0 – 1600 ng mL -1 for BAs. The RSD for the BAs SP:SAMPLEPREP_SUMMARY was on average 17.8 % for the QC samples and 19.4 % for the NIST samples. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Agilent 7890B CH:COLUMN_NAME Waters ACQUITY UPLC BEH C18 (100 x 2.1mm,1.7um) CH:SOLVENT_A 70% water/30% methanol CH:SOLVENT_B 100% methanol CH:FLOW_GRADIENT The flow rate was set at 0.4 mLmin-1 with the elution gradient as follows: 0-1.5 CH:FLOW_GRADIENT min, mobile phase B was increased from 5% to 30%; 1.5-4.5 min, mobile phase B CH:FLOW_GRADIENT increased to 70%; 4.5-7.5 min, mobile phase B increased to 100% and held for 5.5 CH:FLOW_GRADIENT min. A post-time of 5 min was used to regain the initial conditions for the next CH:FLOW_GRADIENT analysis. The total run time per sample was 18 min CH:FLOW_RATE 0.4ml/min CH:COLUMN_TEMPERATURE 70 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent 7200 QTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS MassHunter B.06.01 software (Agilent Technologies, Santa Clara, CA, USA) was MS:MS_COMMENTS used for all data acquisition. Identification of compounds was done by in-house MS:MS_COMMENTS spectral library using MS (and retention time), MS/MS information. Quantitation MS:MS_COMMENTS was based on a matrix matched calibration curve spiked with native compounds. MS:MS_COMMENTS The calibration curve consisted of concentrations ranging from 0 – 1600 ng mL MS:MS_COMMENTS -1 for BAs. The RSD for the BAs was on average 17.8 % for the QC samples and MS:MS_COMMENTS 19.4 % for the NIST samples. MS:MS_RESULTS_FILE ST002495_AN004093_Results.txt UNITS:Raw output from software Has m/z:Yes Has RT:Yes RT units:Minutes #END