Summary of Study ST002761

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR001719. The data can be accessed directly via it's Project DOI: 10.21228/M8HF01 This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

Study ID	ST002761
Study Title	Metabolic responses of normal rat kidneys to a high salt intake (Urine)
Study Type	Time-course metabolomics experiment
Study Summary	In this study, novel methods were developed which allowed continuous (24/7) measurement of arterial blood pressure and renal blood flow in freely moving rats and the intermittent collection of arterial and renal venous blood to estimate kidney metabolic fluxes of O2 and metabolites. Specifically, the study determined the effects of a high salt (HS; 4.0% NaCl) diet upon whole kidney O2 consumption and arterial and renal venous plasma metabolomic profiles of normal Sprague-Dawley rats. A separate group of rats was studied to determine changes in the cortex and outer medulla tissue metabolomic profiles before and following the switch from a 0.4% to 4.0% NaCl diet.
Institute	Medical College of Wisconsin
Department	Physiology
Laboratory	Dr. Allen W. Cowley
Last Name	Cowley
First Name	Allen
Address	8701 W. Watertown Plank Rd, Milwaukee, WI 53226
Email	cowley@mcw.edu
Phone	4149558277
Submit Date	2023-06-26
Raw Data Available	Yes
Raw Data File Type(s)	mzML
Analysis Type Detail	LC-MS
Release Date	2023-07-02
Release Version	1

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Project:

Project ID:	PR001719
Project DOI:	doi: 10.21228/M8HF01
Project Title:	SD Rat Metabolomics in Response to Salt
Project Type:	Untargeted Four-Mode Metabolomics
Project Summary:	This study analyzed the effects of a high salt (HS; 4.0% NaCl) diet upon the kidney, arterial plasma, and renal venous plasma metabolomic profiles of normal Sprague-Dawley rats.
Institute:	Medical College of Wisconsin
Department:	Physiology
Laboratory:	Dr. Allen W. Cowley
Last Name:	Cowley
First Name:	Allen
Address:	8701 W. Watertown Plank Rd, Milwaukee, WI 53226
Email:	cowley@mcw.edu
Phone:	414-955-8277
Funding Source:	NHLBI
Contributors:	Satoshi Shimada, Brian R. Hoffmann, Chun Yang, Theresa Kurth, Andrew S. Greene, Mingyu Liang, Ranjan K. Dash, Allen W. Cowley Jr

Subject:

Subject ID:	SU002868
Subject Type:	Mammal
Subject Species:	Rattus norvegicus
Taxonomy ID:	10116

Factors:

Subject type: Mammal; Subject species: Rattus norvegicus (Factor headings shown in green)

mb_sample_id	local_sample_id	Treatment	Source	CF
SA290969	20211015_Cowley3_Urine_14_39-417-HS14-U_C18neg_rep	HS14	Urine	Urine
SA290970	20211015_Cowley3_Urine_14_39-417-HS14-U_C18neg	HS14	Urine	Urine
SA290971	20211015_Cowley3_Urine_12_51-421-HS14-U_C18neg	HS14	Urine	Urine
SA290972	20211015_Cowley3_Urine_15_87-463-HS14-U_C18neg	HS14	Urine	Urine
SA290973	20211015_Cowley3_Urine_12_51-421-HS14-U_C18neg_rep	HS14	Urine	Urine
SA290974	20211015_Cowley3_Urine_17_75-434-HS14-U_C18neg	HS14	Urine	Urine
SA290975	20211101_Cowley3_Urine_20_39-417-HS14-U_HILICneg	HS14	Urine	Urine
SA290976	20211101_Cowley3_Urine_20_39-417-HS14-U_HILICneg_rep	HS14	Urine	Urine
SA290977	20211015_Cowley3_Urine_17_75-434-HS14-U_C18neg_rep	HS14	Urine	Urine
SA290978	20211015_Cowley3_Urine_5_63-429-HS14-U_C18neg_rep	HS14	Urine	Urine
SA290979	20211015_Cowley3_Urine_15_87-463-HS14-U_C18neg_rep	HS14	Urine	Urine
SA290980	20211015_Cowley3_Urine_5_63-429-HS14-U_C18neg	HS14	Urine	Urine
SA290981	20211025_Cowley3_Urine_19_63-429-HS14-U_HILICpos_rep	HS14	Urine	Urine
SA290982	20211025_Cowley3_Urine_19_63-429-HS14-U_HILICpos	HS14	Urine	Urine
SA290983	20211025_Cowley3_Urine_16_51-421-HS14-U_HILICpos_rep	HS14	Urine	Urine
SA290984	20211025_Cowley3_Urine_16_51-421-HS14-U_HILICpos	HS14	Urine	Urine
SA290985	20211025_Cowley3_Urine_6_87-463-HS14-U_HILICpos	HS14	Urine	Urine
SA290986	20211025_Cowley3_Urine_2_39-417-HS14-U_HILICpos_rep	HS14	Urine	Urine
SA290987	20211025_Cowley3_Urine_1_75-434-HS14-U_HILICpos	HS14	Urine	Urine
SA290988	20211025_Cowley3_Urine_1_75-434-HS14-U_HILICpos_rep	HS14	Urine	Urine
SA290989	20211025_Cowley3_Urine_2_39-417-HS14-U_HILICpos	HS14	Urine	Urine
SA290990	20211025_Cowley3_Urine_6_87-463-HS14-U_HILICpos_rep	HS14	Urine	Urine
SA290991	20211101_Cowley3_Urine_14_87-463-HS14-U_HILICneg_rep	HS14	Urine	Urine
SA290992	20211017_Cowley3_Urine_7_39-417-HS14-U_C18pos_rep	HS14	Urine	Urine
SA290993	20211017_Cowley3_Urine_7_39-417-HS14-U_C18pos	HS14	Urine	Urine
SA290994	20211017_Cowley3_Urine_11_87-463-HS14-U_C18pos	HS14	Urine	Urine
SA290995	20211017_Cowley3_Urine_11_87-463-HS14-U_C18pos_rep	HS14	Urine	Urine
SA290996	20211017_Cowley3_Urine_15_51-421-HS14-U_C18pos_rep	HS14	Urine	Urine
SA290997	20211017_Cowley3_Urine_15_51-421-HS14-U_C18pos	HS14	Urine	Urine
SA290998	20211017_Cowley3_Urine_1_75-434-HS14-U_C18pos_rep	HS14	Urine	Urine
SA290999	20211017_Cowley3_Urine_1_75-434-HS14-U_C18pos	HS14	Urine	Urine
SA291000	20211101_Cowley3_Urine_10_75-434-HS14-U_HILICneg	HS14	Urine	Urine
SA291001	20211101_Cowley3_Urine_3_51-421-HS14-U_HILICneg_rep_20211102184214	HS14	Urine	Urine
SA291002	20211101_Cowley3_Urine_10_75-434-HS14-U_HILICneg_rep	HS14	Urine	Urine
SA291003	20211101_Cowley3_Urine_12_63-429-HS14-U_HILICneg	HS14	Urine	Urine
SA291004	20211101_Cowley3_Urine_14_87-463-HS14-U_HILICneg	HS14	Urine	Urine
SA291005	20211101_Cowley3_Urine_12_63-429-HS14-U_HILICneg_rep	HS14	Urine	Urine
SA291006	20211017_Cowley3_Urine_19_63-429-HS14-U_C18pos	HS14	Urine	Urine
SA291007	20211017_Cowley3_Urine_19_63-429-HS14-U_C18pos_rep	HS14	Urine	Urine
SA291008	20211101_Cowley3_Urine_3_51-421-HS14-U_HILICneg_20211102181055	HS14	Urine	Urine
SA291009	20211101_Cowley3_Urine_13_78-434-HS21-U_HILICneg	HS21	Urine	Urine
SA291010	20211101_Cowley3_Urine_13_78-434-HS21-U_HILICneg_rep	HS21	Urine	Urine
SA291011	20211101_Cowley3_Urine_8_54-421-HS21-U_HILICneg_rep	HS21	Urine	Urine
SA291012	20211015_Cowley3_Urine_7_78-434-HS21-U_C18neg_rep	HS21	Urine	Urine
SA291013	20211015_Cowley3_Urine_16_66-429-HS21-U_C18neg	HS21	Urine	Urine
SA291014	20211015_Cowley3_Urine_18_54-421-HS21-U_C18neg	HS21	Urine	Urine
SA291015	20211015_Cowley3_Urine_16_66-429-HS21-U_C18neg_rep	HS21	Urine	Urine
SA291016	20211015_Cowley3_Urine_7_78-434-HS21-U_C18neg	HS21	Urine	Urine
SA291017	20211101_Cowley3_Urine_6_90-463-HS21-U_HILICneg_rep	HS21	Urine	Urine
SA291018	20211015_Cowley3_Urine_4_90-463-HS21-U_C18neg	HS21	Urine	Urine
SA291019	20211101_Cowley3_Urine_4_42-417-HS21-U_HILICneg	HS21	Urine	Urine
SA291020	20211101_Cowley3_Urine_4_42-417-HS21-U_HILICneg_rep	HS21	Urine	Urine
SA291021	20211015_Cowley3_Urine_4_90-463-HS21-U_C18neg_rep	HS21	Urine	Urine
SA291022	20211015_Cowley3_Urine_6_42-417-HS21-U_C18neg	HS21	Urine	Urine
SA291023	20211015_Cowley3_Urine_18_54-421-HS21-U_C18neg_rep	HS21	Urine	Urine
SA291024	20211101_Cowley3_Urine_8_54-421-HS21-U_HILICneg	HS21	Urine	Urine
SA291025	20211015_Cowley3_Urine_6_42-417-HS21-U_C18neg_rep	HS21	Urine	Urine
SA291026	20211025_Cowley3_Urine_11_42-417-HS21-U_HILICpos_rep	HS21	Urine	Urine
SA291027	20211017_Cowley3_Urine_2_90-463-HS21-U_C18pos_rep	HS21	Urine	Urine
SA291028	20211017_Cowley3_Urine_2_90-463-HS21-U_C18pos	HS21	Urine	Urine
SA291029	20211017_Cowley3_Urine_9_42-417-HS21-U_C18pos_rep	HS21	Urine	Urine
SA291030	20211017_Cowley3_Urine_17_66-429-HS21-U_C18pos_rep	HS21	Urine	Urine
SA291031	20211017_Cowley3_Urine_6_54-421-HS21-U_C18pos	HS21	Urine	Urine
SA291032	20211017_Cowley3_Urine_6_54-421-HS21-U_C18pos_rep	HS21	Urine	Urine
SA291033	20211101_Cowley3_Urine_5_66-429-HS21-U_HILICneg_rep_20211102204731	HS21	Urine	Urine
SA291034	20211101_Cowley3_Urine_6_90-463-HS21-U_HILICneg	HS21	Urine	Urine
SA291035	20211017_Cowley3_Urine_9_42-417-HS21-U_C18pos	HS21	Urine	Urine
SA291036	20211025_Cowley3_Urine_3_66-429-HS21-U_HILICpos	HS21	Urine	Urine
SA291037	20211017_Cowley3_Urine_17_66-429-HS21-U_C18pos	HS21	Urine	Urine
SA291038	20211017_Cowley3_Urine_10_78-434-HS21-U_C18pos_rep	HS21	Urine	Urine
SA291039	20211025_Cowley3_Urine_11_42-417-HS21-U_HILICpos	HS21	Urine	Urine
SA291040	20211025_Cowley3_Urine_9_54-421-HS21-U_HILICpos_rep	HS21	Urine	Urine
SA291041	20211025_Cowley3_Urine_9_54-421-HS21-U_HILICpos	HS21	Urine	Urine
SA291042	20211025_Cowley3_Urine_3_66-429-HS21-U_HILICpos_rep	HS21	Urine	Urine
SA291043	20211017_Cowley3_Urine_10_78-434-HS21-U_C18pos	HS21	Urine	Urine
SA291044	20211025_Cowley3_Urine_14_90-463-HS21-U_HILICpos	HS21	Urine	Urine
SA291045	20211025_Cowley3_Urine_17_78-434-HS21-U_HILICpos_rep	HS21	Urine	Urine
SA291046	20211025_Cowley3_Urine_17_78-434-HS21-U_HILICpos	HS21	Urine	Urine
SA291047	20211025_Cowley3_Urine_14_90-463-HS21-U_HILICpos_rep	HS21	Urine	Urine
SA291048	20211101_Cowley3_Urine_5_66-429-HS21-U_HILICneg_20211102201612	HS21	Urine	Urine
SA291049	20211101_Cowley3_Urine_1_60-426-HS7-U_HILICneg_20211102160537	HS7	Urine	Urine
SA291050	20211101_Cowley3_Urine_9_48-421-HS7-U_HILICneg	HS7	Urine	Urine
SA291051	20211101_Cowley3_Urine_7_84-463-HS7-U_HILICneg_rep	HS7	Urine	Urine
SA291052	20211101_Cowley3_Urine_7_84-463-HS7-U_HILICneg	HS7	Urine	Urine
SA291053	20211101_Cowley3_Urine_1_60-426-HS7-U_HILICneg_rep_20211102163657	HS7	Urine	Urine
SA291054	20211025_Cowley3_Urine_20_84-463-HS7-U_HILICpos	HS7	Urine	Urine
SA291055	20211025_Cowley3_Urine_7_48-421-HS7-U_HILICpos	HS7	Urine	Urine
SA291056	20211025_Cowley3_Urine_5_72-434-HS7-U_HILICpos_rep	HS7	Urine	Urine
SA291057	20211025_Cowley3_Urine_5_72-434-HS7-U_HILICpos	HS7	Urine	Urine
SA291058	20211101_Cowley3_Urine_9_48-421-HS7-U_HILICneg_rep	HS7	Urine	Urine
SA291059	20211025_Cowley3_Urine_7_48-421-HS7-U_HILICpos_rep	HS7	Urine	Urine
SA291060	20211025_Cowley3_Urine_10_60-429-HS7-U_HILICpos	HS7	Urine	Urine
SA291061	20211025_Cowley3_Urine_3_36-417-HS7-U_HILICpos_rep	HS7	Urine	Urine
SA291062	20211025_Cowley3_Urine_3_36-417-HS7-U_HILICpos	HS7	Urine	Urine
SA291063	20211025_Cowley3_Urine_10_60-429-HS7-U_HILICpos_rep	HS7	Urine	Urine
SA291064	20211025_Cowley3_Urine_20_84-463-HS7-U_HILICpos_rep	HS7	Urine	Urine
SA291065	20211015_Cowley3_Urine_9_36-417-HS7-U_C18neg	HS7	Urine	Urine
SA291066	20211015_Cowley3_Urine_2_84-463-HS7-U_C18neg	HS7	Urine	Urine
SA291067	20211101_Cowley3_Urine_11_36-417-HS7-U_HILICneg	HS7	Urine	Urine
SA291068	20211015_Cowley3_Urine_2_84-463-HS7-U_C18neg_rep	HS7	Urine	Urine

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Collection:

Collection ID:	CO002861
Collection Summary:	Plasma was collected through an arterial and renal venous catheter throughout the study (200 µL of arterial and renal venous blood were sampled at the day 7, 14, and 21). Overnight urine (18 hours) from the day before the blood draw was collected on ice. The kidneys were collected either at 14 days of HS (HS14) or 21 days of HS (HS21). The kidneys of only LS fed SD rats were also collected for comparison. The collected kidneys (n=5 for each group for metabolomics) were dissected to cortex and outer medulla and snap frozen with liquid nitrogen. Plasma, urine and tissue were stored in -80°C until further analysis.
Sample Type:	Urine
Storage Conditions:	-80?

Treatment:

Treatment ID:	TR002877
Treatment Summary:	Rats (n=7, 10-11 weeks of age) were performed renal blood flow (RBF) probe implantation and femoral arterial catheterization5. Briefly, rats were anesthetized with isoflurane and arterial catheter was inserted. Following an abdominal incision, RBF probe was implanted on left renal artery and the cable was exposed at nape of the neck via the subcutaneous route. In addition to the RBF probe implantation, renal venous catheter was inserted through the femoral vein and placed in the left renal vein and secured to the luminal wall with 10-0 nylon. RBF and BP via arterial line were measured by conscious freely moving rats and recorded on average of every minute for 24 h/day. After 7-10 days of recovery period, 200 µL of arterial and renal venous blood were sampled and that blood was replaced from donor rats before and following 7, 14 and 21 days after the switch in diet from 0.4% (LS) to 4.0% (HS) salt diet (Dyets Inc, Bethlehem, PA). Overnight urine (18 hours) from the day before the blood draw was collected on ice. The kidneys were collected either at 14 days of HS (HS14) or 21 days of HS (HS21). The kidneys of only LS fed SD rats were also collected for comparison. The collected kidneys (n=5 for each group for metabolomics and mRNAseq analysis) were dissected to cortex and outer medulla and snap frozen with liquid nitrogen. Plasma, urine and tissue were stored in -80°C until further analysis.

Treatment ID:

TR002877

Treatment Summary:

Rats (n=7, 10-11 weeks of age) were performed renal blood flow (RBF) probe implantation and femoral arterial catheterization5. Briefly, rats were anesthetized with isoflurane and arterial catheter was inserted. Following an abdominal incision, RBF probe was implanted on left renal artery and the cable was exposed at nape of the neck via the subcutaneous route. In addition to the RBF probe implantation, renal venous catheter was inserted through the femoral vein and placed in the left renal vein and secured to the luminal wall with 10-0 nylon. RBF and BP via arterial line were measured by conscious freely moving rats and recorded on average of every minute for 24 h/day. After 7-10 days of recovery period, 200 µL of arterial and renal venous blood were sampled and that blood was replaced from donor rats before and following 7, 14 and 21 days after the switch in diet from 0.4% (LS) to 4.0% (HS) salt diet (Dyets Inc, Bethlehem, PA). Overnight urine (18 hours) from the day before the blood draw was collected on ice. The kidneys were collected either at 14 days of HS (HS14) or 21 days of HS (HS21). The kidneys of only LS fed SD rats were also collected for comparison. The collected kidneys (n=5 for each group for metabolomics and mRNAseq analysis) were dissected to cortex and outer medulla and snap frozen with liquid nitrogen. Plasma, urine and tissue were stored in -80°C until further analysis.

Sample Preparation:

Sampleprep ID:	SP002874
Sampleprep Summary:	Plasma/Urine Metabolite Extraction. Metabolites were extracted from 20 µL of plasma and 20 µL of urine from each SD rat in the study according to standard operating procedures in the Mass Spectrometry and Protein Chemistry Service at The Jackson Laboratory34. Metabolites were extracted using 500 µL of an ice cold 2:2:1 methanol:acetonitrile:water (MeOH:ACN:H2O) buffer; the sample was part of the water fraction. Caffeine, 1-napthylamine, and 9-anthracene carboxylic acid were all added at 0.5 ng/ µL in the extraction buffer as internal standards. Each sample was then vortexed for 30 seconds on the highest setting, subject to one minute of mixing with the Tissue Lyser II in pre-chilled cassettes, and then sonicated at 30 Hz for 5 minutes of 30 seconds on 30 seconds off in an ice water bath. Samples were then placed in the -20°C freezer overnight (16 hours) for extraction. Following the extraction, samples were centrifuged at 21,000 x g at 4°C and supernatant from each metabolite extract was equally divided into five 2 mL microcentrifuge tubes. Each sample supernatant was divided into five equal volume aliquots, one for each of the four modes and the rest to create equal representation pools of all samples, one for each mode. Each aliquot was then dried down using a vacuum centrifuge for storage at -80°C until further use. Tissue Metabolite Extraction. Metabolites were extracted from 20 mg of kidney cortex and medulla from each SD rat in the study according to standard operating procedures in the Mass Spectrometry and Protein Chemistry Service at The Jackson Laboratory34 as described for the plasma and urine samples with slight modification. Metabolites were extracted using 1000 µL of an ice cold 2:2:1 methanol:acetonitrile:water (MeOH:ACN:H2O) buffer containing internal standards as above per 20 mg of sample to ensure the extraction equivalents were normalized. Each sample had a 5 mm stainless steel bead added, then were pulverized in extraction buffer for two minutes usingTissue Lyser II. Samples were then placed in the -20°C freezer overnight (16 hours) for extraction and the supernatant was collected as with the urine/plasma samples. Each sample supernatant was divided into five equal volume aliquots, one for each of the four modes and the rest to create equal representation pools of all samples, one for each mode. Each aliquot was then dried down using a vacuum centrifuge for storage at -80°C until further use.

Sampleprep ID:

SP002874

Sampleprep Summary:

Plasma/Urine Metabolite Extraction. Metabolites were extracted from 20 µL of plasma and 20 µL of urine from each SD rat in the study according to standard operating procedures in the Mass Spectrometry and Protein Chemistry Service at The Jackson Laboratory34. Metabolites were extracted using 500 µL of an ice cold 2:2:1 methanol:acetonitrile:water (MeOH:ACN:H2O) buffer; the sample was part of the water fraction. Caffeine, 1-napthylamine, and 9-anthracene carboxylic acid were all added at 0.5 ng/ µL in the extraction buffer as internal standards. Each sample was then vortexed for 30 seconds on the highest setting, subject to one minute of mixing with the Tissue Lyser II in pre-chilled cassettes, and then sonicated at 30 Hz for 5 minutes of 30 seconds on 30 seconds off in an ice water bath. Samples were then placed in the -20°C freezer overnight (16 hours) for extraction. Following the extraction, samples were centrifuged at 21,000 x g at 4°C and supernatant from each metabolite extract was equally divided into five 2 mL microcentrifuge tubes. Each sample supernatant was divided into five equal volume aliquots, one for each of the four modes and the rest to create equal representation pools of all samples, one for each mode. Each aliquot was then dried down using a vacuum centrifuge for storage at -80°C until further use. Tissue Metabolite Extraction. Metabolites were extracted from 20 mg of kidney cortex and medulla from each SD rat in the study according to standard operating procedures in the Mass Spectrometry and Protein Chemistry Service at The Jackson Laboratory34 as described for the plasma and urine samples with slight modification. Metabolites were extracted using 1000 µL of an ice cold 2:2:1 methanol:acetonitrile:water (MeOH:ACN:H2O) buffer containing internal standards as above per 20 mg of sample to ensure the extraction equivalents were normalized. Each sample had a 5 mm stainless steel bead added, then were pulverized in extraction buffer for two minutes usingTissue Lyser II. Samples were then placed in the -20°C freezer overnight (16 hours) for extraction and the supernatant was collected as with the urine/plasma samples. Each sample supernatant was divided into five equal volume aliquots, one for each of the four modes and the rest to create equal representation pools of all samples, one for each mode. Each aliquot was then dried down using a vacuum centrifuge for storage at -80°C until further use.

Combined analysis:

Analysis ID	AN004487	AN004488	AN004489	AN004490
Analysis type	MS	MS	MS	MS
Chromatography type	Reversed phase	Reversed phase	HILIC	HILIC
Chromatography system	Thermo Vanquish	Thermo Vanquish	Thermo Vanquish	Thermo Vanquish
Column	Agilent InfinityLab Poroshell 120 EC-C18 (2.1 x 50 mm; 2.7-Micron)	Agilent InfinityLab Poroshell 120 HILIC-Z (2.1 x 50 mm; 2.7 micron; #689775-924)	Agilent InfinityLab Poroshell 120 HILIC-Z (2.1 x 50 mm; 2.7 micron; #689775-924)	Agilent InfiinityLab Poroshell 120 HILIC-Z (2.1 x 50 mm; 2.7 micron; #689775-924)
MS Type	ESI	ESI	ESI	ESI
MS instrument type	Orbitrap	Orbitrap	Orbitrap	Orbitrap
MS instrument name	Thermo Q Exactive Orbitrap	Thermo Q Exactive Orbitrap	Thermo Q Exactive Orbitrap	Thermo Q Exactive Orbitrap
Ion Mode	POSITIVE	NEGATIVE	POSITIVE	NEGATIVE
Units	Area	Area	Area	Area

Chromatography:

Chromatography ID:	CH003372
Chromatography Summary:	This chromatography method was utilized for all C18 positive polarity runs in this study.
Instrument Name:	Thermo Vanquish
Column Name:	Agilent InfinityLab Poroshell 120 EC-C18 (2.1 x 50 mm; 2.7-Micron)
Column Temperature:	25C
Flow Gradient:	0-1 minutes at 98% A1/2% B1, 1-13 minutes from 98% A1/2% B1 to 10% A1/90% B1, 13-15 minutes at 10% A1/90% B1, 15-16 minutes from 10% A1/90% B1 to 98% A1/2% B1, and was re-equilibrated from 16-25 minutes at 98% A1/2% B1
Flow Rate:	0.1 mL/minute
Internal Standard:	Caffeine, 1-napthylamine, and 9-anthracene carboxylic acid were all added at 0.5 ng/ µL in the extraction buffer as internal standards
Solvent A:	100% water, 0.2% acetic acid
Solvent B:	100% acetonitrile, 0.2% acetic acid
Chromatography Type:	Reversed phase

Chromatography ID:	CH003373
Chromatography Summary:	This chromatography method was utilized for all C18 negative polarity runs in this study.
Instrument Name:	Thermo Vanquish
Column Name:	Agilent InfinityLab Poroshell 120 HILIC-Z (2.1 x 50 mm; 2.7 micron; #689775-924)
Column Temperature:	25C
Flow Gradient:	0-1 minutes at 2% A/98% B, 1-11 minutes from 2% A/98% B to 30% A/70% B, 11-12 minutes from 30% A/70% B to 40% A/60% B, 12-16 minutes from 40% A/60% B to 95% A/5% B, was held at 95% A/5% B from 16-18 minutes, 18-20 minutes from 95% A/5% B to 2% A/98% B, and was re-equilibrated from 20-25 minutes at 2% A/98% B
Flow Rate:	0.1 mL/minute
Internal Standard:	Caffeine, 1-napthylamine, and 9-anthracene carboxylic acid were all added at 0.5 ng/ µL in the extraction buffer as internal standards
Solvent A:	100% water, 0.2% acetic acid
Solvent B:	100% acetonitrile, 0.2% acetic acid
Chromatography Type:	Reversed phase

Chromatography ID:	CH003374
Chromatography Summary:	This chromatography method was utilized for all HILIC positive polarity runs in this study.
Instrument Name:	Thermo Vanquish
Column Name:	Agilent InfinityLab Poroshell 120 HILIC-Z (2.1 x 50 mm; 2.7 micron; #689775-924)
Column Temperature:	25C
Flow Gradient:	0-1 minutes at 2% A/98% B, 1-11 minutes from 2% A/98% B to 30% A/70% B, 11-12 minutes from 30% A/70% B to 40% A/60% B, 12-16 minutes from 40% A/60% B to 95% A/5% B, was held at 95% A/5% B from 16-18 minutes, 18-20 minutes from 95% A/5% B to 2% A/98% B, and was re-equilibrated from 20-25 minutes at 2% A/98% B
Flow Rate:	0.1 mL/minute
Internal Standard:	Caffeine, 1-napthylamine, and 9-anthracene carboxylic acid were all added at 0.5 ng/ µL in the extraction buffer as internal standards
Solvent A:	10 mM ammonium formate in H2O with 0.1% formic acid (Solvent A2)
Solvent B:	90% ACN with 10 mM ammonium formate in H2O with 0.1% formic acid (Solvent B2)
Chromatography Type:	HILIC

Chromatography ID:	CH003375
Chromatography Summary:	This chromatography method was utilized for all HILIC negative polarity runs in this study.
Instrument Name:	Thermo Vanquish
Column Name:	Agilent InfiinityLab Poroshell 120 HILIC-Z (2.1 x 50 mm; 2.7 micron; #689775-924)
Column Temperature:	25C
Flow Gradient:	0-1 minutes at 2% A/98% B, 1-11 minutes from 2% A/98% B to 30% A/70% B, 11-12 minutes from 30% A/70% B to 40% A/60% B, 12-16 minutes from 40% A/60% B to 95% A/5% B, was held at 95% A/5% B from 16-18 minutes, 18-20 minutes from 95% A/5% B to 2% A/98% B, and was re-equilibrated from 20-25 minutes at 2% A/98% B
Flow Rate:	0.1 mL/minute
Internal Standard:	Caffeine, 1-napthylamine, and 9-anthracene carboxylic acid were all added at 0.5 ng/ µL in the extraction buffer as internal standards
Solvent A:	10 mM ammonium acetate in H2O, pH 9.0 with 0.1% AffinityLab Deactivator Inhibitor (Agilent, #5191-3940; Solvent A3)
Solvent B:	85% ACN with 10 mM ammonium acetate in H2O with 0.1% AffinityLab Deactivator Inhibitor (Solvent B3)
Chromatography Type:	HILIC

MS:

MS ID:	MS004234
Analysis ID:	AN004487
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	C18 positive urine data: The tandem mass spectrometry RAW data files (consisting of MS1 and MS2 spectra collected) were analyzed using Thermo Compound Discoverer (v3.2.0.421). The MS1 and MS2 data was searched against the Thermo mzCloud database, ChemSpider database, Metabolika Pathways, and mzLogic predicted composition in the Compound Discoverer workflow.
Ion Mode:	POSITIVE

MS ID:	MS004235
Analysis ID:	AN004488
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	C18 negative urine data: The tandem mass spectrometry RAW data files (consisting of MS1 and MS2 spectra collected) were analyzed using Thermo Compound Discoverer (v3.2.0.421). The MS1 and MS2 data was searched against the Thermo mzCloud database, ChemSpider database, Metabolika Pathways, and mzLogic predicted composition in the Compound Discoverer workflow.
Ion Mode:	NEGATIVE

MS ID:	MS004236
Analysis ID:	AN004489
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	HILIC positive urine data: The tandem mass spectrometry RAW data files (consisting of MS1 and MS2 spectra collected) were analyzed using Thermo Compound Discoverer (v3.2.0.421). The MS1 and MS2 data was searched against the Thermo mzCloud database, ChemSpider database, Metabolika Pathways, and mzLogic predicted composition in the Compound Discoverer workflow.
Ion Mode:	POSITIVE

MS ID:	MS004237
Analysis ID:	AN004490
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	HILIC negative urine data: The tandem mass spectrometry RAW data files (consisting of MS1 and MS2 spectra collected) were analyzed using Thermo Compound Discoverer (v3.2.0.421). The MS1 and MS2 data was searched against the Thermo mzCloud database, ChemSpider database, Metabolika Pathways, and mzLogic predicted composition in the Compound Discoverer workflow.
Ion Mode:	NEGATIVE