Summary of Study ST003751

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 PR002335. The data can be accessed directly via it's Project DOI: 10.21228/M8T25X 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	ST003751
Study Title	Comprehensive Lipidomic Analysis Identifies Critical Lipid and Metabolic Pathway Shifts in Alport Syndrome
Study Summary	Alport syndrome (AS) is a hereditary kidney disease caused by COL4A3-5 gene mutations, leading to glomerular basement membrane abnormalities. While the genetic and structural aspects of AS are well established, the mechanisms linking collagen IV defects to podocyte injury remain incompletely understood. Emerging evidence suggests that lipotoxicity and lipid dysregulation may play a pivotal role in mediating podocyte damage in AS, akin to its established role in diabetic kidney disease (DKD). We sought to identify plasma and urine lipid alterations in autosomal dominant and X-linked AS compared with DKD and healthy controls. Using liquid chromatography coupled to mass spectrometry (LC-MS), we annotated 580 and 203 lipid species in plasma and urine, respectively. Volcano plot and ROC analyses (AUC ≥ 0.80) identified key lipids, including urinary HexCer 18:0(3O)/24:0(2OH) and CAR 12:0. These analyses highlighted the most relevant lipotoxic pathways, which may warrant deeper investigation for drug development in AS. Compared to controls, AS exhibited unbalanced sphingolipid catabolism, ceramide overload, and impaired fatty acid β-oxidation, alongside phospholipid and cholesterol imbalances suggestive of compromised ABCA1-mediated lipid efflux and mitochondrial dysfunction. Comparisons with DKD indicated a shared lipotoxic environment with ceramide elevation and disrupted fatty acid metabolism. However, disease-specific adaptations emerged, with severe ABCA1 dysfunction and marked phospholipid/cholesterol derangements in DKD, whereas AS showed pronounced sphingomyelin depletion. These findings demonstrate that AS involves distinct lipidomic disruptions and underscore shared lipotoxic mechanisms. This improved understanding of disease-specific lipid imbalances provides new potential therapeutic targets to mitigate podocyte injury and slow progression of AS.
Institute	Universidad CEU San Pablo
Department	Centro de Metabolómica y Bioanálisis (CEMBIO)
Last Name	González
First Name	Carolina
Address	km 0, Universidad CEU-San Pablo Urbanización Montepríncipe. M-501
Email	carolina.gonzalezriano@ceu.es
Phone	646251045
Submit Date	2025-02-21
Raw Data Available	Yes
Raw Data File Type(s)	d
Analysis Type Detail	LC-MS
Release Date	2025-03-17
Release Version	1

Select appropriate tab below to view additional metadata details:

Project:

Project ID:	PR002335
Project DOI:	doi: 10.21228/M8T25X
Project Title:	Comprehensive Lipidomic Analysis Identifies Critical Lipid and Metabolic Pathway Shifts in Alport Syndrome
Project Summary:	Alport syndrome (AS) is a hereditary kidney disease caused by COL4A3-5 gene mutations, leading to glomerular basement membrane abnormalities. While the genetic and structural aspects of AS are well established, the mechanisms linking collagen IV defects to podocyte injury remain incompletely understood. Emerging evidence suggests that lipotoxicity and lipid dysregulation may play a pivotal role in mediating podocyte damage in AS, akin to its established role in diabetic kidney disease (DKD). We sought to identify plasma and urine lipid alterations in autosomal dominant (ADAS) and X-linked AS (XLAS) compared with DKD and healthy controls. Using liquid chromatography coupled to mass spectrometry (LC-MS), we annotated 580 and 203 lipid species in plasma and urine, respectively. Volcano plot and ROC analyses (AUC ≥ 0.80) identified key lipids, including urinary HexCer 18:0(3O)/24:0(2OH) and CAR 12:0. These analyses highlighted the most relevant lipotoxic pathways, which may warrant deeper investigation for drug development in AS. Compared to controls, AS exhibited unbalanced sphingolipid catabolism, ceramide overload, and impaired fatty acid β-oxidation, alongside phospholipid and cholesterol imbalances suggestive of compromised ABCA1-mediated lipid efflux and mitochondrial dysfunction. Comparisons with DKD indicated a shared lipotoxic environment with ceramide elevation and disrupted fatty acid metabolism. However, disease-specific adaptations emerged, with severe ABCA1 dysfunction and marked phospholipid/cholesterol derangements in DKD, whereas AS showed pronounced sphingomyelin depletion. These findings demonstrate that AS involves distinct lipidomic disruptions and underscore shared lipotoxic mechanisms. This improved understanding of disease-specific lipid imbalances provides new potential therapeutic targets to mitigate podocyte injury and slow progression of AS.
Institute:	Universidad CEU San Pablo
Department:	Centro de MEtabolómica y Bioanálisis (CEMBIO)
Last Name:	Gonzalez-Riano
First Name:	Carolina
Address:	Facultad de Farmacia, Universidad CEU San Pablo, Campus Monteprincipe, Boadilla del Monte, Boadilla del Monte, Madrid, 28668, Spain
Email:	car.gonzalez@ceindo.ceu.es
Phone:	00 34 91 3724753
Funding Source:	This work was supported by grants from the following entities: The Ministry of Science and Innovation of Spain (MICINN) and the European Regional Development Fund FEDER, grant number PID2021-122490NB-I00 (CGR, BF, SM and CB). Instituto de Salud Carlos III and the European Union’s European Regional Development Fund grants PI19/01624 and PI24/01711. European Union-Next generation EU, Mechanism for Recovery and Resilence (MRR) RICORS (RD21/0005/0030; RD24/0004/0014) (GFJ, ASF). Spanish Society of Nephrology (AS, TBB).

Subject:

Subject ID:	SU003884
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Gender:	Male and female

Factors:

Subject type: Human; Subject species: Homo sapiens (Factor headings shown in green)

mb_sample_id	local_sample_id	Sample source	Condition
SA408391	CA_2_5_Plasma	Plasma	ADAS
SA408392	CA_2_0_Plasma	Plasma	ADAS
SA408393	CA_2_1_Plasma	Plasma	ADAS
SA408394	CA_2_2_Plasma	Plasma	ADAS
SA408395	CA_2_3_Plasma	Plasma	ADAS
SA408396	CA_2_4_Plasma	Plasma	ADAS
SA408397	CA_2_6_Plasma	Plasma	ADAS
SA408398	CA_2_7_Plasma	Plasma	ADAS
SA408399	CA_2_8_Plasma	Plasma	ADAS
SA408400	CA_2_9_Plasma	Plasma	ADAS
SA408401	CA_2_10_Plasma	Plasma	ADAS
SA408402	CA_2_11_Plasma	Plasma	ADAS
SA408403	CO_4_7_Plasma	Plasma	Control
SA408404	CO_4_13_Plasma	Plasma	Control
SA408405	CO_4_12_Plasma	Plasma	Control
SA408406	CO_4_11_Plasma	Plasma	Control
SA408407	CO_4_10_Plasma	Plasma	Control
SA408408	CO_4_8_Plasma	Plasma	Control
SA408409	CO_4_0_Plasma	Plasma	Control
SA408410	CO_4_6_Plasma	Plasma	Control
SA408411	CO_4_5_Plasma	Plasma	Control
SA408412	CO_4_4_Plasma	Plasma	Control
SA408413	CO_4_3_Plasma	Plasma	Control
SA408414	CO_4_2_Plasma	Plasma	Control
SA408415	CO_4_1_Plasma	Plasma	Control
SA408416	CO_4_15_Plasma	Plasma	Control
SA408417	CO_4_14_Plasma	Plasma	Control
SA408418	CO_4_9_Plasma	Plasma	Control
SA408419	CO_4_16_Plasma	Plasma	Control
SA408420	CO_4_18_Plasma	Plasma	Control
SA408421	CO_4_19_Plasma	Plasma	Control
SA408422	CO_4_17_Plasma	Plasma	Control
SA408423	CO_1_14_Plasma	Plasma	DKD
SA408424	CO_1_7_Plasma	Plasma	DKD
SA408425	CO_1_6_Plasma	Plasma	DKD
SA408426	CO_1_5_Plasma	Plasma	DKD
SA408427	CO_1_4_Plasma	Plasma	DKD
SA408428	CO_1_3_Plasma	Plasma	DKD
SA408429	CO_1_2_Plasma	Plasma	DKD
SA408430	CO_1_1_Plasma	Plasma	DKD
SA408431	CO_1_0_Plasma	Plasma	DKD
SA408432	CO_1_11_Plasma	Plasma	DKD
SA408433	CO_1_12_Plasma	Plasma	DKD
SA408434	CO_1_13_Plasma	Plasma	DKD
SA408435	CO_1_10_Plasma	Plasma	DKD
SA408436	CO_1_8_Plasma	Plasma	DKD
SA408437	CO_1_9_Plasma	Plasma	DKD
SA408438	QC_11_Plasma	Plasma	Quality Control
SA408439	QC_1_Plasma	Plasma	Quality Control
SA408440	QC_2_Plasma	Plasma	Quality Control
SA408441	QC_3_Plasma	Plasma	Quality Control
SA408442	QC_4_Plasma	Plasma	Quality Control
SA408443	QC_5_Plasma	Plasma	Quality Control
SA408444	QC_6_Plasma	Plasma	Quality Control
SA408445	QC_7_Plasma	Plasma	Quality Control
SA408446	QC_23_Plasma	Plasma	Quality Control
SA408447	QC_22_Plasma	Plasma	Quality Control
SA408448	QC_12_Plasma	Plasma	Quality Control
SA408449	QC_20_Plasma	Plasma	Quality Control
SA408450	QC_19_Plasma	Plasma	Quality Control
SA408451	QC_18_Plasma	Plasma	Quality Control
SA408452	QC_17_Plasma	Plasma	Quality Control
SA408453	QC_16_Plasma	Plasma	Quality Control
SA408454	QC_15_Plasma	Plasma	Quality Control
SA408455	QC_14_Plasma	Plasma	Quality Control
SA408456	QC_13_Plasma	Plasma	Quality Control
SA408457	QC_8_Plasma	Plasma	Quality Control
SA408458	QC_9_Plasma	Plasma	Quality Control
SA408459	QC_10_Plasma	Plasma	Quality Control
SA408460	QC_21_Plasma	Plasma	Quality Control
SA408461	CA_1_1_Plasma	Plasma	XLAS
SA408462	CA_1_0_Plasma	Plasma	XLAS
SA408463	CA_1_50_Plasma	Plasma	XLAS
SA408464	CA_1_13_Plasma	Plasma	XLAS
SA408465	CA_1_22_Plasma	Plasma	XLAS
SA408466	CA_1_21_Plasma	Plasma	XLAS
SA408467	CA_1_20_Plasma	Plasma	XLAS
SA408468	CA_1_19_Plasma	Plasma	XLAS
SA408469	CA_1_18_Plasma	Plasma	XLAS
SA408470	CA_1_17_Plasma	Plasma	XLAS
SA408471	CA_1_16_Plasma	Plasma	XLAS
SA408472	CA_1_15_Plasma	Plasma	XLAS
SA408473	CA_1_14_Plasma	Plasma	XLAS
SA408474	CA_1_12_Plasma	Plasma	XLAS
SA408475	CA_1_24_Plasma	Plasma	XLAS
SA408476	CA_1_11_Plasma	Plasma	XLAS
SA408477	CA_1_10_Plasma	Plasma	XLAS
SA408478	CA_1_9_Plasma	Plasma	XLAS
SA408479	CA_1_8_Plasma	Plasma	XLAS
SA408480	CA_1_7_Plasma	Plasma	XLAS
SA408481	CA_1_6_Plasma	Plasma	XLAS
SA408482	CA_1_5_Plasma	Plasma	XLAS
SA408483	CA_1_3_Plasma	Plasma	XLAS
SA408484	CA_1_2_Plasma	Plasma	XLAS
SA408485	CA_1_49_Plasma	Plasma	XLAS
SA408486	CA_1_23_Plasma	Plasma	XLAS
SA408487	CA_1_4_Plasma	Plasma	XLAS
SA408488	CA_1_25_Plasma	Plasma	XLAS
SA408489	CA_1_38_Plasma	Plasma	XLAS
SA408490	CA_1_47_Plasma	Plasma	XLAS

Showing page 1 of 3 Results: 1 2 3 Next Showing results 1 to 100 of 241

Collection:

Collection ID:	CO003877
Collection Summary:	We collected urine and plasma samples from 63 case subjects with a diagnosis of ADAS (n=51) or XLAS (n=12). The inclusion criteria were a genetic analysis with pathogenic or likely pathogenic variants in COL4A3-5 with the presence of hematuria. The same samples were collected from 15 patients with biopsy-proven diabetic kidney disease (DKD) and from 20 healthy volunteers, who served as control groups. Clinical and demographic data were collected from the medical records. The study was conducted in accordance with the principles outlined in the Declaration of Helsinki with approval from the ethics committee of Hospital Universitario 12 de Octubre. Written consent was obtained from patients at the time of sample collection.
Sample Type:	Plasma and urine

Treatment:

Treatment ID:	TR003893
Treatment Summary:	No treatment.

Sample Preparation:

Sampleprep ID:	SP003890
Sampleprep Summary:	PLASMA SAMPLES: Plasma samples underwent deproteinization and lipid extraction using an all-in-one single extraction method employing a solvent mixture composed of MeOH/MTBE/CHCl3 (4:3:3, v/v/v) for the isolation of non-polar lipids(1). The lipid extraction method started with thawing the samples on ice followed by a 2 min vortex homogenization. Subsequently, 40 µL of the plasma sample was mixed with 800 µL of the solvent mixture – also containing the internal standards (IS): 1 ppm C17-sphinganine and 2 ppm d31-palmitic acid –. The resulting mixture was vortex-mixed for 20 min followed by sample centrifugation at 16,100 x g for 10 min at 15 °C. Then, 300 µL of the supernatant were transferred to LC Chromacol (Thermo Fisher Scientific, Madrid, Sain) vials with insert and centrifuged at 16,100 x g for 10 min at 15 °C prior to the analysis. Blank solutions were prepared containing 40 µL of H2O and 800 µL of the solvent mixture and same procedure was followed. For quality control (QC) samples, three independent pools were prepared by pipetting 10 µL of each sample – for QCTOTAL –, 10 µL of Alport samples (cases 1 and 2) – for QCCASES –, and 10 µL of control samples (DM patients and healthy individuals) – for QCCONTROL–. Then 40 µL of each of the pools were transferred to Eppendorf™ tubes and 800 µL of the solvent mixture were added. The same procedure as in sample preparation was followed. Both blank and QC samples were processed in parallel with the rest of plasma samples. URINE SAMPLES: Urine samples were prepared at the "Centro de Metabolómica y Bioanálisis, CEMBIO" (Madrid, Spain), following a monophasic extraction method (MeOH:EtOH (1:1, v/v)), tested and developed in our laboratory to obtain the urine lipidomics fingerprint. Briefly, the samples were thawed on ice and then vortex-mixed for 2 min. On ice, 100 µL of urine sample was mixed with 300 µL of a cold (-20 °C) mixture of MeOH:EtOH (1:1, v/v), and a mixture of nonendogenous internal standards (C17-Sphinganine at 1 ppm and d31-palmitic acid at 2 ppm). Samples were vortex-mixed for 30 minutes. After centrifugation for 10 min at 16.1 rpm at 15 °C, 100 µL of the supernatant was collected and transferred into an HPLC-MS vial with a glass insert. Finally, all the vials were centrifuged at 2000g at 15 °C for 10 min before injecting into the system.

Sampleprep ID:

SP003890

Sampleprep Summary:

PLASMA SAMPLES: Plasma samples underwent deproteinization and lipid extraction using an all-in-one single extraction method employing a solvent mixture composed of MeOH/MTBE/CHCl3 (4:3:3, v/v/v) for the isolation of non-polar lipids(1). The lipid extraction method started with thawing the samples on ice followed by a 2 min vortex homogenization. Subsequently, 40 µL of the plasma sample was mixed with 800 µL of the solvent mixture – also containing the internal standards (IS): 1 ppm C17-sphinganine and 2 ppm d31-palmitic acid –. The resulting mixture was vortex-mixed for 20 min followed by sample centrifugation at 16,100 x g for 10 min at 15 °C. Then, 300 µL of the supernatant were transferred to LC Chromacol (Thermo Fisher Scientific, Madrid, Sain) vials with insert and centrifuged at 16,100 x g for 10 min at 15 °C prior to the analysis. Blank solutions were prepared containing 40 µL of H2O and 800 µL of the solvent mixture and same procedure was followed. For quality control (QC) samples, three independent pools were prepared by pipetting 10 µL of each sample – for QCTOTAL –, 10 µL of Alport samples (cases 1 and 2) – for QCCASES –, and 10 µL of control samples (DM patients and healthy individuals) – for QCCONTROL–. Then 40 µL of each of the pools were transferred to Eppendorf™ tubes and 800 µL of the solvent mixture were added. The same procedure as in sample preparation was followed. Both blank and QC samples were processed in parallel with the rest of plasma samples. URINE SAMPLES: Urine samples were prepared at the "Centro de Metabolómica y Bioanálisis, CEMBIO" (Madrid, Spain), following a monophasic extraction method (MeOH:EtOH (1:1, v/v)), tested and developed in our laboratory to obtain the urine lipidomics fingerprint. Briefly, the samples were thawed on ice and then vortex-mixed for 2 min. On ice, 100 µL of urine sample was mixed with 300 µL of a cold (-20 °C) mixture of MeOH:EtOH (1:1, v/v), and a mixture of nonendogenous internal standards (C17-Sphinganine at 1 ppm and d31-palmitic acid at 2 ppm). Samples were vortex-mixed for 30 minutes. After centrifugation for 10 min at 16.1 rpm at 15 °C, 100 µL of the supernatant was collected and transferred into an HPLC-MS vial with a glass insert. Finally, all the vials were centrifuged at 2000g at 15 °C for 10 min before injecting into the system.

Combined analysis:

Analysis ID	AN006158	AN006159	AN006160	AN006161
Analysis type	MS	MS	MS	MS
Chromatography type	Reversed phase	Reversed phase	Reversed phase	Reversed phase
Chromatography system	Agilent 1290 Infinity II	Agilent 1290 Infinity II	Agilent 1290 Infinity II	Agilent 1290 Infinity II
Column	Agilent InfinityLab Poroshell 120 EC-C18 (100 x 3mm,2.7um)	Agilent InfinityLab Poroshell 120 EC-C18 (100 x 3mm,2.7um)	Agilent InfinityLab Poroshell 120 EC-C18 (100 x 3mm,2.7um)	Agilent InfinityLab Poroshell 120 EC-C18 (100 x 3mm,2.7um)
MS Type	ESI	ESI	ESI	ESI
MS instrument type	QTOF	QTOF	QTOF	QTOF
MS instrument name	Agilent 6545 QTOF	Agilent 6545 QTOF	Agilent 6546 QTOF	Agilent 6546 QTOF
Ion Mode	POSITIVE	NEGATIVE	POSITIVE	NEGATIVE
Units	peak area	peak area	peak area	peak area

Chromatography:

Chromatography ID:	CH004678
Chromatography Summary:	The Agilent 1290 Infinity II Multisampler system was employed with a multiwash option, and 1 µL of extracted samples was injected. The multisampler temperature was maintained at 15 °C to ensure the stability of compounds and prevent lipid precipitation. For chromatographic separation, an Agilent InfinityLab Poroshell 120 ECC18 (3.0 × 100 mm, 2.7 µm) (Agilent Technologies) column and a compatible guard column (Agilent InfinityLab Poroshell 120 ECC18, 3.0 × 5 mm, 2.7 µm) were employed and held at 50 °C. The chromatography gradient was initiated at 70% of B at 0–1 min, increased to 86% at 3.5–10 min, and reached 100% B at 11–17 min. The initial conditions were restored by minute 17, followed by a 2-minute re-equilibration, resulting in a total running time of 19 min. The mobile phases for positive and negative ionization modes comprised (A) 10 mM ammonium acetate, 0.2 mM ammonium fluoride in a 9:1 water/methanol ratio and (B) 10 mM ammonium acetate, 0.2 mM ammonium fluoride in a 2:3:5 acetonitrile/methanol/isopropanol ratio. The flow rate was maintained at 0.6 mL/min. The multisampler's multiwash strategy involved a methanol:isopropanol (50:50, v/v) mixture with a 15-second wash time and an aqueous:organic phases (30:70, v/v) mixture to achieve the initial conditions.
Instrument Name:	Agilent 1290 Infinity II
Column Name:	Agilent InfinityLab Poroshell 120 EC-C18 (100 x 3mm,2.7um)
Column Temperature:	50 °C
Flow Gradient:	Started at 70% of B at 0–1 min, 1-3.5min linear from 70% B to 86% B, 86% B at 3.5–10 min, 10-11min linear from 86% B to 100% B, and 100% B at 11–17 min
Flow Rate:	0.6 mL/min
Solvent A:	90% water/10% methanol; 10 mM ammonium acetate; 0.2 mM ammonium fluoride
Solvent B:	20% acetonitrile/30% methanol/50% isopropanol; 10 mM ammonium acetate; 0.2 mM ammonium fluoride
Chromatography Type:	Reversed phase

MS:

MS ID:	MS005863
Analysis ID:	AN006158
Instrument Name:	Agilent 6545 QTOF
Instrument Type:	QTOF
MS Type:	ESI
MS Comments:	150 V fragmentor, 65 V skimmer, 3500 V capillary voltage, 750 V octopole radio frequency voltage, 10 L/min nebulizer gas flow, 200 °C gas temperature, 50 psi nebulizer gas pressure, 12 L/min sheath gas flow, and 300 °C sheath gas temperature. Data were collected in positive and negative ESI modes in separate runs, operated in full scan mode from 40 to 1700 m/z with a scan rate of 3 spectra/s. A solution consisting of two reference mass compounds was infused throughout the whole analysis: purine (C5H4N4) at m/z 121.0509 and HP-0921 (C18H18O6N3P3F24) at m/z 922.0098. These masses were continuously infused into the system through an Agilent 1260 Iso Pump at a 1 mL/min (split ratio 1:100) to provide a constant mass correction.
Ion Mode:	POSITIVE

MS ID:	MS005864
Analysis ID:	AN006159
Instrument Name:	Agilent 6545 QTOF
Instrument Type:	QTOF
MS Type:	ESI
MS Comments:	150 V fragmentor, 65 V skimmer, 3500 V capillary voltage, 750 V octopole radio frequency voltage, 10 L/min nebulizer gas flow, 200 °C gas temperature, 50 psi nebulizer gas pressure, 12 L/min sheath gas flow, and 300 °C sheath gas temperature. Data were collected in positive and negative ESI modes in separate runs, operated in full scan mode from 40 to 1700 m/z with a scan rate of 3 spectra/s. A solution consisting of two reference mass compounds was infused throughout the whole analysis: purine (C5H4N4) at m/z 119.0363 and HP-0921 (C18H18O6N3P3F24) at m/z 980.0163 (HP-0921 + acetate). These masses were continuously infused into the system through an Agilent 1260 Iso Pump at a 1 mL/min (split ratio 1:100) to provide a constant mass correction.
Ion Mode:	NEGATIVE

MS ID:	MS005865
Analysis ID:	AN006160
Instrument Name:	Agilent 6546 QTOF
Instrument Type:	QTOF
MS Type:	ESI
MS Comments:	150 V fragmentor, 65 V skimmer, 3500 V capillary voltage, 750 V octopole radio frequency voltage, 10 L/min nebulizer gas flow, 200 °C gas temperature, 50 psi nebulizer gas pressure, 12 L/min sheath gas flow, and 300 °C sheath gas temperature. Data were collected in positive and negative ESI modes in separate runs, operated in full scan mode from 40 to 1700 m/z with a scan rate of 3 spectra/s. A solution consisting of two reference mass compounds was infused throughout the whole analysis: purine (C5H4N4) at m/z 121.0509 and HP-0921 (C18H18O6N3P3F24) at m/z 922.0098. These masses were continuously infused into the system through an Agilent 1260 Iso Pump at a 1 mL/min (split ratio 1:100) to provide a constant mass correction.
Ion Mode:	POSITIVE

MS ID:	MS005866
Analysis ID:	AN006161
Instrument Name:	Agilent 6546 QTOF
Instrument Type:	QTOF
MS Type:	ESI
MS Comments:	150 V fragmentor, 65 V skimmer, 3500 V capillary voltage, 750 V octopole radio frequency voltage, 10 L/min nebulizer gas flow, 200 °C gas temperature, 50 psi nebulizer gas pressure, 12 L/min sheath gas flow, and 300 °C sheath gas temperature. Data were collected in positive and negative ESI modes in separate runs, operated in full scan mode from 40 to 1700 m/z with a scan rate of 3 spectra/s. A solution consisting of two reference mass compounds was infused throughout the whole analysis: purine (C5H4N4) at m/z 119.0363 and HP-0921 (C18H18O6N3P3F24) at m/z 980.0163 (HP-0921 + acetate). These masses were continuously infused into the system through an Agilent 1260 Iso Pump at a 1 mL/min (split ratio 1:100) to provide a constant mass correction.
Ion Mode:	NEGATIVE