Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA333530

Local Sample ID:	L77
Subject ID:	SU003218
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606

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Subject:

Subject ID:	SU003218
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
L77	SA333530	FL039607	Plasma	Sample source
L77	SA333530	FL039607	Long COVID	Factor

Collection:

Collection ID:	CO003211
Collection Summary:	Samples were collected after fasting conditions. 1,500 μL of cold MeOH/EtOH (1:1, v/v) were added to 500 μL of plasma for virus inactivation. Afterward, samples were vigorously mixed using vortex for 1 min, followed by incubation on ice for 5 min, and subsequent centrifugation at 16,000 x g for 20 min at 4 °C to eliminate proteins by precipitation. The resulting extract, containing the metabolites of interest, was transferred to EppendorfTM tubes, and stored at –80 °C until analysis.
Sample Type:	Blood (plasma)
Storage Conditions:	-80℃

Treatment:

Treatment ID:	TR003227
Treatment Summary:	N/A

Sample Preparation:

Sampleprep ID:	SP003224
Sampleprep Summary:	For CE-MS analysis 200 µL of frozen plasma extract (MeOH/EtOH 1:1, v/v) were thawed on ice and dried completely using a SpeedVac Concentrator System. The resulting residue was then reconstituted in 100 µL of 0.2 mM MetS dissolved in 0.1 M FA. After vortex-mixing for 1 min, the samples were transferred to a Millipore filter with a 30 kDa protein cutoff and centrifuged at 2,000 x g for 40 min at 4 °C. Finally, the resulting ultrafiltrate was transferred to a CE-MS vial for further analysis. Prior to the analysis vials were centrifuged at 2,000 x g for 10 min at 4 °C to ensure that any possible sediment remained at the bottom of the vial. For GC-MS analysis 200 µL of each plasma extract was thawed on ice to room temperature and 30 µL of the internal standard (IS), palmitic acid-d31 in MeOH (80 mg/mL) was added to it. The mixture was vortexed for 5 min and 200 µL of the solution were transferred to a GC-MS vial. Then, samples were evaporated to dryness using a SpeedVac Concentrator System and maintained at 8 °C in the Gerstel Multiple Purpose Sample (MPS) Preparation Station. An automated two step derivatization process was performed before sample injection using a protocol adjusted from a previous reported method. First, each precipitate was redissolved in 20 µL of O-methoxyamine solution (15 mg/mL in pyridine) for the methoximation process, mixed 10 min at 1,000 rpm and incubated for 90 min at 60 °C at 750 rpm. Second, and after waiting 5 min, 40 µL of BSTFA with 1% TMCS were added for the silylation process. Then, samples were mixed for 10 min at 1,000 rpm and incubated for 60 min at 60 °C at 750 rpm. After waiting 30 min at 8 °C, 80 µL of heptane containing 20 mg/mL of tricosane (IS) were added and mixed for 5 min at 1,000 rpm. Finally, samples were maintained at 8 °C for 30 min before injection. For LC-MS 200 µL of each plasma extract was thawed on ice to room temperature and centrifuged for 10 min at 16,100 x g at 4 °C, transferred to a LC-MS vial and directly injected into the system.

Sampleprep ID:

SP003224

Sampleprep Summary:

For CE-MS analysis 200 µL of frozen plasma extract (MeOH/EtOH 1:1, v/v) were thawed on ice and dried completely using a SpeedVac Concentrator System. The resulting residue was then reconstituted in 100 µL of 0.2 mM MetS dissolved in 0.1 M FA. After vortex-mixing for 1 min, the samples were transferred to a Millipore filter with a 30 kDa protein cutoff and centrifuged at 2,000 x g for 40 min at 4 °C. Finally, the resulting ultrafiltrate was transferred to a CE-MS vial for further analysis. Prior to the analysis vials were centrifuged at 2,000 x g for 10 min at 4 °C to ensure that any possible sediment remained at the bottom of the vial. For GC-MS analysis 200 µL of each plasma extract was thawed on ice to room temperature and 30 µL of the internal standard (IS), palmitic acid-d31 in MeOH (80 mg/mL) was added to it. The mixture was vortexed for 5 min and 200 µL of the solution were transferred to a GC-MS vial. Then, samples were evaporated to dryness using a SpeedVac Concentrator System and maintained at 8 °C in the Gerstel Multiple Purpose Sample (MPS) Preparation Station. An automated two step derivatization process was performed before sample injection using a protocol adjusted from a previous reported method. First, each precipitate was redissolved in 20 µL of O-methoxyamine solution (15 mg/mL in pyridine) for the methoximation process, mixed 10 min at 1,000 rpm and incubated for 90 min at 60 °C at 750 rpm. Second, and after waiting 5 min, 40 µL of BSTFA with 1% TMCS were added for the silylation process. Then, samples were mixed for 10 min at 1,000 rpm and incubated for 60 min at 60 °C at 750 rpm. After waiting 30 min at 8 °C, 80 µL of heptane containing 20 mg/mL of tricosane (IS) were added and mixed for 5 min at 1,000 rpm. Finally, samples were maintained at 8 °C for 30 min before injection. For LC-MS 200 µL of each plasma extract was thawed on ice to room temperature and centrifuged for 10 min at 16,100 x g at 4 °C, transferred to a LC-MS vial and directly injected into the system.

Combined analysis:

Analysis ID	AN005077	AN005078	AN005079	AN005080	AN005081
Analysis type	MS	MS	MS	MS	MS
Chromatography type	Reversed phase	Reversed phase	GC	CE	CE
Chromatography system	Agilent 1290 Infinity II	Agilent 1290 Infinity II	Agilent 8890 GC System	Agilent 7100 CE	Agilent 7100 CE
Column	Agilent InfinityLab Poroshell 120 EC-C18 (100 x 3mm,2.7um)	Agilent InfinityLab Poroshell 120 EC-C18 (100 x 3mm,2.7um)	GC DB5-MS column (40 m length, 0.25 mm inner diameter, and 0.25 µm film of 95% dimethyl/5% diphenylpolysiloxane)	Agilent Technologies fused silica capillary (total length, 100 cm; internal diameter, 50 µm)	Agilent Technologies fused silica capillary (total length, 100 cm; internal diameter, 50 µm)
MS Type	ESI	ESI	EI	ESI	ESI
MS instrument type	QTOF	QTOF	Single quadrupole	TOF	TOF
MS instrument name	Agilent 6545 QTOF	Agilent 6545 QTOF	Agilent 5977B	Agilent 6230 TOF	Agilent 6230 TOF
Ion Mode	POSITIVE	NEGATIVE	UNSPECIFIED	POSITIVE	NEGATIVE
Units	Area	Corrected areas	Corrected areas	Corrected areas	Corrected areas

Chromatography:

Chromatography ID:	CH003834
Chromatography Summary:	RP-UHPLC-ESI(+)-QTOF-MS
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:	The chromatographic gradient started at 70 % of B at 0 - 1 min, 86 % B at 3.5 - 10 min, 100% B at 11 - 17 min. The starting conditions were recovered by min 17, followed by a 2 min re-equilibration time, reaching a total running time of 19 min
Flow Rate:	0.6 mL/min
Solvent A:	10 mM CH3COONH4 and 0.2 mM NH4F in H2O/MeOH (9:1, v/v)
Solvent B:	10 mM CH3COONH4 and 0.2 mM NH4F in ACN/MeOH/IPA (2:3:5, v/v/v)
Analytical Time:	19 min
Chromatography Type:	Reversed phase

Chromatography ID:	CH003835
Chromatography Summary:	RP-UHPLC-ESI(-)-QTOF-MS
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:	The chromatographic gradient started at 70 % of B at 0 - 1 min, 86 % B at 3.5 - 10 min, 100% B at 11 - 17 min. The starting conditions were recovered by min 17, followed by a 2 min re-equilibration time, reaching a total running time of 19 min
Flow Rate:	0.6 mL/min
Solvent A:	10 mM CH3COONH4 and 0.2 mM NH4F in H2O/MeOH (9:1, v/v)
Solvent B:	10 mM CH3COONH4 and 0.2 mM NH4F in ACN/MeOH/IPA (2:3:5, v/v/v)
Analytical Time:	19 min
Chromatography Type:	Reversed phase

Chromatography ID:	CH003836
Chromatography Summary:	GC-Q-MS
Instrument Name:	Agilent 8890 GC System
Column Name:	GC DB5-MS column (40 m length, 0.25 mm inner diameter, and 0.25 µm film of 95% dimethyl/5% diphenylpolysiloxane)
Column Temperature:	The column temperature gradient was programmed as follows: it started at 60 °C for 1 min, then increased by 10 °C/min until reaching 325 °C and held at this temperature for 10 min before cooling down
Flow Gradient:	Constant
Flow Rate:	0.5658 mL/min
Solvent A:	Helium
Solvent B:	N/A
Analytical Time:	37 min followed by 5 min of post-run period
Chromatography Type:	GC

Chromatography ID:	CH003837
Chromatography Summary:	CE-ESI(+)-TOF-MS
Instrument Name:	Agilent 7100 CE
Column Name:	Agilent Technologies fused silica capillary (total length, 100 cm; internal diameter, 50 µm)
Column Temperature:	20°C
Flow Gradient:	N/A
Flow Rate:	N/A
Solvent A:	BGE (1 M FA in 10% MeOH)
Solvent B:	N/A
Analytical Time:	26 min
Capillary Voltage:	30kV
Sheath Liquid:	MeOH: H2O (1:1, v/v) with 1mM FA containing two reference masses (purine: 121.0509 and HP-0922: 922.0098) at a flow rate of 0.6 mL/min (1:100 of split ratio)
Chromatography Type:	CE

Chromatography ID:	CH003838
Chromatography Summary:	CE-ESI(-)-TOF-MS
Instrument Name:	Agilent 7100 CE
Column Name:	Agilent Technologies fused silica capillary (total length, 100 cm; internal diameter, 50 µm)
Column Temperature:	20°C
Flow Gradient:	N/A
Flow Rate:	N/A
Solvent A:	BGE (1 M FA in 10% MeOH)
Solvent B:	N/A
Analytical Time:	40 min
Capillary Voltage:	-30kV
Sheath Liquid:	MeOH: H2O (1:1, v/v) with 1mM FA containing two reference masses (purine: 121.0509 and HP-0922: 922.0098) at a flow rate of 1.0 mL/min (1:100 of split ratio)
Chromatography Type:	CE

MS:

MS ID:	MS004815
Analysis ID:	AN005077
Instrument Name:	Agilent 6545 QTOF
Instrument Type:	QTOF
MS Type:	ESI
MS Comments:	The mass spectrometer operated in full scan mode, scanning from m/z 40 - 1700 at a scan rate of 3 spectra/s. During the analysis, a solution containing two reference mass compounds was continuously infused to the system at a flow rate of 1 mL/min to provide mass correction. The reference masses used were m/z 121.0509 (purine detected as [C5H4N4 + H]+) and m/z 922.0098 (HP-0921 detected as [C18H18O6N3P3F24 + H]+) for ESI(+) and m/z 119.0363 (purine detected as [C5H4N4 - H]-) and m/z 1033.9881 (HP-0921 detected as [C18H18O6N3P3F24 + CF3COOH-H]-) for ESI(–). At the end of the analysis, ten iterative-MS/MS runs were performed for both, positive and negative ionization modes using a QC sample. They were operated with a MS and MS/MS scan rates of 3 spectra/s, 3 precursors per cycle, a mass range of m/z 40 - 1700, a narrow (~ 1.3 amu) MS/MS isolation width, and 5000 counts and 0.001 % of MS/MS threshold. The collision energy for the first five iterative-MS/MS runs was set at 20 eV, and the subsequent five runs were performed at 40 eV. To ensure accuracy, reference masses and contaminants detected in blank samples were excluded from the analysis. This prevented thein inclusion in the iterative-MS/MS runs. Data was acquired using MassHunter Workstation Software LC-MS Data Acquisition v B.09.00 (Agilent Technologies, Waldbronn, Germany).
Ion Mode:	POSITIVE

MS ID:	MS004816
Analysis ID:	AN005078
Instrument Name:	Agilent 6545 QTOF
Instrument Type:	QTOF
MS Type:	ESI
MS Comments:	The mass spectrometer operated in full scan mode, scanning from m/z 40 - 1700 at a scan rate of 3 spectra/s. During the analysis, a solution containing two reference mass compounds was continuously infused to the system at a flow rate of 1 mL/min to provide mass correction. The reference masses used were m/z 121.0509 (purine detected as [C5H4N4 + H]+) and m/z 922.0098 (HP-0921 detected as [C18H18O6N3P3F24 + H]+) for ESI(+) and m/z 119.0363 (purine detected as [C5H4N4 - H]-) and m/z 1033.9881 (HP-0921 detected as [C18H18O6N3P3F24 + CF3COOH-H]-) for ESI(–). At the end of the analysis, ten iterative-MS/MS runs were performed for both, positive and negative ionization modes using a QC sample. They were operated with a MS and MS/MS scan rates of 3 spectra/s, 3 precursors per cycle, a mass range of m/z 40 - 1700, a narrow (~ 1.3 amu) MS/MS isolation width, and 5000 counts and 0.001 % of MS/MS threshold. The collision energy for the first five iterative-MS/MS runs was set at 20 eV, and the subsequent five runs were performed at 40 eV. To ensure accuracy, reference masses and contaminants detected in blank samples were excluded from the analysis. This prevented thein inclusion in the iterative-MS/MS runs. Data was acquired using MassHunter Workstation Software LC-MS Data Acquisition v B.09.00 (Agilent Technologies, Waldbronn, Germany).
Ion Mode:	NEGATIVE

MS ID:	MS004817
Analysis ID:	AN005079
Instrument Name:	Agilent 5977B
Instrument Type:	Single quadrupole
MS Type:	EI
MS Comments:	For ionization, the EI source was used with the following settings: filament source temperature at 230 °C and electron ionization energy at 70 eV. Mass spectra were collected in a mass range of m/z 50 - 600 at a scan rate of 2 spectra/s. Mass calibration was performed after every injection with internal reference masses m/z 68.9947, 263.9866 and 501.9706. Data acquisition was performed using the Agilent MassHunter Workstation GC-MS Data Acquisition v 10.0 (Agilent Technologies, Waldbronn, Germany).
Ion Mode:	UNSPECIFIED

MS ID:	MS004818
Analysis ID:	AN005080
Instrument Name:	Agilent 6230 TOF
Instrument Type:	TOF
MS Type:	ESI
MS Comments:	The mass spectra data were acquired in positive polarity mode with a full scan range of m/z 70 - 1000 at a rate of 1.36 spectra/s. The following MS parameters were employed: fragmentor set to 125 V, skimmer to 65 V, OCT RF Vpp to 750 V, drying gas temperature to 200 °C ESI(+), flow rate to 10 L/min, nebulizer to 10 psi, and capillary voltage to 3500 V ESI(+). The Agilent MassHunter Workstation v B.09.00 (Agilent Technologies, Waldbronn, Germany) was used for CE-MS data acquisition.
Ion Mode:	POSITIVE

MS ID:	MS004819
Analysis ID:	AN005081
Instrument Name:	Agilent 6230 TOF
Instrument Type:	TOF
MS Type:	ESI
MS Comments:	The mass spectra data were acquired in negative polarity mode with a full scan range m/z 60 - 1000 at a rate of 1 spectra/s. The following MS parameters were employed: fragmentor set to 125 V, skimmer to 65 V, OCT RF Vpp to 750 V, drying gas temperature to 275 °C ESI(–), flow rate to 10 L/min, nebulizer to 10 psi, and capillary voltage to 2000 V ESI(-). The Agilent MassHunter Workstation v B.09.00 (Agilent Technologies, Waldbronn, Germany) was used for CE-MS data acquisition.
Ion Mode:	NEGATIVE