Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA249255

Local Sample ID:	sample_0028
Subject ID:	SU002588
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Gender:	Male and female

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Subject:

Subject ID:	SU002588
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Gender:	Male and female

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
sample_0028	SA249255	FL031489	2 - Anorexia	Category

Collection:

Collection ID:	CO002581
Collection Summary:	Analysis of serum polar metabolites by gas chromatography - time-of-flght mass spectrometry The metabolites listed as gut microbiota-related metabolites is based on literature mining 77,78 . Serum samples were randomized, and the sample preparation was done as described previously 43,79 . Shortly, 400 μL of MeOH containing internal standards (heptadecanoic acid, deuterium-labeled DL-valine, deuterium- labeled succinic acid, and deuterium-labeled glutamic acid, c= 1 μg/ml) was added to 30 μl of the serum samples which were vortex mixed and incubated on ice for 30 min after which they were centrifuged (9400 × g, 3 min) and 350 μL of the supernatant was collected after centrifugation. The solvent was evaporated to dryness and 25 μL of MOX reagent was added and the sample was incubated for 60 min at 45 °C. 25 μL of N-Methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) was added and after 60 min incubation at 45 °C 25 μL of the retention index standard mixture (n-alkanes, c=10 μg/ml) was added. The analyses were done by Agilent 7890B gas chromatography (GC) coupled to Agilent 7200 auadrupole time-of-flight mass spectrometry (Q-TOF MS) (Agilent Technologies, Palo Alto, CA, USA). Injection volume 1 μL with 100:1 split on PTV at 70 °C, heating to 300 °C at 120 °C /min. Column: Zebron ZB- SemiVolatiles. Length: 20m, I.D. 0.18mm, film thickness: 0.18 μm. With initial Helium flow 1.2 mL/min, increasing to 2.4 mL/min after 16 mins. Oven temperature program: 50 °C (5 min), then to 270°C at 20 °/min and then to 300 at 40 °/min (5 min). EI source: 250 °C, 70 eV electron energy, 35μA emission, solvent delay 3 min. Mass range 55 to 650 amu, acquisition rate 5 spectra/s, acquisition time 200 ms/spectrum. Quad at 150 °C, 1.5 mL/min N2 collision flow, aux-2 temperature: 280 °C. Calibration curves were constructed using alanine, citric acid, fumaric acid, glutamic acid, glycine, lactic acid, malic acid, 2-hydroxybutyric acid, 3-hydroxybutyric acid, linoleic acid, oleic acid, palmitic acid, stearic acid, cholesterol, fructose, glutamine, indole-3-propionic acid, isoleucine, leucine, proline, succinic acid, valine, asparagine, aspartic acid, arachidonic acid, glycerol-3-phosphate, lysine, methionine, ornithine, phenylalanine, serine and threonine purchased from Sigma-Aldrich (St. Louis, MO, USA) at concentration range of 0.1 to 80 μg/ml. An aliquot of each sample was collected and pooled and used as quality control samples, together with National Institute of Standards and Technology (NIST) CRM1950 serum sample, an in-house pooled serum sample. The relative standard deviation of the concentrations was on average 16% for the pooled QC samples, 10% for the NIST samples. Analysis of serum bile acids and serum semipolar metabolites The sample preparation procedure was performed as described previously 80 . The plate was preconditioned with 450 μL acetonitrile before the addition of 100 μL of sample and 10 μL of PFAS and BA internal standard mixture (200 ng/mL and 1000 ng/mL respectively). Thereafter, 450 μL of acetonitrile containing 1% formic acid were added to each well and the samples extracted using a 10” vacuum manifold. The eluate was evaporated to dryness under nitrogen gas flow and reconstituted to 80 μL of MeOH/2 mM aqueous NH4AC. Chromatographic separation was carried out using an Acquity UPLC BEH C18 column (100 mm × 2.1 mm i.d., 1.7 μm particle size), fitted with a C18 precolumn (Waters Corporation, Wexford, Ireland). Mobile phase A consisted of H2O:MeOH (v/v 70:30) and mobile phase B of MeOH with both phases containing 2mM ammonium acetate as an ionization agent. The flow rate was set at 0.4 mLmin-1 with the elution gradient as follows: 0-1.5 min, mobile phase B was increased from 5% to 30%; 1.5-4.5 min, mobile phase B increased to 70%; 4.5-7.5 min, mobile phase B increased to 100% and held for 5.5 min. A post-time of 5 min was used to regain the initial conditions for the next analysis. The total run time per sample was 18 min. The dual ESI ionization source was settings were as follows: capillary voltage was 4.5 kV, nozzle voltage 1500 V, N2 pressure in the nebulized was 21 psi and the N2 flow rate and temperature as sheath gas was 11 L min-1 and 379 °C, respectively. In order to obtain accurate mass spectra in MS scan, the m/z range was set to 100-1700 in negative ion mode. MassHunter B.06.01 software (Agilent Technologies, Santa Clara, CA, USA) was used for all data acquisition. Identification of compounds was done by in-house spectral library using MS (and retention time), MS/MS information. Quantitation was based on a matrix matched calibration curve spiked with native compounds. The calibration curve consisted of concentrations ranging from 0 – 1600 ng mL -1 for BAs. The RSD for the BAs was on average 17.8 % for the QC samples and 19.4 % for the NIST samples.
Sample Type:	Blood (serum)
Storage Conditions:	-80℃

Treatment:

Treatment ID:	TR002600
Treatment Summary:	No treatment was applied.

Sample Preparation:

Sampleprep ID:	SP002594
Sampleprep Summary:	Analysis of serum polar metabolites by gas chromatography - time-of-flght mass spectrometry The metabolites listed as gut microbiota-related metabolites is based on literature mining 77,78 . Serum samples were randomized, and the sample preparation was done as described previously 43,79 . Shortly, 400 μL of MeOH containing internal standards (heptadecanoic acid, deuterium-labeled DL-valine, deuterium- labeled succinic acid, and deuterium-labeled glutamic acid, c= 1 μg/ml) was added to 30 μl of the serum samples which were vortex mixed and incubated on ice for 30 min after which they were centrifuged (9400 × g, 3 min) and 350 μL of the supernatant was collected after centrifugation. The solvent was evaporated to dryness and 25 μL of MOX reagent was added and the sample was incubated for 60 min at 45 °C. 25 μL of N-Methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) was added and after 60 min incubation at 45 °C 25 μL of the retention index standard mixture (n-alkanes, c=10 μg/ml) was added. The analyses were done by Agilent 7890B gas chromatography (GC) coupled to Agilent 7200 auadrupole time-of-flight mass spectrometry (Q-TOF MS) (Agilent Technologies, Palo Alto, CA, USA). Injection volume 1 μL with 100:1 split on PTV at 70 °C, heating to 300 °C at 120 °C /min. Column: Zebron ZB- SemiVolatiles. Length: 20m, I.D. 0.18mm, film thickness: 0.18 μm. With initial Helium flow 1.2 mL/min, increasing to 2.4 mL/min after 16 mins. Oven temperature program: 50 °C (5 min), then to 270°C at 20 °/min and then to 300 at 40 °/min (5 min). EI source: 250 °C, 70 eV electron energy, 35μA emission, solvent delay 3 min. Mass range 55 to 650 amu, acquisition rate 5 spectra/s, acquisition time 200 ms/spectrum. Quad at 150 °C, 1.5 mL/min N2 collision flow, aux-2 temperature: 280 °C. Calibration curves were constructed using alanine, citric acid, fumaric acid, glutamic acid, glycine, lactic acid, malic acid, 2-hydroxybutyric acid, 3-hydroxybutyric acid, linoleic acid, oleic acid, palmitic acid, stearic acid, cholesterol, fructose, glutamine, indole-3-propionic acid, isoleucine, leucine, proline, succinic acid, valine, asparagine, aspartic acid, arachidonic acid, glycerol-3-phosphate, lysine, methionine, ornithine, phenylalanine, serine and threonine purchased from Sigma-Aldrich (St. Louis, MO, USA) at concentration range of 0.1 to 80 μg/ml. An aliquot of each sample was collected and pooled and used as quality control samples, together with National Institute of Standards and Technology (NIST) CRM1950 serum sample, an in-house pooled serum sample. The relative standard deviation of the concentrations was on average 16% for the pooled QC samples, 10% for the NIST samples. Analysis of serum bile acids and serum semipolar metabolites The sample preparation procedure was performed as described previously 80 . The plate was preconditioned with 450 μL acetonitrile before the addition of 100 μL of sample and 10 μL of PFAS and BA internal standard mixture (200 ng/mL and 1000 ng/mL respectively). Thereafter, 450 μL of acetonitrile containing 1% formic acid were added to each well and the samples extracted using a 10” vacuum manifold. The eluate was evaporated to dryness under nitrogen gas flow and reconstituted to 80 μL of MeOH/2 mM aqueous NH4AC. Chromatographic separation was carried out using an Acquity UPLC BEH C18 column (100 mm × 2.1 mm i.d., 1.7 μm particle size), fitted with a C18 precolumn (Waters Corporation, Wexford, Ireland). Mobile phase A consisted of H2O:MeOH (v/v 70:30) and mobile phase B of MeOH with both phases containing 2mM ammonium acetate as an ionization agent. The flow rate was set at 0.4 mLmin-1 with the elution gradient as follows: 0-1.5 min, mobile phase B was increased from 5% to 30%; 1.5-4.5 min, mobile phase B increased to 70%; 4.5-7.5 min, mobile phase B increased to 100% and held for 5.5 min. A post-time of 5 min was used to regain the initial conditions for the next analysis. The total run time per sample was 18 min. The dual ESI ionization source was settings were as follows: capillary voltage was 4.5 kV, nozzle voltage 1500 V, N2 pressure in the nebulized was 21 psi and the N2 flow rate and temperature as sheath gas was 11 L min-1 and 379 °C, respectively. In order to obtain accurate mass spectra in MS scan, the m/z range was set to 100-1700 in negative ion mode. MassHunter B.06.01 software (Agilent Technologies, Santa Clara, CA, USA) was used for all data acquisition. Identification of compounds was done by in-house spectral library using MS (and retention time), MS/MS information. Quantitation was based on a matrix matched calibration curve spiked with native compounds. The calibration curve consisted of concentrations ranging from 0 – 1600 ng mL -1 for BAs. The RSD for the BAs was on average 17.8 % for the QC samples and 19.4 % for the NIST samples.

Sampleprep ID:

SP002594

Sampleprep Summary:

Analysis of serum polar metabolites by gas chromatography - time-of-flght mass spectrometry The metabolites listed as gut microbiota-related metabolites is based on literature mining 77,78 . Serum samples were randomized, and the sample preparation was done as described previously 43,79 . Shortly, 400 μL of MeOH containing internal standards (heptadecanoic acid, deuterium-labeled DL-valine, deuterium- labeled succinic acid, and deuterium-labeled glutamic acid, c= 1 μg/ml) was added to 30 μl of the serum samples which were vortex mixed and incubated on ice for 30 min after which they were centrifuged (9400 × g, 3 min) and 350 μL of the supernatant was collected after centrifugation. The solvent was evaporated to dryness and 25 μL of MOX reagent was added and the sample was incubated for 60 min at 45 °C. 25 μL of N-Methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) was added and after 60 min incubation at 45 °C 25 μL of the retention index standard mixture (n-alkanes, c=10 μg/ml) was added. The analyses were done by Agilent 7890B gas chromatography (GC) coupled to Agilent 7200 auadrupole time-of-flight mass spectrometry (Q-TOF MS) (Agilent Technologies, Palo Alto, CA, USA). Injection volume 1 μL with 100:1 split on PTV at 70 °C, heating to 300 °C at 120 °C /min. Column: Zebron ZB- SemiVolatiles. Length: 20m, I.D. 0.18mm, film thickness: 0.18 μm. With initial Helium flow 1.2 mL/min, increasing to 2.4 mL/min after 16 mins. Oven temperature program: 50 °C (5 min), then to 270°C at 20 °/min and then to 300 at 40 °/min (5 min). EI source: 250 °C, 70 eV electron energy, 35μA emission, solvent delay 3 min. Mass range 55 to 650 amu, acquisition rate 5 spectra/s, acquisition time 200 ms/spectrum. Quad at 150 °C, 1.5 mL/min N2 collision flow, aux-2 temperature: 280 °C. Calibration curves were constructed using alanine, citric acid, fumaric acid, glutamic acid, glycine, lactic acid, malic acid, 2-hydroxybutyric acid, 3-hydroxybutyric acid, linoleic acid, oleic acid, palmitic acid, stearic acid, cholesterol, fructose, glutamine, indole-3-propionic acid, isoleucine, leucine, proline, succinic acid, valine, asparagine, aspartic acid, arachidonic acid, glycerol-3-phosphate, lysine, methionine, ornithine, phenylalanine, serine and threonine purchased from Sigma-Aldrich (St. Louis, MO, USA) at concentration range of 0.1 to 80 μg/ml. An aliquot of each sample was collected and pooled and used as quality control samples, together with National Institute of Standards and Technology (NIST) CRM1950 serum sample, an in-house pooled serum sample. The relative standard deviation of the concentrations was on average 16% for the pooled QC samples, 10% for the NIST samples. Analysis of serum bile acids and serum semipolar metabolites The sample preparation procedure was performed as described previously 80 . The plate was preconditioned with 450 μL acetonitrile before the addition of 100 μL of sample and 10 μL of PFAS and BA internal standard mixture (200 ng/mL and 1000 ng/mL respectively). Thereafter, 450 μL of acetonitrile containing 1% formic acid were added to each well and the samples extracted using a 10” vacuum manifold. The eluate was evaporated to dryness under nitrogen gas flow and reconstituted to 80 μL of MeOH/2 mM aqueous NH4AC. Chromatographic separation was carried out using an Acquity UPLC BEH C18 column (100 mm × 2.1 mm i.d., 1.7 μm particle size), fitted with a C18 precolumn (Waters Corporation, Wexford, Ireland). Mobile phase A consisted of H2O:MeOH (v/v 70:30) and mobile phase B of MeOH with both phases containing 2mM ammonium acetate as an ionization agent. The flow rate was set at 0.4 mLmin-1 with the elution gradient as follows: 0-1.5 min, mobile phase B was increased from 5% to 30%; 1.5-4.5 min, mobile phase B increased to 70%; 4.5-7.5 min, mobile phase B increased to 100% and held for 5.5 min. A post-time of 5 min was used to regain the initial conditions for the next analysis. The total run time per sample was 18 min. The dual ESI ionization source was settings were as follows: capillary voltage was 4.5 kV, nozzle voltage 1500 V, N2 pressure in the nebulized was 21 psi and the N2 flow rate and temperature as sheath gas was 11 L min-1 and 379 °C, respectively. In order to obtain accurate mass spectra in MS scan, the m/z range was set to 100-1700 in negative ion mode. MassHunter B.06.01 software (Agilent Technologies, Santa Clara, CA, USA) was used for all data acquisition. Identification of compounds was done by in-house spectral library using MS (and retention time), MS/MS information. Quantitation was based on a matrix matched calibration curve spiked with native compounds. The calibration curve consisted of concentrations ranging from 0 – 1600 ng mL -1 for BAs. The RSD for the BAs was on average 17.8 % for the QC samples and 19.4 % for the NIST samples.

Combined analysis:

Analysis ID	AN004092
Analysis type	MS
Chromatography type	Reversed phase
Chromatography system	Agilent 7890B
Column	Waters ACQUITY UPLC BEH C18 (100 x 2.1mm,1.7um)
MS Type	EI
MS instrument type	QTOF
MS instrument name	Agilent 7200 QTOF
Ion Mode	NEGATIVE
Units	Raw output from software

Chromatography:

Chromatography ID:	CH003030
Chromatography Summary:	The plate was preconditioned with 450 μL acetonitrile before the addition of 100 μL of sample and 10 μL of PFAS and BA internal standard mixture (200 ng/mL and 1000 ng/mL respectively). Thereafter, 450 μL of acetonitrile containing 1% formic acid were added to each well and the samples extracted using a 10” vacuum manifold. The eluate evaporated to dryness under nitrogen gas flow and reconstituted to 80 μL of MeOH/2 mM aqueous NH4AC. Chromatographic separation was carried out using an Acquity UPLC BEH C18 column (100 mm × 2.1 mm i.d., 1.7 μm particle size), fitted with a C18 precolumn (Waters Corporation, Wexford, Ireland). Mobile phase A consisted of H2O:MeOH (v/v 70:30) and mobile phase B of MeOH with both phases containing 2mM ammonium acetate as an ionization agent. The flow rate was set at 0.4 mLmin-1 with the elution gradient as follows: 0-1.5 min, mobile phase B was increased from 5% to 30%; 1.5-4.5 min, mobile phase B increased to 70%; 4.5-7.5 min, mobile phase B increased to 100% and held for 5.5 min. A post-time of 5 min was used to regain the initial conditions for the next analysis. The total run time per sample was 18 min. The dual ESI ionization source was settings were as follows: capillary voltage was 4.5 kV, nozzle voltage 1500 V, N2 pressure in the nebulized was 21 psi and the N2 flow rate and temperature as sheath gas was 11 L min-1 and 379 °C, respectively.
Instrument Name:	Agilent 7890B
Column Name:	Waters ACQUITY UPLC BEH C18 (100 x 2.1mm,1.7um)
Column Temperature:	70
Flow Gradient:	The elution gradient was as follows: 0-1.5 min, mobile phase B was increased from 5% to 30%; 1.5-4.5 min, mobile phase B increased to 70%; 4.5-7.5 min, mobile phase B increased to 100% and held for 5.5 min. A post-time of 5 min was used to regain the initial conditions for the next analysis. The total run time per sample was 18 min.
Flow Rate:	0.4ml/min
Solvent A:	70% water/30% methanol
Solvent B:	100% methanol
Chromatography Type:	Reversed phase

MS:

MS ID:	MS003839
Analysis ID:	AN004092
Instrument Name:	Agilent 7200 QTOF
Instrument Type:	QTOF
MS Type:	EI
MS Comments:	MassHunter B.06.01 software (Agilent Technologies, Santa Clara, CA, USA) was used for all data acquisition. Identification of compounds was done by in-house spectral library using MS (and retention time), MS/MS information. Quantitation was based on a matrix matched calibration curve spiked with native compounds. The calibration curve consisted of concentrations ranging from 0 – 1600 ng mL -1 for BAs. The RSD for the BAs was on average 17.8 % for the QC samples and 19.4 % for the NIST samples.
Ion Mode:	NEGATIVE