Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA127701

Local Sample ID:	Control_Urine_LC_NEG
Subject ID:	SU001592
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606

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Subject:

Subject ID:	SU001592
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
Control_Urine_LC_NEG	SA127701	FL015710	Control	Subject

Collection:

Collection ID:	CO001587
Collection Summary:	Metabolite extraction was performed in plasma and urine samples from the patient and a healthy control, matched for age, sex and weight, according to standard protocols with some modifications.
Sample Type:	Blood (plasma)
Storage Conditions:	-80℃

Treatment:

Treatment ID:	TR001607
Treatment Summary:	Metabolite extraction was performed in plasma and urine samples from the patient and a healthy control, matched for age, sex and weight, according to standard protocols with some modifications.

Sample Preparation:

Sampleprep ID:	SP001600
Sampleprep Summary:	Plasma sample preparation for liquid chromatography–mass spectrometry (LC–MS) entailed the following steps: (1) thaw sample on ice, vortex for 2 min and transfer 100 µL of plasma into an Eppendorf tube; (2) for protein precipitation, add 300 µL of cold (-18˚C) methanol/ethanol (1:1 v/v), vortex for 1 min, incubate on ice for 5 min and vortex again briefly; (3) centrifuge at 13000 rpm, 4˚C, for 20 min; and (4) transfer the supernatant into the LC–MS system. Plasma sample preparation for gas chromatography–mass spectrometry (GC–MS) entailed the following steps: (1) thaw sample on ice, vortex for 2 min and transfer 40 µL of plasma into an Eppendorf tube; (2) for protein precipitation, add 120 µL of cold acetonitrile, vortex for 2 min and incubate on ice for 5 min; (3) centrifuge at 15400 rpm, 4˚C, for 10 min; and (4) transfer the supernatant into a GC vial with insert; (5) evaporate in a Speedvac at 30°C until dry; (6) add 10 µL of O-methoxyamine hydrochloride (15 mg/mL) in pyridine and vortex for 5 min; (7) sonicate for 2 min and vortex for 2 min (repeat step a total of three times); (8) incubate at room temperature for 16 h in darkness for the reaction of the methoxymation; (9) add 10 µL of N,O-Bis(trimethylsilyl)trifluoroacetamide (BSTFA) with 1 % trimethylchlorosilane (TMCS) and incubate 1 h at 70 ˚C for the silylation; (10) add 100 µL of 10 ppm C18:0 methyl ester in n-heptane as internal standard, vortex for 2 min and centrifuge at 2500 rpm, 20˚C, for 15 min; and (11) transfer into the GC–MS system. Plasma sample preparation for capillary electrophoresis–mass spectrometry (CE–MS) entailed the following steps: (1) thaw sample on ice, vortex for 1 min and transfer 100 µL of plasma into an Eppendorf tube already filled with 100 µL of 0.2 M formic acid with 5 % acetonitrile and 0.4 mM methionine sulfone as internal standard; (2) vortex for 2 min and transfer to a Millipore filter (30 kDa protein cutoff); (3) centrifuge at 2000 rpm, 4˚C, for 70 min; and (4) transfer the filtrate into a CE vial where the volume is directly injected into the CE–MS system. Urine samples for LC–MS were thawed on ice and vortexed prior injection into the LC-MS system (without pre-treatment). For CE–MS, sample procedure entailed: (1) thaw on ice and vortex for 1 min and transfer 200 µL into an Eppendorf tube; (2) centrifuge at 13200 rpm, 4˚C, for 20 min; (3) transfer 100 µL of the supernatant into an Eppendorf tube already filled with 400 µL of 0.125 M formic acid with 0.25 mM methionine sulfone as internal standard; (4) vortex for 1 min and centrifuge at 13200 rpm, 4˚C, for 10 min; and (5) transfer 200 µL of the supernatant into a CE vial where the volume is directly injected into the CE–MS system. Quality control (QC) samples were independently prepared for each technique and followed the same procedure as applied for experimental samples. QC samples were always injected at the beginning of each analytical run, followed by samples randomized independently. A QC sample was rerun after each block of 5 samples.

Sampleprep ID:

SP001600

Sampleprep Summary:

Plasma sample preparation for liquid chromatography–mass spectrometry (LC–MS) entailed the following steps: (1) thaw sample on ice, vortex for 2 min and transfer 100 µL of plasma into an Eppendorf tube; (2) for protein precipitation, add 300 µL of cold (-18˚C) methanol/ethanol (1:1 v/v), vortex for 1 min, incubate on ice for 5 min and vortex again briefly; (3) centrifuge at 13000 rpm, 4˚C, for 20 min; and (4) transfer the supernatant into the LC–MS system. Plasma sample preparation for gas chromatography–mass spectrometry (GC–MS) entailed the following steps: (1) thaw sample on ice, vortex for 2 min and transfer 40 µL of plasma into an Eppendorf tube; (2) for protein precipitation, add 120 µL of cold acetonitrile, vortex for 2 min and incubate on ice for 5 min; (3) centrifuge at 15400 rpm, 4˚C, for 10 min; and (4) transfer the supernatant into a GC vial with insert; (5) evaporate in a Speedvac at 30°C until dry; (6) add 10 µL of O-methoxyamine hydrochloride (15 mg/mL) in pyridine and vortex for 5 min; (7) sonicate for 2 min and vortex for 2 min (repeat step a total of three times); (8) incubate at room temperature for 16 h in darkness for the reaction of the methoxymation; (9) add 10 µL of N,O-Bis(trimethylsilyl)trifluoroacetamide (BSTFA) with 1 % trimethylchlorosilane (TMCS) and incubate 1 h at 70 ˚C for the silylation; (10) add 100 µL of 10 ppm C18:0 methyl ester in n-heptane as internal standard, vortex for 2 min and centrifuge at 2500 rpm, 20˚C, for 15 min; and (11) transfer into the GC–MS system. Plasma sample preparation for capillary electrophoresis–mass spectrometry (CE–MS) entailed the following steps: (1) thaw sample on ice, vortex for 1 min and transfer 100 µL of plasma into an Eppendorf tube already filled with 100 µL of 0.2 M formic acid with 5 % acetonitrile and 0.4 mM methionine sulfone as internal standard; (2) vortex for 2 min and transfer to a Millipore filter (30 kDa protein cutoff); (3) centrifuge at 2000 rpm, 4˚C, for 70 min; and (4) transfer the filtrate into a CE vial where the volume is directly injected into the CE–MS system. Urine samples for LC–MS were thawed on ice and vortexed prior injection into the LC-MS system (without pre-treatment). For CE–MS, sample procedure entailed: (1) thaw on ice and vortex for 1 min and transfer 200 µL into an Eppendorf tube; (2) centrifuge at 13200 rpm, 4˚C, for 20 min; (3) transfer 100 µL of the supernatant into an Eppendorf tube already filled with 400 µL of 0.125 M formic acid with 0.25 mM methionine sulfone as internal standard; (4) vortex for 1 min and centrifuge at 13200 rpm, 4˚C, for 10 min; and (5) transfer 200 µL of the supernatant into a CE vial where the volume is directly injected into the CE–MS system. Quality control (QC) samples were independently prepared for each technique and followed the same procedure as applied for experimental samples. QC samples were always injected at the beginning of each analytical run, followed by samples randomized independently. A QC sample was rerun after each block of 5 samples.

Combined analysis:

Analysis ID	AN002521	AN002522	AN002523	AN002524
Analysis type	MS	MS	MS	MS
Chromatography type	GC	Reversed phase	Reversed phase	CE
Chromatography system	Agilent 7890A	Agilent 1290 Infinity	Agilent 1290	Agilent 7100 CE
Column	Agilent DB5-MS (30m x 0.25mm, 0.25um)	Discovery HS C18 (15cm x 2.1 mm,3um)	Discovery HS C18 (15cm x 2.1 mm,3um)	capillary with an inner diameter of 50um and a total length of 100 cm
MS Type	EI	ESI	ESI	ESI
MS instrument type	Single quadrupole	QTOF	QTOF	Other
MS instrument name	Agilent 5975C	Agilent 6520 QTOF	Agilent 6520 QTOF	Agilent 6224 TOF
Ion Mode	POSITIVE	POSITIVE	NEGATIVE	POSITIVE
Units		area	area	area

Chromatography:

Chromatography ID:	CH001841
Instrument Name:	Agilent 7890A
Column Name:	Agilent DB5-MS (30m x 0.25mm, 0.25um)
Chromatography Type:	GC

Chromatography ID:	CH001842
Chromatography Summary:	Positive ionization
Instrument Name:	Agilent 1290 Infinity
Column Name:	Discovery HS C18 (15cm x 2.1 mm,3um)
Chromatography Type:	Reversed phase

Chromatography ID:	CH001843
Chromatography Summary:	Negative ionization
Instrument Name:	Agilent 1290
Column Name:	Discovery HS C18 (15cm x 2.1 mm,3um)
Chromatography Type:	Reversed phase

Chromatography ID:	CH001844
Instrument Name:	Agilent 7100 CE
Column Name:	capillary with an inner diameter of 50um and a total length of 100 cm
Chromatography Type:	CE

MS:

MS ID:	MS002339
Analysis ID:	AN002521
Instrument Name:	Agilent 5975C
Instrument Type:	Single quadrupole
MS Type:	EI
MS Comments:	None
Ion Mode:	POSITIVE

MS ID:	MS002340
Analysis ID:	AN002522
Instrument Name:	Agilent 6520 QTOF
Instrument Type:	QTOF
MS Type:	ESI
MS Comments:	None
Ion Mode:	POSITIVE

MS ID:	MS002341
Analysis ID:	AN002523
Instrument Name:	Agilent 6520 QTOF
Instrument Type:	QTOF
MS Type:	ESI
MS Comments:	None
Ion Mode:	NEGATIVE

MS ID:	MS002342
Analysis ID:	AN002524
Instrument Name:	Agilent 6224 TOF
Instrument Type:	Other
MS Type:	ESI
MS Comments:	None
Ion Mode:	POSITIVE