Return to study ST001518 main page
MB Sample ID: SA127697
Local Sample ID: | Control_Plasma_LC_NEG |
Subject ID: | SU001592 |
Subject Type: | Human |
Subject Species: | Homo sapiens |
Taxonomy ID: | 9606 |
Select appropriate tab below to view additional metadata details:
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_Plasma_LC_NEG | SA127697 | 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. |
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 |