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MB Sample ID: SA125873

Local Sample ID:PlasmaIS1_66
Subject ID:SU001567
Subject Type:Human
Subject Species:Homo sapiens
Taxonomy ID:9606

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Subject:

Subject ID:SU001567
Subject Type:Human
Subject Species:Homo sapiens
Taxonomy ID:9606

Factors:

Local Sample IDMB Sample IDFactor Level IDLevel ValueFactor Name
PlasmaIS1_66SA125873FL015453Plasma + IS 1Treatment

Collection:

Collection ID:CO001562
Collection Summary:LC-MS grade acetonitrile (ACN), methanol (MeOH), isopropanol (IPA) and chloroform were purchased from Biosolve BV (Valkenswaard, The Netherlands). Ammonium formate (AmF), formic acid (FA) and tert-Butyl methyl ether (MTBE) were purchased from Sigma Aldrich (St. Louis, MO). Various lipid standards were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL). Heparin-anticoagulated plasma samples were obtained from adult patients at the University Medical Center Groningen (UMCG) in an anonymous manner and were combined to generate a standard plasma sample.
Sample Type:Blood (plasma)
Collection Method:Anonymous
Collection Location:University Medical Center Groningen (UMCG)

Treatment:

Treatment ID:TR001582
Treatment Summary:60 µl of plasma was mixed with 300 µl of MeOH and sonicate for 10 min. Subsequently, 1000 µl MTBE was added and the mixture was kept under 25 °C on a shaker (900 rpm) for 30 min. Phase separation was induced by adding 190 µl ultrapure water. Then the mixture was centrifuged at 3000 RCF for 10 min and the 850 µl upper phase were transferred to a new tube. The re-extraction was performed by adding 600 µl MTBE/MeOH/ultrapure water (10:3:2.5, v/v/v) into the lower phase and 500 µl were collected after centrifugation to combine with the previous organic phase. The combined lipid extract solution was aliquoted into 6 tubes (190 µl per tube) to generate plasma lipid matrix. Different concentrations of lipid standard mixture were added to the plasma lipid extract aliquots and dried in a vacuum centrifuge under 45 °C. The dried lipid extracts were resuspended with 30 µl chloroform:MeOH:MQ (60:30:4.5, v/v/v) and further diluted with 90 µl (IPA:ACN:MQ 2:1:1 v/v/v) for LC-MS analysis.

Sample Preparation:

Sampleprep ID:SP001575
Sampleprep Summary:20 different deuterium-labelled lipid IS and 4 deuterium-labelled lipid IS premix were selected to cover the major lipid classes and distributed evenly in mz and retention time range. All lipid standard stock solutions were diluted with chloroform: MeOH (1:1, v/v) and mixed to generate a lipid IS mixture with optimized concentrations for each standard to acquire adequate signal intensity. The lipid IS mixture was used to create a dilution series where concentration ratios were set to a factor of two starting from concentration 1 up to concentration 1/16.

Combined analysis:

Analysis ID AN002475
Analysis type MS
Chromatography type Reversed phase
Chromatography system Waters Acquity
Column Waters Acquity CSH C18 (100 x 2.1mm, 1.7um)
MS Type ESI
MS instrument type Orbitrap
MS instrument name Thermo Q Exactive Orbitrap
Ion Mode POSITIVE
Units Peak area

Chromatography:

Chromatography ID:CH001813
Chromatography Summary:LC-MS lipid analysis was performed on an Ultimate 3000 High-Performance UPLC. Chromatography separation was achieved with an Acquity UPLC CSH column [1.7 μm, 100 × 2.1 mm, (Waters Corporation, Milford, MA)] under 55°C with a flow rate of 0.4 ml/min. Mobile phase A was composed of ultrapure water/acetonitrile 40:60 (v/v), 10 mM ammonium formate and 0.1% formic acid. Mobile phase B contained ACN/IPA 10:90 (v/v) with 10 mM ammonium formate and 0.1% formic acid. The LC gradient started with 40% mobile phase B and raised to 43% mobile phase B in 2 min. The percentage of mobile phase B raised to 50% in the next 0.1 min and increased to 54% in next 9.9 min. Mobile phase B raised to 70% in 0.1 min and increased to 99% in 5.9 min and maintained at 99% for 1 min. Then the percentage of mobile phase B went back to 40% in 0.1 min and the system was equilibrated for 3.9 min before the next run started.
Instrument Name:Waters Acquity
Column Name:Waters Acquity CSH C18 (100 x 2.1mm, 1.7um)
Column Temperature:under 55°C
Flow Rate:0.4 ml/min
Solvent A:ultrapure water/acetonitrile 40:60 (v/v), 10 mM ammonium formate and 0.1% formic acid
Solvent B:ACN/IPA 10:90 (v/v) with 10 mM ammonium formate and 0.1% formic acid
Chromatography Type:Reversed phase

MS:

MS ID:MS002295
Analysis ID:AN002475
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:The MS was set for positive mode and data-dependent acquisition. A full MS scan the range from 250-1750 Da was acquired at resolution 70,000 FWHM at 200 Da. (AGC target was set to 1·106, maximum injection time was 50 ms, MS1 scan was followed by up to 8 MS/MS events with a collision energy of 25 eV at resolution 17,500 FWHM at 200 Da. The precursor isolation window was set to 1.5 Da with the dynamic exclusion time of 6 s. The ionization settings were as follows: capillary voltage, +3.2 kV; capillary temperature: 320 °C; sheath gas/auxiliary gas: 60/20.
Ion Mode:POSITIVE
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