Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA151896

Local Sample ID:	P16_t0
Subject ID:	SU001733
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Gender:	Male and female

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Subject:

Subject ID:	SU001733
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
P16_t0	SA151896	FL017854	Food	Trigger
P16_t0	SA151896	FL017854	Moderate	Severity
P16_t0	SA151896	FL017854	Time 0	Time

Collection:

Collection ID:	CO001726
Collection Summary:	A prospective clinical and observational study of patients with anaphylactic reactions was performed. Patients of all ages and both sexes were recruited at outpatient clinics and the departments of Emergency, and other services at Hospital La Fe. All fulfilled clinical criteria of anaphylaxis and severity was graded following the classification by Brown, et al3. Patients were classified as food, drug or idiopathic origin, as well as in mild, moderate or severe according to the number of organs affected and clinical symptoms. The allergy evaluation was conducted by the Allergology Service of Hospital La Fe. The ethical committee approved the study protocol and all subjects were informed and provided written consent. Serum samples were taken during the acute moment of the reaction at the first moment of medical attention (< 2h, hereafter referred as ‘T1’), and after clinical recovery (approximately 2-4h later, referred to as ‘T2’). Patients were treated according to the Galaxy 2016 practical guide, using all necessary drugs to rescue them. Samples were collected, following specific standard operating procedures (SOPs)31-33. Briefly, these were collected in a vacutainer tube (Ref. 368965) and processed within the first 30 to 60 min after blood extraction. Sample aliquots were stored at -80ºC until further analyses. Subsequently, between 2-3 months after the anaphylaxis, a serum sample was taken when the allergy evaluation was performed (basal state, called ‘T0’).
Sample Type:	Blood (serum)
Storage Conditions:	-80℃

Treatment:

Treatment ID:	TR001746
Treatment Summary:	Patients were treated according to the Galaxy 2016 practical guide, using all necessary drugs to rescue them.

Sample Preparation:

Sampleprep ID:	SP001739
Sampleprep Summary:	After thawing the samples, 150 µL of cold acetonitrile (0.1% formic acid, v/v) was added to 50 µL of serum, vortex and kept at -20ºC for 30 min for protein precipitation. Then, 25 µl of cleaned supernatant were transferred to a 96 well-plate for UPLC-MS analysis. Finally, 125 µL of H2O (0.1% formic acid, v/v), and 2 µL of internal standard (IS) mix solution, containing reserpine, leucine enkephaline, caffeine-d9 and phenylalanine-d5 in H20:CH3OH (1:1, 0.1% v/v formic acid) at 20 µM were added to each sample. Blank samples were prepared by replacing serum by ultrapure H20 in order to identify potential artefacts from the tube, reagents and other materials. A quality control (QC) was prepared by mixing 10 µL from each prepared sample. Samples were randomly injected in the chromatographic system (UPLC-ToF-MS) injection a QC every 6 serum samples in order to avoid intra-batch variability, as well as to enhance quality and reproducibility. Blank analysis was performed at the beginning and at end of the sequence. Sample stability and analytical drift were investigated through the internal standard intensities.

Sampleprep ID:

SP001739

Sampleprep Summary:

After thawing the samples, 150 µL of cold acetonitrile (0.1% formic acid, v/v) was added to 50 µL of serum, vortex and kept at -20ºC for 30 min for protein precipitation. Then, 25 µl of cleaned supernatant were transferred to a 96 well-plate for UPLC-MS analysis. Finally, 125 µL of H2O (0.1% formic acid, v/v), and 2 µL of internal standard (IS) mix solution, containing reserpine, leucine enkephaline, caffeine-d9 and phenylalanine-d5 in H20:CH3OH (1:1, 0.1% v/v formic acid) at 20 µM were added to each sample. Blank samples were prepared by replacing serum by ultrapure H20 in order to identify potential artefacts from the tube, reagents and other materials. A quality control (QC) was prepared by mixing 10 µL from each prepared sample. Samples were randomly injected in the chromatographic system (UPLC-ToF-MS) injection a QC every 6 serum samples in order to avoid intra-batch variability, as well as to enhance quality and reproducibility. Blank analysis was performed at the beginning and at end of the sequence. Sample stability and analytical drift were investigated through the internal standard intensities.

Combined analysis:

Analysis ID	AN002703	AN002704
Analysis type	MS	MS
Chromatography type	Reversed phase	Reversed phase
Chromatography system	Agilent 6550	Agilent 6550
Column	Waters Acquity BEH C18 (100 x 2mm,1.7um)	Waters Acquity BEH C18 (100 x 2mm,1.7um)
MS Type	ESI	ESI
MS instrument type	QTOF	QTOF
MS instrument name	Agilent 6550 QTOF	Agilent 6550 QTOF
Ion Mode	POSITIVE	NEGATIVE
Units	Intensity	intensity

Chromatography:

Chromatography ID:	CH001995
Chromatography Summary:	The chromatographic separation was performed by using an UPLC BEH C18 (100 x 2.1 mm, 1.7 µm, Waters, Wexford, Ireland) column from Waters (Wexford, Ireland). Autosampler and column temperatures were set to 4°C and 40°C, respectively, and the injection volume was 5 µL. Mobile phase A and B consisted of H20 and acetonitrile, respectively, both containing 0.1% formic acid. The gradient elution lasted 14 minutes at a flow rate of 400 µl min-1. The mobile phase A was maintained at 98% for 1 min, and then decreased until 75% in 2 minutes, 50% in 3 minutes and 5% in 3 more minutes. 95% of mobile phase B was held for 3 min and then, a 0.55 min gradient was used to return to the initial conditions, which were held for 2.5 min to full column recovery.
Instrument Name:	Agilent 6550
Column Name:	Waters Acquity BEH C18 (100 x 2mm,1.7um)
Column Temperature:	40
Flow Gradient:	The mobile phase A was maintained at 98% for 1 min, and then decreased until 75% in 2 minutes, 50% in 3 minutes and 5% in 3 more minutes. 95% of mobile phase B was held for 3 min and then, a 0.55 min gradient was used to return to the initial conditions, which were held for 2.5 min to full column recovery.
Flow Rate:	400 ul/min
Solvent A:	100% water; 0.1% formic acid
Solvent B:	100% acetonitrile; 0.1% formic acid
Chromatography Type:	Reversed phase

MS:

MS ID:	MS002500
Analysis ID:	AN002703
Instrument Name:	Agilent 6550 QTOF
Instrument Type:	QTOF
MS Type:	ESI
MS Comments:	Full scan MS data from 50 to 1700 m/z with a scan frequency of 6 Hz was collected both in positive and negative electrospray ionization modes (ESI + and ESI -, respectively). The following ESI parameters were used: gas temperature, 200°C; drying gas, 14 l min-1; nebulizer, 60 psi; sheath gas temperature, 350°C; sheath gas flow, 11 l min-1. Automatic MS spectra recalibration was carried out introducing a reference standard containing m/z 149.0233, m/z 121.0508 and m/z 922.0097 into the source via a reference sprayer valve during the analysis.
Ion Mode:	POSITIVE

MS ID:	MS002501
Analysis ID:	AN002704
Instrument Name:	Agilent 6550 QTOF
Instrument Type:	QTOF
MS Type:	ESI
MS Comments:	Full scan MS data from 50 to 1700 m/z with a scan frequency of 6 Hz was collected both in positive and negative electrospray ionization modes (ESI + and ESI -, respectively). The following ESI parameters were used: gas temperature, 200°C; drying gas, 14 l min-1; nebulizer, 60 psi; sheath gas temperature, 350°C; sheath gas flow, 11 l min-1. Automatic MS spectra recalibration was carried out introducing a reference standard containing m/z 149.0233, m/z 121.0508 and m/z 922.0097 into the source via a reference sprayer valve during the analysis.
Ion Mode:	NEGATIVE