Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA025745

Local Sample ID:	Sa_pre_36
Subject ID:	SU000517
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Species Group:	Human

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Subject:

Subject ID:	SU000517
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Species Group:	Human

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
Sa_pre_36	SA025745	FL006058	No	debridement

Collection:

Collection ID:	CO000511
Collection Summary:	The subjects were asked to refrain from brushing and using mouthwash for at least 1 hour prior to sample collection and periodontal examinations. We collected a minimum of 3 mL of unstimulated whole saliva between 1:00 and 3:00 p.m. Thereafter, saliva was sampled again at 15 minutes following removal of supragingival plaque and calculus with an ultrasonic scaler (debridement). Finally, 1 ml of each sample was immediately frozen with liquid nitrogen and stored at -80°C until analysis.
Sample Type:	Saliva

Treatment:

Treatment ID:	TR000531
Treatment Summary:	Debridement was performed with an ultrasonic scaler, as described in “Collection Summary”. To maintain the subgingival microbiota and avoid bleeding, we avoided insertion of the scalar tip into the gingival sulcus.

Sample Preparation:

Sampleprep ID:	SP000524
Sampleprep Summary:	Frozen saliva samples were freeze-dried overnight using a freeze-dryer (VD-800F; TAITEC, Saitama, Japan), and metabolites were extracted with 1 ml of MeOH/H2O/CHCl3 (5/2/2, v/v/v). As an internal standard, 60 µl of ribitol (0.2 mg/ml) was added to the mixture. The sample was centrifuged at 16,000 g for 3 min at 4 °C, and the supernatant (900 µl) was transferred to another microtube and mixed with 400µl of water. After centrifugation at 16,000 g for 3 min at 4 °C, 800µl of the supernatant was transferred to another microtube whose cap was pierced. The extract was evaporated using a vacuum centrifuge dryer for 2 h in order to remove methanol, then freeze-dried overnight. For derivatization, 100 µl of methoxyamine hydrochloride (20 mg/ml in pyrimidine) and 50 µl of N-methyl-N-(trimethylsilyl)trifluoroacetamide were employed. The sample was then used for GCMS analysis, and a 1 µL of sample was injected in split mode (25/1, v/v).

Sampleprep ID:

SP000524

Sampleprep Summary:

Frozen saliva samples were freeze-dried overnight using a freeze-dryer (VD-800F; TAITEC, Saitama, Japan), and metabolites were extracted with 1 ml of MeOH/H2O/CHCl3 (5/2/2, v/v/v). As an internal standard, 60 µl of ribitol (0.2 mg/ml) was added to the mixture. The sample was centrifuged at 16,000 g for 3 min at 4 °C, and the supernatant (900 µl) was transferred to another microtube and mixed with 400µl of water. After centrifugation at 16,000 g for 3 min at 4 °C, 800µl of the supernatant was transferred to another microtube whose cap was pierced. The extract was evaporated using a vacuum centrifuge dryer for 2 h in order to remove methanol, then freeze-dried overnight. For derivatization, 100 µl of methoxyamine hydrochloride (20 mg/ml in pyrimidine) and 50 µl of N-methyl-N-(trimethylsilyl)trifluoroacetamide were employed. The sample was then used for GCMS analysis, and a 1 µL of sample was injected in split mode (25/1, v/v).

Combined analysis:

Analysis ID	AN000762
Analysis type	MS
Chromatography type	GC
Chromatography system	Shimadzu GCMS-QP2010 ultra
Column	a 30 m × 0.25 mm i.d. fused silica capillary column coated with 0.25 µm CP-SIL 8 CB low bleed (Varian Inc., Palo Alto, CA)
MS Type	EI
MS instrument type	GC-TOF
MS instrument name	Shimadzu QP2010 Ultra
Ion Mode	POSITIVE
Units	Peak area

Chromatography:

Chromatography ID:	CH000548
Chromatography Summary:	The device used was GCMS-QP2010 ultra (Shimadzu Inc., Kyoto, Japan) equipped with a 30 m × 0.25 mm i.d. fused silica capillary column coated with 0.25 µm CP-SIL 8 CB low bleed (Varian Inc., Palo Alto, CA) and AOC-20i (Shimadzu) as an autosampler. The injection temperature was 230 °C. The helium gas flow rate through the column was 1.12 ml/min. The column temperature was held at 80 °C for 2 min and then raised by 15 °C /min to 330 °C and held for 6 min. The transfer line and the ion source temperatures were 250 °C and 200 °C, respectively. Ions were generated by a 70 kV electron impact (EI), and 20 scans/sec were recorded over the mass range 85-500. A standard alkane mixture (C8-C40) was injected at the beginning of the analysis for subsequent identification of metabolites.
Instrument Name:	Shimadzu GCMS-QP2010 ultra
Column Name:	a 30 m × 0.25 mm i.d. fused silica capillary column coated with 0.25 µm CP-SIL 8 CB low bleed (Varian Inc., Palo Alto, CA)
Column Temperature:	The column temperature was held at 80 °C for 2 min and then raised by 15 °C /min to 330 °C and held for 6 min.
Flow Rate:	The helium gas flow rate through the column was 1.12 ml/min.
Sample Injection:	1 µL
Chromatography Type:	GC

MS:

MS ID:	MS000674
Analysis ID:	AN000762
Instrument Name:	Shimadzu QP2010 Ultra
Instrument Type:	GC-TOF
MS Type:	EI
Ion Mode:	POSITIVE