GC Technical Note

GC/MS Metabolome analyses(Miso, Sake, Syoyu)

GT084

GL Sciences Inc.

Fermented foods like Miso, Sake, Syoyu made from cereals contain amino acids, organic acids,
sugar and so on. In this study, they were extracted, derivatized, and analysed with gas
chromatography/mass spectrometry. The silylated compounds in Miso, Sake and Syoyu extracts
were identified using InertCap 5MS Metabolomics metabolites Library (No.81).
Procedure of sample preparation

Sample 10 mg

Miso : Weigh out ten grams in a microtube.
* 50 μL
Shoyu : Weigh out twenty microliter of ten fold dilution
Syoyu in a microtube.
Sake : Measure twenty microliter in a microtube.
Add Internal Standard.
Follow the procedure from the “Dry inRibitol (I.S.)
a vacuum-centrifuge dryer and freeze dryer.
1000 μL of Methanol/H2O/Chloroform(2.5/1/1/,v/v/v)
Vortex 20 s Incubate (30 min)Centrifuge (16000 g, 3 min)

800 μL aliquot of supernatant
400 μL of distilled water
Vortex 20 sCentrifuge(16000 g, 3 min)
800 μL aliquot of supernatant*
Dry in a vacuum-centrifuge dryer and freeze dryer
100 µL of Methoxyamine hydrochloride (20 mg/mL pyridine solution)
Incubate(30 ºC, 90 min)
50 µL of MSTFA (Cat.No. 1022-11061)
Incubate(37 ºC, 30 min)GC/MS

GL Sciences GC Technical Note
Analysis of Miso
GC/MS Conditions
SystemColumn: GC-MS: InertCap 5MS Metabolomics 0.25 mm I.D. × 30 m df =0.25 µm (Cat.No. 1010-18952): Split Constant Pressure 75 kPa: 230 ºC
: 80 ºC (2 min) - 15 ºC /min - 330 ºC (13 min): Helium: 5 mL/min: 25:1The silylated compounds in Miso extract: 250 ºC: 200 ºC
were identified based on retention index: 70 eVand mass spectrum using InertCap: m/z 85 - 500
metabolites library (Technical note No.81): 1 μL

Injection
Injection Temp.
Oven Temp.
Carrier gas
Septum purge flow
Split ratio
Interface Temp.
Ion source Temp.
Ionization Vortage
Scan range
Inj. Vol.

Isocitric acid + Citricacid

Arabitol
Ribitol(I.S.)

Glutamic acid_3TMS
Phenylalanine_2TMS

4-Aminobutyric acid

Erythritol
Aspartic acid_3TMS
Pyroglutamic acid_2TMS

Unknown_Amine likeThreonine_3TMS

Serine_3TMS + Alanine_3TMSThreonine_2TMS + Isoleucine_2TMS
Proline_2TMS
Succinic acid(or anhydride) + Glycine_3TMS

Serine_2TMS
Leucine_2TMS

Valine_2TMS

Isoleucine_1TMS

Leucine_1TMS

Lactic acid

Phosphate + Glycerol

and NIST.

GL Sciences GC Technical Note
Analysis of Sake
GC/MS Conditions
System Column

Injection
Inj. Temp.
Oven Temp.
Carrier gas
Septum purge flow
Split ratio
Interface Temp.
Ion source Temp.
Ionization Vortage
Scan range
Inj. Vol.

: GC-MS: InertCap 5MS Metabolomics 0.25 mm I.D. × 30 m df =0.25 µm (Cat.No. 1010-18952): Split Constant Pressure 75 kPa: 230 ºC
: 80 ºC (2 min) - 15 ºC /min - 330 ºC (13 min): Helium: 5 mL/min: 25:1The silylated compounds in Sake extract: 250 ºC
: 200 ºC
were identified based on retention index: 70 eVand mass spectrum using InertCap: m/z 85 - 500
metabolites library (Technical note No.81): 1 μL

Ribitol(I.S.)

Glycylglycine+Isocitric acid + Citric acid

Xylitol

Asparagine_3TMS
RiboseGlutamic acid_3TMS
Phenylalanine_2TMS

4-Hydroxyphenylethanol

Arabitol

Erythritol
Pyroglutamic acid_2TMS
4-Aminobutyric acid

Aspartic acid_3TMS
Malic acidUnknown_Amine likeThreonine_3TMS

Succinic acid(or anhydride) + Glycine_3TMS
UracilFumaric acid
Serine_3TMS + Alanine_3TMS

Phosphate + Glycerol
Isoleucine_2TMS
Proline_2TMS

2-Aminoethanol
Serine_2TMS

Valine_2TMS
Urea_2TMS

Leucine_1TMS
Isoleucine_1TMS

Alanine_2TMS

Leucine_2TMS

Lactic acidand NIST.

GL Sciences GC Technical Note
Analysis of Syoyu
GC/MS Conditions
System Column

Glycylglycine
Glycylglycine+Isocitric acid + Citric acid
Hypoxanthine

Ribitol(I.S.)

Arabitol
Xylitol

XyloseArabionose

Putrescine

Glutamic acid_3TMS
Phenylalanine_2TMS

Methionine_2TMS+Aspartic acid_3TMS
Pyroglutamic acid_2TMS

4-Aminobutyric acid

Erythritol
Malic acidThreitolThymineb-Alanine_3TMS

Serine_3TMS + Alanine_3TMS
Threonine_3TMS

Leucine_2TMS
Serine_2TMS
2-Aminoethanol

Valine_2TMS
Malonic acid

Isoleucine_1TMS

Alanine_2TMS

Leucine_1TMS

Glycolic acid
Valine_1TMS

Lactic acidThreonine_2TMS+Isoleucine_2TMS

Injection
Inj. Temp.
Oven Temp.
Carrier gas
Septum purge flow
Split ratio
Interface Temp.
Ion source Temp.
Ionization Vortage
Scan range
Inj. Vol.

Phosphate + Glycerol
Proline_2TMS
Succinic acid(or anhydride) + Glycine_3TMS
Glyceric acid
Uracil: GC-MS: InertCap 5MS Metabolomics 0.25 mm I.D. × 30 m df =0.25 µm (Cat.No. 1010-18952): Split Constant Pressure 75 kPa: 230 ºC
: 80 ºC (2 min) - 15 ºC /min - 330 ºC (13 min): Helium: 5 mL/min: 25:1The silylated compounds in Syoyu extract: 250 ºC
: 200 ºC
were identified based on retention index and: 70 eV
mass spectrum using InertCap metabolites: m/z 85 - 500
library (Technical note No.81) and NIST.
: 1 μLGL Sciences GC Technical NoteAIoutput is available for peak identification. In that case, it is convenient to use
the n-alkane standard solution C9-C40 in n- Hexane.
What is AIoutput?
•Software for metabolome analyses based on Excel (upto 2007)•Peak identification based on retention indices and mass spectrum•
Auto peak identification, PCA analysis, PLS analysis, t-test•
Free software!
•Everybody can download, now!
http://prime.psc.riken.jp/Metabolomics_Software/AIoutput/index.html
n-Alkane standard solution C9-C40 in n-HexaneConcentration: 50 μg/mL each component of n-Hexane※C10,C20,C30,C40 are 100 μg/mLConditions
Volume: 1 mL
: GC-FID
System: InertCap 5MS Mtabolomics
ColumnPackaging: amplue of 1 mL
0.25 mm I.D.×30 m, df = 0.25 μm

C20C10C30

Injection Inj.
Temp.
Oven Temp.
Carrier gas
Split flow
Det. Temp.
Inj. Vol.

: Split 120 kPa: 230ºC: 100 ºC (2 min) -20 ºC/min - 330 ºC (17 min hold): Helium: 25 mL/min: 330 ºC
: 1 μL

C40
01020Description
n-Alkane standard solution C9-C40 in C6 (coldstrage)

Volume
1 mL
1 mL ×5

3 0 [min]Cat.No.
1021-58321
1021-58325

This product contains hazardous material which requires special freight handling, and thenadditional charges apply. Please contact your local distributor.

GL Sciences disclaims any and all responsibility for any injury or damage which may becaused by this data directly or indirectly. We reserve the right to amend this information
or data at any time and without any prior announcement.

GL Sciences disclaims any and all responsibility for any injury or damage which may be caused by this data directly or indirectly.
We reserve the right to amend this information or data at any time and without any prior announcement.

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