SPL-M100 Application
SPL-M100
ApplicationNoteNoteNo. AS240506EN

Metabolome analysis of saliva
by online solid-phase analytical derivatization GC-MS system

for SPE-GC system

Introduction
Solid-phase derivatization (SPD) is a techniqueofderivatization

without

theSample

time-consuming

centrifugal concentration and lyophylization, by

Spiral insert

retaining the target compounds on a solid phase

Flash-SPESalivaand dewatering it by passing a organic solvent
throughitthenSPL-M100
SPL-M100

infiltrating

thederivatization

reagent and performing the reaction on the solidphase. The example of pretreatment method andanalytical condition for metabolome analysis ofInformation
Information

saliva are shown below.
Online SPE-GC-MS system

SPD pretreatment workflow
Sample 0.5 mL

Online SPE-GC: SPL-M100

Vials

Water 0.5 mL
Flash-SPE ACXs

Transfer 200 µL
ACN 800 µL

Automation

Conditioning
ACN-Water (1/1)

Loading [Retaintion] Extract 50 µL
Washing ACN-Water (1/1)

Shaking (37 ℃, 30 min)Dewater ACN

Centrifuge (14000 rpm, 3 min)Infiltration 0.5% Methyoxyamine/Pyridine

Derivtization (Methoximation), 3 min

Supernatant 500 µL

Infiltration MSTFA/Hexane (1/1)

Analytical solutionDerivatization (TMS), 1 min

pH adjustment (8-9)

Elution Hexane
GC-MS

Analytical condition
SPE-GC interface
SPE cartridge
PTV injection port

SPL-M100 (AiSTI SCIENCE)
Flash-SPE
LVI-S250 (AiSTI SCIENCE)

Insert typeSpiral insert

Temp.

220℃(0.5 min)-50℃/min-290℃(23 min)Gas chromatograph
Inlet mode

Split 1:50

Flow mode

Constant flow, 1.0 mL/min

Pre-column

0.25 mm i.d. x 0.5 m

ColumnVf-5ms, 0.25 mm i.d. x 30 m, df=0.25 μm

Oven Temp.

100℃(2 min)-10℃/min-320℃(2min)

Transfer line Temp.

290℃

Mass spectrometer
Acquisition mode

Scan (m/z 70-600)

Data acquisition

3.0-26 min

AiSTI SCIENCEAiSTI SCIENCE
Product

Online SPE-GC

SPL-M100
Solid-phasecartridge

ProductFlash-SPE
GC large volume
injection port

LVI-S250

AiSTI SCIENCE Co., Ltd.
TEL: +81-73-475-0033
E-mail: as@aisti.co.jp
Tel：+81-73-475-0033
www.aisti.co.jp

AiSTI SCIENCE CO.,Ltd.

E-mail：as@aisti.co.jp
HP：www.aisti.co.jp

No. AS240506EN

AiSTI Application Note
Results
Phosphoric
acid_3TMS

Citric acid_4TMS

Expanded

サンプルNo.
Norleucine_2TMS Adipic acid_2TMS
内標S12,448,000
111,000
バイアル中20µM
S22,374,000
110,200
Figure: Total ion current
chromatogram
S32,313,000
102,700
S42,383,000
110,300
S52,349,000
103,400
Table: Result of recovery test (n=5)
サンプルSample
内標Standard solution
バイアル中20µM
IS concentrations:

20 μM in vials

唾液
10D
内標
除タンパク後添加
バイアル中20µM(K/Sx100)
唾液
10D
内標
唾液に添加
試料中200µM(バイアル中20µM)

No.
S1
S2
S3
S4
S5

Norleucine_2TMS Adipic acid_2TMS
2,448,000
111,000
2,374,000
110,200
2,313,000
102,700
2,383,000
110,300
2,349,000
103,400

Ave.
2,373,400
107,520
No. %Norleucine_2TMS
Adipic acid_2TMS
RSD,2.1
3.8
唾液-K1
2,310,000
126,700
S1
2,448,000
111,000
Feces_K1
Feces_K1
唾液-K2
2,025,000
115,300
S2
2,374,000
110,200
Feces_K2
Feces_K2
10 times
唾液-K3
2,134,000
117,700
S3
2,313,000
102,700
Feces_K3
Feces_K3
Spike
内標 period of IS: Feces_K4
唾液-K4
2,063,000
109,300
S4
2,383,000
110,300
Feces_K4
After
除タンパク後添加
唾液-K5
2,197,000
120,900
Feces_K5
S52,349,000
103,400
Feces_K5
deprotenization,
バイアル中20µM
Ave.
2,145,800
117,980
2,373,400
107,52020 μM in vials
RSD, %
5.3
5.5
2.1
3.8(K/Sx100) Recovery,唾液唾液-K1
2,310,000
126,700
REC, % %90110
唾液唾液_A1
1,978,000
121,700
10D
唾液-K2
2,025,000
115,300
SalivaFeces_A1
10D
唾液_A2
1,961,000
113,900
唾液-K3
2,134,000
117,700
Dilution:
Feces_A2
10 times
内標
唾液_A3
2,080,000
122,200
唾液-K4
2,063,000
109,300
Feces_A3
Spikeperiod of IS: Feces_A4
唾液に添加
唾液_A4
1,968,000
111,400
除タンパク後添加
唾液-K5
2,197,000
120,900
Before extraction, Feces_A5
試料中200µM
唾液_A5
2,054,000
122,300
バイアル中20µM
Ave.
2,145,800
117,980
200 μM in saliva(バイアル中20µM)
Ave.%
2,008,200
118,300
RSD,5.3
5.5(20 μM (K/Sx100)
in vials)
RSD,
2.7
4.4
REC, %
90110(A/Sx100) Recovery,唾液唾液_A1
1,978,000
121,700
回収率,%%
85110(A/Kx100) Recovery,
10D
唾液_A2
1,961,000
113,900
回収率,%%
94100
サンプルSample唾液
内標
Saliva10D
バイアル中20µM
Dilution:

Ave.
RSD, %

2,373,400
2.1

107,520
3.8

唾液-K1
唾液-K2
唾液-K3
唾液-K4
唾液-K5

2,310,000
2,025,000
2,134,000
2,063,000
2,197,000

126,700
115,300
117,700
109,300120,900

Ave.
RSD, %

2,145,800
5.3

117,980
5.5

REC, %
唾液_A1
唾液_A2
唾液_A3
唾液_A4
唾液_A590110
1,978,000
121,700
1,961,000
113,900
2,080,000
122,200 内標
唾液_A3
1,968,000
111,400 唾液に添加
唾液_A4
Table:of repeatability(RSD%)
2,054,000 Result 122,300
試料中200µM test 唾液_A5
2,008,200
118,300(バイアル中20µM)
Ave.

Ave.

2,080,000
1,968,000
2,054,000

2,008,200

RSD,%
2.7 唾液-1
4.4 2
1
34 %
5Metabolites
RSD,2.7Ave.
No.
化合物名
唾液-2
唾液-3
唾液-4
唾液-5(A/Sx100) 113,300
回収率, %85回収率, %1 (A/Sx100)
Alanine_2TMS
124,100 110
108,500
112,100
114,200 85
114,440(A/Kx100) 回収率, %(A/Kx100) 回収率, %
94
100
942
Urea_3TMS
59,95053,27065,70054,05070,00060,594
3
Proline_2TMS
481,000
418,600
437,000
432,700
454,600 444,780
4Glycine_3TMS
354,300
319,700
323,500
322,100
340,900 332,100
5Succinic acid_2TMS
49,02046,33045,91044,52046,79046,514
6
5-Aminovaleric acid_3TMS
4,982,000 4,212,000 4,519,000 4,623,000 4,786,000 4,624,400
7Putrescine_4TMS
457,300
404,100
422,900
413,200
438,100 427,120
8Citric acid_4TMS
9,392
8,965
8,049
8,148
7,767
8,464
9
Ornithine_4TMS
74,06062,22063,89063,74068,90066,562
10
Cadaverine_4TMS
131,400
113,400
119,100
116,700
124,200 120,960
11
Myristic acid_TMS
15,19014,16014,43012,53014,17014,096
12
Lysine_4TMS
15,05012,85013,01013,89014,08013,77613
Tyrosine_3TMS
105,300
90,52096,26091,42096,84096,06814
Palmitic acid_TMS
73,91072,73074,01063,60075,89072,02815
Uritic acid_4MS
122,400
105,400
111,000
111,300
108,700 111,760
16
Oleic Acid_TMS
17,69018,57016,44015,330
16,25016,856
17
Stearic acid_TMS
40,93039,76038,73034,41039,65038,696122,200
111,400
122,300

118,300

RSD,4.4%
110
5.1
100
12.0
5.4
4.5
3.5
6.3
4.9
8.1
7.3
5.8
6.9
6.5
6.1
6.7
5.7
7.6
6.5