SPL-M100 Application
SPL-M100
ApplicationNoteNoteNo. AS240507EN

Metabolome analysis of mouse fecesby online solid-phase analytical derivatization GC-MS system
Introduction

for SPE-GC system

Solid-phase derivatization (SPD) is a techniqueofderivatization

centrifugal

SPL-M100
SPL-M100

without

concentration

theandSample

time-consuming
lyophylization,by

Spiral insert

retaining the target compounds on a solid phase andFlash-SPE

dewatering it by passing a organic solvent through it
theninfiltrating

thederivatization

reagentandFeces (mouse)

performing the reaction on the solid phase. The
example of pretreatment method and analyticalcondition for metabolome analysis of mouse feces

Information
Information

are shown below.
Online SPE-GC-MS system

SPD pretreatment workflow
Sample 10 mg

Online SPE-GC: SPL-M100

Vials

ACN-Water (1/1) 500 µL
Crashing (4000 rpm, 1 min ×2)

Flash-SPE ACXs

Centrifuge (14000 rpm, 3 min)Loading [Retaintion] Extract 50 µL

Supernatant 500 µL

Washing ACN-Water (1/1)

ACN 500 µL

Dewater ACN

Shaking (37 ℃, 30 min)Infiltration 0.5% Methyoxyamine/Pyridine

Centrifuge (14000 rpm, 3 min)Derivatization (Methoximation), 3 min

Supernatant 500 µL

Infiltration MSTFA/Hexane (1/1)

ACN-Water (1/1) 500 µL

Derivatization (TMS), 1 min

Analytical solution

Elution Hexane

pH adjustment (8-9)

GC-MS

Analytical condition
SPE-GC interface
SPE cartridge
PTV injection port

Automation

Conditioning
ACN-Water (1/1)

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

Online SPE-GC

SPL-M100
Solid-phasecartridge

Insert typeSpiral insert

Temp.

220℃(0.5 min)-50℃/min-290℃(23 min)Product
Flash-SPE

Inlet mode

Split 1:50

LVI-S250

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℃

Gas chromatograph

Mass spectrometer
Acquisition mode

Scan (m/z 70-600)

Data acquisition

3.0-26 min

GC large volume
injection port

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

AiSTI Application NoteNo. AS240507EN

Results

Expanded
サンプル
No.
Norleucine_2TMS Adipic acid_2TMS
内標
S1
1,431,000
83,510Figure: Total ion current
chromatogram
バイアル中20µM
S2
1,409,000
80,230S3
1,371,000
79,830S4
1,327,000
74,290
Table 1: Result of recovery test (n=5)
S51,292,000
75,900サンプル
Sample
内標Standard solution
バイアル中20µM
IS concentrations:
20 μM in vials

マウス糞便
200D
内標
除タンパク後添加
バイアル中20µM(K/Sx100)
マウス糞便
200D
内標
糞便に添加
試料中2mM(バイアル中20µM)

No.
12345678910111213141516

No.
S1S2S3S4S5Norleucine_2TMS Adipic acid_2TMS
1,431,000
83,5101,409,000
80,2301,371,000
79,8301,327,000
74,2901,292,000
75,900Ave.
RSD, %

1,366,000
4.2

78,752
4.7

糞便-K1
糞便-K2
糞便-K3
糞便-K4
糞便-K5

1,556,000
1,597,000
1,607,000
1,638,000
1,612,000

94,92093,00095,60096,67097,420Ave.
RSD, %

1,602,000
1.9

95,522
1.8

REC, %
糞便_A1
糞便_A2
糞便_A3
糞便_A4
糞便_A5

Ave.

117
121
1,864,000
95,2501,876,000
106,400
内標
糞便_A3
1,869,000
1,869,000
103,300糞便に添加
糞便_A4
1,937,000
1,937,000
107,900
Table1,926,000
2 : Result ofrepeatability
試料中2mMtest (n=5,糞便_A5RSD%)
1,926,000
107,700(バイアル中20µM)
Ave.
1,894,400
1,894,400
104,110

Metabolites
化合物名
RSD, %(A/Sx100)
回収率, %
Alanine_2TMS(A/Kx100) 回収率, %Ave.
1,366,000
78,752
No. % Norleucine_2TMS
RSD,4.2 Adipic acid_2TMS
4.7
S1
1,431,000
83,510
糞便-K1
1,556,000
94,920Feces_K1
S2
1,409,000
80,230
糞便-K2
1,597,000
93,000Feces_K2
S3
1,371,000
79,830200 times糞便-K3
1,607,000
95,600Feces_K3
Spike
Feces_K4
S4
1,327,000
74,290
内標 period of IS:
糞便-K4
1,638,000
96,670After deprotenization, Feces_K5
S51,292,000
75,900除タンパク後添加
糞便-K5
1,612,000
97,42020 μM in vials
Ave.
1,366,000
78,752
バイアル中20µM
1,602,000
95,522
RSD, %
4.2
4.7
1.9
1.8
マウス糞便(K/Sx100) Recovery,
糞便-K1
1,556,000
94,920REC, % %
117
121
200D
糞便-K2
1,597,000
93,000
マウス糞便
糞便_A1
1,864,000
95,250
Mouse Feces
Feces_A1
糞便-K3
1,607,000
95,600200D
糞便_A2
1,876,000
106,400
Dilution:
Feces_A2
内標
糞便-K4
1,638,000
96,670200 times
糞便_A3
1,869,000
103,300
Feces_A3
Spike period of IS:
除タンパク後添加
糞便-K5
1,612,000
97,420Feces_A4
糞便に添加
糞便_A4
1,937,000
107,900
Before extraction
Feces_A5
バイアル中20µM
Ave.
1,602,000
95,522
試料中2mM
糞便_A5
1,926,000
107,700
2 mM in samples
RSD,1.9
1.8(バイアル中20µM)
Ave.%
1,894,400
104,110(20 μM (K/Sx100)
in vials)REC, %
117
121
RSD,1.85.1
マウス糞便(A/Sx100) 回収率,
糞便_A1% %1,864,000
95,250
139
132
Recovery,200D
糞便_A2% %1,876,000
106,400(A/Kx100) 回収率,
118
109
Recovery,
サンプル
Sample内標
マウス糞便
MouseFeces
バイアル中20µM
200D
Dilution:

1
1.8糞便-1

139
3,665,000
118
Valine_2TMS
1,036,000
Glycine_3TMS
834,500
Glyceric acid_3TMS
46,900
Uracil_2TMS
170,700
Serine_3TMS
379,400
Threonine_3TMS
266,400
γ-Aminobutyric acid_3TMS 599,200
Phenethylamine_2TMS
424,000
Ornithine_4TMS
529,800
Lysine_4TMS
336,000
Gluconic acid_6TMS
44,270Isoferulic acid_2TMS
85,120
Stearolic acid_TMS
156,300
Oleic acid_TMS
160,500
Stearic acid_TMS81,640

2
3
4 RSD, % 糞便-5
5
糞便-2
糞便-3
糞便-4Ave.
1.8
5.1(A/Sx100)
回収率, %
139132
3,692,000
3,688,000
3,774,000
3,788,000 3,721,400(A/Kx100) 回収率, %118
109
1,049,000
1,044,000 1,072,000
1,077,000 1,055,600
821,000
828,300
830,500
868,700 836,600
55,45059,53066,66067,39059,186
186,000
175,700
181,500
182,500 179,280
396,500
395,100
406,400
408,100 397,100
266,800
273,900
273,700
276,600 271,480
598,900
595,000
596,000
615,400 600,900
444,700
430,100
430,200
438,600 433,520
538,400
553,400
554,100
580,800 551,300
352,900
369,600
371,000
393,200 364,540
47,43047,08050,95049,49047,844
93,23093,49096,30096,57092,942
170,600
168,000
167,200
163,500 165,120
174,900
161,500
168,800
169,000 166,940
96,58090,46089,64089,50089,564

103,300
107,900
107,700

104,110

RSD,5.1%
132
1.5
109
1.7
2.2
14.3
3.4
2.9
1.7
1.4
1.9
3.5
5.9
5.3
5.0
3.4
3.6
5.9