SPL-M100 Application
Application Note
SPL-M100
Note

No. AS240504EN

Metabolome analysis of serumby online solid-phase analytical derivatization GC-MS system
Introduction
Solid-phasetechniqueofconsuming

SPL-M100
SPL-M100for SPE-GC systemderivatization

(SPD)

isa

derivatization

withoutthetime-centrifugal

concentration

andSampleSpiral insert

Flash-SPElyophylization, by retaining the target compounds
on a solid phase and dewatering it by passing aSerumorganic solvent through it then infiltrating thederivatization reagent and performing the reaction
on the solid phase. The example of pretreatment
method and analytical condition for metabolome

Information
Information

analysis of serum are shown below.
Online SPE-GC-MS system

SPD pretreatment workflow
Online SPE-GC: SPL-M100

Vials

Automation

Sample 50 µL
Water 150 µL

Conditioning
ACN-Water (1/1)

Flash-SPE ACXs

ACN 800 µL

Loading [Retaintion] Extract 50 µL

Shaking (37 ℃, 30 min)Washing

Centrifuge (14000 rpm, 3 min)

ACN-Water (1/1)

Dewater ACN
Infiltration 0.5% Methyoxyamine/Pyridine

Supernatant 500 µL

Derivatization (Methoximation), 3 min

Water 500 µL
pH adjustment (8-9)

Infiltration MSTFA/Hexane (1/1)
Derivatization (TMS), 1 min

Extract

ElutionHexaneGC-MS

AiSTI SCIENCE

Analytical condition
SPE-GC interface
SPE cartridge
PTV injection port

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

No. AS240504EN

AiSTI Application Note
Results

Expanded

サンプルNo.

Norleucine_2TMS Adipic acid_2TMS
1,309,000
76,2201,315,000
76,5401,331,000
75,5901,264,00075,570
1,305,000
73,340内標
S1Figure: Total ion current
chromatogram
バイアル中20µM

S2S3
S4Table: Result of recovery test (n=5)
S5Ave.
サンプルSample
内標Standard solution
バイアル中20µM
IS concentrations:
20 μM in vials

血清50µL
20D x 2D =40 D
内標
除タンパク後添加
バイアル中20µM(K/Sx100)
血清50µL
20D x 2D =40 D
内標
血清に添加
試料中800µM(バイアル中20µM)

No.
S1S2
S3
S4S5

Norleucine_2TMS Adipic acid_2TMS
1,309,000
76,2201,315,000
76,5401,331,000
75,5901,264,000
75,5701,305,000
73,340Ave.
RSD, %

1,304,800
1.975,452
1.7

血清-K1
血清-K2
血清-K3
血清-K4
血清-K5

1,158,000
1,220,000
1,229,000
1,204,000
1,186,000

86,25085,48084,95089,98091,570Ave.
RSD, %

1,199,400
2.4

87,646
3.4

REC, %
血清_A1
血清_A2
血清_A3
血清_A4
血清_A5

RSD, %
血清-K1
REC, % %Recovery,
血清_A1
血清-K2
Serum_A1
血清_A2
血清-K3
Serum_A2
血清_A3
血清-K4
Serum_A3
血清_A4
Serum_A4
血清-K5
Serum_A5
血清_A5
Ave.
Ave.%
RSD,
RSD,REC, %%%血清50µL (A/Sx100) 回収率,
血清_A1
Recovery,(A/Kx100)
Recovery,
回収率,%%
20D x 2D =40D
血清_A2

血清50µL (K/Sx100)
血清50µL
20Dx 2D =40 D
Serum
20Dx 2D =40 D
Dilution:
40 times
内標
Spike period of IS:
血清に添加
除タンパク後添加
Before extraction
試料中800µM
バイアル中20µM
800 μM in serum(バイアル中20µM)(20 μM (K/Sx100)
in vials)

3.2
78
85

1

3.1
286
74

75,452

2.4
1.9

3.4
1.7

1,158,000
92
1,064,000
1,220,000
1,010,000
1,229,000
1,023,000
1,204,000
974,600
1,186,000
1,028,000
1,199,400

86,250116
66,51085,48066,89084,95063,60089,98064,24091,57062,16087,646

1,019,920
2.4

64,680
3.4
3.1
116
86
66,51074
66,89063,60064,24062,16064,680
3.1
86
74

3.2
92116
92
1,064,000
66,51078
1,064,000
1,010,000
66,89085
1,010,000
1,023,000
63,600
内標
血清_A3
1,023,000
974,600
64,240
血清に添加
血清_A4
974,600
Table:
Result of repeatability
test (RSD%,
1,028,000
62,160試料中800µM
血清_A5 n=5)1,028,000
1,019,920
64,680(バイアル中20µM)
Ave.
1,019,920

Ave.
RSD,%
Metabolites(A/Sx100) 回収率, %(A/Kx100) 回収率, %1,304,800

サンプル
No. %Norleucine_2TMS
Adipic acid_2TMS
RSD,1.9
1.7Sample
血清50µL
血清-K1
1,158,000
86,250内標S1
1,309,000
76,220
Serum
Serum_K1
20Dx 2D =40 D
血清-K2
1,220,000
85,480
バイアル中20µM
S2
1,315,000
76,540
Dilution:
Serum_K2
40 times
血清-K3
1,229,000
84,950S31,331,000
75,590
Serum_K3
Spike
内標 period of IS:
血清-K4
1,204,000
89,980S4
1,264,000
75,570
Serum_K4
After deprotenization, Serum_K5
除タンパク後添加
血清-K5
1,186,000
91,570S51,305,000
73,34020 μM in vials
バイアル中20µM
Ave.
1,199,400
87,646
1,304,800
75,45234 RSD, %(A/Sx100) 回収率, %(A/Kx100) 回収率, %53.2
7885