SPL-M100 Application Note

No. AS200605EN

Evaluation of On-Line Solid Phase Derivatization
SPE-GC/MS Analysis of Mouse SerumIntroduction
In conventional GC/MS analysis of metabolomics, extraction, lyophilization, and derivatization werecomplicated and time-consuming, and the data obtained tended to be highly inconsistent. We havedramatically shortened the time required, simplified the process, and improved the accuracy by using
our proprietary “solid-phase derivatization” technique. Metabolites and other substances are held inthe solid phase by ion-exchange interactions, and then passed through acetonitrile to dehydrate andwash the solid phase. In this study, we attempted to analyze metabolites in mouse serum using theSPL-M100 system, which fully automates these processes.

Pretreatment flow

SPL-M100for SPE-GC system

SampleMouse serum

Online SPE-GC：SPL-M100

Sample 50 µL mouse serum

Vials

Water 150 µL

Automation
Pretreatment：15 min.

ACN 800 µL
Flash-SPE ACXs

Shaking 37 ℃, 30 min

Conditioning
ACN-Water(1/1)
ACN

Information【Sample】
・ Mouse serum

Loading[Retention] Extract 50 µL

Centrifuge 14000 rpm, 3 min

Washing ACN-Water(1/1)

Supernatant 500 µL【TargetComponent】
・ Amino acid・ Amine・ Organic acid

Dewater ACN

Water 500 µL

Infiltration Methoxyamine /Pyridine

0.1N NaOH

Derivatization (Methoximation), 3 min

Extract

Infiltration MSTFA-Toluene(3/1)
Derivatization (TMS), 1 min
Elution Hexane

Analytical condition

GC-MS Split 1:50

PTV Injector
LVI-S250 (AiSTI Science)
Insert Type
Spiral Insert
Temp.
220℃(0.5min)-50℃/min-290℃(16min)
GC-MS
Injection mode Split 1:50
Flow mode
Constant Flow, 1 ml/min
Pre-column
0.25mm i.d. x 1m
ColumnVf-5ms, 0.25mm i.d. x 30m, df;0.25µm
Oven Temp.
100℃(2min)-10℃/min-220℃-30℃/min-320℃
Trans. Temp. 290℃MS
SCAN, m/z;70-470

SPE-GC-MS system for metabolome analysis

SPL-M100 / GCMS-TQ8040NXAiSTI SCIENCE

51Product16Online SPE-GC

SPL-M100

4Solid Phase cartridge

2717① Lactic acid-2TMS② Alanine-2TMS③ Valine-2TMS④ Leucine-2TMS⑤ Phosphoric acid-3TMS⑥ Isoleucine-2TMS⑦ Proline-2TMS⑧ Glycine-3TMS⑨ Succinic acid-2TMS⑩ Glyceric acid-3TMS2115910LVI-S250

18,19146GC Large VolumeInjection

20

11 12
3Flash-SPE

22813⑪ Serine-3TMS⑫ Threonine-3TMS⑬ Malic acid-3TMS⑭ Aspartic acid-3TMS⑮ Pyroglutamic acid-2TMS⑯ Glutamic acid-3TMS⑰ Phenylalanine-2TMS⑱ Ornithine-4TMS⑲ Citric acid-4TMS⑳ Lysine-4TMS㉑ Tyrosine-3TMS㉒ Gluconic acid-6TMS

Figure1 SCAN total ion chromatogram of mouse serum using this method

AiSTI SCIENCE CO.,Ltd.
Tel：+81-73-475-0033
as@aisti.co.jp

E-mail：as@aisti.co.jp
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AiSTI Application Note
Table 1Recovery Test of Mouse Serum with this SystemNo.

成分名 names
Compound12
6910111214151617182021222325272831333637384143

Lactic acid-2TMS
Alanine-2TMS
Valine-2TMS
Leucine-2TMS
Isoleucine-2TMS
Proline-2TMS
Glycine-3TMS
Succinic acid-2TMS
Fumaric acid-2TMS
Serine-3TMS
Threonine-3TMS
Malic acid-3TMS
Aspartic acid-3TMS
Methionine-2TMS
4-Hydroxyproline-3TMS
GABA-3TMS
Threonic acid-4TMS
Glutamic acid-3TMS
Phenylalanine-2TMS
Putrescine-4TMS
Citric acid-4TMS
Lysine-4TMS
Histidine-3TMS
Tyrosine-3TMS
Tryptophan-3TMS
Cystine-4TMS

Standard
ST
2,641,331
2,526,424
3,223,917
3,806,169
3,483,014
3,349,273
2,822,020
5,442,569
1,886,256
1,763,290
584,437
610,728
2,929,674
321,4032,400,683
808,381
331,4482,350,172
1,457,813
4,356,542
911,581
1,587,702
2,194,472
4,650,501
230,794
2,142,088

Operation
Blank マウス血清
操作Blank
Mouse serum+ST
Mouse serum マウス血清＋STB
2,044,267
84,451
8,177
9,760
5,009
6,613
38,90351,2603,365
19,550
2,454
1,773
5,116
1,578
122
5,804
417
2,555
1,404
1,610
660
1,259
1,959
0
0

M
14,805,790
4,971,432
2,224,994
3,940,497
958,6813,008,108
3,192,716
607,407
11,533
1,451,277
484,176
18,302784,543
77,62754,733
9,875
27,1253,437,586
690,541
9,512
216,437
940,190
10,446
1,175,034
29,096
14,728A
11,895,241
6,847,093
5,182,605
7,217,079
4,444,077
5,928,827
5,175,424
6,159,930
1,967,128
3,032,036
957,064
629,151
3,822,406
321,294
1,997,473
776,441
202,330
6,150,075
2,098,341
4,597,459
1,160,422
2,675,748
3,240,581
5,939,201
398,017
2,196,172

Recovery%
回収率,%(A-M)/(ST-B)
7792
8610087
71103
104
91
81100
104
76819653116
97105
104
109
147
102160102

Table２ Reproducibility of mouse serum by this system
No.
1269101112
14
15
1617182021222325272831
33
36
37
38
41
43

Compoundnamesv
成分名
U1
U2
U3
U4
U5
Ave.
RSD,%
Lactic acid-2TMS
14,649,682 15,089,600 13,841,394 15,639,698 14,808,578 14,805,790
4.4
Alanine-2TMS
5,118,252 4,719,988 4,790,287 5,126,762 5,101,869 4,971,432
4.0
Valine-2TMS
2,261,504 2,125,119 2,152,889 2,285,552 2,299,904 2,224,994
3.6
Leucine-2TMS
4,005,604 3,817,067 3,780,734 4,039,212 4,059,866 3,940,497
3.3
Isoleucine-2TMS
971,384928,534
931,475
980,243
981,767
958,6812.8
Proline-2TMS
3,078,401 2,818,958 2,910,867 3,100,657 3,131,659 3,008,108
4.5
Glycine-3TMS
3,243,322 2,973,200 3,083,005 3,312,366 3,351,687 3,192,716
5.0
Succinic acid-2TMS
609,722610,940
585,493
627,168
603,713607,407
2.5
Fumaric acid-2TMS
11,626
11,14910,691
11,947
12,254
11,533
5.4
Serine-3TMS
1,466,003 1,327,848 1,366,049 1,533,536 1,562,951 1,451,277
7.1
Threonine-3TMS
497,778
441,900
461,299
506,740
513,163484,176
6.4
Malic acid-3TMS
19,076
18,062
17,17318,72818,47018,302
4.0
Aspartic acid-3TMS
745,837
821,214711,854825,260
818,549784,543
6.7
Methionine-2TMS
71,25566,98376,05992,88980,95077,627
12.9
4-Hydroxyproline-3TMS
54,58347,943
51,23858,56961,33354,733
9.9
GABA-3TMS
10,265
8,742
9,675
10,265
10,4309,875
7.0
Threonic acid-4TMS
28,17728,501
22,54529,63826,76527,125
10.2
Glutamic acid-3TMS
3,365,311 3,486,693 3,156,198 3,603,886 3,575,843 3,437,586
5.3
Phenylalanine-2TMS
682,545669,938
666,324718,207715,692
690,541
3.6
Putrescine-4TMS
9,970
9,716
9,256
8,922
9,694
9,512
4.4
Citric acid-4TMS
212,800
218,960
209,116
224,337
216,971
216,437
2.7
Lysine-4TMS
926,298
906,996922,350974,013
971,295
940,190
3.2
Histidine-3TMS
10,802
9,595
9,636
10,237
11,95910,446
9.4
Tyrosine-3TMS
1,192,371 1,093,010 1,123,091 1,241,557 1,225,141 1,175,034
5.5
Tryptophan-3TMS
29,18729,591
28,39329,02729,28229,096
1.5
Cystine-4TMS
15,113
14,20913,34915,634
15,333
14,728
6.4【Results and Considerations】
The RSD. (reproducibility) of each component is less than 13%, indicating high accuracy. Peak shape and separation are also
excellent in TIC. From these results, it can be inferred that washing and dehydration by solid-phase extraction functionallywork, and derivatization is efficiently performed in the solid phase. The good results obtain even with samples containing alarge amount of lipids, etc., will reduce GC/MS maintenance labor. The solid-phase derivatization of this analytical method
improves the accuracy and shortens the time required for analysis, and automation of the method is expected to increase
efficiency. The method is expected to be applied to the analysis of metabolites in biological samples in the future, as it canmaximize the advantages of GC/MS, such as high resolution, high qualitative ability, and a full database, by overcoming thecomplexity and long time required for pretreatment, which is a weak point of the method.