Development of online SPE-GC-MS system with automatedSPE-based derivatization method for metabolome analysis.
Ryoichi SASANO1, Koji MACHITANI2, Shusuke OSAKI2, Masahiro FURUNO3, Eiichiro FUKUSAKI3
1; Aisti Science co., Ltd., 2; Industrial Technology Center of Wakayama prefecture, 3; Osaka University【Abstract】
Due to complicated sample preparation procedures that include centrifugal concentration, freeze-drying and derivatization, the current metabolomics protocol using
GC-MS, requires not only longer processing time but also advanced technical skill. In addition, some silylated metabolites are unstable and the time required forcomplete silylation varies depending on the metabolite. Therefore, the time gap between derivatization and actual GC-MS analysis can potentially be a source of error.
In this study, automated SPE-based derivatization method coupled with GC-MS was developed to accomplish an extremely rapid sample preparation. The derivatization
method using SPE-gel has a high reaction efficiency and can provide sequential sample introduction to keep the interval from silylation to GC-MS injection constant.
【Experimental】■ Online SPE-GC-MS system with automated SPE-based derivatization method■ SPE-based derivatization method

Step 1

Step 2

Step 3

Step 4

Washing

Dehydrating

Derivatization

Water-ACN

ACN

Loading &Adsorbent
SampleAmino group-SO3⊝ ⊕H3N-R

1 min

Automated pre-treatment time: 10 min

Step 5MSTFA

Elution2 min

Hexane-SO3⊝ ⊕H3N-R-SO3⊝ ⊕H3N-R-SO3⊝ TMS-HN-R

-SO3⊝

-N⊕

-N⊕ ⊝OOC-R

-N⊕

-N⊕

The time fromderivatization to
injecting is always
fixed.

Flash-SPE
AOS

Carboxyl group⊝OOC-R-N⊕(CH3)3⊝OOC-R(CH3)3(CH3)3

Nonionic
compound

TMS-OOC-R(CH3)3

LVI-S250
Spiral Insert(CH3)3

GC-MS

R-NH-TMS
R-COO-TMS

H2O

An amino group and Carboxyl group are
adsorbed in SPE by ion exchange interaction

SampleLoading

Conditioning

TMS derivatization is done by permeating MSTFA reagent
directly into the sorbent, and is detached from resin

The remaining water in the sorbent
was removed with acetonitrile.

Elution &Injection

Washing
Dehydrating

Adding MSTFA
Derivatization

Amino acids and organic acids from the extracted samples were loaded into the SPE column filled with a mixture of ion-exchange sorbent.
After washing with acetonitrile/water, the remaining water in the sorbent was removed with acetonitrile. TMS derivatization was done bypermeating MSTFA reagent directly into the sorbent. A needle was automatically connected to the SPE cartridge and inserted into the GC-MS.
Then, the TMS-derivatized metabolites were eluted with n-hexane, and injected directly into GC-MS with LVI spiral insert.
■ Large Volume Injection-Split mode

Automation
Sample： 50µL（50mg）

Online SPE-GC-MS System

Vial

Added Water 150µL■ Condition of SPE-GC-MS System

Aliquot of extract 40µL

Added ACN 800µL

Conditioning with Water-ACN 100µL
Flash-SPE AOS-3mg

Shaking （37℃, 30min）

Wash with Water-ACN 100µL ：Cleaning

Centrifuging（14000rpm, 5min）

Sampling

Wash with ACN 100µL ：Dehydration

Flash-SPE

Injection

1st Stage

Pre-treatment
Unit

Added Methoxyamine-Pyridine-MSTFA 8µL

Aliquot of upper extract： 250µL

Derivatization in SPE 2min

Added Water 250μL

Pump Unit

Elute Acetone-Hexane 40µL

Vial

2nd Stage

3rd Stage

4th Stage

Injection

Concentration

Transfer

Elimination

The injector is kept at
a temperature lower
than the boiling pointof sample solvent.

While the evaporated
sample solvent isexhausted, the sample
is concentrated.

Target compounds aretransferred to thecolumn at an elevatedtemperature.

Matrix compounds are
eliminated from theinsert at further
elevated temperature.

The spiral shaped insert makes it possible toaccept all of the injected eluate(40μL) from the SPE
cartridge.

GC-MS： LVI-Split（1:50）
Online SPE-GC-MS System【Results】[ｸﾛﾏﾄｸﾞﾗﾑ]

TIC
3,4

:

4331844-0

Table 3. Recoveries of metabolites added to mouse serum

4.0E+06
11

Relative peak area

10

3.0E+0618
61

2.0E+06

2
1
9

5

23

20,21

24

1.0E+06

15
8

12

13

0.0E+00
R.T-->
07:00 08:00 09:00 10:00 11:00 12:00 13:00 14:00

Fig. 1. Relations of derivatization time in sorbent and the relative peak area.

Fig. 2. Relations of the derivatization reagent and therelative peak area.

1.Alanine-2TMS
2. Valine-2TMS
3. Phosphate-3TMS
4. Leucine-2TMS
5. Isoleucine-2TMS

O
O[ｸﾛﾏﾄｸﾞﾗﾑ]

TIC:

5977585

-

Si

0

Si

24,25

Si

Si

NH
O

NH

N

O

O

5.0E+06
113,4
10

4.0E+06

6

17
9

Si

28

O
N

14

29

Si

Si

Si

N
N

O

Si

Si

Putrescine-4TMS

Aminobutyric acid-3TMS

27

1.0E+0622

0.0E+00
R.T-->
07:00
08:00
09:00
10:00
11:00
12:00
13:00
14:00
15:00
6. Proline-2TMS
7. Glycine-3TMS
8. Succinic acid-2TMS
9. Fumaric acid-2TMS
10. Serine-3TMS

11. Threonine-3TMS
12. Malic acid-3TMS
13. Aspartic acid-3TMS
14. Methionine-2TMS
15. Proline-oxo-2TMS

16. Cytosine-2TMS
17. Aminobutyric acid-3TMS
18. Ketoglutaric acid-3TMS
19. Glutamic acid-3TMS
20. Phenylalanine-2TMS

21. Asparagine-3TMS
22. Putrescine-4TMS
23. Aconitic acid-3TMS
24. Citric acid-4TMS
25. Ornithine-4TMS

26. Adenine-2TMS
27. Lysine-4TMS
28. Tyrosine-3TMS
29. Guanine-3TMS

Fig. 3. The SCAN total ion chromatogram of standard solution using SPE-GC-MS systemwith automated SPE-based derivatization method.

◆ Linearity
6.E+06

Alanine-2TMS

7.E+06

Succinic acid-2TMS

5.E+06

R² = 0.9985

6.E+06

R² = 0.9972

5.E+06

4.E+06

4.E+06

Peak Area

13

18
19 21

Peak Area1

3.E+06

3.E+06

2.E+06

2.E+06

1.E+06＊Concentration of standard solution is 0.01nmol/μL

1.E+06
0.E+000.E+00
0

5

10

15

20

Concentration (pmol/μL)

23. Lysine-4TMS
24. Tyrosine-3TMS

0

25

5

10

15

Standard
ST

Mause
M

Mause +ST
A

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29

Alanine-2TMS
Valine-2TMS
Phosphate (3:1)-3TMS
Norleucine-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
Proline-oxo-2TMS
Cytosine-2TMS
Aminobutyric acid-3TMS
Ketoglutaric acid-3TMS
Glutamic acid-3TMS
Phenylalanine-2TMS
Asparagine-3TMS
Putrescine-4TMS
Aconitic acid-3TMS
Citric acid-4TMS
Ornithine-4TMS
Adenine-2TMS
Lysine-4TMS
Tyrosine-3TMS
Guanine-3TMS

2,395,769
2,857,898
1,691,961
3,402,608
2,801,029
3,037,141
1,800,878
2,958,640
1,676,379
1,769,642
900,834
475,088
440,006
1,186,002
1,684,508
1,069,383
1,425,778
162,941
381,767
1,361,973
212,637
928,721482,874
2,365,829
741,337
1,710,221
344,472
2,171,566
1,112,060

5,539,074
2,759,114
4,279,367
4,067,823
1,086,415
2,998,133
2,966,859
224,256
13,946
1,608,041
852,911
20,319190,845
93,758740,135
5,948
82,936

7,703,074
5,744,538
6,787,045
7,424,476
4,096,438
6,301,408
4,603,320
3,375,441
1,869,217
3,632,037
1,865,424
551,360
795,480
1,417,378
3,085,018
1,182,673
1,774,441
163,614
1,524,345
2,285,882
291,982
1,124,501
544,525
3,143,411
1,295,449
1,966,746
680,1113,535,152
1,479,513◆ Reproducibility
Table 2. Reproducibility of peak area with the mouse serum using SPE-GC-MS system.

Si26

2.0E+06

20

25

Concentration (pmol/μL)

No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25

CompoundAlanine-2TMS
Valine-2TMS
Phosphate (3:1)-3TMS
Norleucine-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
Proline-oxo-2TMS

Cytosine-2TMS
Aminobutyric acid-3TMS
Glutamic acid-3TMS
Phenylalanine-2TMS
Citric acid-4TMS
Ornithine-4TMS

Adenine-2TMS
Lysine-4TMS
Tyrosine-3TMS

Guanine-3TMS

1
5,541,262
2,755,712
4,201,912
4,056,211
1,078,007
2,990,521
3,037,839
29,04014,4001,617,736
854,196
20,457
195,51293,568719,078
5,878
87,027
1,268,042
699,562
335,441
663,367
4,077
384,118
739,5513,773

25,554,574
2,771,037
4,356,822
4,080,919
1,080,660
3,000,081
2,895,879
29,234
13,605
1,601,793
855,270
20,641
183,707
94,791
784,164
5,728
83,832
1,206,401
714,084
333,534
673,4443,243
400,505
763,6703,789

35,750,943
2,893,545
4,434,444
4,276,463
1,131,523
3,191,334
3,107,951
29,61014,528
1,691,622
914,762
21,151
208,840
100,427
740,721
5,647
70,197
1,296,449
762,498
337,541
787,729
3,358
466,485
838,1103,207

45,697,229
2,911,101
4,412,647
4,306,038
1,141,625
3,154,192
3,217,887
29,11813,7571,701,792
923,062
21,808210,072
104,122869,323
5,592
83,924
1,342,892
773,628
334,091
789,718
3,410
466,911
848,3093,438

5
5,588,344
2,803,596
4,201,988
4,157,663
1,094,518
3,053,991
3,354,424
26,403
13,4691,664,881
882,377
19,071
204,684
101,698
610,496
5,629
75,857
1,281,052
724,391
312,166
771,4703,217
449,562
773,7372,485

65,721,789
2,871,371
4,259,611
4,270,224
1,124,087
3,162,046
3,293,368
27,322
13,211
1,718,251
911,832
20,283
225,449
103,386
641,127
5,516
76,002
1,393,273
758,118
321,946
808,655
2,885
483,398
850,3072,720

75,850,742
2,926,797
4,389,792
4,312,737
1,139,671
3,244,535
3,059,023
28,53012,847
1,759,025
928,177
20,751
240,856
105,816
674,841
4,974
85,355
1,513,486
786,916
322,681
771,975
3,216
450,203875,8072,968

8
5,681,853
2,853,359
4,266,977
4,223,433
1,121,838
3,101,102
3,070,554
28,003
12,142
1,685,417
920,805
19,941
233,191
97,718868,861
5,028
82,175
1,405,403
753,925
314,421652,659
2,978
364,748
790,0953,136

95,706,369
2,867,084
4,210,194
4,227,870
1,118,968
3,103,402
3,113,071
27,16911,996
1,696,241
914,839
19,094
238,539
99,320
839,473
5,413
87,546
1,516,263
773,827
317,960
737,745
3,005
433,653
852,336
2,457

Ave.
RSD, %
5,677,012
1.8
2,850,400
2.1
4,303,821
2.2
4,212,395
2.2
1,114,544
2.2
3,111,245
2.8
3,127,777
4.5
28,2703.9
13,3286.7
1,681,862
2.9
900,591
3.2
20,355
4.4
215,650
9.3
100,094
4.2
749,787
13.0
5,489
5.6
81,3247.3
1,358,140
8.0
749,661
4.0
325,5313.0
739,640
8.2
3,265
10.8
433,287
9.5
814,658
5.9
3,108
16.1

1.E+07

Alanine-2TMS

1.E+07

R² = 0.9985

8.E+06
6.E+06
4.E+06

62,676,876
3,132,760
1,658,840
3,798,730
3,105,804
3,272,235
2,288,729
2,677,718
1,608,388
1,978,512
1,028,004
446,942
689,805
1,299,449
2,218,260
1,179,900
1,983,878
156,600
585,775
1,564,042
264,791
1,218,672
1,009,638
2,410,546
1,025,487
1,839,583
469,2632,300,204
1,292,009

7
2,632,581
3,054,963
1,585,771
3,712,869
3,057,182
3,230,054
2,328,562
2,840,826
1,635,905
1,918,379
988,509451,563
622,430
1,267,142
2,414,150
1,130,354
1,952,195
151,045
510,207
1,507,985
258,735
1,113,605
1,007,640
2,372,287
1,049,537
1,690,694
485,851
2,231,995
1,214,782

8
2,692,127
3,085,131
1,679,471
3,729,371
3,062,330
3,247,932
2,291,359
2,917,558
1,710,549
1,950,455
997,181459,884
590,812
1,274,606
2,348,929
1,140,346
1,884,295
147,256
483,426
1,508,373
249,746
1,075,243
1,025,144
2,395,838
1,005,050
1,722,793
449,843
2,184,577
1,217,067

Ave.
9
RSD, %
2,718,118 2,706,045
3.0
3,160,769 3,144,454
3.4
1,991,253 1,799,098
8.3
3,834,508 3,801,752
3.4
3,129,208 3,124,590
3.4
3,297,886 3,288,018
3.6
2,432,007 2,270,036
4.3
3,180,044 2,950,192
5.2
1,825,272 1,719,697
4.9
1,968,792 1,982,700
3.9
1,024,209 1,024,597
3.8
497,251475,484
4.5
358,848544,997
18.3
1,279,034 1,281,506
4.6
2,386,591 2,232,380
6.1
1,191,227 1,161,433
3.5
2,036,145 1,922,516
5.6
178,112
163,861
10.1
320,272
474,03016.8
1,520,941 1,540,630
4.2
263,337
259,998
7.9
1,130,409 1,112,462
5.0
1,085,668 1,041,941
4.1
2,470,814 2,451,140
3.5
1,047,923
992,5626.3
1,876,276 1,801,518
5.0
475,516
447,9517.8
2,239,834 2,254,717
4.4
1,316,201 1,276,678
4.5

2.E+06

3.E+06

1.E+06

0.E+000.E+00
0

20

40

60

Sample loading volume (μL)

Methionine-2TMS

3.E+05

R² = 0.9944

3.E+05

5

10

15

20

Concentration (pmol/μL)

25

Tyrosine-3TMS

5.E+06

3.E+06
2.E+06
1.E+06
0.E+00
0

5

10

15

20

Concentration (pmol/μL)

25

40

60

80

Lysine-4TMS

1.E+06

R² = 0.9965

1.E+06
8.E+05

2.E+05

6.E+05

2.E+05

4.E+05

1.E+055.E+04

2.E+050.E+000.E+00
0

20

40

60

0

80

20

40

60

80

Sample loading volume (μL)

Sample loading volume (μL)

Tyrosine-3TMS

9.E+05Citric acid-4TMS

R² = 0.996

8.E+05

R² = 0.9967

7.E+05

2.E+06

5.E+05
4.E+05

1.E+06

3.E+05
2.E+050.E+000.E+000Alanine-2TMS
0

5

10

15

20

Concentration (pmol/μL)

25

Leucine-2TMS

Lysine-4TMS

Citric acid-4TMS

Fig. 6. Reproducibility of ion chromatogram of the mouse serum using SPE-GC-MS system( n = 9 ).

20

40

60

80

Sample loading volume (μL)

0

20

40

60

80

Sample loading volume (μL)

Fig. 7. Relations of sample loading volume andthe peak area.

Lysine-4TMS

9.E+058.E+05
7.E+05
6.E+055.E+05
4.E+05
3.E+05
2.E+05
1.E+050.E+00R² = 0.9952

4.E+0620

Sample loading volume (μL)

1.E+050.E+000080

5.E+05

1.E+06

0.E+002.E+05
1.E+05

2.E+06

5.E+05

3.E+05

6.E+05

R² = 0.9956

4.E+06

2.E+06

4.E+05

Peak Area

52,663,543
3,107,653
1,746,163
3,750,253
3,112,611
3,264,503
2,229,077
2,874,284
1,697,384
1,969,420
1,019,290
471,484
548,152
1,233,662
2,171,464
1,154,429
1,911,954
169,160
494,429
1,528,499
260,091
1,140,680
1,026,875
2,437,909
963,445
1,831,552
423,849
2,220,032
1,273,918

R² = 0.9985

5.E+05

2.E+06

Peak Area

42,570,446
2,966,049
1,762,489
3,572,341
2,942,720
3,055,493
2,118,024
2,891,328
1,626,601
1,857,176
963,085
464,603439,870
1,165,099
2,213,001
1,081,564
1,718,333
138,034
375,004
1,422,103
215,342
1,069,833
973,865
2,326,885
889,675
1,670,688
389,2632,070,195
1,201,496

5.E+06

Succinic acid-2TMS

6.E+05

2.E+06

Peak Area

32,805,838
3,270,689
1,842,146
3,961,145
3,263,756
3,452,215
2,219,378
3,047,906
1,790,816
2,078,505
1,075,400
505,695
605,941
1,320,877
2,188,232
1,211,399
1,832,270
167,292
528,880
1,616,874
269,785
1,022,892
1,085,018
2,551,370
928,962
1,930,628
411,966
2,339,399
1,372,469

R² = 0.997

35,714
3,083
334,488
668,406
3,660
389,645748,544
3,595

1.E+07

Citric acid-4TMS

R² = 0.9489【Conclusion】

Peak Area

22,814,678
3,290,271
1,945,488
3,995,800
3,281,486
3,445,278
2,352,541
3,101,538
1,813,725
2,110,285
1,085,291
496,725
521,172
1,376,552
2,178,513
1,199,619
2,080,359
187,293482,193
1,642,952
293,568
1,143,662
1,095,208
2,585,329
1,074,181
1,859,990
487,673
2,415,910
1,325,553

3.E+06

Peak Area

12,780,202
3,231,804
1,980,261
3,860,754
3,166,213
3,326,569
2,170,649
3,020,526
1,768,634
2,012,774
1,040,407
485,209527,945
1,317,135
1,972,283
1,164,055
1,903,218
179,954
486,088
1,553,897
264,587
1,097,163
1,068,411
2,509,279
948,801
1,791,455
438,332
2,290,304
1,276,608

6.E+06

Peak Area

Table 1. Reproducibility of peak area with standard solution using SPE-GC-MS system.

3.E+06

Methionine-2TMS

1,237,713
705,325

REC, %(A-M)/ST
90
104
148
99
107
109
91
107
111
114
112
112
137
112
139
110
119
100
75
116
137
117
112
119
85
115
84
128
133

＊Sample were spiked at 0.01 nmol/μL of each metabolites.

3.E+06◆ Reproducibility
CompoundAlanine-2TMS
Valine-2TMS
Phosphate (3:1)-3TMS
Norleucine-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
Proline-oxo-2TMS
Cytosine-2TMS
Aminobutyric acid-3TMS
Ketoglutaric acid-3TMS
Glutamic acid-3TMS
Phenylalanine-2TMS
Asparagine-3TMS
Putrescine-4TMS
Aconitic acid-3TMS
Citric acid-4TMS
Ornithine-4TMS
Adenine-2TMS
Lysine-4TMS
Tyrosine-3TMS
Guanine-3TMS

Si

Lysine-4TMS

Ornithine-4TMS

23

2
0

18. Glutamic acid-3TMS
19. Phenylalanine-2TMS
20. Citric acid-4TMS
21. Ornithine-4TMS

N
Si

12

2

No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29

N
Si

Glycine-3TMS

15,16

5 8
7

3.0E+06

1.Alanine-2TMS
2. Valine-2TMS
3. Phosphate-3TMS
4. Leucine-2TMS
5. Isoleucine-2TMSSi

11. Threonine-3TMS
12. Malic acid-3TMS
13. Aspartic acid-3TMS
14. Methionine-2TMS
15. Proline-oxo-2TMS

Fig. 5. The SCAN total ion chromatogram of the mouse serum using SPE-GC-MS systemwith automated SPE-based derivatization method.

Si

Si

O

Si

6. Proline-2TMS
7. Glycine-3TMS
8. Succinic acid-2TMS
10. Serine-3TMS

CompoundPeak Area

Relative peak area

7

No.

Peak Area◆ Derivatization Reagent◆ Recovery

Peak Area◆ Derivatization Time

Evaluation with the mouse serum

Peak Area

Evaluation with the standard solution

0

5

10

15

20

Concentration (pmol/μL)

Fig. 4. Relations of concentration in vial andthe peak area.

25

Pre-treatment, dehydration and derivatization of the sample was done in 10 minutes. Applying thesystem to metabolite standard solutions and mouse serums resulted in good reproducibility androbustness. Thus, online SPE-GC-MS system with automated SPE-based derivatization method
enables the rapid and reliable analysis of metabolites.
Acknowledgments
We thank Takeshi Bamba, Toshiyuki Yamashita and Kuniyo Sugitate for technical advice
and discussions. This work was financially supported by subsidies for projects to promote
the enhancement of manufacturing technology for small and medium enterprises.

Metabolomics 2017

Development of online SPE-GC-MS system with
automated SPE-based derivatization method
for metabolome analysis.
Ryoichi SASANO1, Koji MACHITANI2, Shusuke OSAKI2, Masahiro FURUNO3, Eiichiro FUKUSAKI3
1; Aisti Science co., Ltd., 2; Industrial Technology Center of Wakayama prefecture,
3; Osaka University「メタボローム分析のための自動固相誘導体化法
を用いたオンラインSPE-GC-MSシステムの開発」

目的
従来のメタボローム分析は、採取した試料を溶媒抽出、遠心濃縮、凍結乾燥、誘導体化等の前処理工程が煩雑であるため、長時間を要するとともに熟練された技術が必須となる．
また、多検体の前処理をバッチ処理した場合、GC/MSでの測定はそれらの検体を順次測定していくため、それらの検体において誘導体化後から測定までの時間が異なってしまう。これらは分析バッチ間の系統誤差の要因となり、多サンプルのメタボローム解析を遂行する上で致命的な短所である．
そこで、本研究では、迅速な自動誘導体化とその誘導体化からGCMSへの直接試料導入を目指して、固相誘導体化法を用いたオンラインSPE-GC/MSシス
テムの開発を行ったので報告する。

固相誘導体化法

Step 1

Step 2

Step 3

Step 4

Step 5

試料負荷 & 保持

洗浄

脱水

誘導体化

溶出

試料

Water-ACN

ACN

Amino group-SO3⊝ ⊕H3N-R-SO3⊝ ⊕H3N-R

1 min-SO3⊝ ⊕H3N-R

2 min
MSTFA

ヘキサン

-SO3⊝ TMS-HN-R-SO3⊝

Carboxyl group-N⊕⊝OOC-R(CH3)3-N⊕(CH3)3⊝OOC-R

非イオン性化合物
アミノ酸と有機酸をイオン交換相互作用により固相に保持

水-ACNで固相に残存している非イオン性化合物を除去-N⊕ ⊝OOC-R(CH3)3-N⊕(CH3)3

TMS-OOC-R

H2O
ACNで固相に残存している水分を除去

誘導体化試薬を固相に含侵させて、目的成分を固相中で誘導体化-N⊕(CH3)3

R-NH-TMS
R-COO-TMS

自動固相誘導体化オンラインSPE-GC-MSシステム
自動前処理時間 （精製・脱水・誘導体化）: 10 min
誘導体化から注入までの時間が
常に一定

溶出・注
入

Flash-SPE
AOS

LVI-S250
Spiral Insert
GC-MS
コンディ
ショニング

試料負荷

洗浄・脱水

誘導体化

前処理フロー
Automation

試料： 50µL（50mg）
バイアル

添加 水 150µL

分取 : 40µL

添加 ACN 800µL
振とう （37℃, 30min）
遠心分離（14000rpm, 5min）
分取：上澄み液： 250µL
添加 水 250μL
バイアル

Flash-SPE AOS-3mg

Online SPE-GC-MS System
コンディショニング
水 100μL
アセトニトリル 100µL

洗浄 水-ACN（1/4）100µL
脱水 ACN 100µL
含侵 Methoxyamine-Pyridine-MSTFA 8µL
固相誘導体化反応 2min
溶出 アセトン-ヘキサン(1/4) 40µL

GC-MS： LVI-Split（1:50）

オンラインSPE-GC-MSシステム
前処理ユニット

SGI-M100

Sampling

送液ユニットOnline SPE-GC-MS System

Injection

Flash-SPE

SPE-GC-MS条件

大量注入-スプリット モード

1st Stage

2nd Stage

3rd Stage

注入

濃縮

スプリット導入

注入口温度を溶出溶媒の沸点より低く設定し、溶出液を注入する。

溶出溶媒を気化してさせながら排出し、目的成分を濃縮していく。

注入口温度を上げて、ス
プリットしながら目的成分をカラムへ導入する。

4th Stage

除去
高沸点の夾雑成分はスプリットで除去する。

スパイラルインサートの形状は固相カートリッジからの溶出液全量を一時的に受け止めることができる。

Relative peak area

固相誘導体化時間

Fig. 1. Relations of derivatization time in sorbent and the relative peak area.

Relative peak area

誘導体化試薬について

Fig. 2. Relations of the derivatization reagent and the relative peak area.

Methoxyamin/Pyridine-MSTFAの効果
O
O

Si
Si

Si

NH
O

NH

Si

O

Si

Si

N
O

O

Si

Si

N

N

Si

Si

Si

Glycine-3TMS

Ornithine-4TMS

Lysine-4TMS
Si

Si

O
N
Si

Si

O

Aminobutyric acid-3TMS

Si

N
N

Si

Si

Putrescine-4TMS

Methoxyamin/pyridineをMSTFAに混合することで誘導体化が促進された物質-CH2-NH2の基に対してしっかりと-CH2-N-2TMSに、グリシンの立体障害を受けない-C(-NH2)-についても-C(-N-2TMS)-になりました。

本法による標準溶液のSCANトータルイオンクロマトグラム[ｸﾛﾏﾄｸﾞﾗﾑ]

TIC:

5977585

-

0

24,25

5.0E+06
113,4
10

4.0E+06

15,16
12

5 8
7

3.0E+06

6

17

20

23
28

9

2

26
14

2.0E+06

29
27

1

13

1.0E+06

18
19 21

22

0.0E+00
R.T-->
07:00
08:00
09:00
10:00
11:00
12:00
13:00
14:00
15:00
1.Alanine-2TMS
2. Valine-2TMS
3. Phosphate-3TMS
4. Leucine-2TMS
5. Isoleucine-2TMS

6. Proline-2TMS
7. Glycine-3TMS
8. Succinic acid-2TMS
9. Fumaric acid-2TMS
10. Serine-3TMS

11. Threonine-3TMS
12. Malic acid-3TMS
13. Aspartic acid-3TMS
14. Methionine-2TMS
15. Proline-oxo-2TMS

16. Cytosine-2TMS
17. Aminobutyric acid-3TMS
18. Ketoglutaric acid-3TMS
19. Glutamic acid-3TMS
20. Phenylalanine-2TMS

21. Asparagine-3TMS
22. Putrescine-4TMS
23. Aconitic acid-3TMS
24. Citric acid-4TMS
25. Ornithine-4TMS

26. Adenine-2TMS
27. Lysine-4TMS
28. Tyrosine-3TMS
29. Guanine-3TMS

Fig. 3. The SCAN total ion chromatogram of standard solution using SPE-GCMS system with automated SPE-based derivatization method.
＊標準溶液バイアル中濃度：0.01nmol/μL本法による標準溶液の再現性
Table 1. Reproducibility of peak area with standard solution using SPE-GC-MS system.
N o. C om pound
1 A lanine-2TM S
2 V aline-2TM S
3 P hosphate (3:1)-3TM S
4 N orleucine-2TM S
5 Isoleucine-2TM S
6 P roline-2TM S
7 G lycine-3TM S
8 Succinic acid-2TM S
9 Fum aric acid-2TM S
10 Serine-3TM S
11 Threonine-3TM S
12 M alic acid-3TM S
13 A spartic acid-3TM S
14 M ethionine-2TM S
15 P roline-oxo-2TM S
16 C ytosine-2TM S
17 A m inobutyric acid-3TM S
18 K etoglutaric acid-3TM S
19 G lutam ic acid-3TM S
20 P henylalanine-2TM S
21 A sparagine-3TM S
22 P utrescine-4TM S
23 A conitic acid-3TM S
24 C itric acid-4TM S
25 O rnithine-4TM S
26 A denine-2TM S
27 Lysine-4TM S
28 Tyrosine-3TM S
29 G uanine-3TM S

12,780,202
3,231,804
1,980,261
3,860,754
3,166,213
3,326,569
2,170,649
3,020,526
1,768,634
2,012,774
1,040,407
485,209527,945
1,317,135
1,972,283
1,164,055
1,903,218
179,954
486,088
1,553,897
264,587
1,097,163
1,068,411
2,509,279
948,801
1,791,455
438,332
2,290,304
1,276,608

2
2,814,678
3,290,271
1,945,488
3,995,800
3,281,486
3,445,278
2,352,541
3,101,538
1,813,725
2,110,285
1,085,291
496,725
521,172
1,376,552
2,178,513
1,199,619
2,080,359
187,293482,193
1,642,952
293,568
1,143,662
1,095,208
2,585,329
1,074,181
1,859,990
487,673
2,415,910
1,325,553

32,805,838
3,270,689
1,842,146
3,961,145
3,263,756
3,452,215
2,219,378
3,047,906
1,790,816
2,078,505
1,075,400
505,695
605,941
1,320,877
2,188,232
1,211,399
1,832,270
167,292
528,880
1,616,874
269,785
1,022,892
1,085,018
2,551,370
928,962
1,930,628
411,966
2,339,399
1,372,469

42,570,446
2,966,049
1,762,489
3,572,341
2,942,720
3,055,493
2,118,024
2,891,328
1,626,601
1,857,176
963,085
464,603439,870
1,165,099
2,213,001
1,081,564
1,718,333
138,034
375,004
1,422,103
215,342
1,069,833
973,865
2,326,885
889,675
1,670,688
389,2632,070,195
1,201,496

5
2,663,543
3,107,653
1,746,163
3,750,253
3,112,611
3,264,503
2,229,077
2,874,284
1,697,384
1,969,420
1,019,290
471,484
548,152
1,233,662
2,171,464
1,154,429
1,911,954
169,160
494,429
1,528,499
260,091
1,140,680
1,026,875
2,437,909
963,445
1,831,552
423,849
2,220,032
1,273,918＊標準溶液バイアル中濃度：0.01nmol/μL

62,676,876
3,132,760
1,658,840
3,798,730
3,105,804
3,272,235
2,288,729
2,677,718
1,608,388
1,978,512
1,028,004
446,942
689,805
1,299,449
2,218,260
1,179,900
1,983,878
156,600
585,775
1,564,042
264,791
1,218,672
1,009,638
2,410,546
1,025,487
1,839,583
469,2632,300,204
1,292,009

7
2,632,581
3,054,963
1,585,771
3,712,869
3,057,182
3,230,054
2,328,562
2,840,826
1,635,905
1,918,379
988,509451,563
622,430
1,267,142
2,414,150
1,130,354
1,952,195
151,045
510,207
1,507,985
258,735
1,113,605
1,007,640
2,372,287
1,049,537
1,690,694
485,851
2,231,995
1,214,782

8
2,692,127
3,085,131
1,679,471
3,729,371
3,062,330
3,247,932
2,291,359
2,917,558
1,710,549
1,950,455
997,181459,884
590,812
1,274,606
2,348,929
1,140,346
1,884,295
147,256
483,426
1,508,373
249,746
1,075,243
1,025,144
2,395,838
1,005,050
1,722,793
449,843
2,184,577
1,217,067

A ve.
92,718,118 2,706,045
3,160,769 3,144,454
1,991,253 1,799,098
3,834,508 3,801,752
3,129,208 3,124,590
3,297,886 3,288,018
2,432,007 2,270,036
3,180,044 2,950,192
1,825,272 1,719,697
1,968,792 1,982,700
1,024,209 1,024,597
497,251475,484
358,848544,997
1,279,034 1,281,506
2,386,591 2,232,380
1,191,227 1,161,433
2,036,145 1,922,516
178,112
163,861
320,272
474,030
1,520,941 1,540,630
263,337
259,998
1,130,409 1,112,462
1,085,668 1,041,941
2,470,814 2,451,140
1,047,923
992,562
1,876,276 1,801,518
475,516
447,951
2,239,834 2,254,717
1,316,201 1,276,678

R S D ,%
3.0
3.4
8.3
3.4
3.4
3.6
4.3
5.2
4.9
3.9
3.8
4.5
18.3
4.6
6.1
3.5
5.6
10.1
16.8
4.2
7.9
5.0
4.1
3.5
6.3
5.0
7.8
4.4
4.5

濃度とピーク面積値の関係（直線性）
Alanine-2TMS

6.E+06

Methionine-2TMS

3.E+06

R² = 0.9985

R² = 0.997

0.E+00

1.E+06

0.E+00
0

5

10

15

20

25

0

Concentration (pmol/μL)

8.E+06

Peak Area

2.E+06

5

10

15

20

6.E+06

Citric acid-4TMS

2.E+06
0.E+002.E+0615

20

25

Concentration (pmol/μL)

10

15

20

25

Lysine-4TMS

8.E+050.E+00105Concentration (pmol/μL)

1.E+06

Peak Area

4.E+065

1.E+060

4.E+06

Peak Area

Peak Area

R² = 0.99720

2.E+06

0.E+0025

R² = 0.9956
6.E+06

3.E+06

Concentration (pmol/μL)

Succinic acid-2TMS

R² = 0.9952

4.E+06

2.E+06

Peak Area

Peak Area

4.E+06

Tyrosine-3TMS

5.E+06

R² = 0.9489

6.E+05

4.E+05
2.E+050.E+00

0

5

10

15

20

25

Concentration (pmol/μL)

0

5

10

15

20

25

Concentration (pmol/μL)

Fig. 4. Relations of concentration in vial and the peak area.

本法によるマウス血清のトータルイオンクロマトグラム[ｸﾛﾏﾄｸﾞﾗﾑ]

TIC
3,4

: 4331844-0

4.0E+06711
10

3.0E+0618
61

2.0E+06

2
19

20,21

23

5

24

1.0E+06

15
8

12

13

0.0E+00
R.T-->
07:00 08:00 09:00 10:00 11:00 12:00 13:00 14:00
1.Alanine-2TMS
2. Valine-2TMS
3. Phosphate-3TMS
4. Leucine-2TMS
5. Isoleucine-2TMS

6. Proline-2TMS
7. Glycine-3TMS
8. Succinic acid-2TMS
10. Serine-3TMS

11. Threonine-3TMS
12. Malic acid-3TMS
13. Aspartic acid-3TMS
14. Methionine-2TMS
15. Proline-oxo-2TMS

18. Glutamic acid-3TMS
19. Phenylalanine-2TMS
20. Citric acid-4TMS
21. Ornithine-4TMS

23. Lysine-4TMS
24. Tyrosine-3TMS

Fig. 5. The SCAN total ion chromatogram of the mouse serum using SPE-GC-MS systemwith automated SPE-based derivatization method.

本法によるマウス血清のピーク面積値の再現性
Table 2. Reproducibility of peak area with the mouse serum using SPE-GC-MS system.
N o. C om pound
1 A lanine-2TM S
2 V aline-2TM S
3 P hosphate (3:1)-3TM S
4 N orleucine-2TM S
5 Isoleucine-2TM S
6 P roline-2TM S
7 G lycine-3TM S
8 Succinic acid-2TM S
9 Fum aric acid-2TM S
10 Serine-3TM S
11 Threonine-3TM S
12 M alic acid-3TM S
13 A spartic acid-3TM S
14 M ethionine-2TM S
15 P roline-oxo-2TM S
16 C ytosine-2TM S
17 A m inobutyric acid-3TM S
18 G lutam ic acid-3TM S
19 P henylalanine-2TM S
20 C itric acid-4TM S
21 O rnithine-4TM S
22 A denine-2TM S
23 Lysine-4TM S
24 Tyrosine-3TM S
25 G uanine-3TM S

15,541,262
2,755,712
4,201,912
4,056,211
1,078,007
2,990,521
3,037,839
29,04014,4001,617,736
854,196
20,457
195,51293,568719,078
5,878
87,027
1,268,042
699,562
335,441
663,367
4,077
384,118
739,5513,773

25,554,574
2,771,037
4,356,822
4,080,919
1,080,660
3,000,081
2,895,879
29,234
13,605
1,601,793
855,270
20,641
183,707
94,791
784,164
5,728
83,832
1,206,401
714,084
333,534
673,4443,243
400,505
763,6703,789

35,750,943
2,893,545
4,434,444
4,276,463
1,131,523
3,191,334
3,107,951
29,61014,528
1,691,622
914,762
21,151
208,840
100,427
740,721
5,647
70,197
1,296,449
762,498
337,541
787,729
3,358
466,485
838,1103,207

45,697,229
2,911,101
4,412,647
4,306,038
1,141,625
3,154,192
3,217,887
29,11813,7571,701,792
923,062
21,808210,072
104,122869,323
5,592
83,924
1,342,892
773,628
334,091
789,718
3,410
466,911
848,3093,438

5
5,588,344
2,803,596
4,201,988
4,157,663
1,094,518
3,053,991
3,354,424
26,403
13,4691,664,881
882,377
19,071
204,684
101,698
610,496
5,629
75,857
1,281,052
724,391
312,166
771,4703,217
449,562
773,7372,485

65,721,789
2,871,371
4,259,611
4,270,224
1,124,087
3,162,046
3,293,368
27,322
13,211
1,718,251
911,832
20,283
225,449
103,386
641,127
5,516
76,002
1,393,273
758,118
321,946
808,655
2,885
483,398
850,3072,720

75,850,742
2,926,797
4,389,792
4,312,737
1,139,671
3,244,535
3,059,023
28,53012,847
1,759,025
928,177
20,751
240,856
105,816
674,841
4,974
85,355
1,513,486
786,916
322,681
771,975
3,216
450,203875,8072,968

8
5,681,853
2,853,359
4,266,977
4,223,433
1,121,838
3,101,102
3,070,554
28,003
12,142
1,685,417
920,805
19,941
233,191
97,718868,861
5,028
82,175
1,405,403
753,925
314,421652,659
2,978
364,748
790,0953,136

95,706,369
2,867,084
4,210,194
4,227,870
1,118,968
3,103,402
3,113,071
27,16911,996
1,696,241
914,839
19,094
238,539
99,320
839,473
5,413
87,546
1,516,263
773,827
317,960
737,745
3,005
433,653
852,336
2,457

A ve.
R S D ,%
5,677,012
1.8
2,850,400
2.1
4,303,821
2.2
4,212,395
2.2
1,114,544
2.2
3,111,245
2.8
3,127,777
4.5
28,2703.9
13,3286.7
1,681,862
2.9
900,591
3.2
20,355
4.4
215,650
9.3
100,094
4.2
749,787
13.0
5,489
5.6
81,3247.3
1,358,140
8.0
749,661
4.0
325,5313.0
739,640
8.2
3,265
10.8
433,287
9.5
814,658
5.9
3,108
16.1

本法によるマウス血清の添加回収試験
N o.

C om pound

StandardST

M auseM

M ause + ST
A

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29

A lanine-2TM S
V aline-2TM S
P hosphate (3:1)-3TM S
N orleucine-2TM S
Isoleucine-2TM S
P roline-2TM S
G lycine-3TM S
Succinic acid-2TM S
Fum aric acid-2TM S
Serine-3TM S
Threonine-3TM S
M alic acid-3TM S
A spartic acid-3TM S
M ethionine-2TM S
P roline-oxo-2TM S
C ytosine-2TM S
A m inobutyric acid-3TM S
K etoglutaric acid-3TM S
G lutam ic acid-3TM S
P henylalanine-2TM S
A sparagine-3TM S
P utrescine-4TM S
A conitic acid-3TM S
C itric acid-4TM S
O rnithine-4TM S
A denine-2TM S
Lysine-4TM S
Tyrosine-3TM S
G uanine-3TM S

2,395,769
2,857,898
1,691,961
3,402,608
2,801,029
3,037,141
1,800,878
2,958,640
1,676,379
1,769,642
900,834
475,088
440,006
1,186,002
1,684,508
1,069,383
1,425,778
162,941
381,767
1,361,973
212,637
928,721482,874
2,365,829
741,337
1,710,221
344,472
2,171,566
1,112,060

5,539,074
2,759,114
4,279,367
4,067,823
1,086,415
2,998,133
2,966,859
224,256
13,946
1,608,041
852,911
20,319190,845
93,758740,135
5,948
82,936

7,703,074
5,744,538
6,787,045
7,424,476
4,096,438
6,301,408
4,603,320
3,375,441
1,869,217
3,632,037
1,865,424
551,360
795,480
1,417,378
3,085,018
1,182,673
1,774,441
163,614
1,524,345
2,285,882
291,982
1,124,501
544,525
3,143,411
1,295,449
1,966,746
680,1113,535,152
1,479,513

1,237,713
705,325
35,714
3,083
334,488
668,406
3,660
389,645748,544
3,595

R E C ,%(A -M )/ST
90
104
148
99
107
109
91
107
111
114
112
112
137
112
139
110
119
100
75
116
137
117
112
119
85
115
84
128
133

Table 3. Recoveries of metabolites
added to mouse serum＊添加濃度：0.01nmol/μL（バイアル中換算）

サンプル抽出液の負荷量とピーク面積値
Alanine-2TMS

1.E+07

R² = 0.9985

Peak Area

6.E+06
4.E+06

R² = 0.996
2.E+06

2.E+05
2.E+05
1.E+050.E+000.E+000.E+00
0

20

40

60

80

Succinic acid-2TMS

6.E+050

Sample loading volume (μL)

20

40

60

Lysine-4TMS

1.E+06

R² = 0.9965

Peak Area

1.E+05

2.E+050.E+000.E+006080

9.E+05Citric acid-4TMS

8.E+05

R² = 0.9967

5.E+05

4.E+05

4.E+05

2.E+0540

6.E+05

6.E+05

3.E+0520

Sample loading volume (μL)

7.E+058.E+05

4.E+05080

Sample loading volume (μL)

1.E+06

R² = 0.9985

5.E+05

1.E+06
5.E+055.E+04

2.E+06

2.E+06

Peak Area

Peak Area

8.E+06

Tyrosine-3TMS

3.E+06

R² = 0.9944

3.E+05

1.E+07

Peak Area

Methionine-2TMS

3.E+05

Peak Area

1.E+07

3.E+05
2.E+05
1.E+05

0

20

40

60

80

Sample loading volume (μL)

0

20

40

60

80

Sample loading volume (μL)

0.E+00
0

20

40

60

80

Sample loading volume (μL)

Fig. 7. Relations of sample loading volume and the peak area.

本法によるマウス血清の定量イオンクロマトグラムの再現性（重ね描き, n=9）

Alanine-2TMS

Leucine-2TMS

Lysine-4TMS

Citric acid-4TMS

Fig. 6. Reproducibility of ion chromatogram of the mouse serum using
SPE-GC-MS system( n = 9 ).

まとめ
 抽出後の本システムによる誘導体化を含めた前処理時間は10分で、かつ、その前処理からGC-MSによる測定まで完全に自動で行うことができた。
 誘導体化してから注入するまでの時間が常に一定であるため、多検体においても安定した結果を得られると考えられる。
 本システムを用いて、スタンダードおよびマウス血清による再現性、直線性を評価したところ、共に良好な結果を得た。

 開発したオンライン自動固相誘導体化SPE-GC/MSシステムはメタボローム
分析に有効であることがわかった。