Expansion of Analyte Metabolites Using Online
SPE-GC/MS System with Solid-phase Derivatization〇Ryoichi Sasano1, Shoya Arakawa1, Tomonori Asai1, Chiaki Maruyama1, Tsuyoshi Masuda2, Syusuke Osaki2, Hitomi Yadori3,
Masahiro Furuno3, Yuki Miyoshi4, Masahide Hamaguchi4, Kozo Nishida5, Hiroshi Tsugawa5, Eiichiro Fukusaki3
1 AiSTI SCIENCE Co., Ltd., 2 Industrial Technology Center of Wakayama Prefecture, 3 Osaka University,
4 Kyoto Prefectural University of Medicine, 5 Tokyo University of Agriculture and Technology

Abstract
We have developed an online SPE-GC/MS system with solid-phase derivatization in which a solid-phase retaining metabolite is dehydrated with acetonitrile, impregnated thesolid-phase with a derivatization reagent (MSTFA or MTBSTFA) to derivatize the metabolites, and injected directly into GC while eluting the derivatized metabolites (TMS ortBDMS). The system has made it possible to shorten (15-25 minutes) and automate pre-processing, which used to be a lengthy (20 hours) and manual process. In addition,
the time between derivatization and injection is constant, enabling stable analysis even in multiple samples. This study aimed to expand the number of target metabolites andoptimize the solid-phase derivatization method by their types using the online SPE-GC/MS.

Automated SPE Device
SPL-M100(AiSTI SCIENCE)

Amino acids, amines, polyamines, nucleobases, nucleosides, catecholamines, polyvalent carboxylic acids, oxocarboxylic acids, hydroxycarboxylic acids, aromatic fatty acids,
short-chain fatty acids, long-chain fatty acids, polyunsaturated fatty acids, steroids, bile acids, isoflavonoids, sugars, sugar acids, sugar phosphates, etc., were selected basedon commonly reported metabolites and metabolic circuits. They were then measured with this system. The solid phases were ion-exchange resins for ionic compoundswith carboxyl and amino groups, HILIC for non-ionic hydrophilic compounds, and ODS for non-ionic hydrophobic compounds. As a result, derivatization of 250
metabolites was confirmed. Those metabolites were grouped by type, good reproducibility was obtained by optimizing each process such as solid phase type, amount ofderivatization reagent added, elution volume, and elution flow rate. Volatile components such as short-chain fatty acids were likely to be lost by freeze-drying in conventional
methods, however, in this method, they were held in the solid phase and dehydrated so that they could be analyzed without loss. In addition, for metabolites such as sterols
and isoflavonoids, high-sensitivity analysis was possible using the large-volume injection method.

Online Solid-Phase Derivatization

Conventional
Derivatization

Sample Load& Wash

Lyophilization,Derivatization

Sample solution■ Solid-Phase
Cartridge
Flash-SPE

Constant

Solidphase

Flash-22mm

Precision& AccuracySPE

De-proteinization

Sugars,Triglycerides

Matrices

Analytes（CH3）3

Non-ioniccompounds(Sugars, Glycerides)H2Oand organic acids
on solid-phase byion-exchangeinteraction

Automated pretreatment

1 min

Wash out remained Dehydrated insolid-phase by ACN
other compounds
by ACN-water

MSTFA

NH3-R

-SO3

OOC-R

-N

（CH3）3（CH3）3

Retain amino acids

NH3-R

-SO3

OOC-R

-N

Methoxyamine

Acetonitrile

NH3-R

-SO3

Organic acids
OOC-R

-N

Solid Phase・1.4 mm x 5 mm
・3 mg

GC/MS

Derivatives

Acetonitrle-Warer

Amino acids
NH3-R

-SO3

Reaction Time

20 hours

Online SPE-GC/MS system■ Solid-Phase Derivatization

Dehydration Derivatization Elute & Injection(1 min)MO (3～15 min)
TMS (1 min)Manual operation

GC/MS

TMS-NH-R-SO3

OOC-R-N

Hexane-SO3TMS-OOC-R-N（CH3）3（CH3）33 min

1 min

Infiltrate the reagent

Infiltrate MSTFA andand methoximize
analytes in solidphase

trimethylsilylate
analytes in solidphase-N（CH3）3

R-NH-TMS
R-OOC-TMS
Elute derivatizedcompounds fromsolid-phase byHexane

15～25 min
AMI-a

14
13

127

3 4 56289AMI-b15

1220

17310210AMI-c

18941113 1413 14641131978

117101915118

6167111291585162

1.
2.
3.
4.
5.

Alanine_2TMS
Valine_2TMS
Leucine_2TMS
Isoleucine_2TMS
Proline_2TMS

6. Glycine_3TMS
7. Serine_3TMS
8. Threonine_3TMS
9. Aspartic acid_3TMS
10. Methionine_2TMS

AMI-Ph

84

12
6

1.
2.
3.
4.
5.
6.
7.
8.

1.
2.
3.
4.
5.
6.
7.

11. Glutamic acid_3TMS
12. Phenylalanine_2TMS
13. Lysine_4TMS
14. Tyrosine_3TMS
15. Cystine_4TMS

5320

15

N-methylphenetyramine_2TMS
Nicotinamide
Phenethylamine_2TMS
N-methyltyramine_2TMS
Synephrine_3TMS
Histamine_3TMS
Tyramine_3TMS
3-Methoxytyramine_3TMS

2-Aminobutyric acid_2TMS
Norvaline_2TMS
Norleucine_2TMS
Homoserine_3TMS
Pyroglutamic acid_2TMS
Cysteine_3TMS
Ornithine_3TMS

PAM

8. Asparagine_4TMS
9. Asparagine_3TMS
10. 2-Aminoadipic acid_3TMS
11. 1-Methylhistidine_2TMS
12. Ornithine_4TMS
13. Hydroxylysine_4TMS
14. Histidine_3TMS

AMI-CT

13

1.
2.
3.
4.
5.

15. Kynurenine_3TMS
16. Tryptophan_2TMS
17. Tryptophan_3TMS
18. Monoiodotyrosine_3TMS
19. 5-Hydroxytryptophan_4TMS
20. Diiodotyrosine_3TMS

Putrescine_4TMS
Cadaverine_4TMS
Spermidine_5TMS
Spermine_5TMS
Spermine_6TMS

5

1.
2.
3.
4.

24

N-Methylalanine_2TMS
Urea_2TMS
Ethanolamine_3TMS
beta-Alanine_3TMS
Cysteamine_2TMS
Pyroglutamic acid_2TMS
Anthranilic acid_2TMS

8. Alanyl-glycine_2TMS
9. N-Acetylglutamine_3TMS
10. Glycylglycine_4TMS
11. Glycylglycine_3TMS
12. Allantoic acid_4TMS from Allantoin
13. Carbamoylaspartic acid_3TMS?
14. N2-acetylornithine_3TMS

CA-CT

4

23

1.
2.
3.
4.
5.
6.
7.

Adrenaline_4TMS
Dopamine_4TMS
Dopa_4TMS
Noradrenaline_5TMS

2SUP

2,3453

1. Dihydroxyphenylacetic acid_3TMS
2. Dihydroxymandelic acid_4TMS
3. Gallic acid_4TMS

1

1.
2.
3.
4.
5.

11

15. N2-acetylornithine_4TMS
16. Cysteinylglycine_3TMS from Glutathione
17. Tryptamine_2TMS
18. Tryptamine_3TMS
19. Serotonin_4TMS
20. Serotonin_3TMS

Ribose-5-phosphate_5TMS_MO
Fluctose-6-phosphate_6TMS_MO
Glucopyranose-1-phosphate_6TMS
Glucose-6-phosphate_6TMS_MO
6-Phosphogluconic acid_7TMS

4”

1”

7CA-aCA-Ph

1813924 56

331612106CA-AM

9476311711117951210810144171513115287281211”

Oxalic acid_2TMS
Malonic acid_2TMS
Methylmalonic acid_2TMS
Ethylmalonic acid_2TMS
Succinic acid_2TMS
Maleic acid_2TMS
Fumaric acid_2TMS

CAH

8. Glutaric acid_2TMS
15. 2-Oxoadipic acid_MO_2TMS_2
9. Malic acid_3TMS
16. cis-Aconitic acid_3TMS
10. Adipic acid_2TMS
17. Azelaic acid_2TMS
11. α-Ketoglutaric acid_MO_2TMS 18. Citric acid_4TMS
12. Pimelic acid_2TMS
19. Isocitric acid_4TMS
13. Tartaric acid_4TMS
14. 2-Oxoadipic acid_MO_2TMS_1
1. Lactic acid_2TMS
2. 2-Hydroxyisobutyric acid_2TMS
3. Glycolic acid_2TMS
4. 2-Hydroxybutyric acid_2TMS
5. 2-Hydroxy-2-methylbutyric acid_2TMS
6. Hydroxypropionic acid_2TMS9
7. 3-Hydroxybutyric acid_2TMS
8. 3-Hydroxyisovaleric acid_2TMS
9. Mevalonic acid_3TMS
10. Shikimic acid_4TMS43578216V-EK
1.
2.
3.
4.

1 2 334”

3”

1 2”

1Androsterone_TMS
Dehydroepiandrosterone_MS
Dihydrotestosterone_TMS
Estrone_TMS
Androstenedione

34 565863FA-L9356 7 8,910

6. Stigmasterol_TMS
7. Dihydrolanosterol_TMS
8. β-Sitosterol_TMS
9. Lanosterol_TMS
10. 7-Dehydrocholesterol_TMS

16110C21 24536127 9 1011814137”

1.
2.
3.
4.
5.

A

2

Propanoic acid(C3)_tBDMS
Isobutyric acid(C4)_tBDMS
Butyric acid(C4)_tBDMSIsovaleric acid(C5)_tBDMS
Valeric acid_tBDMS

6. Hexanoic acid(C6)_tBDMS
7. Heptanoic acid(C7)_tBDMS
8. Octanoic acid(C8)_tBDMS
9. Nonanoic acid(C9)_tBDMS
10. Decanoic acid(C10)_tBDMS

3SUG-2Cholecarciferol_TMS
Cholesterol_TMSBrassicasterol_TMS
Ergosterol_TMS
Campesterol_TMS

15

5

1.
2.
3.
4.
5.

1.
2.
3.
4.
5.
6.
7.
8.

Undecanoic acid(C11)_tBTMS
9. Oleic acid(C18:1, n-9)_tBTMS
Dodecanoic acid(C12)_tBTMS
10. alpha-Linolenic acid_tBDMS
Tridecanoic acid(C13)_tBTMS
11. Stearic acid(C18)_tBTMS
Myristic acid(C14)_tBTMS
12. Nonadecanoic acid(C19)_tBTMS
Pentadecanoic acid(C15)_tBTMS 13. Arachidonic acid_tBTMS
Palmitic acid(C16)_tBTMS
14. Eicosapentaeoic acid_tBDMS
Heptadecanoic acid(C17)_tBTMS 15. Arachidic acid(C20)_tBTMS
Linoleic acid(C18:2, n-6)_tBTMS 16. Heneicosanoic acid(C21)_tBTMS

Formononetin-TMS
Genistein-3TMS
Daidzein-2TMS
Glycitein-2TMS
Quercetin-5TMS

SUG-1

4

1.
2.
3.
4.
5.

42

9 10

6. Estradiol_2TMS
7. Testosterone_TMS
8. Pregnenolone_TMS
9. Progesterone
10. Estriol_3TMS

FLV

13. Carbamoylaspartic acid_3TMS
14. 3-Indoleacetic acid-2TMS
15. trans-Urocanic acid_2TMS
16. 5-Methoxyindoleacetic acid_2TMS
17. 5-Hydroxyinlodeacetic acid_3TMS
18. Biotin_3TMS

B711
1.
2.
3.
4.
5.

Glyoxylic acid_MO_TMS
Pyruvic acid _MO_TMS
2-Oxobutyric acid_MO_TMS
Ketoisoleucine_MO_TMS
2-Oxoisocaproic acid_MO_TMS
Phenylpyruvic acid_MO_TMS
Hydroxyphenylpyruvic
acid_MO_2TMS6”

7. 3-Ureidopropionic acid_3TMS
8. N-Acetylaspartic acid_2TMS
9. N-Acetylaspartic acid_3TMS
10. 5-Aminolevulinic acid_3TMS_2
11. Hippuric Acid_2TMS
12. Hippuric Acid_TMS

64A

752

1.
2.
3.
4.
5.
6.
7.

B

3210

Lithocholic acid_2TMS
Deoxycholic acid_3TMS
Cholic acid_4TMS
Chenodeoxycholic acid_3TMS
Hyodeoxycholic acid_3TMSUrsodeoxycholic acid_3TMS

8,B

A

5

Sarcosine_2TMS
3-Aminoisobutyric acid_2TMS
Pipecolic acid_2TMS
3-Aminoisobutyric acid_3TMS
Hydroxyproline_3TMS
γ-Aminoisobutyric acid_3TMS

FA-S A

74+5”

Sterols-2

64

CAMO

1.
2.
3.
4.
5.
6.

7. Homovanilic acid_2TMS
8. 4-Hydroxyphenylpyruvic acid_3TMS
9. Vanillylmandelic acid_3TMS
10. Hydroxyphenyllactic acid_3TMS
11. Kynurenic acid_2TMS
12. Xanthurenic acid_3TMS

2

1.
2.
3.
4.
5.
6.

41

Nicotinic acid_1TMS
Picolinic acid_1TMS
p-Hydroxyphenylactic acid_2TMS
Phthalic acid_2TMS
Quinolinic acid_2TMS
Orotic acid_3TMSBA

δ-Tocopherol-TMS
γ-Tocopherol-TMS
α-Tocopherol
Phylloquinone

Sterols-1

1.
2.
3.
4.
5.
6.

18

16

1114

1.
2.
3.
4.
5.
6.
7.

15

1.
2.
3.
4.
5.
6.

Sucrose_8TMS
Lactose_MO_8TMS
Maltose_MO_8TMS
Trehalose_8TMS
Melibiose_MO_8TMS
Galactinol_9TMS

12 13
1474

115 16
175

5632

1011
12,34

3”
2”

689185”

1.
2.
3.
4.
5.
6.

Xylose_MO_4MTS
Lyxose_MO_4TMS
Arabinose_MO_4TMS
Ribose_MO_4TMS
Xylitol_5TMS
Rhamnose_MO_4TMS

7. Ribitol_5TMS
8. Fucose_MO_4TMS
9. 2-Deoxyglucose_MO_4TMS
10. Fructose_MO_5TMS_1
11. Fructose_MO_5TMS_2
12. Mannose_MO_5TMS

13. Galactose_MO_5TMS
14. Glucose_MO_5TMS
15. Mannitol_6TMS
16. Glucitol_6TMS
17. Galactitol_6TMS
18. Glucopyranose_5TMS