Poster Number 1448

METABOLOMICS 2024(6.16-20, OSAKA, JAPAN)

Examination of extraction method for metabolites appropriate to solid-phase derivatization
by online SPE-GC system〇Tomonori Asai1, Shoya Arakawa1, Chiaki Maruyama1, Ryoichi Sasano1, 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 Department of Biotechnology, Graduate School ofEngineering, Osaka University, 4 Department of Endocrinology and Metabolism, Graduate School of Medical Science, Kyoto Prefectural Universityof Medicine, 5 Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology※ E-mail: asai-to@aisti.co.jp【Introduction】
Overview of Solid Phase Derivatization (SPD) method by Online SPE-GC system
SPD pretreatment process

Online SPE-GC system

Dehydration is achieved by passing acetonitrile ornitrogen gas through the solid phase while themetabolite is retained on the solid phase, andderivatization can then be quickly performed byreacting with a reagent on the solid phase after
dehydration.

Automatic pretreatment time （Purification, Dewater, Derivatization）: 15 min

Conditioning

Sampleloading

WashingSPDElution and Injection

Automated SPE Device
SPL-M100
Large Volume Injector
LVI-S250

After reacting, the target compounds retained on
the solid phase with a derivatization reagent, they
can be eluted with a solvent and directly injected
into a GC.

Flash-SPE

GC/MS

GC/MS

Online SPE-GC equipped with automatic solid phase extraction device and large volume
injection port device

Purpose of this study
Examination of extraction methods suitable for hydrophilic metabolites (such as amino acids) and fat-soluble metabolites(such as steroids) for analysis using the SPD method.

The process from solid phase extraction to GC/MS measurement can be fully automated.

【Material and Method】
Sample: Chicken Liver
Water

Water and Acetone Standard (Recovery Test)

Collect two portions of supernatant[Online SPE-GC]
Analyzing the SPD M01
method[Preparing for hydrophilic metabolites]
900 µL of dilution solvent*1

100 µL of supernatant

pH adjustment to 8-9Freeze crushed sample
1g

Added water sameamount of the sampleand mixing

Transferred 100 µL ofthe sample to a ballmill tube, then added
100 µL of water and800 µL of acetone

Ball-mill crushing(4000 rpm, 90 sec)e.g.)：0.2 mol/L NaOH
20 µL

Centrifuge(15,700 g, 5min)[Preparing for fat-soluble metabolites]
960 µL of dilution solvent*1 or *2
40 µL of supernatant

Transferred the supernatant
to another tube, then added
200 µL of water and 800 µLof acetone to the residue,
and re-crushed it

SPD method M01 (for hydrophilic metabolites)

Flash-SPE ACXs

SPD method M06 (for fat-soluble metabolites)

Conditioning
・Acetonitrile/Water=1/1 240 µL
Flash-SPE C18Loading [Retained], Extract 50µL
Washing, Acetonitrile/Water=1/1 240µL
Dewater, Acetonitrile 400µL

Washing, Water 50µL
Drying, N2 purge 2 min

Infiltration, 0.5% Methoxyamine-Pyridine 4 µL
SPD reaction Methoximation, 3 min

Infiltration, MSTFA/Hexane=1/1 8 µL
SPD reaction Trimethylsilylation, 1min sec

Drying, N2 purge 15 sec

Elution, Hexane 35 µL

Infiltration, MSTFA/Hexane=1/1 7 µL
SPD reaction Trimethylsilylation , 60 sec

GC/MS

Elution, Hexane 25 µL
GC/MS

GC/MS method M06

GC/MS method M01

DB-5MS [30 m x 0.25 mm (0.25 µm)]
100°C(2 min)–10°C/min–320°C(2 min)Split (1:50)
220°C(0.5 min)–70°C/min–290°C(26min)
1 mL/min (He)
Scan (m/z 70–470)

Column: DB-5MS [30 m x 0.25 mm (0.25 µm)]
Oven Temp.: 170°C(4 min)–10°C/min–320°C(6 min)Inje. Mode: Solvent vent (150 mL/min(0.3 min)–splitless–50mL/min(4 min)Inje. Temp.: 80°C(0.4 min)–120°C/min–300°C(20min)
Flow rate: 1 mL/min (He)
MS: Scan (m/z 70–700)【Results】
M01 SPD methoda)*1 Dilution solvent: Acetonitrile/Water=4/1(v/v)*2 Dilution solvent: Acetonitrile/Water=1/4(v/v)

Recovery results of representative compounds

M01 SPD method

M06 SPD method

Compounds

Category

LogPow

Recovery%

Norleucine_2TMS

Amino acid-1.53

Adipic acid_2TMS

Organig acid0.08

Loading [Retained], Extract 100µL

Drying, N2 purge 15 sec

Column:Oven Temp.:
Inje. Mode:
Inje. Temp.:
Flow rate:
MS:

Conditioning
・Hexane 100 µL
・Acetone 230 µL
・Water 220 µL of Water

Table 1

M06 SPD methodc)

Analyzing the SPD M06
methodRecovery%Compounds

Category

LogPow

124

Daidzein_2TMSFlavonoid

2.63

*2

105

118

Estradiol_2TMS

Steroid

4.01

*2120

Chenodeoxycholic acid_3TMS

Bile acid

4.15

*294

Stigmasterol_1TMSSteroid

6.95

*199

Phylloquinone

Vitamin (fat-soluble)

8.48

*185

Dilution solvent

The recovery rates of representative compounds in the extraction procedure using chicken liver samples areindicated. The hydrophilic components were measured using the M01 SPD method, while the fat-solublecomponents were measured using the M06 SPD method. Good recovery results were obtained for compoundswith a wide range of polarities.
Table 2Reproducibility results of compounds detected in chicken liver

M01 SPD method

M06 SPD method

No.

Compounds

RSD% (n=5)

1Alanin_2TMS

6.9

22

Aspartic acid_3TMS

23-Hydroxybutyric acid_2TMS

8.1

23

3

2-Aminobutyric acid_2TMS

6.1

4

beta-Alanine_2TMS

5

No. Compounds

RSD% (n=5)

Dilution solvent

RSD% (n=5)

No.

Compounds

6.9

1

alpha-Tocopherol_1TMS

*1

12.0

Pyroglutamic acid_2TMS

12.1

2

Cholesterol_1TMS

*1

9.7

24

Threonic acid_4TMS

11.3

3

Campesterol_1TMS

*1

3.6

11.2

25

Glutamic acid_3TMS

12.5

4

beta-Sitosterol_1TMS

*1

6.726

Phenylalanine_2TMS

5.0

12.5

Valine_2TMS

6Ethanolamine_3TMS

5.327

Tartaric acid_4TMS

8.47

Phosphoric acid_3TMS

6.528

Myristic acid(C14)_1TMS

3.1

8

Leucine_2TMS

6.629

Hydroxyphenyllactic acid_3TMS

5.99

Isoleucine_2TMS

7.2

30

Tyrosine_3TMS

10.7

10

Maleic acid_2TMS

6.931

Glucuronic acid_5TMS_1

9.4

11

Proline_2TMS

7.632

Galacturonic acid_1MO_5TMS

13.2

12

Glycine_3TMS

8.033

Gluconic acid_6TMS

11.013

Succinic acid_2TMS

12.734

Palmitic acid(C16)_1TMS

2.414

Glyceric acid_3TMS10.835Heptadecanoic acid(C17)_1TMS

4.615

Uracil_2TMS

4.536

gamma-Linolenic acid(C18:3, n-6)_1TMS

6.416

Fumaric acid_2TMS

5.737

Linoleic acid(C18:2, n-6)_1TMS

4.9

17

Serine_3TMS

7.438

Oleic acid(C18:1, n-9)_1TMS

1.518

Pipecolic acid_2TMS

4.439

Stearic acid(C18)_1TMS

4.519

Threonine_3TMS

7.3

40

Arachidonic acid(C20:4, n-6)_1TMS

6.2

20

beta-Alanine_3TMS

8.3

41

Eicosapentaenoic acid(C20:5, n-3)_1TMS

6.521

Malic acid_3TMS

6.242Arachidic acid(C20)_1TMS

8.7

The reproducibility of extracting and measuring compounds detected in chicken liver was indicated. TheM01 method provided stable and reproducible results for 42 compounds, including amino acids, organicacids, and fatty acids. The M06 method provided good reproducibility for four compounds, including
steroids and fat-soluble vitamins.

【Discussion】b)d)

Under the extraction conditions examined this study, it was possible to detect compounds with a wide range ofpolarities, but the number of fat-soluble compounds detected was limited. This is because the amount ofcholesterol present in chicken liver is high, making it difficult to detect compounds with low concentrations.
Therefore, it was considered necessary to examine conditions that would allow for a good balance between
measuring compounds with high and low amounts.

【Conclusion】
Fig. 1

Correlation between sample loading volume and peak area

Figure 1 shows the correlation between sample load and peak area value for representative compounds. a) andb) show data measured by the M01 SPD method, while c) and d) show data measured by the M06 SPD method.
Each SPD method also demonstrated a good correlation between the loading amount and area value forcompounds with varying abundances. Subsequent tests were performed at loading amounts indicated by red
arrows that did not exceed the retention capacity of the solid phase.

The extraction conditions examined in this study were adaptable to the SPD method, enabling highthroughput analysis from extraction to GC/MS measurement.

【Acknowledgment】
Part of this research was conducted with the support of the “Go-Tech program”.