Development of in-SPE derivatization method for GC-MS metabolome analysis
Ryoichi SASANO1, Yoko KOJIMA1, Shusuke OSAKI2, Yoshie TANAKA2, Akihiro MATSUMOTO2, Toshiyuki YAMASHITA3, Takeshi BAMBA3, Eiichiro FUKUSAKI3
1;AiSTI SCIECE co., Ltd., 2;Industrial Technology Center of Wakayama prefecture, 3;Osaka University【Introduction】
Due to complex and long-time sample preparations, including solvent extraction, centrifugal
concentration, freeze-drying and derivatization, technical skills have been required in aconventional metabolome analysis for GC-MS. It causes discrepancies in the acquired datafrom multiple experiments, especially between in analysis batches. Furthermore, it causes a
critical problem when the statistical analysis is done because a lot of samples should be
handled in the metabolome analysis. The purpose of this study was to develop a rapid androbust method of sample preparation for metabolomics using solid phase extraction (SPE).

【Methods】
Extracted samples with acetonitrile were loaded on an ion-exchange cartridge SPE. Ion-exchange
SPEs were used depending on what kind of chemical functional group the compounds have. The
target compounds were retained in the SPE, then the cartridge was washed by acetonitrile fordehydration. Derivatization was done by methoxyamine/pridine and MSTFA which were directly
added sequentially on the SPE. After derivatization, the object substances were eluted byacetone/n-hexane. The 10μL of derivatized compounds were injected in the GC-MS with a Large
Volume Injection system equipped with a spiral shaped liner.

The derivatization processing 8〜19 Hour

The derivatization processing

Extract fractionation： 400µL

AX-SPE derivatization method
for Organic acid

6 min

The derivatization processing

6 min

AX-SPE derivatization method
for saccharides
The derivatization processing

CX-SPE derivatization method

Amino acid
R- C – COO⊖–

CX-SPE derivatization method
for Amino acid

The conventional derivatization method【Preliminary Results】
A cation-exchange column, CXi, was used for amino acids which have cationic amino group, and an anion-exchange column, AXi, wasapplied for the compounds which have carbonyl and hydroxyl group, such as organic acids and sugars. Washing with acetonitrile after
loading the target compounds enabled not only to eliminate other interferences but also to dehydrate at the same time. It took several
hours to dry the samples in the conventional method, while dehydration was done in a few minutes using SPE. In this method, target
metabolites which were retained in the SPEs by ion-exchange interaction were methoxyaminated by methoxyamine hydrochlolide inpyridine followed by trimethlysilylation effectively. Derivatized metabolites were changed into less-polar compounds and easily eluted
using organic solvent, acetone/n-hexane. The total preparation time from sample loading on the column to derivatization was within 10
minutes, and good reproducibility was obtained.

⊕NH
3

MSTFA

10 minR- C – COO⊖

R- C – COO⊖

Extract fractionation： 20µL

－SO3⊖

–

Extract fractionation： 50µL–

Extract fractionation： 50µL

Vacuum concentration（ 1～2 Hour）－SO3⊖ ⊕NH3

－SO3⊖ ⊕NH3

－SO3⊖
R- C – COO－TMS

Freeze-drying（5～16Hour）

Hybrid-SPE CXi3-2mg ⊖Added 2%Methoxyamin/pyridine 100uL

Hybrid-SPE AXi3-2mg ⊕Wash with ACN 100µL（Dehydration）

Incubation（30℃, 90min）：Derivatization
Added MSTFA 50uL

Centrifugal separation（16000 rpm, 4℃, 3min）
Vial

Wash with ACN 100µL（Dehydration）
Added MSTFA 20µL in SPE
Derivatization in SPE

Elute Hexane 100µL

Elute Hexane 100µL

Added Hexane 400µL

Added Hexane 400µL

Organic acid

Added 20%Methoxyamin/pyridine 5uL in SPE
Derivatization in SPE 3 min

Elute Hexane 100µL

GC/MS：Injection 10µL (LVI)

GC/MS：Injection 10µL (LVI)

Added Hexane 400µL⊖

AX-SPE derivatization method for saccharides

Examination of the optimum when a sample is loaded

Examination of the optimum when a sample is loaded

Examination of the optimum when a sample is loaded

100

100

100

100

40
20

2.5%

5%

10%
20%
Water concentration

Ala-2TMS
Pro-2TMS
Glt-3TMS

Gly-2TMS
Ser-3TMS
Phe-2TMS50%

Val-2TMS
Thr-3TMS
Lys-3TMS75%

Leu-2TMS
Asp-3TMS
Tyr-3TMS

1%

9

7 8
3
1

45

Pyruvate-2TMS
Fumaric-2TMS

10%

50%

75%

Oxalic-2TMS
Malic-3TMS

Maleic-2TMS
Tartaric-4TMS

40
20

6

11

－Ｎ⊕
(CH3)3
TMS－OOC－Ｒ

Carbonyl and hydroxyl
group are derivatized andis detached from SPE

80
60
40

Xylose-4TMS
Fructose1-5TMS
Glucose-5TMS

2%
5%
10%
Water concentration
Arabinose-4TMS
Rhamnose-4TMS
Fructose2-5TMS
Mannose-5TMS
Sucrose-8TMS
Lactose-8TMS

20%

50%

2%

5%

10%

20%

Methoxyamin concentration in pyridine

Fucose-4TMS
Galactose-5TMS
Maltose-8TMS

Fig. 3-1 Relations of water conc. in solvent and the relative peak area, whensaccharides in solvent are loaded through the AX-SPE and are adsorbed by the SPE.

Xylose-4TMS
Fructose1-5TMS
Glucose-5TMS

Arabinose-4TMS
Fructose2-5TMS
Sucrose-8TMS

Rhamnose-4TMS
Mannose-5TMS
Lactose-8TMS

Fucose-4TMS
Galactose-5TMS
Maltose-8TMS

Fig. 3-4 Relations of Methoxyamin conc. in pyridine and the relative
peak area.

10

8

Table 4: GC/MS condition

7
5

4

2

⊖

0
1%

3
13

－Ｎ⊕ OOC－Ｒ(CH3)320

90%

Succinic-2TMS
Citric-4TMS

7

2

6
1

2 34

8
9

11 12

5

1

Fig. 2-2 The GC/MS-SCAN total ion chromatogram of organic acids with inSPE derivatization method

Table 1 : A coefficient of correlation and RSD
No. Amino acidR2

2.E+06

2.E+06

1.E+06

5.E+050.E+00
0.0

0.5

1.0

1.5

2.0

Quantity of amino acid (nmol)
Val-2TMS

Ile-2TMS

Tyr-3TMS

Fig. 1-3 Relations of quantity of Amino acids
and the relative peak area with in-SPE
derivatization method.

Table 2: A coefficient of correlation and RSD

1.E+07RSD

No.

n=6, %

1

Ala-2TMS

0.9996

7.4

2

Gly-2TMS

0.995813.1

3

Val-2TMS

0.9993

7.5

4

Leu-2TMS

0.9992

6.7

5

Ile-2TMS

0.9995

6.2

6

Pro-2TMS

0.9982

12.5

7

Ser-3TMS

0.99885.5

8Thr-3TMS

0.99904.7

9

Asp-3TMS

0.99894.9

10 Met-2TMS

0.9991

7.5

11

Glt-3TMS

0.9911

8.8

12 Phe-2TMS

0.9996

5.6

13

Lys-3TMS

0.9927

32.2

14

Tyr-3TMS

0.9977

4.8

15

Cys-4TMS

0.995029.2

8.E+06

Peak area

3.E+06

Fig. 3-2 The GC/MS-SCAN total ion chromatogram of saccharides with in-SPE
derivatization method

6.E+06

4.E+06

2.E+06

0.E+00
0.0

0.5

1.0

1.5

2.0

Organic acid

R2

Table 3: A coefficient of correlation and RSD

RSD

3.E+06n=6, %

1 Pyruvate-2TMS

0.999815.1

2 Oxalic-2TMS

0.9946

6.2

3 Maleic-2TMS

0.9991

1.8

4 Succinic-2TMS

0.958822.0

5 Fumaric-2TMS

0.9991

2.2

6 Malic-3TMS

0.99972.1

7 Tartaric-4TMS

0.99981.9

8 Citric-4TMS

0.99982.5

RSD;Reproducibility parameters for the peak areawith in-SPE derivatization method

Peak area

Fig. 1-2 The GC/MS-SCAN total ion chromatogram of amino acids with in-SPE
derivatization method

Peak area

5%

Fig. 2-1 Relations of water conc. in solvent and the relative peak area, when organic
acids in solvent are loaded through the AX-SPE and adsorbed by the SPE.

12

10

6

2%

15

14

60

0.5%

Water concentrationIle-2TMS
Met-2TMS
Cys-4TMS

Fig. 1-1 Relations of water conc. in solvent and the relative peak area, when amino acids
in solvent are loaded through the CX-SPE and adsorbed by the SPE.

80

0

0

97.5%

Relative peak area

120

Rerative peak area

120

Rerative peak area

120

60

⊖

Examination of the optimum for methoxym derivatization

120

80

－Ｎ⊕ OOC－Ｒ(CH3)3interaction or polar interaction.

AX-SPE derivatization method for Organic acid

0

MSTFA

Methoxym
Carbonyl and hydroxyl group are（Saccharides
included）
adsorbed in SPE by ion exchange

CX-SPE derivatization method for Amino acid20

Methoxyamin－Ｎ⊕ OOC－Ｒ(CH3)3－Ｎ⊕(CH3)3

GC/MS：Injection 10µL (LVI)

Rerative peak Area

－OH

－COO⊖

Vial

GC/MS：Injection 1µL（Split 25:1)

40

AX-SPE derivatization method

Saccarides

Added MSTFA 25µL in SPE
Derivatization in SPE

Vial

60

Amino group is derivatized
and is detached from SPE

Wash with ACN 100µL（Dehydration）

Vial

80

TMS－NHAn amino group is adsorbed in
SPE by ion exchange interaction

Hybrid-SPE AXi3-2mg ⊕Added MSTFA 20µL in SPE
Derivatization in SPE

Incubation（37℃, 30min）：Derivatization–

Added ACN 100µL

2.E+06

1.E+06

0.E+00
0.0

Tartaric-4TMS

0.4

Fructose1-5TMS

Citric-4TMS

Fig. 2-3 Relations of quantity of Organic acids
and the relative peak area with in-SPE
derivatization method.

0.2

0.6

0.8

1.0

Quantity of saccharides (nmol)

Quantity of orano acid (nmol)
Fumaric-2TMS

No.

Mannose-5TMS

Fig. 3-3 Relations of quantity of Saccharides
and the relative peak area with in-SPE
derivatization method.

AiSTI SCIENCE

Saccharides

R2

RSD
n=6, %

1 Xylose-4TMS

0.9998

5.4

2 Arabinose-4TMS

0.9996

7.5

3 Rhamnose-4TMS

0.997410.8

4 Fucose-4TMS

0.99889.8

5 Fructose1-5TMS

1.0000

3.3

6 Fructose2-5TMS

0.99983.2

7 Mannose-5TMS

0.99822.5

8 Galactose-5TMS

0.9995

1.8

9 Glucose-5TMS

0.9778

2.3

10 Sucrose-8TMS

0.9993

2.9

11 Lactose-8TMS

0.99977.2

12 Maltose-8TMS

0.9999

6.5

RSD;Reproducibility parameters for the peak areawith in-SPE derivatization method【Conclusion】
By means of development of in-SPE derivarization method,
we can achieve rapid and robust method of sample preparation
for GC/MS metabolome analysis.
By using this solid phase derivatization ,1. With holding targeted compounds into solid phase and washingwith ACN, we could achieve dehydration process become quite easyand quickly.
2. As Methoxyamine and MSTFA directly added into the strong
ionic exchange solid phase which hold the targeted compounds, thederivatization reaction was proceeding promptly on the solid phase.
3. The operating time of dehydration and derivatization was greatly
reduced from 19 hours (conventional method ) to 10 minutes .

GC/MS対象メタボローム分析のための固相誘導体化法の開発
2015年7月1日
株式会社アイスティサイエンス
佐々野僚一

従来の誘導体化前処理法
誘導体化前処理時間
減圧濃縮遠心分離（1600rcf, 4℃, 3min）

凍結：液体窒素
凍結乾燥（一晩：16時間）
誘導体化試薬添加
メトキシアミン/ピリジン溶液 20mg/mL 100uL
インキュベーション（30℃, 90min）：誘導体化反応
誘導体化試薬添加：MSTFA 50uL
インキュベーション（37℃, 30min）：誘導体化反応

遠心分離（16000 rcf, 4℃, 3min）
分取：上澄み 100µL

→

バイアル瓶

測定：GC/MS：注入1µL（スプリット 25:1)

9〜19時間CX固相誘導体化前処理法（アミノ酸）
誘導体化前処理時間

6分

抽出液分取 50µL
試料（抽出液）負荷時の水-アセトニトリルの比率を検討
Hybrid-SPE CXi3-2mg

コンディショニング
水 100µL
ACN 100µL

洗浄：ACN 100µL（脱水効果）
誘導体化試薬を含浸 MSTFA 20µL
固相誘導体化反応
溶出：ヘキサン 100µL
添加：ヘキサン 400µL
検液

GC/MS：大量注入10µL

AX固相誘導体化前処理法（有機酸）
誘導体化前処理時間

6分

抽出液分取 50µL
試料（抽出液）負荷時の水-アセトニトリルの比率を検討
Hybrid-SPE AXi3-2mg

コンディショニング
水 100µL
ACN 100µL

洗浄：ACN 100µL（脱水効果）
誘導体化試薬を含浸 MSTFA 20µL
固相誘導体化反応
溶出：ヘキサン 100µL
添加：ヘキサン 400µL
検液

GC/MS：大量注入10µL

AX固相誘導体化前処理法（糖類）10分

誘導体化前処理時間
抽出液分取 20µL
試料（抽出液）負荷時の水-アセトニトリルの比率を検討

添加 ACN 100µL
Hybrid-SPE AXi3-2mg

コンディショニング
水 100µL
ACN 100µL

洗浄：ACN 100µL （脱水効果）
誘導体化試薬を含浸
20%メトキシアミン溶液5µL

メトキシム化時のメトキシアミン濃度の検討

固相上誘導体化反応：3min
誘導体化試薬を添加 MSTFA 25µL
固相誘導体化反応
溶出：ヘキサン 100µL
添加：ヘキサン 400µL
検液

GC/MS：大量注入10µL

試料負荷時の水濃度とアミノ酸のCX固相への保持について

Rerative peak Area

120
100
80
60
40
20

0
2.5%

5%

10%

20%

50%

75%

97.5%

水濃度
Ala-2TMS

Gly-2TMS

Val-2TMS

Leu-2TMS

Ile-2TMS

Pro-2TMS

Ser-3TMS

Asp-3TMS

Met-2TMS

Glt-3TMS

Phe-2TMS

Lys-3TMS

Tyr-3TMS

Cys-4TMS

Thr-3TMS

試料負荷時の水濃度と相対ピーク面積値の関係
アミノ酸とCX固相において、試料負荷時は水濃度が75%以下であれば保持できることがわかった。イオン交換相互作用により保持されていると考えられる。

試料負荷時の水濃度と有機酸のAX固相への保持について
120

Rerative peak area

100

80
60
40
20
0
1%

2%

5%

10%

50%

75%

90%

水濃度
Pyruvate-2TMS
Fumaric-2TMS

Oxalic-2TMS
Malic-3TMS

Maleic-2TMS
Tartaric-4TMS

Succinic-2TMS
Citric-4TMS

試料負荷時の水濃度と相対ピーク面積値の関係
有機酸とAX固相において、試料負荷時は水濃度に依存することなく保持できることがわかった。イオン交換相互作用により保持されていると考えられる。

試料負荷時の水濃度と糖類のAX固相への保持について
120

Rerative peak area

100
80
60
40
20
0
0.5%

1%

2%

5%

10%

20%

50%

Water concentration
Xylose-4TMS
Fructose1-5TMS
Glucose-5TMS

Arabinose-4TMS
Fructose2-5TMS
Sucrose-8TMS

Rhamnose-4TMS
Mannose-5TMS
Lactose-8TMS

Fucose-4TMS
Galactose-5TMS
Maltose-8TMS

試料負荷時の水濃度と相対ピーク面積値の関係
糖類とAX固相において、試料負荷時は水濃度が1%が最適な保持条件であることがわかった。極性相互作用により保持されていると考えられる。
また、0.5%以下になるとSucrose,Lactose,Maltoseは溶液に溶けきれなくなり、
カートリッジ壁面等に析出していることが考えられる。

メトキシアミン濃度と固相メトキシム化について
120

Relative peak area

100
80
60
40
20
0
2%

5%

10%

20%

Methoxyamin concentration in pyridine
Xylose-4TMS
Fructose1-5TMS
Glucose-5TMS

Arabinose-4TMS
Fructose2-5TMS
Sucrose-8TMS

Rhamnose-4TMS
Mannose-5TMS
Lactose-8TMS

Fucose-4TMS
Galactose-5TMS
Maltose-8TMS

固相誘導体化時のメトキシアミン濃度と相対ピーク面積値の関係

CX固相誘導体化前処理法（アミノ酸）
14

7

3

45

1

8

9
12

13

10
6

15

11

2

1. Alaninse-2TMS
5. Isoleucine-2TMS
9. Aspartic acid-3TMS
12. Phenylalanine-2TMS

2. Gliycine-2TMS
6. Proline-2TMS
10. Methionine-2TMS
13. Lysine-3TMS

3. Valine-2TMS
4. Leucine-2TMS
7. Serine-3TMS
8. Threonine-3TMS
11. Glutamic acid-3TMS
14. Tyrosine-3TMS 15. Cystine-4TMS

SCANトータルイオンクロマトグラム

AX固相誘導体化前処理法（有機酸）

6

7

8

3
5
2

4

1

1. Pyruvate acid-2TMS
5. Fumaric acid-2TMS

2. Oxalic acid-2TMS
6. Malic acid-3TMS

3. Maleic acid-2TMS
7. Tartaric acid-4TMS

SCANトータルイオンクロマトグラム

4. Succinic acid-2TMS
8. Citric acid-4TMSAX固相誘導体化前処理法（糖類）
10

7
6
1

2 34

1. Xylose-4TMS
5. Fructose1-5TMS
9. Glucose-5TMS

8
9

11

5

2. Arabinose-4TMS
6. Fructose2-5TMS
10. Sucrose-8TMS

3. Rhamnose-4TMS
7. Mannose-5TMS
11. Lactose-8TMS

SCANトータルイオンクロマトグラム

12

4. Fucose-4TMS
8. Galactose-5TMS
12. Maltose-8TMSCX固相誘導体化前処理法（アミノ酸）の評価
直線性（R2）と再現性（RSD, n=6）
3.E+06

No. Amino acidPeak area

2.E+06

2.E+06

1.E+06

5.E+050.E+00
0.0

0.5

1.0

1.5

2.0

Quantity of amino acid (nmol)
Val-2TMS

Ile-2TMS

Tyr-3TMS

アミノ酸量とピーク面積値の関係

R2RSD
n=6, %

1

Ala-2TMS

0.9996

7.4

2

Gly-2TMS

0.995813.1

3

Val-2TMS

0.9993

7.5

4

Leu-2TMS

0.9992

6.7

5

Ile-2TMS

0.9995

6.2

6

Pro-2TMS

0.9982

12.5

7

Ser-3TMS

0.99885.5

8Thr-3TMS

0.99904.7

9

Asp-3TMS

0.99894.9

10 Met-2TMS

0.9991

7.5

11

Glt-3TMS

0.9911

8.8

12 Phe-2TMS

0.9996

5.6

13

Lys-3TMS

0.9927

32.2

14

Tyr-3TMS

0.9977

4.8

15

Cys-4TMS

0.995029.2

AX固相誘導体化前処理法（有機酸）の評価
直線性（R2）と再現性（RSD, n=6）
1.E+07

No.

Peak area

8.E+06

6.E+06

4.E+06

2.E+06

0.E+00
0.0

0.5

1.0

1.5

2.0

Quantity of orano acid (nmol)
Fumaric-2TMS

Tartaric-4TMS

Citric-4TMS

有機酸量とピーク面積値の関係

Organic acidR2

RSD
n=6, %

1 Pyruvate-2TMS

0.999815.1

2 Oxalic-2TMS

0.9946

6.2

3 Maleic-2TMS

0.9991

1.8

4 Succinic-2TMS

0.958822.0

5 Fumaric-2TMS

0.9991

2.2

6 Malic-3TMS

0.99972.1

7 Tartaric-4TMS

0.99981.9

8 Citric-4TMS

0.99982.5

AX固相誘導体化前処理法（糖類）の評価
直線性（R2）と再現性（RSD, n=6）No.

Peak area

3.E+06

2.E+06

1.E+06

0.E+00
0.0

0.2

0.4

0.6

0.8

1.0

Quantity of saccharides (nmol)
Fructose1-5TMS

Mannose-5TMS

Sucrose-8TMS

糖類量とピーク面積値の関係

Saccharides

R2

RSD
n=6, %

1 Xylose-4TMS

0.9998

5.4

2 Arabinose-4TMS

0.9996

7.5

3 Rhamnose-4TMS

0.997410.8

4 Fucose-4TMS

0.99889.8

5 Fructose1-5TMS

1.0000

3.3

6 Fructose2-5TMS

0.99983.2

7 Mannose-5TMS

0.99822.5

8 Galactose-5TMS

0.9995

1.8

9 Glucose-5TMS

0.9778

2.3

10 Sucrose-8TMS

0.9993

2.9

11 Lactose-8TMS

0.99977.2

12 Maltose-8TMS

0.99996.5