SPL-M100 Application Note

No. AS211101EN

Optimization of metabolome analysis
by using Fast GC/MS 【Intuvo9000GC-5977MSD】
coupled with online solid-phase analytical derivatization technique(1) Repeatability and Durability test of Amino acid and Organic acid

Introduction
Conventional metabolome analysis in GC/MS requires from 4 hours to overnight for drying and 2hours for derivatization after sample extraction. Online solid-phase derivatization (SPD)dramatically reduces the pretreatment time and stabilizes the time finishing derivatization until thestart of a GC/MS analysis, thus it is expected to more stable data acquisition.
In addition, an easy-to-use instrument is desirable as it is possible to perform high-speed analysis
and measure multiple samples in line with the speeding up of pretreatment especially for beginners ofGC/MS analysis. In this note, reproducibility and durability tests of amino acids and organic acids wereconducted using a system that connects SPL-M100 as an online SPAD system and Intuvo 9000GC +5977B MSD capable of high-speed analysis.

Instruments【Intuvo9000GC】
・Fast analysis
・Easy handling
・Using Guard chip

Guard chip・Role as precolumn・Easy changing
・Not required
column cutting【SPL-M100】
・Extremely shorter pretreatment time・Sequenced pretreatment and injection
Intuvo 9000GC-5977B MS
with SPL-M100

Pretreatment workflow

Online SPE-GC：SPL-M100

Add Water 150 µL
Add ACN 800 µL

Flash-SPE ACXs

SampleMouse serum

第15回 メタボロ―ムシ
ンポジウム「高速GC/MSとオンライン固相誘導体化を用いたメタボローム分析の最適化」
杉立久仁代1,佐々野僚
一2,佐久井徳広1,大塚
剛史1,中村貞夫1
1アジレント・テクノロジー
株式会社、 2株式会社
アイスティサイエンス

Automation
Pretreatment：15.3 minVials

Sample 50 µL

for SPE-GC system

Information

BenefitFast GC/MS andOnline SPAD systemSPL-M100

Conditioning
ACN-Water (1/1)
ACN

Loading [Retaintion] Extract 50 µL

Shaking (37 ℃, 30 min)Washing ACN-Water (1/1)
Centrifuge (14000 rpm, 5 min)Dewater ACN
Infiltration 0.5% Methyoxyamine/Pyridine

Supernatant 250 µL
Add Water 250 µL

SPD (Methoximation), 3 min
Infiltration MSTFA/Toluene (3/1)
SPD (TMS), 1 min

Extract

Elution Toluene

Analytical condition

AiSTI SCIENCE

GC-MS

ProductOnline SPE-GC

SPL-M100
Solid phasecartridge

Flash-SPE
GC large volume
injection port

GC Intuvo9000C
ColumnOven Temp.

DB-5MS, 15 m × 0.25 mm I.D. , df=0.25 µm
80℃ (1 min)-20℃/min-220℃-30℃/min-310℃ (3 min) (total 14 min)

MMI Temp.
LinerInjection mode

220℃ (0.5 min)-70℃/min-290℃
Ultra inert liner, single tapered, low pressure drop, glass wool
Split (50:1)

Guard chip Temp.
Carrier gas flow
Transfer line Temp.

Oven track
1.0 mL/min (Constant flow)
290℃

MS 5977B

LVI-S250

AiSTI SCIENCE CO.,Ltd.

ModeIon source Temp.

Scan (m/z 70-470)
250℃

Tel：+81-73-475-0033

Quadropole Temp.

150℃

E-mail：as@aisti.co.jp

HP：www.aisti.co.jp

AiSTI Application Note

No. AS211101EN

Results and Discussion(1) Repeatability test
Total ion chromatograms and the result of the repeatability test are shown by analysis of standard
solution (amino acids and organic acids) and mouse serum.

Standardsolution
0.02 mM

Serum(mouse)

1. Lactic acid 2TMS
2. Alanine 2TMS
3. Oxalic acid 2TMS
4. Malonic acid 2TMS
5. Valine 2TMS
6. Benzoic acid TMS
7. Leucine 2TMS
8. Isoleucine 2TMS
9. Proline 2TMS
10. Maleic acid 2TMS
11. Glycine 3TMS
12. Succinic acid 2TMS
13. Fumaric acid 2TMS
14. Serine 3TMS
15. Threonine 3TMS
16. Malic acid 2TMS
17. Methionine 2TMS

18. Aspartic acid 3TMS
19. Pyroglutamic acid 2TMS
20. Glutamic acid 3TMS
21. Phenylalanine 2TMS
22. Tartaric acid 4TMS
23. Shikimic acid 4TMS
24. Citric acid
25. Quinic acid
26. Lysine 4TMS
27. Tyrosine 3TMS
28. Cysteine 3TMS
1. Gluconic acid 6TMS
2. Palmitelaiic acid TMS
3. Palmitic acid TMS
4. 9,12-Octadecadienoic acid (Z,Z)-, TMS
5. 9-Octadecenoic acid (E), TMS
6. Arachidonic acid TMS

Retention time (min)Total ion chromatograms of standard solution (amino acids and organic acids) and mouse serum

Repeatability (RSD) distributions of standard solution and mouse serum
Serum (mouse)
Standard solution (0.02mM)

First run (lot 1)

12

Second run (lot 2)

1
Less than 10%
10%未満

Less than 10%
10%未満

27 compounds

10-15%
10-15％

26 compounds

More than 15%
15％以上

The successful result was obtained in 16 sequential
analyses (a mixture of 28 standards).

29 compounds

10-15%
10-15％
More than 15%
15％以上

100 sequential analyses were performed twice,using different lots and on different dates.
No change of liners or guard chips was performed during this period.

(2) Durability test
The change in detection response is shown below (the result of the first run). No particulartrends were observed in these responses. A stable result was also obtained in the second run.
These results show that this is a very robust system that does not require liner replacement in 200
sequential analyses of real samples.
Serum (mouse, lot 1)