SIMULITANEOUS IDENTIFICATION AND
DETERMINATION OF FATTY ACIDS, SUGARS, AND
PHYTOCHEMICALS IN JATROPHA CURCAS SEEDS AS
THEIR TMS DERIVERTIVES BY GC/MS
Kiyoshi Imamura (1), Hoa Thi Truong (2), Phuong Duc Luu (1),
Santi Kongmany(1), Yasuaki Maeda(1), and Luu Van Boi(3)
• 1 Osaka Prefecture University, Japan
• 2 DaNang Environmental Technology Center, IET, VAST
• 3 Vietnam National University, Hanoi, Vietnam
Question: What is the best raw biomass which can be cultivated in
the wasteland without deforestation and does not compete with
other crops?
Answer: Jatropha curcas
• The seeds of Jatropha curcas contain more than 30% (W/W) oil
• Be able to grow up in dry, isolated area
• Inedible oil (phorbol esters: toxic)
• It is a multi-purpose tree because of its industrial use and
medicinal use
JATROPHA CURCAS
PHYTOCHEMICAL
S
BIODIESEL SUGARS
+
Clean-up Homogenized
process Sample
Extraction (3times with MeOH)
Concentration
Concentrate
MeOH layer White Precipitate
EtoAC/ Water Distribution
TMS
Ethyl acetate L ayer Aqueous L ayer deravatives
F ig. 1 Scheme of Clean-up process
GC-MS
Tri-methyl silyl derivative reaction
- An aliquot of 0.1mL of each extracts is evaporated to
dryness under the gentle stream of nitrogen gas.
- Added 0.2 mL of pyridine and BSTFA reagent (60C, 1
hour).
- After reaction, the mixture is diluted with a 2mL of ethyl
acetate for analysis of GC/MS
H3C CH3 δ- CH3
R-O + CH3- Si -X R - O - Si -X R - O - Si - CH3 + HX
δ+H CH CH3
H CH3 3
X : -O-C (CH3) = N-Si (CH3) 3
R : R-OH or R-(CO)OH
E: Ethyl acetate phase
D: Aqueous phase
C: Crystal of sugar
B: Authentic FFAs
A: MeOH extracts
Phytochemicals
Fig.2. TICs of TMS derivatives of extracted samples, and authentic FFAs
For the purpose of identification and quantitative analysis:
• A HP-1 capillary column (15m x 0.25mm I.D. and film
thickness of 0.25 um, Agilent, Palo Alto, CA, USA)
A DB-5-MS capillary column (30m x 0.25mm I.D. and
film thickness of 0.25 um, Agilent, Palo Alto, CA, USA)
FFAs
Fig.3. TICs of TMS derivatives of ethyl acetate layer (A) and authentic FFAs (B)
1: palmitic acid, 2: oleic acid, 3: linoleic acid, 4: stearic acid
Table 1 Components of FFAs contained in J apropha seeds
Molecular Retention Time Ratio of
No. Compounds MW MW (TMS)
Fomula tR (min) Concentration
1 Palmitic
parmiticacid
acid (0, C16) C16H 32O2 30.48 256 328 1.00
2 linoleic acid (2, C18) C18H 32O2 33.44 280 352 1.80
3 oleic acid (1, C18) C18H 34O2 33.55 282 354 2.02
4 stearic acid (0, C18) C18H 36O2 33.98 284 356 0.50
Phytochemicals
• Phytochemicals are chemical compounds that occur naturally in
plants
• The term is generally used to refer to those chemicals that have
biological significance (antioxidants, medicinal compoiunds,..)
Phytosterols Vitamin E
(tocopherols and tocotrienols)
Gamma tocopherol
Gamma tocotrienol
Phyto-
chemicals
Fig.4. TICs of TMS derivatives of ethyl acetate layer extracted with MeOH (A), and with EtOH (B)
1: squalene, 2: gamma-tocopherol, 3: gamma-tocotrienol, 4: campesterol,
5: stigmasterol, 6: beta-sitosterol, 7: unknown
Table 2 Components of tocoherols, tocotrienols, and steroidal alkohols in J atropha seeds
Retention time Molecular Ratio of
No. Compounds MW MW(TMS)
(tR) min Fomula Contents
1 squalene 42.87 C30H 50 410 - 0.53
2 gannma-tocopherol-TMS 45.28 C28H 48O2 416 488 0.09
3 gannma-tocotrienol-TMS 46.81 C28H 42O2 410 482 0.54
4 campesterol-TMS 48.47 C28H 48O 400 472 0.10
5 stigmasterol-TMS 48.85 C29H 48O 412 484 0.18
6 beta-sitosterol-TMS 49.55 C29H 50O 414 486 1.00
7 unknown 49.73 C29H 48O 412 484 0.03
Sugars
Fig.5. TICs of TMS derivatives of aqueous layer extracted with MeOH (A), and with EtOH (B)
2: Xylose, 3: Adonitol, 4: Fructose,5: Pinitol, 6: Glucose, 7: Sorbitol (glucitol)
8: Inositol, 9: Sucrose
Table 3 Components of saccharides identified in J atropha seeds
MW Molecular Kovats Index Kovats Index Retention Ratio of
No Compounds MW *1
(TMS) Fomula (obs) (ref.) time(mim) Content
2 Xylose 150 438 C5H 10O5 1747 1734, 1787 24.42 0.00
3 Adonitol 152 512 C5H 12O5 1759 1756, 24.68 0.01
4 Furctose 180 540 C6H 12O6 1842 1832, 1847 26.46 0.01
5 Pinitol 194 554 C7H 14O6 1850 1869 26.62 0.02
6 Gulcose 180 540 C6H 12O6 1926 1932, 2024 28.20 0.03
7 Sorbitol(Glucitol) 182 614 C6H 14O6 1973 1981 29.17 0.29
8 Inositol 180 612 C6H 12O6 2119 2130 31.90 0.04
9 Sucrose 342 918 C12H 22O11 2695 2712 41.25 1.00
COMPARISON
Conventional methods TMS derivatization-
GCMS
FFAs Titration TMS derivatives: more
stable and easily
Phytosterols LCMSMS (not easy) evaporated able to
or GCMS (easy) be analysed by GC-MS
Vitamin E FL-HPLC and LCMSMS (sharp peaks).
Simultaneously
Sugars Thin layer chromatography quantitative analysis
CONCLUSION
We successfully make a survey on the
distribution of tocopherols, tocotrienols, FFAs,
phytosterols, and mono-, and di-saccharides in
Jatropha curcas seeds using by GC/MS
technique after convention of their TMS-
derivatives.
THANK YOU FOR YOUR ATTENTION!