3. Policosanol and long chain aldehyde identification by GC-MS
The silylated derivatization was used for mass spectrum analysis of policosanol, however mass spectrum of aldehyde was analyzed without
silylation. In this case, policosanol was identified as its trimethylsilyl derivates. N-Methyl-N-trimethylsilyl trifluoroacetamide MSTFA was
used as silylation reagent. The derivatization solution was made by mixing 0.5 ml of sample in chloroform with 250 µl MSTFA. The solution was
heated at 50°C for 15 min. The volume of the mixed solution was made up to 1 ml with chloroform for GC-MS analysis. Derivatization was also
applied for policosanol standard solution at concentration of 0.1 mgml. The analysis was performed using Shimadzu GC-MS QP-2010 with
a fused capillary column DB-5 MS 0.25 mm i.d. × 30 m × 0.25 µm film thickness from JW Scientific Folsom, CA under the same GC
conditions. The samples 0.3 µl were injected with split ratio of 1:10. For MS detection, the electron impact EI ion source and transfer line
temperature were set at 200°C and 280°C. The ionization energy was mode at 70 eV. The mass acquisition scan range and rate were 30–500
amu and 2 scanss. Identification of each policosanol or aldehyde was conducted by comparing its retention time and mass spectrum directly to
those of its respective standard. The mass spectrums were also confirmed with NIST 2005 Mass Spectral Library by GCMSsolution software
Shimadzu.
4. Statistical analysis
All extraction runs and analysis were carried out in triplicate and analyzed statistically using ANOVA with completely randomized design.
The mean values of analysis results were reported. The data were analyzed by SPSS Version 13.0 for Windows SPSS Inc., USA, 2003. Univariate
comparisons of the various means were carried out by post hoc test of Duncan at p = 0.05. Nine experimental designs were attempted with
statistical analysis, there were: a. The effect of rind parts of sugarcane cultivars on wax composition 3
samples, i.e. NiF 8, Ni 15 and Ni 22 cultivars. b. The effect of different parts of sugarcane of Ni 15 cultivar on the
policosanol and long chain aldehyde contents 3 samples, i.e. rind, pith and whole stalk.
c. The effect of extraction times of rind part of Ni 15 cultivar on the policosanol and long chain aldehyde contents 5 extraction times, i.e. 2, 4,
8, 16 and 24 h. d. The effect of sugarcane cultivars on the policosanol and long chain
aldehyde contents of the sugarcane rinds 7 samples, i.e. Ni 13, Ni 15, Ni 17, NiF 8, NCo 310, F 161 and F 177 cultivars.
e. The effect of sugarcane harvesting time on the increasing of total policosanol and long chain aldehyde contents of the sugarcane rinds 5
samples, i.e. NCo 310, Ni 15, Ni 17, NiF 8 and Ni 22. f. The effect of extraction methods of Kokuto A on the policosanol and
long chain aldehyde contents 6 extraction methods, i.e. a liquid-liquid extraction method and 5 soxhlet extraction methods.
g. The effect of extraction times of Kokuto A on the policosanol and long chain aldehyde contents 5 extraction times, i.e. 4, 8, 16, 24 and 32 h.
h. The effect of Kokuto types on the policosanol and long chain aldehyde contents of the Kokuto 9 samples, i.e. type A
–
G, P and Q. i. The effect of Kokuto production types on the policosanol and long chain
aldehyde contents 5 samples, i.e. raw juice and end product of Kokuto factory A; and raw juice, clear juice and end product of Kokuto factory
Non-A.
IV. RESULTS AND DISCUSSION
A. Sugarcane Wax Composition
The qualitative separation of most of the major classes of aliphatic wax components could be achieved by silica gel of thin layer chromatography
Figure 7. Compositions of the waxy materials from sugarcane cultivars were all similar. They were composed of sterol, alcohol, acid,
triacylglycerol, methyl ester, aldehyde and sterol ester. Thin layer chromatography had been widely used for qualitative analysis in waxy
materials Hwang et al. 2002; Adhikari et al. 2006; Webster et al. 2006.
Figure 7 Thin layer chromatography of waxes of several sugarcane cultivars. S: Standard.
S NiF 8 Ni 22 Ni 15
Plant sterol Policosanol
Acid Triacylglycerol
Sterol ester
Methyl ester aldehyde
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