Concentration mg100g = Content
� mg
ml �
∗
× Volume of extract ml × 100 Sample weight g
where content was calculated by calibration curve of its standard.
2. Policosanol and long chain aldehyde analysis by GC-FID
a. Standard preparation
Mixed policosanol standards for policosanol compounds quantification were prepared in toluene. Aldehyde standards were
synthesized from their alcohols form by oxidation with pyridinium chlorochromate, as described by Pérez-Camino et al. 2003. Each 1
mM corresponding alcohols standards 19.14 mg hexacosanol, 20.54 mg octacosanol, 21.94 mg triacontanol and 9 mM pyridinium
chlorochromate 97.5 mg were stirred in 50 ml dichloromethane for 1.5 h at room temperature. The reaction mixture was eluted with
dichloromethane through a short column 6 × 2 cm I.D., packed with silica gel 60. Then the reaction product was dried by N
2
gas and diluted in toluene. The synthesized long chain aldehyde standards were
checked with GC-FID analysis.
b. Sample extraction
Kokuto A samples were extracted with two extraction methods in
order to investigate the optimum extraction method; they were liquid-liquid extraction LLE by the method of Irmak et al. 2006 and
solid-liquid extraction SLE, soxhlet method. The optimum extraction
method was applied to policosanol and long chain aldehyde contents analysis in sugarcane and other Kokuto samples.
Liquid-liquid extraction LLE method . Briefly, 6 g of Kokuto
sample was mixed with 50 ml of 1.0 N NaOH and 50 ml of methanol and subsequently hydrolyzed by refluxing in a heater for 30 min. After
cooling, the mixture was filtered through Advantec No. 5A filter paper under vacuum condition. Then Millipore water was added to the filtrate.
The solution was extracted three times with 50 ml diethyl ether. Combined diethyl ether phases from three extractions were neutralized
with Millipore water until pH of water phase reached 7. The extract was dried over 50 g of anhydrous sodium sulfate by storing at 4°C for
one night. Then the solvent was removed from the extract with rotary-evaporator under vacuum condition. The dry extract was diluted
in toluene or chloroform and made up to 2 ml prior to analysis. Solid-liquid extraction SLE method
. Briefly, 10 g of freeze-dried sugarcane or 6 g of Kokuto were placed in Advantec No. 84
thimble filter and extracted using soxhlet with about 150 ml of several systems of organic solvent. The solvent systems were Soxhlet A
chloroform, methanol 2:1 vv; Soxhlet B hexane, methanol 10:1 vv; Soxhlet C hexane, methanol 20:1 vv; Soxhlet D hexane,
methanol 30:1 vv; and Soxhlet E hexane. Several extraction times were also attempted in order to investigate the heat stability and optimum
extraction condition of policosanol and long chain aldehyde compounds in sugarcane and Kokuto samples. The solvent solution was removed
from the extract with rotary-evaporator under vacuum condition at 40°C.
Then the dry extract was diluted in toluene and made up to 2 ml for GC-FID analysis, whereas for GC-MS analysis, the extract was diluted in
chloroform.
c. Gas chromatography analysis GC-FID
