Vol. 26, No. 9 (2014)
Determination of Total Cholesterol in Serum by Gas Chromatography-Mass Spectrometry 2647
99 % bis(trimethylsilyl)trifluoroacetamide (BSTFA) plus 1 %
trimethylchlorosilane (TMCS) and amino acid standard solution
and 10 % boron trifluoride methanol solution (BF3/MeOH
solution) were also purchased from Sigma-Aldrich (St Louis,
MO, USA). Potassium hydroxide (Guanghua ChemicalAgent
Com., China). The solvents used in this study were liquid
chromatographic grade and purchased from Tianjin Chemical
Agent Com., China). All chemicals used in this study were
analytical-reagent grade.
was made at 260 °C. The GC oven was operated with the
following temperature program: initial temperature 120 °C held
for 1 min, ramped at 10 °C min-1 to 250 °C and held for 5 min.
Total run time was 19 min. The MS was operated in electronic
impact (EI) mode and selected ion monitoring (SIM) mode
was used for the identification and quantification of cholesterol.
The MS transfer line temperature was held at 280 °C. Mass
spectrometric parameters were set as follows: electron impact
ionization with 70 eV energy; ion source temperature, 230 °C;
MS quadrupole temperature, 150 °C and solvent delay 3 min.
The experimental protocols for this study were approved
by the Institutional Animal Care of Hebei 16 male Sprague-
Dawley rats were purchased (Shijiazhuan, China) and housed
individually in stainless steel cages in a room maintained at
18-25 °C with a 12:12-h light-dark cycle. After one week of
acclimatization, the rats were divided randomly into a control
group (CON) and a high cholesterol diet group (CHO) for 3
months. The control group fed a diet containing 1 % cholesterol
and 0.5 % cholic acid, i.e. cholesterol diet group fed the high
cholesterol. Cholic acid was added to diets to improve choles-
terol absorption by the intestine. The composition of high
cholesterol diets was detailed in Table-1. Diets and tap water
were freely available.
RESULTS AND DISCUSSION
Optimization of sample preparation procedure: Standard
addition of 5 mmol L-1 cholesterol were pre-treated with three
methods including silanization, methylation and saponification
followed by GC-MS analysis for determining the peak areas.
BSTFA silanization: The BSTFA silanization procedure
described in section extraction and derivatization procedure.
BF3 methylation: Pre-treatment with BF3 methylation was
adopted by the method described by literature12. 200 µL choles-
terol solution was transferred to a 1.5 mL centrifuge tube and
then 4 mL 0.5 n KOH/CH3OH solution was added to initiate
reaction. The sample was then refluxed at 90-100 °C for 13
min, followed by supplementing with 3 mL BF3/CH3OH
solution and continuing to reflux for 2 min. Finally, 2 mL
ether was added to the mixture and further refluxed for another
1 min. The reaction was terminated by adding 10 mL saturated
NaCl solution and cooled down to room temperature. The ether
layer was carefully collected and subjected to GC analysis.
Saponification: Pre-treatment with saponification was
adopted by the method described by literature13. 200 µL choles-
terol solution was transferred to a 1.5 mL centrifuge tube, then
1 mL 1 mol L-1 KOH/MeOH were added to initiate saponifi-
cation reaction. The sample was then refluxed at 50 °C for 60
min, Finally, 1 mL n-hexane was added to the mixture and
further refluxed for another 1 min. the n-hexane layer was
carefully collected and subjected to GC analysis.
TABLE-1
COMPOSITION OF EXPERIMENTAL DIETS (g/kg diet)
Ingredient
Casein
Sucrose
Corn
starch
Cholesterol
Cholic
acid
200
50
500
10
5
Body weight and food intake were monitored in all rats
throughout the experimental period. On the final day of 1st
month, 2nd month and 3rd months, blood was taken from tail
vein and allowed to coagulate on ice, serum was recovered by
centrifugation. The serum samples were frozen and stored at
-80 °C until analysis.
Extraction and derivatization procedure: 200 µL serum
sample was transferred to a 1.5 mL centrifuge tube and submerge
in 1 mL mixture solution of chloroform followed by addition
of 100 µL 5α-cholestane as internal standard. The mixture
was then sonicated for 60 min to extract cholesterol and
precipitate proteins. The precipitated proteins were separated
out by centrifugation at 12,000 rpm for 10 min, with other
components remaining in the solution. Then 600 µL super-
natant was collected from each sample into a vial with PTFE-
lined screw cap and evaporated under nitrogen gas at 50 °C to
dryness. After the sample evaporated to dryness for derivati-
zation, 100 µL of BSTFA with 1 % TMCS was added into vial
and the derivatization reaction was carried out under 70 °C
for 40 min. After derivatization and cooling to room tempera-
ture, 1 µL derivative was injected in the GC-MS for analysis.
GC-MS analysis: GC-MS analysis were performed on
an Agilent (Little Falls, DE, USA) gas chromatograph 7890
equipped with an electronically controlled split/splitless injec-
tion port, an inert 5975C mass selective detector with electron
impact (EI) ionization chamber and a 7683B Series injector/
autosampler. The GC separation was conducted with an HP-
5MS 30 m × 0.25 mm I.D., 0.25 µm film thickness column
(Agilent, CA, USA). Helium was used as the carrier gas at a
constant flow rate of 1 mL min-1. A splitless injection of 1 µL
Using three different pre-treatments, the determination
of total cholesterol in serum samples were also compared. As
shown in Fig. 1, the cholesterol peak areas were higher in those
obtained from the pre-treatment with BSTFA silanization than
those from BF3 methylation or saponification. Therefore,
BSTFA silanization was selected in the experiments. The total
ion current chromatogram of the cholesterol and 5α-cholestane
derivatives utilized BSTFA were shown in Fig. 2. Mass spectra
of the cholesterol derivative utilized BSTFA were shown in
Fig. 3.
5
4.5
4
3.5
3
2.5
2
1.5
1
0.5
0
BSTFA
silamization
Fig. 1. Peak areas of cholesterol using different pre-treatments
BF3 methylation Saponification
Lian, Kaoqi
