1198 J. Agric. Food Chem., Vol. 54, No. 4, 2006
Zhang et al.
4.0 V; scan rate, 1 s; trap, manifold, and the transfer line temperature,
respectively, 150, 40, and 270 °C; and electronmultiplier voltage, 1800
V) with full scan mode between 40 and 600 m/z. The data were acquired
using the Auto Gain Control (AGC: 20000 ions) feature of the Saturn
2000 mass spectrometer. Data acquisition and processing and instru-
mental control were performed by Varian Saturn WS software. The
separation of phytosterol mixture was carried on a VF-5ms capillary
column (stationary phase: 5% phenyl-95% dimethylpolysiloxane,
thickness of 0.1 µm, 60 m × 0.25 mm, Varian). The column temperature
was programmed from 80 to 280 °C at 20 °C/min, then to 292 °C at
0.5 °C/min, and to 300 °C at 2 °C/min. The injector temperature was
set at 250 °C for 40 min. The detector temperature was 120 °C. Helium
(purity 99.9995%, Air Liquide, Paris-La-De´fense, France) was used
as a carrier gas with a flow rate of 1 mL/min. The injection volume
was 1 µL.
Other Apparatus. NMR spectra were recorded on a Bruker AC-
300 spectrometer (1H NMR at 300 MHz and 13C NMR at 75 MHz) in
CDCl3 as the solvent at ambient temperature. The chemical shifts were
reported in units of δ. Melting points (mp) were measured with a SMP3
melting apparatus (Bibby Stuart Scientific, United Kingdom). Optical
rotations were measured with a Perkin-Elmer 341 polarimeter (Bod-
enseewerk, Perkin-Elmer & Co GmbH, Germany).
Figure 2. Total analytical HPLC chromatogram of Generol 95S on a
Polaris C8-A column (250 mm
× 4.6 mm i.d., 5 µm particles); mobile
phase, acetonitrile:2-propanol:water (2:1:1, v/v/v); flow rate, 0.5 mL/min;
UV detection, 208 nm; 0.1 AUFS. Peaks are identified as in Table 1: 1,
∆
7-avenasterol; 2, brassicasterol; 3,
5, campesterol; 6, citrostadienol; 7, stigmasterol; 8,
-sitosterol.
∆
5-avenasterol; 4,
∆7-campesterol;
∆
7-sitosterol; and 9,
Citrostadienol. White powder (10 mg, purity 90%); mp 161.6-
1
163.6 °C. H NMR (300 MHz, CDCl3): δ 5.16 (1H, br d, H-7), 5.15
â
(1H, br d, H-241), 3.59 (1H, m, H-3), 2.04-1.21 [29H, m, including
1d (3H, H-242) at 1.55 ppm], 0.91 (3H, d, J ) 6.5 Hz, H-26), 0.85
(3H, d, J ) 6.8 Hz, H-27), 0.81 (3H, d, J ) 6.9 Hz, H-21), 0.79 (3H,
s, H-19), 0.77 (3H, d, J ) 6.6 Hz, H-28), 0.53 (3H, s, H-18). 13C NMR
(75 MHz, CDCl3): δ 145.84, 139.13, 117.46, 116.46, 76.22, 56.00,
54.94, 49.63, 46.64, 43.37, 40.25, 39.53, 36.99, 36.58, 35.90, 34.83,
30.97, 28.60, 27.98, 26.63, 22.92, 21.36, 21.08, 21.00, 18.92, 15.14,
∆7-Sitosterol. White powder (23 mg, purity 95%); mp 144-145
°C. 1H NMR (300 MHz, CDCl3): δ 5.16 (1H, br d, H-7), 3.59 (1H, m,
H-3), 2.04-1.19 (30H, m), 0.92 (3H, d, J ) 6.4 Hz, H-21), 0.84 (3H,
d, J ) 7.2 Hz, H-242), 0.83 (3H, d, J ) 6.5 Hz, H-26), 0.81 (3H, d, J
) 6.5 Hz, H-27), 0.79 (3H, s, H-19), 0.53 (3H, s, H-18). 13C NMR (75
MHz, CDCl3): δ 139.62, 117.42, 71.06, 56.07, 55.04, 49.44, 45.83,
43.38, 40.25, 39.55, 37.99, 37.14, 36.58, 34.20, 33.89, 31.48, 29.64,
29.14, 27.97, 26.17, 23.06, 22.96, 21.55, 19.83, 19.03, 18.90, 13.04,
20
14.14, 12.76, 11.84. [R]D +8° (c 1, CHCl3).
Campesterol. White powder (20 mg, purity 98%); mp 157-158
°C [literature mp value, 158 °C (19)]. 1H NMR (300 MHz, CDCl3): δ
5.35 (1H, br d, H-6), 3.53 (1H, m, H-3), 2.27 (2H, m), 2.00 (2H, m),
1.85 (3H, m), 1.56-1.07 (21H, m), 1.01 (3H, s, H-19), 0.91 (3H, d, J
) 6.5 Hz, H-21), 0.85 (3H, d, J ) 6.5 Hz, H-26), 0.80 (3H, d, J ) 6.5
Hz, H-27), 0.77 (3H, d, J ) 6.5 Hz, H-241), 0.68 (3H, s, H-18). 13C
NMR (75 MHz, CDCl3): δ 140.76, 121.72, 71.80, 56.76, 56.09, 50.12,
42.31, 39.77, 38.83, 37.25, 36.50, 35.88, 33.70, 32.42, 31.90, 31.67,
30.26, 28.23, 24.29, 21.08, 20.20, 19.40, 18.70, 18.25, 15.37, 11.86.
20
11.97, 11.84. [R]D +7° (c 1, CHCl3).
Soxhlet Extraction of Phytosterols from Chocolates. Chocolate
samples (5 g) were frozen under liquid nitrogen, finely ground, and
placed in extraction thimbles (22 mm × 80 mm, Schleicher&Schuell
MicroScience GmbH, Dassel, Germany) with 250 mL of chloroform
in a Soxhlet apparatus (2 cycles h-1) (Verrerie Striegel, Strasbourg,
France). Extractions lasted 16 h (further cycles did not lead to more
phytosterol extractions). The extracts (about 2 g) were dried with a
rotary evaporator (35 °C, 200 mbar) and stored at 4 °C until further
analysis.
Isolation and Purification of Phytosterols from Food Samples.
The procedure for phytosterol preparation used here was adapted from
the analytical method described in NF EN ISO 12228 (20) and the one
used for BCR-633 (21).
The protocol was as follows: 20 µg of cholestanol (40 µL of 0.5
mg/mL in ethyl acetate) used as an internal standard was spiked into
100 mg of BCR-633, oils, or chocolate extracts in a 100 mL flask.
After removal of ethyl acetate under nitrogen, 5 mL of 0.5 M KOH
ethanolic solution was added, and the mixture was stirred and refluxed
at 90 °C for 60 min. After the reaction system was cooled to room
temperature, the solution was extracted with diethyl ether (3 × 20 mL).
The combined organic extracts were washed with 20 mL of 0.5 M
aqueous KOH solution and 0.2 M aqueous Na2SO4 solution (3 × 15
mL). The organic solvents were removed with a rotary vacuum
evaporator (30 °C, 400 mbar), and the residue was dissolved in 1 mL
of a solution of cyclohexane/diethyl ether (9:1, v/v). The sterol frac-
tion was purified by SiOH solid phase extraction (SPE) cartridge (3
mL/500 mg, Chromabond, Macherey-Nagel, Du¨ren, Germany). The
cartridges were first conditioned with 5 mL of cyclohexane before the
samples were loaded. Low-polarity lipids were eluted with 5 mL of
cyclohexane/diethyl ether (9:1, v/v) and discarded, and the sterol
fractions were then eluted with 4.5 mL of cyclohexane/diethyl ether
(1:1, v/v) in test tubes and dried under gentle nitrogen flow. The purified
sterols were converted to TMS ethers as mentioned above before GC
analysis.
20
[R]D -34° (c 1, CHCl3).
â-Sitosterol. White solid (70 mg, purity 95%); mp 136-138 °C
[literature mp value, 138-139 °C (19)]. 1H NMR (300 MHz, CDCl3):
δ 5.34 (1H, br d, H-6), 3.51 (1H, m, H-3), 2.27-1.08 (30H, m), 1.00
(3H, s, H-19), 0.92 (3H, d, J ) 6.5 Hz, H-21), 0.84 (3H, t, J ) 7.2 Hz,
H-242), 0.83 (3H, d, J ) 6.5 Hz, H-26), 0.80 (3H, d, J ) 6.6 Hz, H-27),
0.68 (3H, s, H-18). 13C NMR (75 MHz, CDCl3): δ 140.75, 121.71,
71.79, 56.76, 56.05, 50.13, 45.83, 42.30, 39.77, 37.25, 36.50, 36.14,
33.94, 31.90, 31.65, 29.14, 28.24, 26.07, 24.30, 23.06, 21.08, 19.82,
19.39, 19.03, 18.77, 11.98, 11.85. [R]D20 -34° (c 2, CHCl3) [literature
25
value, [R]D -38° (19)].
∆7-Avenasterol. White powder (27 mg, purity 98%); mp 143.8-
1
144.8 °C. H NMR (300 MHz, CDCl3): δ 5.15 (1H, br d, H-7), 5.10
(1H, d, J ) 6.8 Hz, H-241), 3.59 (1H, m, H-3), 2.83 (1H, m, H-25),
2.42-1.24 [27H, m, including 1d (3H, J ) 6.6 Hz, H-242) at 1.59
ppm], 0.98 (3H, d, J ) 7.0 Hz, H-26), 0.96 (3H, d, J ) 7.0 Hz, H-27),
0.94 (3H, d, J ) 7.0 Hz, H-21), 0.79 (3H, s, H-19), 0.54 (3H, s, H-18).
13C NMR (75 MHz, CDCl3): δ 145.83, 139.59, 117.44, 116.46, 71.05,
56.01, 55.03, 49.44, 43.40, 40.25, 39.55, 37.99, 37.14, 36.58, 35.91,
34.14, 31.48, 29.64, 28.60, 28.00, 27.95, 22.96, 21.55, 21.08, 21.00,
20
18.92, 13.04, 12.76, 11.84. [R]D +9° (c 1, CHCl3).
∆7-Campesterol. White powder (3 mg, purity 85%); mp 129.6-
1
131.8 °C. H NMR (300 MHz, CDCl3): δ 5.16 (1H, br d, H-7), 3.59
(1H, m, H-3), 2.09-1.20 (28H, m), 0.91 (3H, d, J ) 6.5 Hz, H-21),
0.85 (3H, d, J ) 6.8 Hz, H-26), 0.81 (3H, d, J ) 6.6 Hz, H-27), 0.79
(3H, s, H-19), 0.76 (3H, d, J ) 6.6 Hz, H-241), 0.53 (3H, s, H-18). 13
C
NMR (75 MHz, CDCl3): δ 139.63, 117.41, 71.06, 56.12, 55.04, 49.44,
43.38, 40.25, 39.56, 38.83, 37.99, 37.13, 36.31, 34.20, 33.65, 32.41,
31.48, 30.37, 29.64, 27.95, 22.95, 21.55, 20.20, 18.82, 18.26, 15.37,
Quantitative Analysis. Quantitation was performed by GC-MS with
total ion count (TIC) mode against cholestanol as the internal standard.
The MS RRF values of each sterol TMS ether were determined toward
20
13.04, 11.84. [R]D +9° (c 1, CHCl3).