´ ´
D. Derewiaka, E. Molinska (née Sosinska) / Food Chemistry 171 (2015) 233–240
234
unsaturation. The characteristics of sitosterol oligomers produced
and samples were derivatized for 24 h at room temperature due
to addition of 100 L of anhydrous pyridine and 100 L of sylila-
´
during thermal processing were presented by Sosinska,
l
l
Przybylski, Hazendonk, Zhao, and Curtis (2013). They applied spec-
troscopic methods such NMR, IR, Raman and SEC/APCI/MS to
establish sitosterol dimers structure.
tion agents. Cholesterol, oxysterols and cholestadienes were ana-
lysed on gas chromatograph equipped with a mass spectrometer
(GCMS-QP2010S) Shimadzu Corporation (Shim-Pol A. M. Borzy-
There are only a few studies describing formation of volatile
compounds due to thermal-oxidation of sterols. In Van Lier, de
Costa, & Smith, 1975 determined at least fourteen volatile com-
pounds that were produced during decomposition of cholesterol.
The amount of volatiles, formed during heating of phytosterol in
mowski, Poland) using. DB5ms (30 m ꢀ 0.25 mm ꢀ 0.25
lm) capil-
lary column Phenomenex (Torrance, CA, USA). Helium was used as
a carrier gas at a flow rate of 0.61 mL/min. The injector tempera-
ture was set at 250 °C, and the column temperature was pro-
grammed as follows: 200 °C for 1 min, subsequent increase to
250 °C at the rate of 15 °C/min, then to 310 °C at the rate of 3 °C/
min for 6 min. The interface temperature for GC–MS was 260 °C.
Temperature of ion source was 250 °C and ionisation energy was
70 V. The split ratio was 50:1. The total ion monitoring (TIC) was
used to detect sterols, oxysterols and cholestadienes (m/z ranged
´
different temperatures, was discussed by Rudzinska et al. (2009).
They found mainly hydrocarbons, ketones, aledehydes and acids
in the profile of volatile fraction formed during heating.
The aim of the study was to evaluate the intensity of cholesterol
degradation, oxidation, polymerisation and formation of volatile
compounds during thermal treatment at temperatures typical for
food processing.
100–600). The internal standard 5a-cholestane was used to quan-
tify cholesterol and cholestadienes, 19-hydroxycholesterol to
quantify oxysterols. Regression coefficient of oxysterols curves
were between R2 = 0.94 for 5b,6b-epoxycholesterol to R2 = 0.997
for triol. Cholesterol and cholestadienes content was expressed as
2. Materials and methods
equivalents of 5a-cholestane in mg per g of heated cholesterol
2.1. Materials
standard, where as oxysterols content was expressed as mg of
19-hydroxycholesterol per g of heated cholesterol standard. Identi-
fication of compounds was made on the basis of mass spectral
libraries (NIST 47, NIST 147 and Wiley 175) as well as data from lit-
erature and by comparison of their retention times with authentic
Cholesterol and 5a-cholestane standards were purchased from
Sigma–Aldrich (St. Louis, MO, USA) and 19-hydroxycholesterol
from Steraloids (Newport, RI, USA). Solvents (acetone, hexane,
methanol, diethyl ether, potassium hydroxide, tetrahydrofuran)
were purchased from POCH (Gliwice, Poland), while 1,2-dichlo-
rometane from Aldrich (Dorset, England). A sylilation agents BSTFA
(N,O-Bis(trimethylsilyl) trifluoroacetamide) with 1% TMCS
(trimethylchlorosilane) and anhydrous pyridine were purchased
from Sigma-Aldrich (St. Louis, MO, USA). Cholesterol oxidation
standards, including cholesterol, 7b-hydroxycholesterol, 5a,6a-
epoxycholesterol, 5b,6b-epoxycholesterol, triol, 7-ketocholesterol,
25-hydroxycholesterol. Three replicates per each sample were
analysed.
standards: 7b-hydroxycholesterol, 5
epoxycholesterol, 5 -cholestane-3b, 5b,6b-triol (triol), 7-ketocho-
lesterol, 25-hydroxycholesterol came from Sigma-Aldrich Co.
a,6a-epoxycholesterol, 5b,6b-
2.4. Determination of fragmented cholesterol molecules
a
Fragmented cholesterol molecules were separated on SPE car-
tridges. 2 mg of heated cholesterol sample, diluted in hexane,
was applied on SPE column (conditioned with 2 mL of hexane).
Column was washed firstly with 4 mL of hexane: diethyl ether
(75:25; v/v) and 15 mL of hexane: diethyl ether (60:40; v/v) was
used to collect sterol (I fraction) and than fragmented cholesterol
molecules were eluted with 10 mL of acetone (II fraction). To the
´
(Poznan, Poland). SPE DSC-Si Silica tubes (1 g/6 mL) and SPME
Fibre divinylbenzene/carboxene/.
polydimtheylsiloxane (DVB/CAR/PDMS) (2 cm) were purchased
from Supelco (Bellefonte, PA, USA).
2.2. Sample heat treatment
second fraction 0.2 mL of 5a-cholestane solution (10 mg/25 mL of
Cholesterol standard (10–20 mg) was placed in a glass ampoule
of 20 mL capacity and the glass neck of the ampoule was closed
over the burner flame. Closed ampoules were heated at 120, 150,
180 and 220 °C for 30, 60, 120 and 180 min. Heating procedures
were done in triplicate. A control sample was a sample of non-
heated cholesterol standard.
chloroform) was added Fraction was evaporated under a stream
of nitrogen. Samples were dissolved in 2 mL of hexane and sapon-
ified with addition of 0.5 mL of 2 N potassium methoxide for 1 h at
room temperature. Afterwards, solvents were removed under a
stream of nitrogen and samples were derivatized for 24 h at room
temperature due to addition of 100
lL of anhydrous pyridine and
100 L of sylilation agents. Fragmented cholesterol molecules
l
2.3. Determination of cholesterol, oxysterols and cholestadienes
were analysed on gas chromatograph equipped with a mass spec-
trometer (GCMS-QP2010S) Shimadzu Corporation (Shim-Pol A. M.
Cholesterol, oxysterols and cholestadienes were separated on
SPE cartridges. 2 mg of heated cholesterol sample, diluted in hex-
ane, was applied on SPE column (conditioned with 2 mL of hex-
ane). Column was washed with 4 mL of hexane:diethyl ether
(75:25; v/v), cholesterol was eluted with 15 mL of hexane: diethyl
ether (60:40; v/v) (I fraction) whereas oxysterols and cholestadi-
enes with 10 mL of acetone (II fraction). To the first fraction
Borzymowski, Poland) using. DB5ms (30 m ꢀ 0.25 mm ꢀ 0.25
lm)
capillary column Phenomenex (Torrance, CA, USA). Helium was
used as a carrier gas at a flow rate of 0.61 mL/min. The injector
temperature was set at 250 °C, and the column temperature was
programmed as follows: 200 °C for 1 min, subsequent increase to
250 °C at the rate of 15 °C/min, then to 310 °C at the rate of 3 °C/
min for 6 min. The interface temperature for GC–MS was 260 °C.
Temperature of ion source was 250 °C and ionisation energy was
70 V. The split ratio was 50:1. The total ion monitoring (TIC) was
used to detect fragmented cholesterol molecules (m/z ranged
0.2 mL of 5
was added and to the second fraction 0.2 mL of 19-hydroxycholes-
terol solution (8.5 mg/25 mL of chloroform) and 0.2 mL of -cho-
a-cholestane solution (10 mg/25 mL of chloroform)
a
lestane solution (10 mg/25 mL of chloroform) was added.
Fractions were evaporated under a stream of nitrogen. Samples
were dissolved in 2 mL of hexane and saponified with addition of
0.5 mL of 2 N potassium methoxide for 1 h at room temperature.
Afterwards, solvents were removed under a stream of nitrogen
100–600). The internal standard
semi-quantify fragmented cholesterol molecules. Fragmented ste-
rol molecules content was expressed as equivalents of -choles-
tane in mg per g of heated cholesterol standard. Identification of
compounds was made on the basis of mass spectral libraries (NIST
5a-cholestane was used to
a