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j o u r n a l o f f o o d a n d d r u g a n a l y s i s x x x ( 2 0 1 5 ) 1 e9

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1 ScienceDirect 66
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5 journal homepage: www.jfda-online.com 71
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9 Original Article 75
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Determination of cholesterol and four phytosterols 77
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in foods without derivatization by gas 79
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16 chromatography-tandem mass spectrometry 81
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19 Q7 Yan-Zong Chen a, Shih-Yao Kao a, Hao-Cheng Jian a, Yu-Man Yu a, 85
20 Ju-Ying Li a, Wei-Hsien Wang b, Chung-Wei Tsai a,* 86
21 87
22 a
Department of Food Services, SGS Taiwan Ltd., Kaohsiung, Taiwan, ROC 88
23 b
Department of Marine Biotechnology and Resources, National Sun Yat-Sen University, Kaohsiung, Taiwan, ROC 89
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26 article info abstract 92
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28 Article history: In this study, a method for determination of cholesterol and four phytosterols by gas 94
29 95
Received 14 October 2014 chromatography coupled with electron impact ionization modeetandem mass spectrom-
30 96
Received in revised form etry without derivatization in general food was developed. The sample was saponified with
31 97
21 December 2014 7.5% KOH in methanol. After heating on hot plate and reflux for 60 minutes, the saponified
32 98
33 Accepted 2 January 2015 portion was extracted with n-hexane/petroleum ether (50:50, v/v). The extracts were
99
34 Available online xxx evaporated with rotary evaporator and then redissolved with tetrahydrofuran. The tetra-
100
35 hydrofuran layer was transferred into an injection vial and analyzed by gas chromatog-
101
36 Keywords: raphy on a 30 m VF-5 column. Limit of quantification was 2 mg/kg. Recoveries of 102
37 cholesterol cholesterol and four phytosterols from general food were between 91% and 100%. 103
38 gas chromatographyetandem mass Copyright © 2015, Food and Drug Administration, Taiwan. Published by Elsevier Taiwan 104
39 spectrometry LLC. All rights reserved. 105
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phytosterol
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47 chains, cholesterol increases membrane packing, which re-
1. Introduction 113
48 duces membrane fluidity [4]. The structure of the tetracyclic
114
49 ring of cholesterol contributes to the decreased fluidity of the
115
50 Sterols are tetracyclic lipid components found in animals, cell membrane as the molecule is in a trans conformation,
plants, and microorganisms. Several hundred different 116
51 making all but the side chain of cholesterol rigid and planar 117
52 structures have been identified to date. While cholesterol is [5]. In this structural role, cholesterol reduces the permeability 118
53 the major sterol in animals [1], the most common represen- of the plasma membrane to neutral solutes [6], protons (pos- 119
54 tatives in the plant kingdom are b-sitosterol [2], campesterol itive hydrogen ions), and sodium ions [7]. 120
55 [3], and stigmasterol [3]. 121
Phytosterols are plant compounds that have similar
56 Cholesterol is required to build and maintain membranes. 122
chemical structure and biological functions as cholesterol [8].
57 Through the interaction with the phospholipid fatty-acid 123
58 Phytosterols contain an extra methyl group, ethyl group, or
124
59 125
60 * Corresponding author. Department of Food Services, SGS Taiwan Ltd., 61 Kai-Fa Road, Nanzih Export Processing Zone, Kaohsiung 126
61 81170, Taiwan, ROC. Q1 127
62 E-mail address: [email protected] (C.-W. Tsai). 128
63 http://dx.doi.org/10.1016/j.jfda.2015.01.010
129
64 1021-9498/Copyright © 2015, Food and Drug Administration, Taiwan. Published by Elsevier Taiwan LLC. All rights reserved.

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 JFDA214_proof ■ 21 April 2015 ■ 2/9

2 j o u r n a l o f f o o d a n d d r u g a n a l y s i s x x x ( 2 0 1 5 ) 1 e9

1 double bond. The suggested daily dietary intake of phytos- heptane at 25 C. Intermediate single standards solutions of 66
2 terols is from 160 mg to 400 mg for different races of humans cholesterol, brassicasterol, stigmasterol, b-sitosterol, and 67
3 [9e16]. Phytosterols are known to have hypocholesterolemic campesterol were prepared in THF at a concentration of 10 mg/ 68
4 properties. Phytosterols analogs are suggested to lower L and stored in a refrigerator at 25 C (stable for 1 month). 69
5 70
cholesterol absorption and the lower the serum cholesterol Mixtures of all chemicals were freshly made at five different
6 71
Q2 level, leading to cardiologic health benefits [1,7]. concentrations (2 mg/L, 5 mg/L, 10 mg/L, 20 mg/L, 50 mg/L, and
7 72
Cholesterol, corresponding precursors, and phytosterols in 100 mg/L) for the preparation of calibration standards.
8 73
9 human blood have been determined by gas chromatography THF, n-heptane, n-hexane, petroleum ether, and methanol 74
10 (GC)emass spectrometry (MS) [17]. GC-MS is also executed to were analytical grade and supplied by Merck (Billerica, MA, 75
11 analyze cholesterol, corresponding precursors and phytos- USA). Potassium hydroxide (KOH), anhydrous sodium sulfate 76
12 terols in cultured cells [18]. Rocco and Fanali [19] tried to were supplied by Macron (Center Valley, PA, Mexico). Deion- 77
13 determine phytosterols by nanoliquid chromatographyeMS. ized water was obtained using a Millipore purification system 78
14 AOAC published an official method 994.10 as analysis of (Millipore, Billerica, MA, USA) with a specific resistance of 79
15 cholesterol in foods by GCeflame ionization detector after 18.2 MU cm. 7.5% KOH in methanol was prepared by adding 80
16 saponification and derivatization with trimethylchlorosilane 75 g KOH in 750 mL methanol. The extraction solvent con- 81
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[20]. However, determination of cholesterol has been treated sisted of n-hexane:petroleum ether (50:50, v/v).
18 83
with derivatization during sample preparation in past reports,
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20 and there was no research to show an assay of cholesterol and 2.3. Food samples 85
21 phytosterols by GCetandem MS (MS/MS). In this study, we 86
22 aimed to develop a method of determination of cholesterol Plant oil (containing olive oil and grape seed oil), chicken eggs, 87
23 and four phytosterols without derivatization by GC-MS/MS in milk powder, beverages (milk, tea, and juice), and dietary 88
24 20 minutes. supplement foods (for elderly people or patients) were 89
25 collected as testing samples. All were bought from super- 90
26 markets and then stored at 20 C. All samples were well ho- 91
27 2. Methods mogenized with a blender. 92
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2.1. Apparatus 2.4. General procedure
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32 The GCeelectron impacteMS/MS system consisted of a Well-homogenized food samples (1 g pure oil and 5 g general 97
33 Bruker456-GC system (Bruker, Singapore) connected to a Scion materials) were accurately weighed into 250-mL Erlenmeyer 98
34 TQseries triple-stage quadrupole mass spectrometer (Bruker, flask and spiked with 0.2 mL 1000 mg/L internal standard into 99
35 Philadelphia, PA, USA). GC analysis was performed on a VF- the matrix, then added to a flask containing 50 mL 7.5% KOH in 100
36 5ms (30 m
0.25 mm, film thickness ¼ 0.25 mm; Agilent methanol. The flask was placed on a hot plate, a condenser 101
37 Technologies, Amstelveen, The Netherlands) at 280 C. N2 was attached, the hot plate turned on with the controller, and the 102
38 applied as carrier gas. Total running time was 20 minutes. The mixture refluxed for 60 ± 10 minutes to ensure complete 103
39 injection volume was 1 mL. saponification. 104
40 105
The MS detection system included an electron impact After cooling the solution to room temperature, the
41 106
ionization. Its energy was fixed at 70 eV. Temperatures of ion saponified test portion was transferred to a Rohrig extraction
42 107
43 source and transfer line were set at 200 C and 300 C, respec- tube. The saponified test portion was extracted with 50 mL n- 108
44 tively. Argon was used as the collision-induced dissociation hexane:petroleum ether (50:50, v/v) three times. The upper 109
45 gas at a pressure of 1.5 mTorr. layer (organic phase, about 150 mL) was collected into a sep- 110
46 Heating plates contain heat controls. Rotary evaporator aratory funnel and the lower layer discarded. The collected 111
47 with glass condenser flask between concentration flask and organic phase was washed with 40 mL H2O in a gently rotating 112
48 metal shaft were applied. Glassware used included 250-mL separatory funnel. After allowing layers to separate the lower 113
49 Q3 Erlenmeyer flasks, 250-mL separatory funnel, volumetric aqueous phase was discarded. The H2O wash step was 114
50 flasks, pipets, 250-mL Rohrig extraction tubes, glass funnels, repeated at least three times until the layers were neutral 115
51 116
and graduated cylinders. (pH ¼ 7). The upper organic phase from the separatory funnel
52 117
was poured through a glass funnel containing 20 g sodium
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2.2. Reagents and solutions sulfate in a filter paper into another clean 250-mL Erlenmeyer
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55 flask, and the funnel rinsed twice with 5 mL n-hexane:petro- 120
56 Cholesterol standard (purity > 99%) was purchased from Sig- leum ether (50:50, v/v). All eluates were evaporated to dryness 121
57 maeAldrich (St Louis, MO, USA). Brassicasterol (pu- on a rotary evaporator at 40 ± 1 C. The residues were recon- 122
58 rity > 92.5%), stigmasterol (purity > 89%), and b-sitosterol stituted with 5 mL THF. The final solution was filtered using a 123
59 (purity > 92%) were supplied by ChromaDEX (Irvine, CA, USA). 0.22-mm filter and the sample was transferred into a vial. An 1- 124
60 Campesterol (purity > 99%) was purchased from Sigma- mL aliquot was injected onto the GC column. 125
61 eAldrich (Munich, Germany). As an internal standard, 5a- 126
62 cholestane (purity > 97%) was provided by SigmaeAldrich 2.5. Method performance and validation 127
63 128
(USA). Individual stock standard solutions were prepared at a
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concentration of 1000 mg/L in tetrahydrofuran (THF; stable for Validation of this analytical method was performed by
65 130
3 months), apart from 5a-cholestane, which was prepared in n- assessment of the specificity, linearity, accuracy, precision,

Please cite this article in press as: Chen Y-Z, et al., Determination of cholesterol and four phytosterols in foods without
derivatization by gas chromatography-tandem mass spectrometry, Journal of Food and Drug Analysis (2015), http://dx.doi.org/
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 JFDA214_proof ■ 21 April 2015 ■ 3/9

j o u r n a l o f f o o d a n d d r u g a n a l y s i s x x x ( 2 0 1 5 ) 1 e9 3

1 and limit of quantification (LoQ). For evaluating specificity of The LoQ was evaluated with spiking the lowest concen- 66
2 testing method, six kinds of common food were selected tration level of calibration curve as 2 mg/kg in a blank sample. 67
3 including plant oil, eggs, milk powder, beverages, and dietary But analyzing cholesterol in eggs and phytosterols in plant oil 68
4 supplement foods. We spiked specific concentrations of whenever a real blank are impossible to obtain. Thus, the Q4 69
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cholesterol, brassicasterol, stigmasterol, b-sitosterol, and lowest standard on the calibration curve should be accepted
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campesterol standards in each kind of testing sample. It was as the LoQ given that the way set by the referred guidance are
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possible to avoid the matrix effects with this procedure. met. The analyte peak should be identifiable, discrete, and
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9 Linearity was evaluated by fresh preparing of standard reproducible with a precision of 20% and accuracy of 80e120% 74
10 solution, at five concentration levels in the interval of [21]. 75
11 2e100 mg/L for all cholesterol and four phytosterols. 76
12 The accuracy was evaluated through participating in pro- 77
13 ficiency testing from the Food Analysis Performance Assess- 3. Results and discussion 78
14 ment Scheme (FAPAS). Certified reference materials such as 79
15 milk powder for cholesterol testing were also executed during 80
3.1. Method development
16 routing tests to maintain reliable quality control. Recovery 81
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data were considered acceptable when the accuracy was The GCeelectron impacteMS/MS method was developed to
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within ± 10% of the target value. Precision (intra- and interday) provide confirmatory data for the analysis of general foods for
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20 was calculated by analysis of samples fortified with each cholesterol, brassicasterol, stigmasterol, b-sitosterol, and 85
21 cholesterol, brassicasterol, stigmasterol, b-sitosterol, and campesterol whose structures are shown in Fig. 1. The MS/MS 86
22 campesterol standards at fortification level (20 mg/kg), and fragmentation conditions were investigated and collision en- 87
23 the experiments were performed by the same operator in ergies were optimized for each individual compound. In all 88
24 triplicate at the same day and on 12 separate occasions in a cases, the tandem mass spectrometer was operated in the 89
25 month by three different operators. Ion ratios (peak area of 90
electron ionization mode at 70 eV. The retention times and the
26 confirmation ion pair/peak area of quantitation ion 91
characteristic fragments of the electron ionization mode mass
27 pair
100%) of the described cholesterol, brassicasterol, 92
spectra were determined by multiple reaction monitoring
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stigmasterol, b-sitosterol, and campesterol were 18 ± 2, 24 ± 1, mode. These ions were selected as the precursor ions, the
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74 ± 10, 51 ± 2, and 62 ± 5 (n ¼ 60). most abundant product ions were selected the most sensitive
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Fig. 1 e Structures of (A) b-sitosterol, (B) stigmasterol, (C) brassicasterol, (D) campesterol, and (E) cholesterol.

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derivatization by gas chromatography-tandem mass spectrometry, Journal of Food and Drug Analysis (2015), http://dx.doi.org/
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Fig. 2 e Q1 scan (MS1) and production scan (MS2; (A) b-sitosterol, (B) stigmasterol, (C) brassicasterol, (D) campesterol, (E)
64 Q8
129
cholesterol, and (F) 5a-cholestane) spectra.
65 130

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1 66
Table 1 e Transition reactions monitored of cholesterol and four phytosterols by gas chromatographyeelectron
2 67
impactetandem mass spectrometry, retention time, and peak area ratio.
3 68
4 Analyte Transition reactions (m/z) Retention time (min) Peak area ratio (%) 69
5 Quantitation ion pair Confirmation ion pair 70
6 71
b-sitosterol 329 / 189 413 / 329 15.253 51 ±2
7 72
Stigmasterol 411 / 211 411 / 379 13.652 74 ± 10
8 73
Brassicasterol 397 / 365 271 / 253 11.475 24 ±1
9 74
Campesterol 315 / 189 399 / 315 12.957 62 ±5
10 75
Cholesterol 386 / 159 386 / 301 10.475 18 ±2
11 5a-cholestanea 372 / 217 d 6.255 d
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a
13 Internal standard. 78
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Fig. 3 e Multiple reaction monitoring chromatogram for each of the target analytes in blank matrix (potato starch) extract
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spiked at 10 mg/kg and 50 mg/kg.

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6 j o u r n a l o f f o o d a n d d r u g a n a l y s i s x x x ( 2 0 1 5 ) 1 e9

1 transition for quantification purposes and a second one for campesterol, and cholesterol, respectively. The average cor- 66
2 confirmation. It shows MS/MS transitions (Q1 scan and prod- rected recovery of cholesterol in different matrix such as bev- 67
3 uct ion scan) for quantification and confirmation for each of erages, dietary supplement, eggs, and milk powder was 100%, 68
4 the selected compounds in Fig. 2. For a method to be deemed 100%, 95%, and 94%, respectively. The usefulness of a suitable 69
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confirmatory one parent ion and two daughter ions must be isotope internal standard was demonstrated in the excellent
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monitored (Table 1). This yielded four identification points, reproducibility and interday and intraday reproducibility is
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which provided a suitable confirmatory method in accordance obtained by using the internal standard of cholesterol (Table 2).
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9 with 2002/657/EC [22]. Although no internal standard (5a-cholestane) is available for 74
10 GC columns and conditions were studied in order to opti- b-sitosterol, stigmasterol, brassicasterol, and campesterol, an 75
11 mize the chromatographic separation in terms of resolution acceptable repeatability about intraday and interday repro- 76
12 and overall analysis time. Due to the different properties of ducibility was obtained. The developed method was evaluated 77
13 compounds under investigation. Helium carrier gas was sub- by comparison of results when this method was performed and 78
14 sequently found to give the most reliable result, good peak then the results were then passed in FAPAS proficiency tests. 79
15 shape, and good resolution on VF-5 ms (30 m
0.25 mm film The test result of 142.2 mg/100 g of cholesterol in mixed fat 80
16 thickness ¼ 0.25 mm (Agilent Technologies). Product ion spread compared with 133 mg/100 g FAPAS assigned value, 81
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spectra resulting from collision-induced dissociation were report No. 14119, 2013 was 0.8 of z-score. In real sample testing,
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examined and suitable ions selected for multiple reaction it was a positive case in terms of residue of cholesterol and
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20 monitoring schemes (Fig. 3). phytosterols in milk powder and grape seed oil, respectively. 85
21 Numerous GC methods for the determination of cholesterol There were no significant matrix effect in this testing method. 86
22 and phytosterols, such as cholesterol in serum or brassicasterol, The chromatogram is shown in Fig. 4. 87
23 stigmasterol, b-sitosterol, and campesterol in plant oil have been LoQs were evaluated by spiking the lowest concentration 88
24 proposed [17,20]. GC-MS has been applied to determine choles- of cholesterol and four phytosterols in blank samples [23,24]. 89
25 terol and phytosterols [18]. Nanoliquid chromatography has All signal to noise ratios of peaks for analytes after sample 90
26 been used to analyze phytosterols in plant oils such as olive oil pretreatment at lowest spiked concentration level have to be > 91
27 [19]. Most of them are applied GCeflame ionization detector or 10 during triplicate tests. LoQs were 2 mg/kg for four phytos- 92
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GC-MS determine the amount of cholesterol in samples with terols and cholesterol.
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derivatization procedure during sample preparation [17e20]. Based on these acceptable results of method validation, the
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Development of determination of cholesterol, brassicasterol, method that we described in this study could be executed in
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32 stigmasterol, b-sitosterol, and campesterol in general foods by the analysis of cholesterol and four phytosterols in plant oil, 97
33 GC-MS/MS without derivatization is required. eggs, milk powder, beverages, and dietary supplement foods. 98
34 Comparison with past method [17e20], we can determine 99
35 3.2. Method validation cholesterol and four phytosterols by GC-MS/MS in 20 minutes 100
36 and get sample pretreatment without derivatization. 101
37 The linearity of the chromatographic response was tested 102
38 using six concentration levels in the range of 2e100 mg/L. The 103
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linear regression (r) for all the calibration curves used in this 4. Conclusion
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study was  0.995.
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42 The recoveries of this method were determined using plant In this work, we have shown that the combination of GC with 107
43 oil (n ¼ 20) fortified at 20 mg/kg for four phytosterols and MS/MS detection provides reliable simultaneous quantitation 108
44 cholesterol. Mean recoveries (interday) of samples of analytes, and confirmation of cholesterol, brassicasterol, stigmasterol, 109
45 determined during 1 year (Table 2), were 93%, 94%, 95%, 91%, b-sitosterol, and campesterol in plant oil, eggs, milk powder, 110
46 and 91% for b-sitosterol, stigmasterol, brassicasterol, beverages, and dietary supplement foods. Good recoveries 111
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50 Table 2 e Results for repeatability of interday and intraday reproducibilities of b-sitosterol, stigmasterol, brassicasterol, 115
51 campesterol, and cholesterol in plant oil, eggs, milk powder, beverages, and dietary supplement foods. 116
52 117
Analyte Matrix Fortification concentration Intraday Interday
53 level (mg/kg) 118
54 Recovery (%) RSD (%) Recovery (%) RSD (%) 119
55 b-sitosterol 20 97 2 93 6 120
56 Stigmasterol Plant oil 20 97 3 94 8 121
57 Brassicasterol (n ¼ 20) 20 95 4 95 6 122
58 Campesterol 20 94 2 91 4 123
59 Cholesterol Beverages (n ¼ 20) 20 99 4 100 6 124
60 Dietary supplement foods (n ¼ 26) 20 98 4 100 8 125
61 Plant oil (n ¼ 20) 20 97 2 91 12 126
62 Eggs (n ¼ 16) 20 98 4 95 9 127
63 Milk powder (n ¼ 14) 20 97 5 94 8 128
64 RSD ¼ relative standard deviation. 129
65 130

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Fig. 4 e Typical chromatograms of spiked fortified concentration at 20 mg/kg for (A) cholesterol, (B) b-sitosterol, (C)
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stigmasterol, (D), brassicasterol, and (E) campesterol in (1) blank starch sample and (2) positive sample including cholesterol
64 129
and phytosterols in milk powder and grape seed oil, respectively.
65 130

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8 j o u r n a l o f f o o d a n d d r u g a n a l y s i s x x x ( 2 0 1 5 ) 1 e9

1 with excellent relative standard deviations were obtained accomplished using MS/MS, by comparison of peak ratios for 66
2 with multi sample matrix. Simpler sample preparation pro- two abundant product ions with those of standard samples. 67
3 cedure was executed without derivatization step. Confirma- The results were satisfactory for the development of a rugged 68
4 tion of the cholesterol and four phytosterols was analytical method in this study. 69
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Fig. 4 e (continued).

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derivatization by gas chromatography-tandem mass spectrometry, Journal of Food and Drug Analysis (2015), http://dx.doi.org/
10.1016/j.jfda.2015.01.010
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Please cite this article in press as: Chen Y-Z, et al., Determination of cholesterol and four phytosterols in foods without
derivatization by gas chromatography-tandem mass spectrometry, Journal of Food and Drug Analysis (2015), http://dx.doi.org/
10.1016/j.jfda.2015.01.010