Improved Analytical Method for Determination of Cholesterol

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New Analytical Methods

An improved analytical method for determination of cholesterol oxidation products in meat and animal fat by QuEChERS coupled with gas chromatography-mass spectrometry Che-Wei Chiu, Tsai Hua Kao, and Bing Huei Chen J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00250 • Publication Date (Web): 10 Mar 2018 Downloaded from http://pubs.acs.org on March 16, 2018

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Journal of Agricultural and Food Chemistry

An Improved Analytical Method for Determination of Cholesterol Oxidation Products in Meat and Animal Fat by QuEChERS Coupled with Gas Chromatography-Mass Spectrometry

Che-Wei Chiu, Tsai-Hua Kao, Bing-Huei Chen* Department of Food Science, Fu Jen University, New Taipei City 24205, Taiwan

*To whom correspondence should be addressed E-mail: [email protected] Tel: +886-2-29053626 Fax: +886-2-22093271 Postal Address: Department of Food Science, Fu Jen Catholic University, No. 510, Zhongzheng Road, Xinzhuang District, New Taipei City 242, Taiwan.

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ABSTRACT

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Cholesterol, widely present in animal fats and meat products, can undergo

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oxidation to form cholesterol oxidation products (COPs) during heating. The objectives

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of this study were to develop a QuEChERS method for reduction of solvent volume and

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extraction time for determination of COPs in edible animal fats and meat products by

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gas chromatography–mass spectrometry. By employing a DB–5MS capillary column (30

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m x 0.25 mm I.D., film thickness 0.25 µm) and a temperature programming method, 7

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COPs, cholesterol and internal standard 5α–cholestane could be separated within 19

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min. Limit of detection and limit of quantitation based on COPs standards ranged from

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0.16–180 ng/mL and 0.32–400 ng/mL, respectively, while the recoveries from 89.1–

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107.6% for boiled pork and 80.5–105.6% for lard. Intra-day variability for boiled pork

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and lard ranged from 2.27–6.87% and 1.52–9.78%, respectively, whereas inter-day

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variability from 1.81–7.89% and 3.57–9.26%. Among various meat samples, fish showed

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the highest level of COPs (31.84 µg/g). For edible fats, the COPs contents in tallow

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(22.79–60.15 µg/g) were much higher than in lard (0.152–2.55 µg/g) and butter (0.526–

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1.36 µg/g). Collectively, this method can be applied to determine COPs in cholesterol-

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containing foodstuffs.

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KEYWORDS: cholesterol oxidation products; QuEChERS; GC-MS; meat; animal fat.

19 20 21 22


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INTRODUCTION

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Cholesterol is needed to maintain normal cell function in humans. Approximately

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70% of the cholesterol needed is synthesized internally, while the remaining 30%

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cholesterol is acquired from animal based foods such as eggs, meat products and edible

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animal fat.1 However, cholesterol can undergo oxidation to form cholesterol oxidation

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products (COPs) during heating, light exposure or storage of cholesterol-containing food

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products.2-4 More than 100 COPs have been identified to date, and many of them are

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reported to be detrimental to human health.5 The formation of different varieties and

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amounts of COPs in foodstuffs can be affected by many factors such as heating

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temperature and time length, radiation, pH value, and presence of antioxidants or

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metals.2,6 The mechanism of cholesterol oxidation is similar to lipid oxidation, including

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autoxidation, thermal oxidation, photooxidation and enzymatic oxidation.7,8 Thus, it is

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imperative to learn more about the presence of different variety and amount of COPs in

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various food commodities.

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Most studies on the potential health hazard of COPs have shown that the intake of

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COPs in excess may lead to atherosclerosis or cardiovascular disease.9,10 In a study

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dealing with the effect of experimental diet containing 1% COPs on rat aorta, the COPs

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accumulation was observed with a plague formation in the artery wall.10 Meynier

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al.11 also demonstrated the toxicity of COPs towards vascular cells in hamsters

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administered with the diet containing various COPs for 15 days. Nevertheless, the

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administration dose for animal study is much higher than that can be consumed by

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humans based on body weight difference. It was estimated that the daily intake of COPs


et


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should be ≥98 mg to be detrimental to human health.11 It seems unlikely that such high

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dose can be consumed by humans. Nonetheless, the possible adverse effect caused by

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long-time accumulation of COPs in vivo cannot be overlooked.

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QuEChERS, a fast extraction and purification method, is mainly composed of

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extraction/partitioning and clean-up steps, with the former involving addition of organic

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solvent and water to samples for liquid-liquid extraction, addition of salts for partition

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through salting-out and isolation of compounds of interest from water, while the latter

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involving addition of dispersive solid phase extraction powder for removal of co-

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extractants or impurities for subsequent analysis by GC-MS or HPLC-MS.12 Ever since the

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invention of the QuEChERS method by Anastassiades et al.13 for analysis of residual

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pesticides in food samples, it was applied to determination of animal drugs in chicken

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meat by UHPLC/MS/MS,14 polychlorinated biphenyls (PCBs) in fish by GC/MS/MS,15

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polycyclic aromatic hydrocarbons (PAHs) in poultry meat by GC-MS,16 cholesterol in

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emulsified confectionaries by UPLC17 and acrylamide in coffee by GC-MS.18 However, no

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information is available as to the extraction and purification of COPs in foodstuffs by

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using a QuEChERS method.

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Accordingly, the analysis of COPs in foodstuffs can be carried out by the

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following

steps:

extraction,

saponification

or

transesterification,

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identification and quantitation by HPLC-MS or GC-MS.19 Compared to HPLC-MS, GC-MS

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was often used to determine COPs in foodstuffs because of its superior resolution

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power and short separation time.7 However, no internationally recognized method of

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COPs analysis in food samples has been established due to the following reasons: (1) the


purification,

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route of cholesterol oxidation is quite complex and can be affected by many factors, (2)

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the intermediate products formed during cholesterol oxidation are susceptible to

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change during analysis, (3) the levels of COPs present in food samples are quite low

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(ppm or ppb) and (4) the experimental error can occur during quantitation of different

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forms of COPs.20 Compared to the traditional solid-phase extraction method, both

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solvent volume and extraction time could be reduced substantially for the QuEChERS

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developed in our study. The objectives of this study were to develop a QuEChERS

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method combined with GC-MS for determination of COPs in meat and animal fat. To the

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best of our knowledge, this is the first report to use QuEChERS for COPs analysis in meat

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and animal fat.

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MATERIALS AND METHODS

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Chemicals. Cholesterol and cholesterol oxidation products (COPs) standards including

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5,6α-epoxycholesterol (5,6α-EP), 5,6β-epoxycholesterol (5,6β-EP), 7-keto cholesterol (7-

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keto), 25-hydroxycholesterol (25-OH), 5α-cholestane-3β,-5α, 6β-triol (triol) and internal

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standard 5α-cholestane were purchased from Sigma Co. (St. Louis, MO, USA). Both 7α-

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hydroxy cholesterol (7α-OH) and 7β-hydroxy cholesterol (7β-OH) standards were

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procured from Steraloids Co. (Wilton, NH, USA). For QuEChERS, the extraction powder

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(product no. UR-EX) containing 4g anhydrous magnesium sulfate (MgSO4) and 1 g

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anhydrous sodium acetate (CH3COONa), and the clean-up powder (product no. UR-

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CLEAN-II) containing 300 mg primary secondary amine (PSA), 900 mg MgSO4, 300 mg

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C18EC (octadecylsiloxane endcapped), and ceramic homogenizer were obtained from

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Yiu-Ho Co. (Taipei, Taiwan). In addition, the other extraction powder (product no. 5 ACS Paragon Plus Environment


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5982 – 1010) containing 1 g EMR-Lipid and purification powder (product no. 5982 –

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0101) containing 2 g NaCl-MgSO4 (1:4) were from Agilent Technologies Co. (Palo Alto, CA,

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USA). Solvents including methanol, acetonitrile, chloroform, acetone, and ethyl acetate

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obtained from Merck Co. (Darmstadt, Germany) were of HPLC grade (>99%). Pyridine

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(99%) was from J.T. Baker Co. (Philipsburg, NJ, USA). Deionized water was made using a

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Milli-Q water purification system from Millipore Co. (Bedford, MA, USA). The COPs

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derivatization agent Sylon BTZ composed of BSA (N,O-bis(trimethylsilyl)acetamide)-

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TMCS (trimethyl chlorosilane)-TMSI (N-trimethylsilylimidazole) (3:2:3, w/w/w) was from

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Supelco Co. (Bellefonte, PA, USA).

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Materials. Meat products including chicken cutlet, pork cutlet, sausage, sauryfish, boiled

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pork and smoked chicken with 6 samples each as well as edible animal fats including

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tallow, lard and butter with 5, 8 and 5 samples, respectively, were purchased from

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several local traditional markets in Taipei City. A total of 36 meat samples and 18 animal

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fat samples were used in this study. Prior to extraction, meat samples were cut into

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pieces, homogenized and stored at -20°C. For animal fat samples, they were stored at

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4°C and then melted at 40°C before extraction.

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For GC-MS analysis, three capillary columns including DB-5MS (30 m x 0.25 mm

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ID, film thickness 0.25 µm, 100% phenyl arylene polymer), DB-5MS (60 m x 0.25 mm ID,

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film thickness 0.25 µm, 100% phenyl arylene polymer), and HP-5MS (30 m x 0.25 mm ID,

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film thickness 0.25 mm, (5%-phenyl)-methyl polysiloxane, were purchased from Agilent

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Technologies Co. (Palo Alto, CA, USA).


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Methods. For COPs extraction and purification by QuEChERS, the four parameters

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including sample weight, solvent type, powder variety and weight, and vibration mode

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were evaluated. After various studies, a standard procedure was developed. Initially 2 g

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of sample (meat or fat) was collected and mixed with 10 mL deionized water and one

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ceramic homogenizer in a 50-mL centrifuged tube, followed by vibration for 1 min using

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a vortex mixer, addition of 10 mL acetone, vibration for 1 min again, addition of 4 g

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MgSO4 and 1 g CH3COONa, vibration again for 1 min, centrifugation at 3200 g (4°C) for

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10 min, and collection of upper organic phase for further purification. Next, 4 mL

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organic phase was mixed with 300 mg PSA, 900 mg MgSO4 and 300 mg C18EC in a 15-mL

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centrifuged tube, followed by vibration for 1 min, centrifugation at 3200 g (4°C) for 10

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min, and collection of supernatant (1 mL) for GC-MS analysis. Prior to GC-MS analysis,

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the supernatant was filtered through a 0.22 µm nylon membrane filter, after which a

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portion (40 µL) was poured into a 2-mL vial containing a 250-µL inner tube and then 20

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µL 5α-cholestane (10 ppm) in pyridine was added. Next, 40 µL Sylon BTZ was added and

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reacted at 25°C in the dark for 1 h for COPs derivatization, after which one µL was

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injected for GC-MS analysis with the following conditions: the injector temperature

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280°C, MS interface temperature 300°C, ionization source temperature 260°C,

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quadrupole temperature 150°C, carrier gas He with flow rate 1 mL/min, splitless mode,

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and the oven temperature was controlled at 250°C in the beginning, raised to 290°C at

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10°C/min, maintained for 5 min, increased to 291°C at 0.1°C/min and maintained for 1

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min. A total of 7 COPs, cholesterol and internal standard 5α-cholestane were separated

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within 19 min (Figure 1). The various COPs in food samples were identified by comparing 7 ACS Paragon Plus Environment


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retention times and MS spectra of unknown peaks with reference standards, and

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addition of standards to sample for co-chromatography. In addition, the selected ion

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monitoring (SIM) was used for detection of COPs to enhance sensitivity. However, we

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have to point out here that the COPs after GC-MS identification are indeed trimethylsilyl

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(TMS) derivatives, not native sterols. Based on several previous studies,2,3 only bis-TMSE

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(bis-trimethylsilyl ether) can be formed using the derivatization condition shown above

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instead of tris-TMSE, which may cause degradation of COPs. In other words, a complete

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derivatization was attained with minimal degradation of COPs. To make it easier for

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further discussion, the common names of COPs were used instead. According to elution

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order, the internal standard 5α-cholestane (5-10 min), 7α-OH (10-12 min), cholesterol

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(12-13 min), 7β-OH (13-14.2 min), 5,6β-EP and 5,6α-EP (14.2-16 min), triol (16-17.2 min),

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25-OH and 7-keto (17.2-21 min) were divided into 7 groups, and their identification was

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based on the m/z value for the abundant ions obtained by SIM mode at 217.2, 456.5,

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329.3, 456.4, 384.3 and 474.5, 403.4 and 456.4, as well as 131.1 and 367.3, respectively

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(Figure 2; Table 1). Perfluorotributylamine was used for autotune with m/z at 69, 219

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and 502.

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Method Validation. From a stock solution containing a mixture of 7 COPs with each COP

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at a concentration of 1000 µg/L, a total of 22 concentrations (0.16, 0.24, 0.32, 0.8, 1.6,

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3.2, 4, 8, 16, 20, 24, 32, 40, 60, 80, 100, 120, 140, 160, 180, 200 and 400 µg/L) of COPs

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standard mixture were prepared using pyridine as solvent and injected into GC-MS for

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SIM detection. The limit of detection (LOD) was based on S/N≥3, while the limit of

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quantitation (LOQ) was based on S/N≥10 for the COPs standards solutions. Similarly, the 8 ACS Paragon Plus Environment

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LOQ in palm oil and boiled pork was based on S/N≥10. Palm oil was selected instead of

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lard as it is difficult to find an animal fat without COPs and its matrix composition is

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similar to lard. Likewise, the LOQ of boiled pork was based on COPs standards as it is

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impossible to find pork without COPs. For the precision study, the repeatability, intra-

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day variability and inter-day variability were determined. The injection repeatability was

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performed by injecting the same sample 3 times, while the analytical repeatability was

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carried out by preparing 3 samples and analyzing each sample in triplicate for a total of

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9 analyses, followed by comparing the relative standard deviation (RSD, %). In addition,

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the intra-day variability was carried out by analyzing samples containing COPs standards

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in the morning, afternoon and evening on the same day in triplicate for a total of 9

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analyses, whereas the inter-day variability was performed by analyzing samples

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containing COPs standards on the 1st, 2nd and 3rd day with 3 times each for a total of 9

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analyses. The variation in levels of COPs were determined and the RSD (%) calculated for

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comparison (Table 2). For the accuracy study (recovery), two concentrations of 7 COPs

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standards each were added separately to 2 g lard or boiled pork for a final concentration

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of 0.5 and 1 mg/L. Then, both extraction and purification by QuEChERS were done for

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analysis by GC-MS and the recovery of each COP was determined based on the relative

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ratio of the concentration of COP standard after GC/MS to the concentration of COP

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standard added before GC-MS.

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Preparation of Standard Curves. Five multilevel (0.4, 1.2, 2, 4 and 10 mg/L) mixed

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standards of 7 COPs with internal standard 5α-cholestane at 2 mg/L were prepared for

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meat samples. But for animal fat samples, 5 concentrations (0.08, 0.2, 0.4, 2, and 4 mg/L)



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were prepared for 7α-OH and 7β-OH, while 0.4, 1.2, 2, 4, and 10 mg/L for 5,6α-EP and

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5,6β-EP, as well as 0.04, 0.08, 0.2, 2, and 4 mg/L for triol. Similarly, 0.08, 0.1, 0.18, 0.2,

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and 0.4 mg/L were prepared for 25-OH and 0.4, 2, 4, 10 and 30 mg/L for 7-keto. Likewise,

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all the COP standard concentrations contained 5α-cholestane at 2 mg/L for animal fat

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samples. Following derivatization, each COP standard was injected into GC-MS. Then the

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standard curve of each COP was obtained by plotting concentration ratio (COP standard

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versus internal standard) against area ratio (COP standard versus internal standard), and

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both the linear regression equations and determination of coefficient (R2) were

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obtained automatically by using an Excel software system. The amount of each COP in

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meat and fat samples was calculated by using the following formula: Amount of COPs

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µg As 1 1 1 = − b x x Ci x extraction volume x dilution factor x x g Ai a recovery sample weight !g"

Where As: peak area of COP

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Ai: peak area of internal standard

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b: intercept of the standard curve

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a: slope of the standard curve

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Ci: concentration of internal standard

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Matrix Effect Determination. Matrix effect (ME) was determined by mixing several

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COPs standard concentrations with sample solution (with matrix components) and with

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solvent (without matrix components) separately and subjected to QuEChERS and GC-MS

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analyses. Then two linear regression equations with and without matrix components

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were obtained. Then the matrix effect was calculated using the following formula:21,22


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ME !%" = ME =

MCC − SCC x 100 SCC

MCC SCC

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where, SCC represents the standard calibration curve slope and MCC represents the

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matrix matched calibration curve slope.

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Statistical Analysis. All the experimental data were analyzed using the statistical analysis

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system23 for ANOVA analysis and Duncan’s multiple range test for significance in mean

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comparison (P40 min). Of the other two

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columns (HP-5MS) and (DB-5MS) with the same length (30 m), the separation time was

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similar, however, the latter provided a better separation efficiency in terms of retention

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factor (k), separation factor (α) and resolution (RS). Thus, a DB-5MS capillary column (30

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m x 0.25 mm ID, film thickness 0.25 µm) could simultaneously separate cholesterol, 7

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COPs standards and internal standard 5α-cholestane within 19 min by using the GC-MS

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condition shown in the method section (Figure 1). All the 9 peaks were adequately

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resolved with the retention time ranging from 8.7–18.2 min, retention factor (k) from

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2.92–5.18, separation factor (α) from 1.03–1.48 and resolution (Rs) from 1.03–9.06



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(Table 1). This result implied that an appropriate column and separation condition was

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established.

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For COPs identification in animal fat and meat samples, the selected ion

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monitoring (SIM) mode was used instead of the full scan (total ion chromatogram, TIC)

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mode as the former was reported to show higher sensitivity than the latter.24,25 Table 1

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shows the mass to charge (m/z) ratio used for identification of 5α-cholestane,

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cholesterol and various COPs, and quantitation of COPs by SIM mode. All the target

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compounds were divided into 7 groups according to elution order, and several

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characteristic ions were selected to detect a specific compound in each group (Figure 2).

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Following this approach, a higher sensitivity and selectivity was obtained as the

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interference caused by impurities in food samples could be reduced to a minimum. In

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the literature reports many authors also used the same method for COPs detection in

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milk,26 lard,27 marinated foods,25 tea-leaf eggs2 and pig feet.3

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Optimization of QuEChERS Method for Extraction and Purification of COPs. Four

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parameters including solvent type, sample weight, powder variety and weight, and

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vibration mode were investigated for evaluation of extraction efficiency of COPs. Initially

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one parameter was changed while the other three parameters fixed. For instance, when

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one solvent was used, the sample weight was controlled at 2 g and the vibration mode

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was vortex mixing, while the extraction powder composed of 4 g magnesium sulfate and

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1 g sodium acetate and the purification powder composed of 300 mg PSA, 300 mg

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C18EC and 900 mg magnesium sulfate. Next, the sample weight 2 g and 10 g were

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compared for extraction efficiency. The selection of an appropriate solvent for


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extraction of target compounds by QuEChERS is vital for high extraction efficiency. Both

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acetonitrile and acetone were shown to extract both polar and non-polar analytes

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effectively.28 However, the former was superior to the latter in terms of oil samples due

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to a less amount of co-extractants. In addition, some other solvents such as ethyl

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acetate could produce more co-extractants.29 Thus, initially in our study four solvents

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including acetonitrile, acetone, ethyl acetate and chloroform/methanol (2:1, v/v) were

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selected to compare extraction efficiency of COPs. In addition, the selection of a suitable

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amount of sample is associated with moisture content in food samples. In other words,

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no water should be added if the sample moisture content is >75%. Conversely, an

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appropriate amount of water should be added or the sample amount be reduced if the

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sample moisture content is 24°C

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for 18 h, the recovery of 7-keto was only 53% (37°C) and 49% (45°C). Also, following a

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rise in the KOH strength from 1 M to 3.6 M (24°C for 18 h), the recovery of 7-keto

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further dropped to 71%.37 Furthermore, some more solvents such as hexane or diethyl

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ether are often used for extraction of COPs from unsaponifiable portion after

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saponification step.38 Collectively, the application of direct saponification without lipid

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extraction remains unsuitable in complex food matrices such as meat due to emulsion

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formation and presence of many interfering peaks from impurities.35-37

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To overcome the drawbacks of artifact generation and long time extraction

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during saponification, the SPE technique is usually employed either as a purification step

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after saponification or as a direct extraction of COPs from crude lipid extract. The

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extraction of COPs directly by SPE technique is simple, cheap and can minimize the

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artifact generation and COPs degradation.13 Also, with the sequential use of different

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solvents, most apolar compounds and cholesterol could be removed while the polar

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COPs eluted with a suitable solvent.13 However, without saponification, the total COPs

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level in food samples can be underestimated as a significant amount of COPs may still

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exist as esters and cannot be quantified.38 Thus, the results of COPs in this study were

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expressed as free COPs, as no saponification step was used for the QuEChERS method.

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Furthermore, the SPE technique usually use two or more cartridges or solvent mixtures. 15 ACS Paragon Plus Environment


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For instance, Ferioli et al.34 used two successive silica cartridges repeating the

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procedures twice by pre-equilibration with hexane, followed by washing with

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hexane/diethyl ether (3:1, v/v) and hexane/diethyl ether (3:2, v/v), and then eluting

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COPs with acetone/methanol (3:2, v/v). Likewise, Janoszka39 employed a multistage SPE

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procedure (3 stages) to concentrate COPs from the lipid extract. However, the

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employment of a low polar solvent can reduce the recovery of polar COPs such as triol.40

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Thus, compared to the traditional solid phase extraction method used by Chen et al.,2,3

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the QuEChERS method developed in this study is relatively simple, fast and low in

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solvent consumption, as evident by a reduction of solvent volume from 121 mL to 20.2

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mL and extraction time from 67.5 min to 33 min.

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Moreover, the presence of prooxidants such as metal ions (Fe2+, Fe3+, Cu+ and

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Cu2+) in meat and animal fat samples should be difficult to coextract with COPs by using

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the QuEChERS method as they are more soluble in aqueous phase due to presence in

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the free form or bound to proteins in meat. Thus, the cholesterol oxidation as affected

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by metal ions can be minimized during extraction. Nevertheless, cholesterol oxidation

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may still occur during analysis depending on extraction, purification, environmental and

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separation conditions. Thus, in this study we try to shorten the extraction, purification

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and separation time to decrease cholesterol oxidation to a minimum. Most importantly,

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the extraction was proceeded under nitrogen and temperature controlled at 25°C to

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minimize cholesterol oxidation rate.

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Method Validation. Table 2 shows the recovery of free COPs in lard and boiled pork by

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using QuEChERS method for extraction. A high recovery ranging from 89.1–107.6% was


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shown for lard and 80.5–105.6% for boiled pork, both of which were similar to that for

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blank samples, demonstrating a high accuracy of this method. The LOD for COPs

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standards including 7α-OH, 7β-OH, 5,6β-EP, 5,6α-EP, triol, 25-OH and 7-keto were 0.16,

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0.16, 100, 100, 16, 32 and 180 ng/mL, respectively, while the LOQ were 0.32, 0.32, 400,

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400, 40, 80 and 400 ng/mL (Table 3). Both LOD and LOQ were much lower than that

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reported by several other authors,2,41 which can be due to difference in sensitivity of GC-

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MS instruments. Comparatively, a very low LOD for both 7α-OH and 7β-OH may be due

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to their higher ion abundance values, while the lower ion abundance values may be

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responsible for high LOD obtained for 5,6β-EP, 5,6α-EP and 7-keto (Table 3; Figure 2).

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Furthermore, the LOQ in palm oil and boiled pork was expressed as ng/g based on

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sample weight, with the former being 2, 2, 400, 400, 50, 100 and 400 ng/g for 7α-OH,

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7β-OH, 5,6β-EP, 5,6α-EP, triol, 25-OH and 7-keto, respectively. However, for boiled pork,

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the LOQ was the same as that based on COPs standards.

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The precision data of free COPs in lard and boiled pork analyzed by GC-MS are

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shown in Table 2. In lard samples, the relative standard deviation (RSD) of the intra-day

340

variability for 7 COPs ranged from 1.81-7.89%, whereas the RSD of the inter-day

341

variability was from 3.57-9.26%. Similarly, in boiled pork samples, the RSD of the intra-

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day variability for 7 COPs ranged from 2.27-6.87%, while the RSD of the inter-day

343

variability was from 1.52-9.78%. All the data demonstrated a high repeatability and

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reproducibility of the QuEChERS method developed in this study.

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The matrix effects of free COPs in animal fat and meat samples determined by

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GC-MS are shown in Table 4. The matrix effect of GC-MS refers to the ionization capacity 17 ACS Paragon Plus Environment


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of analytes caused by enhancement or suppression of signals. Furthermore, the matrix

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effect (%) can be either positive or negative, with the former indicating signal or ion

349

enhancement and the latter signal or ion suppression. Accordingly, the matrix effect

350

between 20% ∼ -20% are designated to be “no matrix interference”.21,42 Alternatively,

351

based on the equation, ME = MCC slope/SCC slope, the matrix effect between 0.8–1.2

352

are designated to be “no matrix interference”.22 In lard samples, the matrix effect of 7

353

COPs ranged from -18.78–9.17% or 0.81–1.09, while in tallow samples, the matrix effect

354

was from -13.84–19.22% or 0.86–1.19 (Table 4). This outcome demonstrated no matrix

355

interference of the QuEChERS method developed in our study for animal fats. Similar

356

results were shown in pork, chicken and sauryfish samples, with the matrix effect

357

ranging from -4.71–23.48% or 0.95–1.23, -3.39–25.58% or 0.97–1.26, -10.25–22.75% or

358

0.9–1.23, respectively (Table 4). However, a slight matrix interference was shown for 7β-

359

OH in pork, 7α-OH, triol and 7-keto in chicken, as well as triol and 7-keto in sauryfish,

360

which may be due to the presence of a more complex matrix of meat compared to

361

animal fat. Collectively, most COPs in lard, tallow, pork, chicken and sauryfish samples

362

showed signal enhancement instead of signal suppression by GC-MS analysis in our

363

experiment. In a previous study Liu43 also reported that with GC-MS analysis of residual

364

pesticides in fruits and vegetables, most target compounds signals were enhanced.

365

Conversely, with HPLC-MS and electrospray ionization (ESI) detection, most target

366

compounds signals were suppressed. Likewise, Georgiou et al.22 used UPLC-MS/MS with

367

ESI made for COPs analysis in various food commodities and observed signal


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suppression for most COPs. Apparently the matrix effect can be varied with ionization

369

mode of HPLC-MS or GC-MS.

370

Contents of COPs in Meat and Animal Fat Samples. Table 5 shows the free COPs levels

371

in chicken cutlet, pork cutlet, sausage, sauryfish, boiled pork and smoked chicken. In

372

chicken cutlet, triol was present in the largest amount followed by 7α-OH, 7-keto, 7β-

373

OH, 5,6β-EP, 25-OH and 5,6α-EP. However, a different trend was found in the other

374

meat samples. Nevertheless, triol was more susceptible to formation than the other

375

COPs in chicken cutlet, pork cutlet, sausage, sauryfish, boiled pork, and smoked chicken.

376

It has been well established that triol can be formed from 5,6α-EP or 5,6β-EP in the

377

presence of water under acidic condition during prolonged heating of meat samples

378

because of liberation of organic acid.3 In addition to triol, both 7α-OH and 7β-OH as well

379

as 5,6β-EP were also generated in higher amount than the other COPs in pork cutlet,

380

sausage, sauryfish, boiled pork and smoked chicken, probably due to reduction of 7α-

381

hydroperoxycholesterol (7α-OOH) and 7β-hydroperoxycholesterol (7β-OOH) as well as

382

oxidation of cholesterol in the presence of cholesterol hydroperoxide, respectively.3

383

Both 7α-OOH and 7β-OOH can be formed during initial oxidation of cholesterol.

384

However, they are undetermined in this study as they are susceptible to degradation,

385

reduction or dehydration to form various types of COPs during heating of cholesterol-

386

rich foods.2,3 Also, the formation of 7-keto should be due to dehydration of 7α-OOH or

387

7β-OOH or oxidation of 7α-OH or 7β-OH, while 25-OH due to side chain oxidation of

388

cholesterol during heating of meat samples.3 For total COPs, saury fish possessed the

389

largest amount, followed by boiled pork, sausage, smoked chicken, pork cutlet and 19 ACS Paragon Plus Environment


390

chicken cutlet. Apparently, the formation of different variety and amount of COPs in

391

meat can be dependent upon heating temperature and time, meat variety, heating

392

method, and surface exposure to heat during processing. The GC-MS chromatograms of

393

COPs in various meat and animal fat samples are shown in Figure 4 and Figure S1

394

(Supplementary information), respectively. Based on the identification criteria shown in

395

the method section and GC-MS-SIM spectra in Figure 2, all the 7 COPs including 7α-OH,

396

7β-OH, 5,6β-EP, 5,6α-EP, triol, 25-OH and 7-keto were present in all the meat samples

397

purchased from Taiwan’s market.

398

The free COPs contents in lard and tallow are also shown in Table 5. In most lard

399

samples purchased from market, only 3 COPs, 7α-OH, 7β-OH and 7-keto were detected,

400

with the 7-keto level being higher than 7α-OH and 7β-OH, while triol was detected in

401

only two lard samples. As most lard sold on Taiwan’s market are processed by wet

402

rendering, 7-keto should be more liable to be formed than the other COPs due to

403

dehydration of 7α-OOH or 7β-OOH during heating of pig adipose tissues.6 The absence

404

of 5,6α-EP and 5,6β-EP in 8 commercial lard samples implied that they may undergo

405

complete degradation or further conversion to triol during heating. The presence of triol

406

in two lard samples indicated that a more drastic heating condition was employed for

407

lard preparation compared to the other lard samples. Interestingly, compared to lard,

408

some more COPs including 7α-OH, 7β-OH, 5,6β-EP, 5,6α-EP, triol, and 7-keto were

409

detected in 5 tallow samples purchased from Taiwan’s market. This outcome implies

410

that the processing condition of tallow should be more drastic than lard, as evident by a

411

much higher level of total COPs of the former (Table 5). Similar to lard, 7-keto was 20 ACS Paragon Plus Environment

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412

present in the largest amount while triol in the least amount. Conversely, both 5,6α-EP

413

and 5,6β-EP were detected in tallow. Also, triol was present at a higher level in tallow

414

than in lard, which may be due to liberation of more free fatty acids from hydrolysis of

415

triacylglycerols in tallow during heating.3 Similar outcomes were reported by Park and

416

Addis44 and Chiu et al.45

417

Comparatively, the level of triol was much higher in meat samples than in animal

418

fat samples (Table 5). Interestingly, in most meat samples the level of triol was higher

419

than 7-keto, while in all the lard and tallow samples, the level of 7-keto was much higher

420

than triol (Table 5). Theoretically, 7-keto should be more susceptible to formation than

421

triol during processing of meat products.3,6 As we purchased both meat and animal fat

422

samples from market, the higher triol level in meat samples indicated that a more

423

severe heating condition was used compared to animal fat samples. Also, in most meat,

424

lard and tallow samples, the level of 7α-OH was higher than 7β-OH, which may be due

425

to the reduction rate of 7α-OOH being faster than 7β-OOH during heating, though 7β-

426

OH is thermodynamically more stable.

427

Table 6 shows the free COPs contents in 5 butter samples purchased from

428

Taiwan’s market. Among the various samples, both 7α-OH and 7β-OH were the major

429

COPs present. Interestingly, a significant level of triol (0.292 µg/g) was found in

430

fermented butter (sample 5), which should be due to conversion of 5,6α-EP or 5,6β-EP

431

under acidic condition in the presence of milk during fermentation. Also, both unsalted

432

butter (samples 1 and 2) and salted butter (samples 3 and 4) showed a similar COPs



433

profile. Comparatively, the level of total COPs in butter was much lower than lard and

434

tallow, which can be due to a less severe processing condition for butter.

435

In conclusion, a QuEChERS method combined with GC-MS was developed for

436

extraction, purification, identification and quantitation of COPs in meat and animal fat.

437

A total of 7 COPs, including 7-keto, 7α-OH, 7β-OH, 5,6α-EP, 5,6β-EP, 25-OH, and triol as

438

well as cholesterol and internal standard 5α-cholestane were adequately separated

439

within 19 min by employing an appropriate temperature programming condition with

440

selected ion monitoring (SIM) detection. The extraction, purification, and separation

441

time as well as solvent volume were reduced substantially, while a high accuracy and

442

precision was obtained with this method. Sauryfish was shown to contain the highest

443

amount of total COPs, followed by boiled pork, sausage, smoked chicken, pork cutlet

444

and chicken cutlet. Also, tallow contained a much higher level of total COPs than lard

445

and butter.

446

ASSOCIATED CONTENT

447

Supporting Information. GC-MS chromatograms of trimethylsilyl derivatives of COPs in

448

lard and tallow fat samples – Figure S1

449

ACKNOWLEDGEMENTS

450

This study was supported by a grant (NHRI-107A1-EMCO-2818181) from the National

451

Health Research Institute, Taiwan.

452

CONFLICT OF INTEREST

453

The authors have no potential conflicts of interest to declare.

454


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45. Chiu, Y.; Chiu, C.; Chen, B. H. Determination of cholesterol oxides in heated lard by liquid chromatography, Food Chem., 1994, 50, 53-58.

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590

FIGURE CAPTIONS

591

Figure 1 GC-MS-SIM chromatogram of trimethylsilyl derivatives of COPs and cholesterol standards. Peaks: 1 (IS), 5α-cholestane (internal standard); 2, 7α-OH; 3, cholesterol; 4, 7β-OH; 5, 5,6β-EP; 6, 5,6α-EP; 7, triol; 8, 25-OH; 9, 7-keto.

592 593 594 595 596 597 598 599 600 601 602 603 604 605 606

Figure 2 GC-MS-SIM spectra showing ion abundance at different m/z values for trimethylsilyl derivatives of 7 COPs, cholesterol and internal standard 5α-cholestane in meat and animal fat samples. A (peak 1), 5α-cholestane (internal standard); B (peak 2), 7α-OH; C (peak 3), cholesterol; D (peak 4), 7β-OH; E (peak 5), 5,6β-EP; F (peak 6), 5,6αEP; G (peak 7), triol; H (peak 8), 25-OH; I (peak 9), 7-keto. Figure 3 GC-MS chromatogram of COPs using acetone and acetonitrile as extraction solvents with a higher peak response shown for acetone. Peaks: 1, 5α-cholestane (internal standard); 2, 7α-OH; 3, cholesterol; 4, 7β-OH; 5, 5,6β-EP; 6, 5,6α-EP; 7, triol; 8, 25-OH, 9, 7-keto. Figure 4 GC-MS chromatograms of trimethylsilyl derivatives of COPs in meat samples including chicken cutlet (A), pork cutlet (B), sausage (C), saury fish (D), boiled pork (E) and smoked chicken (F). Peaks: 1, 5α-cholestane (internal standard); 2, 7α-OH; 3, cholesterol; 4, 7β-OH; 5, 5,6β-EP; 6, 5,6α-EP; 7, triol; 8, 25-OH; 9, 7-keto.

607 608 609 610



(FIGURE – 1)


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(FIGURE – 2) 29 ACS Paragon Plus Environment


(FIGURE –3)


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(FIGURE – 4)



Table 1 Retention time (Rt), retention factor (k), separation factor (α) and resolution (Rs) as well as mass to charge (m/z) ratio used for identification of COPs and cholesterol standards by GC-MSa Rsd m/z ratio (SIM mode) compound peak no. Rt (min) kb αc solvent 2.9 5α-cholestanee 1 8.7 217.2 g, 357.4, 372.4 (Group 1) 7α-OH 2 11.5 2.92 1.48 (1, 2)f 9.06 (1, 2)f 456.5, 458.5 (Group 2) cholesterol 3 12.2 3.15 1.08 (2, 3) 2.48 (2, 3) 329.3, 353.3, 368.4, 443.4, 458.5 (Group 3) 7β-OH 4 13.7 3.67 1.17 (3, 4) 4.62 (3, 4) 456.4, 458.5 (Group 4) 5,6β-EP 5 14.5 3.92 1.07 (4, 5) 2.35 (4, 5) 329.3, 356.4, 368.4, 384.3, 459.4, 474.5 (Group 5) 5,6α-EP 6 14.8 4.02 1.03 (5, 6) 1.03 (5, 6) 329.3, 356.4, 368.4, 384.3, 459.4, 474.5 (Group 5) triol 7 16.4 4.58 1.14 (6, 7) 5.66 (6, 7) 321.3, 403.4, 456.4, 546.5 (Group 6) 25-OH 8 17.8 5.05 1.10 (7, 8) 4.36 (7, 8) 131.1, 271.3, 327.3, 367.3, 456.4 (Group 7) 7-keto 9 18.2 5.18 1.03 (8, 9) 1.15 (8, 9) 131.1, 271.3, 327.3, 367.3, 472.4 (Group 7) a

COPs and cholesterol standards identified by GC-MS are indeed trimethylsilyl derivatives. k=tR-t0 / t0, where t0 and tR are retention time of solvent peak and COPs/cholesterol, respectively. c α=k2/k1, where k1 and k2 are retention factor of peak 1 and 2, respectively. d Rs=2 x (t2-t1)/(w1+w2), where t1 and t2 are retention time of peak 1 and peak 2, respectively, while w1 and w2 are width of peak 1 and peak 2. e internal standard. f values in parentheses are neighboring peak numbers. g underlined values in all groups denote the m/z of major ion peak used for identification of COPs, cholesterol and IS, and quantitation of COPs. b

32

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Table 2 Recovery data of free COPs in lard and boiled pork for QuEChERS extraction method as well as precision data for GC-MS analysisa COPs d

7α-OH 7β-OH 5,6β-EP 5,6α-EP triol 25-OH 7-keto

blank 100.7±1.6 100.0±0.2 94.9±0.1 99.8±1.7 97.6±1.3 96.6±2.6 97.6±6.8

Recovery (%)b lard boiled pork 92.4±9.6 97.1±6.9 107.6±0.7 98.9±1.7 89.1±5.3 105.2±6.3 102.8±0.9 80.5±10.3 99.2±1.3 91.5±11.2 106.4±12.3 90.6±0.4 98.5±2.9 105.6±11.3

Intra-day variability (RSD, %)c Lard (µg/g) boiled pork (µg/g) b 2.03±0.05 (2.46) 1.15±0.05 (3.99) b 1.95±0.04 (2.05) 1.22±0.05 (4.12) 1.90±0.15 (7.89) 1.21±0.08 (6.87) 1.81±0.11 (6.08) 1.01±0.05 (5.17) 1.66±0.03 (1.81) 2.04±0.11 (5.29) 1.81±0.06 (3.31) 1.33±0.03 (2.27) 1.95±0.09 (4.62) 1.66±0.08 (4.93)

a

Inter-day variability (RSD, %)c Lard (µg/g) boiled pork (µg/g) 1.90±0.08 (4.21) 1.25±0.03 (2.14) 1.84±0.07 (3.80) 1.23±0.04 (3.43) 1.65±0.14 (8.48) 1.30±0.13 (9.78) 1.62±0.15 (9.26) 1.14±0.08 (7.03) 1.56±0.05 (5.13) 2.13±0.17 (7.95) 1.68±0.06 (3.57) 1.31±0.02 (1.52) 1.79±0.12 (6.70) 1.63±0.14 (8.32)

COPs in lard and boiled pork identified by GC-MS are indeed trimethylsilyl derivatives. Mean of triplicate analyses ± standard deviation c The value in parentheses indicate relative standard deviation calculated using the formula, RSD (%)=(standard deviation/mean of triplicate analyses)x100 d Blank recovery without sample: only COPs standards added for QuEChERS and GC-MS analysis. b

33



Table 3 Limit of detection (LOD) and limit of quantitation (LOQ) of COPs standards as well as LOQ of COPs in palm oil and boiled pork as determined by QuEChERS coupled with GC-MSa Standards Palm oil Boiled pork COPs LOD (ng/mL)b LOQ (ng/mL)c LOQ (ng/g)d LOQ (ng/g)e 0.16 0.32 2 0.32 7α-OH 0.16 0.32 2 0.32 7β-OH 100 400 400 400 5,6β-EP 100 400 400 400 5,6α-EP Triol 16 40 50 40 25-OH 32 80 100 80 7-keto 180 400 400 400 a

COPs standards and COPs in lard and pork identified by GC-MS are indeed trimethylsilyl derivatives b Based on S/N≥3 of COPs standards c Based on S/N≥10 of COPs standards d Based on S/N≥10 of COPs in palm oil, which was selected instead of lard as it is difficult to find an animal fat without COPs and the matrix composition is similar to lard. e Based on S/N≥10 of COPs in standards as it is difficult to find pork without COPs.


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Table 4 Matrix effect of free COPs in animal fat and meat samples by GC-MSa COPs 7α-OH 7β-OH 5,6β-EP 5,6α-EP triol 25-OH 7-keto

slope 2.3671 2.3691 0.1001 0.1055 0.4414 1.0983 0.4061

R2 b 0.9999 0.9997 0.9986 0.9998 0.9978 0.9998 0.998

COPs 7α-OH 7β-OH 5,6β-EP 5,6α-EP triol 25-OH 7-keto

slope 2.7367 2.8244 0.1025 0.0909 0.4934 0.9814 0.4262

R2 b 1 0.9999 0.9995 0.9994 0.9998 0.9999 0.9978

COPs 7α-OH 7β-OH 5,6β-EP 5,6α-EP triol 25-OH 7-keto

slope 2.9025 2.842 0.1033 0.1177 0.552 1.0611 0.51

R2 b 1 1 0.999 0.9983 1 0.9997 0.9985

COPs standards ME(%)c tallow ME(%)c 15.61% 19.22% 2.40% -13.84% 11.78% -10.64% 4.95% chicken steak ME(%)c 22.63% 19.96% 3.20% 11.56% 25.06% -3.39% 25.58%

lard MEd -

slope 2.5841 2.5853 0.0909 0.1115 0.3585 0.989 0.3333

MEd 1.16 1.19 1.02 0.86 1.12 0.89 1.05

slope 2.7811 2.9253 0.1028 0.1219 0.5082 1.0466 0.485

MEd 1.23 1.20 1.03 1.12 1.25 0.97 1.26

slope 2.7141 2.7335 0.099 0.12 0.5418 0.9857 0.4974

a

R2 b 0.9996 0.9995 0.9995 0.9999 0.9985 0.9999 0.9992

ME(%)c 9.17% 9.13% -9.19% 5.69% -18.78% -9.95% -17.93% boiled pork R2 b ME(%)c 0.9999 17.49% 1 23.48% 1 2.70% 0.9999 15.55% 1 15.13% 0.9997 -4.71% 0.9993 19.43% sauryfish R2 b ME(%)c 1 14.66% 1 15.38% 0.9993 -1.00% 0.9998 13.74% 0.9976 22.75% 1 -10.25% 1 22.48%

MEd 1.09 1.09 0.91 1.06 0.81 0.90 0.82 MEd 1.17 1.23 1.03 1.16 1.15 0.95 1.19 MEd 1.15 1.15 0.99 1.14 1.23 0.90 1.22

COPs identified by GC-MS are indeed trimethylsilyl derivatives; bR2=Coefficient of determination; c Matrix effect (ME, %)=(MCC - SCC) / SCC × 100, where MCC and SCC represent slopes of matrix matched calibration curve and standard calibration curve, respectively; d ME=MCC / SCC.

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Table 5-Contents of free COPs in meat and animal fat samples purchased from Taiwan’s marketa COPs 7α-OH 7β-OH 5,6β-EP 5,6α-EP triol 25-OH 7-keto

chicken cutlet 2.04±0.09B (1.94–2.15) 1.99±0.06C (1.92–2.07) 1.78±1.63C (ND–3.14) 1.08±0.99D (ND–1.89) 4.29±0.33A (3.93–4.65) A 1.75±0.87 (0.26–2.39) B 2.00±0.65 (1.46–3.13)

total

14.93±4.62 (11.22–17.63)

B

pork cutlet 2.06±0.10B (1.94–2.19) 1.98±0.11C (1.86–2.12) 2.52±0.44BC (1.74–2.79) 1.77±0.18BCD (1.53–1.95) 4.29±0.38A (3.87–4.68) A 1.69±0.96 (ND–2.35) B 1.35±0.80 (ND–1.98) B

15.66±2.97 (13.57–17.66)

Free COPs contents (µg/g)b,c,d sausage sauryfish 2.24±0.19B (2.11–2.57) 4.05±0.94A (2.54–4.98) 2.23±0.21BC (2.07–2.55) 3.60±0.84A (2.18– 4.32) B 3.42±2.53 (1.91–7.92) 4.87±1.45A (2.28–5.70) BC 1.86±1.16 (ND–3.15) 3.49±0.34A (3.00–3.96) A 3.56±2.00 (ND–4.62) 4.28±0.30A (3.92–4.54) A A 2.16±0.58 (1.35–2.94) 2.17±0.25 (1.90–2.53) B A 1.74±0.79 (0.43–2.42) 9.38±3.33 (4.52–12.39) B

17.21±7.46 (15.27–18.52)

A

31.84±7.45 (21.14–37.35)

boiled pork 2.28±0.23B (1.95–2.54) 2.29±0.24BC (1.94–2.52) 2.31±0.41BC (1.74–2.90) 2.17±0.24B (1.92–2.43) 4.46±0.34A (3.89–4.77) A 2.12±0.14 (1.97–2.32) B 2.10±0.25 (1.90–2.49) B

17.73±1.85 (15.82–18.45)

smoked chicken 2.43±0.55B (3.34–2.04) 2.57±0.66B (2.04–3.54) 2.31±0.83BC (1.41–3.66) 1.24±1.14CD (ND–2.33) 4.22±0.39A (3.75–4.59) A 2.19±0.09 (2.03–2.28) B 2.25±0.66 (1.66–3.13) B

17.21±4.32 (13.56–22.51)

Free COPs contents in lard (µg/g)b,d 1 2 3 4 5 6 7 8 C C A D B B D D 7α-OH 0.132±0.006 0.127±0.005 0.742±0.060 0.042±0.002 0.264±0.004 0.310±0.004 0.071±0.004 0.037±0.002 C C A D B B CD D 7β-OH 0.136±0.010 0.124±0.003 0.726±0.061 0.050±0.003 0.216±0.040 0.240±0.020 0.081±0.004 0.036±0.003 e 5,6β-EP ND ND ND ND ND ND ND ND 5,6α-EP ND ND ND ND ND ND ND ND triol ND ND ND 0.398±0.030A ND ND ND 0.250±0.005B 25-OH ND ND ND ND ND ND ND ND 7-keto ND ND 1.08±0.12B ND 1.94±0.03A 1.93±0.046A ND ND total 0.268±0.016D 0.251±0.008D 2.55±0.24A 0.490±0.035B 2.42±0.07A 2.48±0.070A 0.152±0.008E 0.323±0.010C b,d Free COPs contents in tallow (µg/g) COPs 1 2 3 4 5 7α-OH 4.45±0.56C 4.03±0.05C 8.47±0.37A 2.83±0.03D 5.42±0.07B BC BC A C B 7β-OH 2.52±0.25 2.26±0.06 4.81±1.09 1.54±0.02 3.13±0.05 B B A B B 5,6β-EP 5.26±0.52 4.64±0.54 11.22±3.38 3.19±0.02 5.56±0.46 B B A B B 5,6α-EP 2.76±0.32 2.65±0.23 6.04±2.09 1.31±0.03 3.57±0.23 B B A B B triol 0.289±0.004 0.219±0.0006 2.74±2.82 0.447±0.008 0.294±0.004 e 25-OH ND ND ND ND ND B B A B B 7-keto 22.63±5.04 19.47±3.48 36.87±8.51 13.47±0.55 20.94±3.77 B B A B B total 37.91±6.69 33.27±4.36 60.15±18.26 22.79±0.66 38.91±4.58 a b c COPs identified by GC-MS are indeed trimethylsilyl derivatives; mean of triplicate analyses ± standard deviation; values in parentheses represent range of COPs contents from 6 samples (n=6); c values within a row with different superscript letters (A-E) are significantly different (p

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