Article pubs.acs.org/JAFC
Formation of 3‑MCPD Fatty Acid Esters from Monostearoyl Glycerol and the Thermal Stability of 3‑MCPD Monoesters Yue Zhao,†,‡ Yaqiong Zhang,†,‡ Zhongfei Zhang,† Jie Liu,§ Yi-Lin Wang,∥,⊥ Boyan Gao,†,# Yuge Niu,† Xiangjun Sun,*,† and Liangli Yu*,# †
Institute of Food and Nutraceutical Science, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, China § Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing Technology & Business University (BTBU), Beijing 100048, China ∥ Department of Hepatic Surgery, Fudan University Shanghai Cancer Center, and ⊥Department of Oncology, Shanghai Medical College, Fudan University, Shanghai 200032, China # Department of Nutrition and Food Science, University of Maryland, College Park, Maryland 20742, United States S Supporting Information *
ABSTRACT: Formation of 3-monochloropropanediol (3-MCPD) esters from monostearoyl glycerol (MSG) was investigated under high temperature and low moisture conditions. Different organic and inorganic chlorides, including lindane, KCl, CaCl2, NaCl, MgCl2, AlCl3, CuCl2, MnCl2, SnCl2, ZnCl2, and FeCl3, were evaluated for their potential to react with MSG to form 3MCPD and glycidyl esters at 120 and 240 °C using a UPLC-Q-TOF MS analysis. The results indicated that different chlorine compounds differed in their capacity to react with MSG and formed different products including 3-MCPD mono- and diesters, distearoylglycerol, and glycidyl esters. According to electron spin resonance (ESR) and Fourier transform infrared (FT-IR) spectroscopies, free radical mediated formation mechanisms involving either five-membered or six-membered cyclic acyloxonium free radicals (CAFR) from monoacylglycerol (MAG) were proposed. Tandem quadrupole-time-of-flight (Q-TOF) MS and MS/ MS analyses confirmed the free radical mechanisms. In addition, the results from the present study showed that 3-MCPD monoester could be degraded upon thermal treatment and suggested a possible catalytic role of Fe3+ under the experimental conditions. KEYWORDS: 3-MCPD esters, free radical mechanism, thermal stability, lipid chemistry, Fe3+
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INTRODUCTION Free 3-monochloropropanediol (3-MCPD), a potential carcinogen, was first found in vegetable protein hydrolysates.1 Later, esters of 3-MCPD (also referred as “‘bound MCPD”’) was also found in protein hydrolysates.2 Accumulating evidence indicated that 3-MCPD esters are nephrotoxic and carcinogenic.3 A recent study has discovered that 3-MCPD esters might induce nephrotoxicity through inducing tubular cell apoptosis via JNK/p53 pathways.4 Human breast milk and many food categories, including bread, coffee, refined vegetable oils, infant formula, salty crackers, and pickled olives, have been found to contain 3-MCPD esters, which raised a food safety concern.5−12 Refined edible oils are commonly consumed food ingredients all over the world. It is believed that 3-MCPD esters in refined oils are mainly generated during the deodorization process, which is usually at a high temperature and low moisture condition.10,13,14 A few studies have investigated the possible mechanisms for 3-MCPD ester formation in refined oils.15−20 Two of the proposed mechanisms involved direct nucleophilic substitution by the chlorine anion at the glycerol carbon replacing either an ester group or a hydroxyl group.15,16 Another two proposed pathways include the formation of an intermediate epoxide ring or acyloxonium cation before their nucleophilic attack by a chlorine anion.15−18 These previously © 2016 American Chemical Society
proposed chemical mechanisms were not confirmed with experimental evidence. Recently, our group reported a free radical mechanism, including the formation of a cyclic acyloxonium free radical intermediate from diacylglycerol (DAG) or triacylglycerol (TAG), followed by its reaction with a chlorine containing compound or other chemical species.19,20 The formation of the cyclic acyloxonium free radical intermediate was confirmed by electron spin resonance and Q-TOF-MS/MS techniques.19 Furthermore, Fe2+ and Fe3+ were able to catalyze the 3-MCPD ester formation from TAG under the experimental conditions.20 However, whether and how 3-MCPD esters may be formed from monoacylglycerols (MAG) still remained unclear. In general, MAG is low in edible oils. However, previous studies have suggested MAG as potent precursors for 3-MCPD ester formation.14,21,22 For instance, 3-MCPD esters were believed mainly from DAG and MAG,21 suggesting the importance of investigating whether and how 3-MCPD esters could be formed from MAG, which may serve as a scientific Received: Revised: Accepted: Published: 8918
September 9, 2016 October 26, 2016 October 28, 2016 October 28, 2016 DOI: 10.1021/acs.jafc.6b04048 J. Agric. Food Chem. 2016, 64, 8918−8926
Article
Journal of Agricultural and Food Chemistry
background for MSG and PBN reactions under the same reaction conditions. FT-IR Measurement of MSG under Thermal Treatment. Fourier transform infrared spectroscopy (Nicolet 6700, Thermo Fisher, Waltham, MA, USA) was employed in FT-IR measurement, based on the classic transmission method. A LinkPad temperature controller (Linkam, Surrey, U.K.) was utilized for temperature programming, and the deviation was within 0.1 °C. MSG was mixed thoroughly with KBr to make the sample film. After the baseline was corrected, the sample film was put into the temperature controller and scanned immediately at ambient temperature. Then the temperature was successively raised to 80, 120, 160, 200, and 240 °C, and held for 20 min at each point. The sample was scanned just before the temperature rose. After heating, the sample was scanned once more when it cooled down to 40 °C. Methyl stearate was analyzed by the same method as a negative control, which cannot form cyclic acyloxonium free radical theoretically for lack of the hydroxyl group. Data were processed and analyzed using OMNIC 8.2 software (Thermo Fisher, Waltham, MA, USA). Q-TOF MS and MS/MS Examination of DMPO Radical Adducts. A Waters Xevo G2 quadrupole-time-of-flight (Q-TOF) mass spectrometer (Waters, Milford, MA, USA) was applied to examine if MSG could generate cyclic acyloxonium free radical upon thermal treatment, by determining the structure of the adduct of cyclic acyloxonium free radical (CAFR) and the free radical trapping agent DMPO. MSG and DMPO were dissolved in paraxylene separately, to a concentration of 0.5 mg/mL and 20 mmol/L, respectively. Another mixture solution of MSG and DMPO was prepared to the same concentration in paraxylene. These three solutions were heated at 120 °C for 20 min in an oil bath and cooled down to ambient temperature. All three samples were diluted 50-fold by MS grade methanol and subjected to TOF-MS analysis. MS conditions were as follows: ESI positive mode with lockspray; flow rate, 10 μL/min; cone voltage, 35.0 V; capillary voltage, 3.0 kV; source temperature, 120 °C; desolvation temperature, 450 °C; desolvation gas flow, 800.0 L/h; and cone gas flow, 50.0 L/h. A MS/MS method was used to confirm the structure of the parent ion, which was selected as 492.3665 Da, and conditions were as follows: mass range, m/z 50−1500; ramp collision energy, set from 20 to 35 V. Data analysis was carried out with Waters MassLynx v4.1 software (Waters, Milford, MA, USA). The Thermal Stability of 3-Chloro-1,2-propanediol 1Stearate. 3-Chloro-1,2-propanediol 1-stearate was used to determine the effect of heat and Fe ions on the thermal stability of 3-MCPD monoester. 3-Chloro-1,2-propanediol 1-stearate was dissolved in hexadecane to a final concentration of 0.5 mg/mL. The solution was heated at 120 and 240 °C for 5 min, 10 min, 20 min, and 40 min, with or without iron(III) sulfate, respectively. Iron(III) sulfate was mixed in isopropanol to approximately 50 mg/mL, and 0.2 mL of the solution was pipetted into the 3-chloro-1,2-propanediol 1-stearate solution just before heating, while 0.2 mL of pure isopropanol was added into the control group. After the reaction, the mixtures were cooled to ambient temperature and diluted 50-fold with LC-MS grade methanol:isopropanol (1:1, v/v). The solution was centrifuged at 10000 rpm for 5 min, and the upper solution was subjected to UPLC-Q-TOF MS analysis. All the tests were performed in triplicate. Inorganic Chloride Detection for the Thermally Treated 3Chloro-1,2-propanediol 1-Stearate Solution. 3-Chloro-1,2-propanediol 1-stearate was dissolved in hexadecane to a final concentration of 50 mg/mL. Three milliliters of the solvent was pipetted into a three-necked flask and heated with stirring at 240 °C for 10 min, 20 min, and 40 min, respectively. The reaction system was sealed, and gas was introduced into 1 mL of ultrapure water in a glass tube, to absorb potential HCl gas. After the reaction, the reaction mixture was transferred to a 15 mL centrifuge tube. Three milliliters of ultrapure water was pipetted into the tube and vortexed for 3 min. After centrifugation at 4000 rpm for 10 min, the lower layer (water phase) was carefully removed from the tube and merged with 1 mL of ultrapure water. The extraction was repeated 3 times, and the extracts were combined to obtain approximately 10 mL of solution in total. After the pH value was measured using a pH meter (Orion 3 Star,
base for mitigating 3-MCPD esters in edible oils and food products. In addition, previous studies have shown that 3-MCPD diesters would be degraded at high temperatures, but have relatively good thermal stability at 180−260 °C.23,24 It is interesting to understand the thermal stability of 3-MCPD monoester and related approaches to reduce their levels in foods. The present study aimed to investigate whether and how 3MCPD esters may be formed from MAG. In addition, the thermal stability of 3-MCPD monoester was examined, along with a possible role of Fe3+ on 3-MCPD monoester decomposition. The results of this study may be used to reduce the level of 3-MCPD ester in refined edible oil and other food products.
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MATERIALS AND METHODS
Materials. 1-Stearoyl-rac-glycerol (MSG), rac-1,2-distearoylglycerol (DSG), lindane, 5,5-dimethyl-1-pyrroline N-oxide (DMPO), N-tertbutyl-α-phenylnitrone (PBN), and iron(III) chloride and iron(III) sulfate hydrate were purchased from Sigma-Aldrich Co., LLC (St. Louis, MO, USA). Hexadecane, tert-butylbenzene, paraxylene, and methyl stearate were bought from Aladdin Reagent (Shanghai, China). Potassium chloride, magnesium chloride hexahydrate, aluminum chloride hexahydrate, copper chloride dihydrate, manganese(II) chloride tetrahydrate, tin(II) chloride dehydrate, zinc chloride, acetone, and glacial acetic acid were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Calcium chloride anhydrous and sodium chloride were purchased from Shanghai Lingfeng Chemical Reagent CO., Ltd. (Shanghai, China). 3-Chloro1,2-propanediol 1-stearate, 3-chloro-1,2-propanediol distearate, and glycidyl stearate standards were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). LC-MS grade methanol, 2-propanol, and HPLC grade methyl tert-butyl ether, ethyl acetate, and isohexane were obtained from Merck (Darmstadt, Germany). Sodium methoxide was bought from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Phenylboronic acid was purchased from J&K Scientific Ltd. (Beijing, China). Ultrapure water was prepared by a Millipore ultra-Genetic polishing system with
