Journal of Food Composition and Analysis 37 (2015) 58–66 A pilot survey of 2- and 3-monochloropropanediol and glycidol fatty acid esters in foods on the Canadian market 2011–2013 A. Becalski *, S. Feng, B. P-Y. Lau, T. Zhao Food Research Division, Bureau of Chemical Safety, Health Products and Food Branch, Health Canada, 251 Banting Dr., Address Locator 2203D, Ottawa, ON, Canada K1A 0K9 A R T I C L E I N F O Article history: Received 14 May 2014 Received in revised form 5 September 2014 Accepted 10 September 2014 Available online 22 October 2014 Keywords: 2-Monochloropropanediol 3-Monochloropropanediol Glycidol Esters Gas chromatography–mass spectrometry (GC–MS) Fats Oils Food processing Food safety Food composition Food analysis A B S T R A C T We are presenting data on the occurrence of 2- and 3-MCPD esters and glycidol esters (MCPDEs and GEs) in more than 100 different edible fats, oils, and related products containing fats/oils, such as cookies and cooking sprays. Most of these products were purchased from retail stores in Ottawa, Canada between 2011 and 2013 in duplicate, thus allowing for evaluation of temporal trends. GEs and MCPDEs were determined by stable isotope dilution analysis using glycidol-d5 labelled standards by LC–MS/MS in APCI mode and GC–MS in SIM mode after derivation with cyclohexanone, respectively. Unprocessed oils did not contain detectable levels of GEs or MCPDEs or contained them in trace amounts. The exception was palm oil, which contained 100–550 ng/g MCPDEs. GEs and MCPDEs content was highly variable in processed oils/fats, reaching 10.6 and 17.1 mg/g (expressed as glycidol and MCPDs equivalents, respectively). Walnut, rice bran, grape seed oils and palm oil shortening were found to have the highest levels of MCPDEs and GEs. Levels in cookies also varied greatly from 5 to 339 ng/g, expressed as glycidol equivalents and from 29 to 510 ng/g expressed as MCPD equivalents. Crown Copyright � 2014 Published by Elsevier Inc. All rights reserved. Contents lists available at ScienceDirect Journal of Food Composition and Analysis jo u rn al ho m epag e: ww w.els evier . c om / lo cat e/ j fc a 1. Introduction In the past few years, studies have identified the presence of 2- and 3-monochloropropanediol and glycidol bound in the form of fatty acid esters (2- and 3-MCPDEs and GEs) in many refined fats and oils and also in food products manufactured with fats and oils, such as cookies. 2- and 3-MCPD and glycidol esters may be formed during processing/refining of commercials oils (Crews, 2012; Weisshaar and Perz, 2010; Zelinkova et al., 2006). 2- and 3-MCPD and glycidol esters are generally considered to be toxicants that form during food processing. (Hamlet and Sadd, 2009; Velišek, 2009) Some fatty acid esters of 2- and 3-MCPD and glycidol can be hydrolysed in vivo to their respective parent compounds, glycidol and 2- and 3-MCPDs (Abraham et al., 2013). Glycidol has a Group 2A designation by IARC (probably carcinogenic to humans) but no epidemiological or clinical studies on glycidol have been reported * Corresponding author. E-mail address: adam.becalski@hc-sc.gc.ca (A. Becalski). http://dx.doi.org/10.1016/j.jfca.2014.09.002 0889-1575/Crown Copyright � 2014 Published by Elsevier Inc. All rights reserved. for humans (International Agency for Research on Cancer, 2000). Glycidol is mutagenic in a wide range of in-vivo and in-vitro test systems. In rodents glycidol is a multisite carcinogen (National Toxicology Program, 1990). The US EPA benchmark dose level (BMDL)10 calculated using the incidences of peritoneal mesotheli- omas of the tunica vaginalis observed in male rats from the National Toxicology Program (1990) study was 0.63 mg/kg bw/ day, equivalent to a cancer slope factor of 0.16 (mg/kg bw/day)�1 (United States Environmental Protection Agency, 2008). The US California Environmental Protection Agency estimated human cancer potency for glycidol to be 1.3 (mg/kg-day)�1 on the basis of the tumour incidences observed in female rats from the same NTP study using a multisite statistical approach (Reproductive and Cancer Hazard Assessment Branch Office of Environmental Health Hazard Assessment California Environmental Protection Agency, 2010). So far, knowledge of the biological effects of glycidyl esters of fatty acids is sparse (Ikeda et al., 2012). No evidence is available of any adverse effects resulting from dietary exposure to these esters (Honda et al., 2012), but the human health significance of these http://crossmark.crossref.org/dialog/?doi=10.1016/j.jfca.2014.09.002&domain=pdf http://crossmark.crossref.org/dialog/?doi=10.1016/j.jfca.2014.09.002&domain=pdf http://dx.doi.org/10.1016/j.jfca.2014.09.002 mailto:adam.becalski@hc-sc.gc.ca http://www.sciencedirect.com/science/journal/08891575 www.elsevier.com/locate/jfca http://dx.doi.org/10.1016/j.jfca.2014.09.002 A. Becalski et al. / Journal of Food Composition and Analysis 37 (2015) 58–66 59 compounds is difficult to assess, due to the lack of toxicological and exposure data (Bakhiya et al., 2011; Schilter et al., 2011). Using gastrointestinal modelling, Frank et al. concluded that GEs are rapidly digested by gut lipases to free glycidol and thus GEs, after ingestion, should be considered as a source of glycidol (Frank et al., 2013). In-vivo studies with rats indicate that exposure to glycidyl fatty acids esters should be regarded as exposure to the same molar quantity of glycidol (Appel et al., 2013). The same conclusion was reached by Wakabayashi et al. (2012), who noticed remark- able species difference in the toxicokinetics of GEs and glycidol between rodents and primates. 3-MCPD is classified as a non-genotoxic carcinogen and its tolerable daily intake (TDI) was set at 2 mg/kg bw/day in the European Union (European Commission Scientific Committee on Food, 2001) This TDI was also recommended by JECFA (World Health Organization, 2002). Others indicated that it may be derived upward up to 7 mg/kg bw/day if a benchmark dose approach is used/included in the calculation (Abraham et al., 2012; Rietjens et al., 2012). On the basis of available occurrence data for free 3- MCPD, the maximum levels in foodstuffs (mostly those containing soy sauce or HVP) in some countries were set from 20 mg/kg to 1 mg/kg (Hamlet & Sadd, 2009; EC, 2006; Health Canada, 2012). These limits were not designed to account for 3-MCPDEs. Till now, most studies on MCPDEs have focused on the presence of 3-MCPDEs; 2-MCPDEs have received limited attention, despite indications that they might be present in some foods at levels comparable to 3-MCPDEs (Becalski et al., 2013; Dubois et al., 2012; Kuhlmann, 2011; Seefelder et al., 2011). While the limited data indicate a different metabolic fate of 2-MCPD as compared to 3-MCPD, the toxicological significance of 2-MCPD is largely unknown due to a lack of data (Schilter et al., 2011). The precautionary principle warrants an investigation of the presence of 2-MCPD esters in foods based on structural similarities with 3-MCPD. Adequate risk assessments for 2-MCPD have not been conducted thus far, due to the limited toxicological and occurrence data (Andres et al., 2013). This paper presents data on levels of both 2- and 3-MCPDEs (as 2- and 3-MCPD equivalents) in foods, together with corresponding levels of GEs. Such datasets, where concentrations of 2-MCPD were determined using the internal standard method based on the responses of 2-MCPD vs. 2-MCPD-d5 are few, and this manuscript addresses this gap. Our survey was a pilot study with a small sample size of edible oils/fats designed so that samples were taken from each of the top 5 types of oil corresponding to a minimum 70% of the oil category cumulative market share (AC Nielson data, 2010). These are in order of total oil market share: generic vegetable (35.8%), olive (32.4%), canola (16.0%), corn (10.3%) and sunflower (3.5%), for a total of 98% of the oil market. In addition to the oils selected, other specialty oils (palm, coconut, grape seed, and similar products such as cooking oils sprays, vegetable shortenings and products made from such oils) were selected, based on known factors influencing the probability of formation of MCPDEs and GEs. These known factors are the degree of oil processing and the oil source. It is also known that oils rich in diacylglycerols (e.g. palm oils) are more susceptible to MCPDs and GEs formation. Therefore, samples of cookies and related products were collected and analysed as a proxy for palm and coconut oil. During a second phase of the pilot survey in 2013, we were able to source several unprocessed palm oils and vegetable shortenings from the Canadian market. While several fairly reliable indirect methods for the determi- nation of GEs are in use (DGF, 2013; Ermacora and Hrncirik, 2012; Crews et al., 2013; Kuhlmann, 2011), direct methods are gaining popularity (Blumhorst et al., 2013; Granvogl and Schieberle, 2011; MacMahon et al., 2013c; Dubois et al., 2011; Shiro et al., 2011), due to the elimination of possibility of analytical artefacts formation during sample work-up. Accordingly, in this study, a set of 5 GEs – esters of palmitic, stearic, oleic, linoleic and linolenic acids – were determined directly without derivatisation, by stable isotope dilution analysis (SIDA) LC–MS/MS in atmospheric pressure chemical ionisation (APCI) mode, using glycidol-d5 labelled standards. (Becalski et al., 2012) Selected samples were also analysed analogously for a set of 7 GEs, which included glycidol esters of myristic and lauric acids in addition to the five esters mentioned above. A reverse state of affairs exists in the approach of analysis of MCPDEs. While direct methods for determination of esters of MCPDs have been developed (Granvogl and Schieberle, 2011; Haines et al., 2011; MacMahon et al., 2013a; Moravcova et al., 2012; Yamazaki et al., 2013) most analyses are carried out by indirect methods, probably due to the complexities associated with use of multiple analytical standards. Accordingly, MCPDEs were determined indirectly in this study by using an acidic hydrolysis/transester- ification (Divinova et al., 2004) to avoid possible analytical artefact formation during sodium methoxide treatment (Sato et al., 2013). Derivatisation was accomplished with cyclohexanone, using a fluorinated sulfonic acid resin (Nafion) as a catalyst (Becalski et al., 2013). Additionally, the direct methods for the determination of 2-MCPD esters published so far provide only limited coverage of possible isomers (MacMahon et al., 2013a) and some 2- and 3- isomers might co-elute, thus precluding isomer speciation (Dubois et al., 2012). While there are some indications that toxicological profiles of 3-MCPD monoesters are somewhat different from those of diesters (Barocelli et al., 2011), a relatively low abundance of monoesters (Seefelder et al., 2008) (on average about 10%) appears to justify, at this time, reporting the concentra- tion of 3-MCPD esters as single value of a total equivalent. Very recently, after this work was completed, MacMahon et al. published data on the presence of 3-MCPD and glycidol esters in edible oils in the United States using a direct analysis approach. In their work they analysed for seven target GEs and 28 di- and 9 mono- esters of 3-MCPD (MacMahon et al., 2013b). 2. Materials and methods 2.1. Standards and reagents 3-Monochloropropanediol 98% was from Alfa Aesar; 2-mono- chloropropanediol 98%, 2-monochloropropanediol-d5 98%, isoto- pic purity >99 atom% D, 3-monochloropropanediol-d5 dipalmitate 97%, isotopic purity 99 atom % D, 2-monochloropropanediol-d5 distearate 98%, isotopic purity 98 atom% D were supplied by Toronto Research Chemicals Inc. (Toronto, ON, Canada) and 3- monochloropropanediol-d5 97%, isotopic purity 98 atom % D was from CDN Isotopes (Pointe-Claire, QC, Canada). Glycidyl palmitate (C16:0) 99% was purchased from Wako (Richmond, VA, USA), glycidyl stearate (C18:0) >96% was purchased from TCI (Portland, OR). Glycidyl laurate (C12:0), glycidyl myristate (C14:0), glycidyl oleate (C18:1), linoleate (C18:2) and linolenate (C18:3), all 98%, D5-labelled glycidyl laurate, 98%, 98% D; glycidyl myristate, 98%, 98% D; glycidyl palmitate, 96%, 98% D; stearate, 98%, 98% D; oleate, 98%, 98% D; linoleate 98%, 98% D; and linolenate 95%, 98% D; labelled in glycidol moiety) were supplied by Toronto Research Chemicals (TRC; Toronto, ON, Canada). Nafion SAC-13, 10–20% on a silica support and cyclohexanone 99.8% were from Sigma-Aldrich (St. Louis, MO). Sulfuric acid 98%+ (trace metal use, A-510-500) was from Fisher Scientific while anhydrous sodium sulfate was from EMD (Gibbstown, NJ, USA). Sodium sulphate was muffled at 650 8C for 12 h before use. All other reagents were of analytical grade. Water was obtained from a Barnstead NANOpure Diamond purification system. Table 1 Concentrations GEs and MCPDEs expressed as glycidol and 2- and 3-MCPD equivalents (ng/g) in oils/fats sampled in 2011 and 2013. Product description 2011 2013 Glycidol Equivalent (ng/g) MCPDs Equivalent (ng/g) 2-MCPD Equivalent (ng/g) 3-MCPD Equivalent (ng/g) Ratio 3/2-MCPD Glycidol Equivalent (ng/g) MCPDs Equivalent (ng/g) 2-MCPD Equivalent (ng/g) 3-MCPD Equivalent (ng/g) Ratio 3/2-MCPD Extra virgin olive oil A ND n.d. n.d. ND n.d. n.d. Extra virgin olive oil B ND n.d. n.d. ND n.d. n.d. Extra virgin olive oil C ND n.d. <100 (76) ND n.d. n.d. Extra virgin olive oil D ND n.d. n.d. ND n.d. n.d. Extra virgin olive oil E ND n.d. n.d. ND n.d. n.d. Extra virgin olive oil F ND n.d. n.d. ND n.d. n.d. Light olive oil A 321 2168 608 1560 2.6 294 1350 427 921 2.2 Light olive oil B 524 861 277 584 2.1 927 1050 313 739 2.4 Vegetable oil spray A 9 n.d. n.d. – – – – – Vegetable oil spray B 289 337 98 239 2.4 Vegetable oil spray C 275 205 61 144 2.4 198 n.d. n.d. Vegetable oil spray D 316 662 181 481 2.7 225 130 n.d. 130 Vegetable oil spray E 80 350 81 269 3.3 Mixvegetable oil A 487 1790 569 1220 2.1 – – – – – Mixvegetable oil B 30 n.d. n.d. 383 0 n.d. Mixvegetable oil C 32 n.d. <100 (90) 268 n.d. <100(50) Coconut oil 1000 490 175 315 1.8 1290 549 216 333 1.5 Coconut oil, unrefined, A 26 n.d. n.d. Coconut oil, unrefined, B ND n.d. n.d. Canola oil, unrefined A ND n.d. n.d. Canola oil, unrefined B ND n.d. n.d. Canola oil A 110 264 73 191 2.6 678 483 180 304 1.7 Canola oil B 26 293 75 218 2.9 453 210 n.d. 210 Canola and extra virgin olive oil 59 428 129 299 2.3 284 351 116 235 2.0 Canola and Sunflower oil A 263 234 69 165 2.4 144 n.d. <100(77) Rice Bran oil A 3790 12,400 4030 8340 2.1 – – – – – Rice Bran oil B 1250 524 156 368 2.4 Com oil 121 345 106 239 2.3 159 201 80 121 1.5 Sunflower oil, unrefined A ND n.d. n.d. n.d. n.d. Sunflower oil A 332 358 113 245 2.2 452 n.d. <100(90) Sunflower oil B 212 214 64 150 2.3 256 408 153 254 1.7 Grapeseed oil A 3910 4670 1480 3190 2.2 537 5260 1660 3600 2.2 Grapeseed oil B 660 2000 619 1380 2.2 2620 3110 964 2140 2.2 Walnut oil 584 17,100 5520 11,600 2.1 555 4200 1330 2870 2.2 Almond oil 594 751 236 515 2.2 441 1510 467 1040 2.2 Peanut oil, unrefined 3 n.d. n.d. n.d. n.d. Peanut oil 469 696 223 473 2.1 658 555 171 384 2.2 Avocado oil, unrefined A 22 n.d. n.d. Avocado oil, unrefined B – – – – – 328 n.d. <100(62) n/a Avocado oil 591 1310 395 912 2.3 761 618 183 435 2.4 Toasted Sesame oil 403 1080 323 757 2.3 – –– - – –– Toasted Seasame oil unrefined 380 1010 305 700 2.3 168 825 238 588 2.5 Sesame oil unrefined A 240 472 141 331 2.3 – – – – – Sesame oil unrefined B 484 1890 604 1290 2.1 Margarine A ND* 694 260 434 1.7 151 627 186 441 2.4 Margarine B ND* 248 64 184 2.9 31 284 71 213 3.0 Margarine C ND* 143 51 92 1.8 192 273 70 203 2.9 Margarine D 557 264 86 178 2.1 216 258 93 164 1.8 Vegetable shortening 244 736 234 502 2.1 1030 799 248 551 2.2 Lard 27 628 196 432 2.2 49 600 188 412 2.2 Palm oil shortening – – – – – 10,600 12,500 4030 8420 2.1 A . B eca lsk i et a l. / Jo u rn a l o f Fo o d C o m p o sitio n a n d A n a ly sis 3 7 (2 0 1 5 ) 5 8 – 6 6 6 0 P a lm o il u n re fi n e d A - - - - - N D 2 5 6 n .d . 2 5 6 P a lm o il u n re fi n e d B - - - - - N D 5 5 8 n .d . 5 5 8 P a lm o il u n re fi n e d C - - - - - N D n .d . < 1 0 0 (8 7 ) n .d .— R e p o rt in g li m it fo r 2 -M C P D : 5 0 n g /g , 3 -M C P D : 1 0 0 n g /g , v a lu e s b e tw e e n 5 0 a n d 1 0 0 a re li st e d . R e p o rt in g li m it s fo r co o k ie s a re 5 – 1 0 ti m e s lo w e r d u e to th e la rg e r sa m p le m a ss . N D — re p o rt in g li m it fo r e a ch G E is 1 0 n g /g w h e n 5 0 0 m g o f sa m p le w e re a n a ly se d . N D *— re p o rt in g li m it fo r e a ch G E is 5 0 0 n g /g a s 1 0 m g o f sa m p le w e re a n a ly se d . ‘‘– ’’ sa m p le w a s n o t a v a il a b le /a n a ly se d . In 2 0 1 3 7 G E s w e re d e te rm in e d , C 1 2 :0 , C 1 4 :0 , C 1 6 :0 , C 1 8 :0 , C 1 8 :1 , C 1 8 :2 , C 1 8 :3 ; in 2 0 1 1 /2 0 1 2 5 G E s w e re d e te rm in e d (n o C 1 2 :0 , o r C 1 4 :0 ). A. Becalski et al. / Journal of Food Composition and Analysis 37 (2015) 58–66 61 2.2. Food samples Foods were collected from retail outlets in Ottawa, Ontario, Canada, with the exception of unrefined palm oil and palm oil shortening samples, which were purchased in the Greater Vancouver area, British Columbia. All products were purchased as is in their retail containers. Most purchased oil and fat samples were packaged in glass bottles/jars with 0.5 to 1 L capacity, and only few varieties were packaged in polyethylene or PETE containers. Cookies were packaged in sealed plastic foil containers. A total of 55 samples were collected in the first stage of the survey; 49 discrete, ready-to-eat, samples were collected in 2011 and included edible oils, margarine, lard, vegetable shortening, frying sprays and four cookie samples. Four additional cookie samples were collected in 2012. Most products were not sampled in replicate to ascertain within-lot and lot-to-lot variability with the exception of one brand of cold pressed virgin olive oil (VOO) which was collected in triplicate (single lot, twice in 2010 and once in 2011) as it had been used for method development. In the second stage of the survey done in 2013, 44 samples of edible oils/fats and 6 samples of cookies were collected. We have tried to collect the same products/brands but, in several instances, we were unable to find them so, in a few cases, a suitable replacement was purchased. After purchase, solid sample samples were homogenised using a Waring blender and packaged in 250 mL pre-cleaned glass jars with Teflon lined covers (I-Chem brand). Liquid samples were sampled directly from the bottles/jars. Any edible fat, which was solid at room temperature, was heated to about 60 8C to liquefy before analysis. Cooking sprays were sampled by discharging the well-mixed canister to a clear 20-mL vial until a few grams of product was present in the vial. Oil samples were stored at 4 8C until analysis while other samples were stored at �18 8C. 2.3. Standards preparation—MCPDs and MCPDEs All stock and spiking deuterated standard solutions of 2- and 3-MCPD were prepared in ethyl acetate and stored at �18 8C. Calibration solutions were prepared in 4-mL vials with septa and contained 2, 5, 25, 100, 500, and 1000 ng of 2- and 3-MCPD and 100 ng 2-MCPD-d5 and 3-MCPD-d5 in each vial and 100 mL of cyclohexanone. The total volume of the organic phase was adjusted to 1.0 mL using isooctane. Sodium sulfate (0.2 g) and Nafion/silica (10 mg) were added, vials were capped and heated in a heater block at 45 8C for 1 h. After cooling to room temperature, the organic phase was pipetted into a 2-mL vial for GC/MS analysis. Internal MCPDE standards used for sample spiking were prepared in ethyl acetate as 102.5 mg/mL rac-bispalmitate-3-MCPD-d5 and 112.2 mg/mL 1,3-distearate-2-MCPD-d5 Stock, spiking and deuterated standard solutions of glycidyl esters were prepared in isopropanol or 1:1 v/v mixture of methanol and isopropanol. Working solutions were stored at �18 8C while stock solutions were kept at �80 8C. Calibration solutions were prepared in 1:1 v/v mixture of methanol and isopropanol and contained 5, 10, 50, 200, 500 and 2000 ng/mL of native glycidyls and 200 ng/mL of deuterated glycidyls. 2.4. Typical sample preparation—MCPDs The procedure described by us previously was generally followed (Becalski et al., 2013). The availability of 2-MCPD esters standards prompted us to replace internal standards of free 2- and 3-MCPD-d5, with its diesters added at the extraction stage in order to increase robustness of the method. The sample was spiked with deuterium labelled analogues of 2- and 3-MCPD diesters, (5 mL of 102.5 mg/mL rac-bispalmitate- 3-MCPD-d5 and 5 mL of 112.2 mg/mL 1,3-distearate-2-MCPD-d5, T a b le 2 C o n ce n tr a ti o n s G E s a n d M C P D E s e x p re ss e d a s g ly ci d o l a n d 2 - a n d 3 -M C P D e q u iv a le n ts (n g /g ) in co o k ie s sa m p le d in 2 0 1 1 /2 0 1 2 a n d 2 0 1 3 . B C D E F G H I J K L M 2 2 0 1 1 /2 0 1 2 2 0 1 3 3 P ro d u ct d e sc ri p ti o n Li p id co n te n t (% ) G ly ci d o l e q u iv a le n t (n g /g ) M C P D s e q u iv a le n t (n g /g ) 2 -M C P D E q u iv a le n t (n g /g ) 3 -M C P D E q u iv a le n t (n g /g ) R a ti o 3 /2 -M C P D G ly ci d o l e q u iv a le n t (n g /g ) M C P D s e q u iv a le n t (n g /g ) 2 -M C P D e q u iv a le n t (n g /g ) 3 -M C P D e q u iv a le n t (n g /g ) R a ti o 3 /2 -M C P D 4 C o o k ie s A 1 1 7 6 3 8 8 1 3 9 2 4 9 1 .8 2 8 1 2 7 4 1 8 6 2 .1 5 C o o k ie s B 5 3 3 9 2 6 9 9 4 1 7 5 1 .9 – – – – – 6 C o o k ie s C 2 1 1 2 6 2 3 0 8 1 1 4 9 1 .8 – – – – – 7 C o o k ie s D 1 3 5 3 2 7 4 1 1 0 1 6 4 1 .5 – – – – – 8 C o o k ie s E 1 5 3 1 2 2 1 7 6 1 4 4 1 .9 7 9 7 2 8 6 9 2 .4 9 C o o k ie s F 2 1 2 4 7 1 2 6 4 6 8 0 1 .7 – – – – – 1 0 C o o k ie s G 1 8 1 7 2 9 7 2 2 3 .1 5 6 4 2 1 4 3 2 .0 1 1 C o o k ie s H 1 9 3 0 4 7 0 1 2 3 3 4 7 2 .8 9 3 5 1 0 1 6 7 3 4 3 2 .0 A. Becalski et al. / Journal of Food Composition and Analysis 37 (2015) 58–6662 equivalent to 100 ng of free 2- and 3-MCPD-d5 in 100 mg of sample lipids). Cookies (1 to 3 g) were extracted twice with ethyl ether/ hexane (1:9, v/v). Margarine samples were extracted in a fashion similar to cookies, and some margarine samples were additionally analysed for the presence of free 2- and 3-MCPD. Lipids (�100 mg) were subjected to acidic hydrolysis and liberated free 2- and 3- MCPD was purified/isolated using diatomaceous earth extraction. For GC–MS analysis, MCPDs were derivatised with cyclohexanone using Nafion resin as acidic catalyst. Analysis was performed on a DB-5 column and detection of target MCPD derivatives was accomplished in selected ion monitoring mode with 4 ion transitions for native analytes and 2 ion transitions for internal standards. 2.5. Typical sample preparation—GEs The procedure described by us earlier for the determination of five target GEs (palmitate, stearate, oleate, linoleate and linolenate) was generally1 followed (Becalski et al., 2012); 10 mg edible oil or fat sample were spiked with deuterium-labelled analogues of glycidol esters (5 mL of the �10 ppm internal standard solution) and purified by a two-step chromatography procedure on C18 and normal silica solid-phase extraction (SPE) cartridges. If the concentration of analytes was expected to be below 500 ng/g, 0.5 g sample of oil were pre-concentrated first using a silica column. Due to varied concentration of analytes, some samples were processed twice, in 10- and 500-mg quantities. Samples of cookies, (1 to 3 g), were extracted twice with ethyl ether/hexane mixture (1:9, v/v) and the solvent was evaporated to yield lipids. A dried final extract was re-dissolved in 250 mL of a mixture of methanol/isopropanol (1:1, v/v); 15 mL were injected on the analytical C18 LC column and analytes were eluted with methanol. Detection of target glycidol fatty acid esters was accomplished by LC–MS/MS operating in multiple reaction monitoring (MRM) mode with 2 ion transitions for each analyte using positive ion APCI. For native glycidol laurate and myristate transitions 257 > 57, 257 > 71 and 285 > 57, 285 > 71 were, respectively, used, while for their d5 internal standards transitions 262 > 57, 262 > 71, 290 > 57 and 290 > 71 were used. Retention times were 4.9 min for glycidol laurate and 5.65 for glycidol myristate. 3. Results and discussion Foods were collected from retail outlets in Ottawa, ON, Canada, with the exception of palm oil/shortening samples, which were purchased in Vancouver, B.C. In 2011, 45 edible oil/fat samples and 4 cookie samples were collected; 4 additional cookie samples were collected in 2012. In 2013, 44 samples of edible oils/fats and 4 samples of cookies were collected. Our pilot study did not focus on role/contribution of the container to the levels of contaminants found in the products. Most purchased oil and fat samples were packaged in glass bottles/jars, and only a few varieties were packaged in polyethylene or PETE containers while cookies were packaged in sealed plastic foil containers. In view of packaging materials, a contribution of the container is likely to be not significant (see Table 1). 1 In samples rich in saturated fatty acid triglycerides (e.g. coconut, palm oils), a precipitate formed upon storage at �18 8C of final extracts destined for LC–MS/MS analysis. That precipitate could not be easily removed by centrifugation at �15 8C and we found syringe filter filtration at �18 8C to be cumbersome. Instead, the formed precipitate was allowed to dissolve upon warming of the vial, and the vial was then centrifuged for at least 2 h at �6000 g in a refrigerated centrifuge (Sorvall 6B) using a temperature gradient from room temperature to �15 8C. Afterwards, the supernatant was transferred quickly to another vial. The removal of the precipitate helped to remove interfering peaks from LC–MS/MS chromatograms. A. Becalski et al. / Journal of Food Composition and Analysis 37 (2015) 58–66 63 In the first phase of the survey (2011–2012), samples were analysed for 5 GEs; palmitic, stearic, oleic, linoleic and linolenic. In the second phase (2013), samples were also analysed for GEs of lauric and myristic acids. Those two analytes were incorporated into our previously published method (Becalski et al., 2012) and method performance for these compounds was very similar to the other five analytes. Recovery of glycidyl laurate and myristate from spiked cookies (n = 3, 25 ng/g spike) was 102% and 109% with respective standard deviations of 7.7% and 6.8%. Recovery of glycidyl laurate and myristate from spiked virgin olive oil (n = 3, 50 ng/g spike) was 103% and 101% with respective standard deviations of 5.6% and 9.4%. GEs of lauric (C12:0) and myristic acids (C14:0) were found in appreciable amounts only in a processed coconut oil purchased in 2013 (49% of total glycidol equivalent). In the vast majority of samples analysed in the second stage of the survey those GEs were not detected; only some margarines and cookies contained those GEs in measurable amounts of �10% or less of the total glycidol equivalent. The detailed breakdown of GEs is not shown, as GE composition generally follows the fatty acid profiles of the individual oils/fats e.g. (Dubois et al., 2011, 2012; Shiro et al., 2011). Use of d5 labelled MCPD esters as internal standards did not alter method performance. When 3-monochloropropanediol-d5 dipalmitate, was spiked in the olive oil at 1000 mg/kg (of 3-MCPD equivalents) at the initial extraction stage with 10% ether/hexane, the average recovery was 81% (standard deviation 4.7%, n = 4). Spiking of olive oil with free 3-MCPD-d5 at 1000 mg/kg at the hydrolysis stage, afforded an average recovery of 78% (standard deviation 3.0%, n = 4) as calculated by external quantification. The completeness of the extraction with 10% ether/hexane was further investigated using native (i.e. non-labelled) 3-MCPD esters spiked at the initial extraction stage at 2500 mg/kg and employing MCPDs Equi valen ts (ng/ MCPDs Equi valen ts 0 200 0 M C P D s E qu iv al en ts (n g/ g) in o ils /fa ts in 2 01 3 0 2000 4000 6000 170 00 180 00 Fig. 1. MCPDs equivalents (ng/g) internal standard quantification with free 3-MCPD-d5 spiked at the hydrolysis stage. When 3-monochloropropanediol dipalmitate was used the average recovery was 93% (standard deviation 2%, n = 4). Spiking with 3-monochloropropanediol-1-palmitate afforded an average recovery of 85% (standard deviation 5%, n = 4), slightly lower than that of the diester due to its higher solubility in water. Recovery of 2-MCPD and 3-MCPD from spiked cookies (n = 4, 500 ng/g spike as MCPD equivalent, in the form of diesters of stearate and palmitate, respectively) was 107.5% and 99% with respective standard deviations of 1.7% and 2.6%. As seen from Table 1, most oils labelled as virgin/unprocessed/ unrefined did not contain detectable levels of GEs or MCPDEs. The two exceptions were palm oil which contained 100–550 ng/g MCPDEs, (but GEs were not detectable), and a sesame oil. Two different brands of sesame oil, one from 2011 and one from 2013, both labelled as unrefined and untoasted, contained considerable amounts of GEs and MCPDs. The reasons for an unrefined oil to contain �200 to 400 ng/g glycidol equivalents and �500 to 2000 ng/g MCPD equivalents are not clear, especially when compared with the data of Kuhlmann (2011), who found undetectable amounts of GEs and MCPDEs in all ‘‘crude/pristine’’ oils tested, including sesame oil. Levels of GEs and MCPDs in toasted unrefined sesame oils found by MacMahon et al. (2013b) are comparable to the levels of GEs and MCPDs found by us in toasted and unrefined sesame oils, thus indicating that roasting process might not be the sole reason for presence of GEs and MCPDEs in processed sesame oils. GEs and MCPDEs levels were highly variable in processed oils/fats, reaching 4 and 17 mg/g, expressed as glycidol and MCPDs equivalents, respectively. Our data underscore variability of GEs and MCPDs content in edible oils/fat as evidenced by differences between brands and temporal variations. For example, concentration of GEs and MCPDs g) in oils/ fats in 2011 vs 201 3 (ng/g ) in oils /fats in 201 1 4000 60 00 1700 0180 00 in oils/fats in 2011 vs 2013). Glycidol Equi valen ts (n g/g) in oils/ fats in 2011 vs 201 3 Glycidol Equi valents (ng/g) in oil s/fats in 201 1 0 500 1000 1500 2000 2500 38004000 G ly ci do l E qu iv al en ts (n g/ g) in o ils /fa ts in 2 01 3 0 500 1000 1500 2000 2500 3800 4000 Fig. 2. Glycidol equivalents (ng/g) in oils/fats in 2011 vs 2013. A. Becalski et al. / Journal of Food Composition and Analysis 37 (2015) 58–6664 in refined peanut oil and lard did not change significantly between 2011 and 2013. However, both canola oils A and B contained significantly more GEs in 2013 than in 2011, while MCPDEs content did not change much. Two grapeseed oils A and B had rather comparable MCPDs content in both 2011 and 2013 but GEs content was significantly higher in 2011 in one oil, and in 2013, in another. The walnut oil sampled in 2013 contained less than 25% of the MCPDs found in a 2011 sample of the same brand, but a vegetable shortening sampled in 2013 contained 4 times more GEs than the same product sampled in 2011. Walnut, rice bran, grape seed oils and palm oil shortening were found to have the highest levels of MCPDEs and GEs. These findings agree with data reported by Kuhlmann who found high levels of MCPDEs and GEs in walnut and grapeseed oils and with results of Razak et al. (2012) who found up to 5800 ng/g of 3-MCPD in palm oils. MacMahon et al. also found that grape seed oils and palm oils have higher concentrations of both GEs and MCPDEs than most oils they had tested. Our findings of a much higher GEs/MCPDEs ratio in coconut oil than in all other oils tested also agree with other findings (Kuhlmann, 2011; MacMahon et al., 2013b). The median level of 3-MCPD in margarines (210 ng/g) and in refined oils (380 ng/g) found in this study is much lower than the corresponding values of 1400 and 1000 reported in 2011 in Germany (Weisshaar, 2011). However, the mean level of 3-MCPDEs of 390 ng/g found in ‘‘fats and oils’’ in a 2013 survey in Hong Kong (Chung et al., 2013) is close to the median of 384 ng/g obtained by us for vegetable oils. In view of the high sodium chloride content of margarines (�1%), which might facilitate formation of MCPDs, margarine samples collected in 2011were also tested for the presence of free MCPDs. No free 3- or 2-MCPD were detected (<3 ng/g) in any of 4 samples tested (details not shown). Levels of glycidol and MCPD equivalents in cookies also varied greatly from 5 to 339 ng/g, and 29 to 510 ng/g, respectively (Table 2). Large variation in MCPDEs and GEs content of cookies parallel the variation found in edible oils. The differences in baking processes are likely to be a source of variability as well (Mogol et al., 2014). The ratio of 3- to 2-MCPD in all samples analysed varied from 1.5 to 3.3 with a median of 2.18 and mean of 2.22. Our results, obtained using the isotope dilution method, parallel the ratios estimated by Kuhlmann, mostly in the range 1.7–2.5. Similarly, the median ratio of 3- to 2-MCPD found in six oils by Ermacora and Hrncirik (2012) was 2.2. For comparison of temporal trends 24 pairs (same brand, product and packaging code) of oils/fats and 4 pairs of cookies were available. There were no overall temporal trends (signed rank test p = 0.09 for GE and p = 0.37 for MCPD) in concentrations of GEs and MCPDs in oils, which were tested in 2011 and 2013. The results are displayed in Figs. 1 and 2, where the solid line represent y = x relationship. While the number of cookie samples is small, it appears that some products contain significantly more MCPDEs and GEs than others. 4. Conclusions Future monitoring of temporal trends concentrations of 2- and 3-MCPDEs together with corresponding levels of GEs is essential in view of the substantial amounts of those substances in foods. In view of the widespread use of refined oils in food products, expansion of the monitoring program appears to be warranted, in order to obtain an accurate estimation of intake of 2- and 3-MCPDEs and GEs. Health Canada’s Food Directorate will use the A. Becalski et al. / Journal of Food Composition and Analysis 37 (2015) 58–66 65 data to update its exposure estimations and risk assessments for 2- and 3-MCPD and glycidol esters in food. Acknowledgements The authors would like to thank D. Sit (Health Canada) for collecting samples of palm oils, and L. Pelletier and Mei-Tein Lo (Health Canada) for comments and a review of the manuscript. References Abraham, K., Appel, K.-E., Berger-Preis, E., Apel, E., Gerling, S., Mielke, H., et al., 2013. 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Food Additives and Contaminants 23, 1290–1298. http://refhub.elsevier.com/S0889-1575(14)00156-2/sbref0230 http://refhub.elsevier.com/S0889-1575(14)00156-2/sbref0230 http://refhub.elsevier.com/S0889-1575(14)00156-2/sbref0235 http://refhub.elsevier.com/S0889-1575(14)00156-2/sbref0235 http://refhub.elsevier.com/S0889-1575(14)00156-2/sbref0240 http://refhub.elsevier.com/S0889-1575(14)00156-2/sbref0240 http://refhub.elsevier.com/S0889-1575(14)00156-2/sbref0240 http://refhub.elsevier.com/S0889-1575(14)00156-2/sbref0240 http://refhub.elsevier.com/S0889-1575(14)00156-2/sbref0245 http://refhub.elsevier.com/S0889-1575(14)00156-2/sbref0245 http://refhub.elsevier.com/S0889-1575(14)00156-2/sbref0245 http://refhub.elsevier.com/S0889-1575(14)00156-2/sbref0245 http://refhub.elsevier.com/S0889-1575(14)00156-2/sbref0250 http://refhub.elsevier.com/S0889-1575(14)00156-2/sbref0250 http://refhub.elsevier.com/S0889-1575(14)00156-2/sbref0250 A pilot survey of 2- and 3-monochloropropanediol and glycidol fatty acid esters in foods on the Canadian market 2011-2013 1 Introduction 2 Materials and methods 2.1 Standards and reagents 2.2 Food samples 2.2 Food samples 2.3 Standards preparation-MCPDs and MCPDEs 2.4 Typical sample preparation-MCPDs 2.5 Typical sample preparation-GEs 3 Results and discussion 4 Conclusions Acknowledgements References