McGill University, Department of Food Science and Agricultural Chemistry 21,111 Lakeshore, Ste. Anne de Bellevue, Quebec, Canada, H9X3V9.

Journal of Oleo Science Copyright 2014 by Japan Oil Chemists Society doi : 10.5650/jos.ess14117 Characterization of Electron Ionization Mass Spectral (EIMS) Fragmentation Patterns of Chloropropanol Esters of Palmitic Acid Using Isotope Labeling Technique Anja K. K. Rahn and Varoujan A. Yaylayan * McGill University, Department of Food Science and Agricultural Chemistry 21,111 Lakeshore, Ste. Anne de Bellevue, Quebec, Canada, H9X3V9. Abstract: Chloropropanol (CP) esters are a class of thermally-induced toxicants that are mainly formed in refined edible oils. The structural diversity of these esters presents significant analytical challenges which have often been overcome through analysis of their corresponding free alcohols after a hydrolysis step. Mass spectrometry-based methodologies incorporating characteristic fragmentation patterns of particular isomers of CP esters greatly facilitates their identification. The electron ionization mass spectra (EIMS) of various isomers of synthetic and commercially available 13 C- and 2 H-labeled CP ester standards of palmitic (C 16 ) and other short chain fatty acids (C 3 to C 10 ) were generated and analyzed using GC/MS. Short chain CP esters were synthesized by reacting their respective acid anhydrides with the corresponding 3-chloroand 2-chloro- propanediols in addition to 1,3-dichloro- and 1,2-dichloropropanols. Five fragmentation pathways were identified. Four of the five pathways, such as α-cleavage, McLafferty rearrangement, α-h rearrangement and cyclic acyloxonium ion formation, were characteristic of CP mono- and diesters. The remaining pathway generating chloronium ion was found only in dichlorinated isomers. The proposed fragmentation pathways for the palmitic acid esters were confirmed through the use of 13 C- and 2 H-labeled CP ester standards of palmitic acid, and the generality of identified fragmentation patterns was confirmed through the identification of equivalent ions in the mass spectra of short chain fatty acids (C 3 to C 16 ). Characteristic ions that were identified in this study retaining the chlorine atom in their structures can be considered as potential markers for the presence of CP esters. Key words: electron ionization fragmentations, 3-chloropropanediol dipalmitate and monopalmitates, 2-chloropropanediol dipalmitate and monopalmitate, dichloropropanol palmitates, chloropropanol esters 1 INTRODUCTION Chloropropanol CP esters are a group of process-induced toxicants generated mainly during the deodorization stage of palm oil. The high temperature of the refining process results in the interaction between triacylglycerides 1 and the naturally occurring organic and/or inorganic chlorine compounds in the crude oil 2 leading to the formation of CP esters. Current practice of CP ester detection consists predominantly of two approaches: direct and indirect 3. Indirect methods were initially developed to avoid the use of large number of CP ester standards necessary 3 for the method development, it involves the initial conversion of CP esters into their corresponding free CPs before analysis. Increased commercial availability of CP ester standards has led to the resurgence of interest in direct methods that are currently becoming a dominant theme in CP ester research 4 7. Although direct detection techniques have the advantage of minimizing chemical manipulation prior to analysis, however, they have been accompanied by their own disadvantages as summarized by Crews et al. 3. Amongst these obstacles are the array of standards required for proper analysis, the matrix effects as well as the inconvenience of frequent instrument clean-up 3. Diagnostic mass spectral fragments such as M-RCO 2 resulting from the loss of a single fatty acid under various ionization conditions 4 7 are currently being utilized for characterization and quantitation of these compounds 8 10 without systematic understanding of their formation pathways or their structures. For example Yamazaki et al. 11 represented the ion at m/z 331, originating from ammoniat- * Correspondence to: Varoujan Yaylayan, McGill University, Department of Food Science and Agricultural Chemistry 21,111 Lakeshore, Ste. Anne de Bellevue, Quebec, Canada, H9X 3V9. E-mail: varoujan.yaylayan@mcgill.ca Accepted July 4, 2014 (received for review June 4, 2014) Journal of Oleo Science ISSN 1345-8957 print / ISSN 1347-3352 online http://www.jstage.jst.go.jp/browse/jos/ http://mc.manusriptcentral.com/jjocs 1045

Anja K. K. Rahn and Varoujan A. Yaylayan ed 3-CP dipalmitate, as an acyclic protonated fragment, whereas, MacMahon, Begley, and Diachenko 7 using a similar approach and identical precursor characterized the ion at m/z 331 as a cyclic acyloxonium ion generated upon elimination of a fatty acid group. Only recently, Rahn and Yaylayan 12 established not only the cyclic nature of these ions M-RCO 2 but also their intrinsic cationic character. Furthermore, the use of a single ion series such as M-RCO 2 is not sufficient for proper characterization of CP esters, the need to properly establish the structures of all specific ions originating from various isomers of CP esters is becoming increasingly important especially for the development of direct methods of detection. This investigation aims to characterize such fragmentation pathways of various CP ester isomers by means of isotope labeling technique to justify the current and future use of these ions in the direct analysis of these food components during mass spectrometric analysis. 2 MATERIALS AND METHODS The following chemicals were used without further purification: 3-chloro-1,2-propanediol 3-CP 98, 2,3-dichloro-1-propanol 2,3-DCP 97.0 ; propionic anhydride 99, butyric anhydride 98, valeric anhydride 97, hexanoic anhydride 97, decanoic anhydride 98 ; palmitic anhydride 97, palmitic acid- 13 C 1 99, 1,2-dipalmitoyl-rac-glycerol 99 and 1-palmitoyl-glycerol 99 from Sigma-Aldrich St. Louis, MO, USA ; 1,3-dichloro-2-propanol 1,3-DCP 98, ACP Chemicals Inc., Montreal, Quebec, Canada. The 2-chloro-1,3-propanediol 2-CP 98, 1,3-dipalmitoyl-2-chloropropanediol 98, 1-palmitoyl-2-chloropropanediol 98, 1-palmit o y l - 3 - c h l o r o p r o p a n e d i o l 9 8, 2 - p a l m i t o y l - 3-chloropropanediol 98, rac 1,2-bis-palmitoyl- 3-chloropropanediol-d 5 97, 1,3-dipalmitoyl- 2-chloropropanediol-d 5 98, rac 1,2-bis-palmitoyl- 3-chloropropanediol- 13 C 3 97, glycidyl palmitate 98 and rac 1,2-bis-palmitoyl-3-chloropropanediol were from Toronto Research Chemicals Ontario, Canada, whereas tripalmitin was obtained from Nu-Chek-Prep Inc. Elysian, MN. Table 1 Model systems employed for the generation of chloropropanol (CP) esters. Model Systems 3 Monochloropropane-1,2-diol + Propionic Anhydride 3 Monochloropropane-1,2-diol + Butyric Anhydride 3 Monochloropropane-1,2-diol + Valeric Anhydride 3 Monochloropropane-1,2-diol + Hexanoic Anhydride 3 Monochloropropane-1,2-diol + Decanoic Anhydride 3 Monochloropropane-1,2-diol + Palmitic Anhydride 2 Monochloropropane-1,2-diol + Valeric Anhydride 2 Monochloropropane-1,2-diol + Hexanoic Anhydride 2 Monochloropropane-1,2-diol + Decanoic Anhydride 2 Monochloropropane-1,2-diol + Palmitic Anhydride 1,3 Dichloropropanol + Propionic Anhydride 1,3 Dichloropropanol + Butyric Anhydride 1,3 Dichloropropanol + Valeric Anhydride 1,3 Dichloropropanol + Hexanoic Anhydride 1,3 Dichloropropanol + Decanoic Anhydride 1,3 Dichloropropanol + Palmitic Anhydride 2,3 Dichloropropanol + Propionic Anhydride 2,3 Dichloropropanol + Butyric Anhydride 2,3 Dichloropropanol + Valeric Anhydride 2,3 Dichloropropanol + Hexanoic Anhydride 2,3 Dichloropropanol + Decanoic Anhydride 2,3 Dichloropropanol + Palmitic Anhydride 2.1 CP ester formation Chloropropanol esters were generated through a known procedure 13 where fatty acid anhydrides and chloropropanols listed in Table 1 were heated at 60 overnight to induce esterification. Four chloropropanols CPs were employed; 3-CP, 2-CP, 1,3-DCP and 2,3-DCP, in conjunction with six acid anhydrides; propionic, butyric, valeric, hexanoic, decanoic and palmitic, as illustrated in Fig. 1. This method proved to be sufficient for the generation of all listed palmitates in Table 2 except CP-dipalmitin. An alternative method employing palmitic acid was developed due to the commercial availability of 13 C 1 palmitic acid. When 2,3-DCP, 3-CP and 1,3-DCP were heated at 80 with excess palmitic acid and aluminum chloride as catalyst for five days, surprisingly, only 2,3-DCP generated 3-CP dipalmitin as evidenced by the retention time and the mass spectrum see Fig. 2, whereas 1,3-DCP generated trace amounts and 3-CP generated only dipalmitin under the experimental conditions see part 1 of the Supplementary materials for more details. The CP esters generated were directly analyzed by GC/MS as described in the methodology section below. Preparation of chloropropanol esters using acid anhydrides of fatty acids C3. Twenty micro-liters of either 3-CP, 2-CP, 1,3-DCP or 2,3-DCP were mixed with 30 microliters of the anhydrides see Table 1 in 1 ml autosampler vials. The reaction mixtures were incubated at 60 overnight 12 h and then diluted with 0.5 ml of a 1:4 v/v 1046

Diagnostic ions of chloropropanol esters 2-propanol/hexane mixture. Standards were not incubated; however, 2 mg of each standard was diluted in 1 ml autosampler vials with 0.5 ml of the same 1:4 v/v 2-propanol/ hexane mixture before analysis. 2.2 Preparation of 13 C-labeled 3-monochloropropanedioldipalmitate Five-fold excess 13 C 1 palmitic acid relative to 2,3-dichloropropanol was heated in a thermostated bath at 80 for 120 hrs in the presence of AlCl 3 molar ratio C 16 H 32 O 2 : C 3 H 6 Cl 2 O: AlCl 3, 5.2:1:1 without solvent. The reaction mixture was diluted in hexane before analysis by GC/MS. Fig. 1 Generation of mono- and dichloropropanol esters from their respective precursors in the presence of acid anhydrides. AA = acid anhydride; 3-CP = 3-monochloropropanediol; 2-CP = 2-monochloropropanediol; 1,3-DCP = 1,3-dichloropropanol; 2,3-DCP = 2,3-dichloropropanol. 2.3 Gas chromatography-mass spectrometry GC/MS analysis of CP esters A Varian GC with a mass selective detector 3900 GC/2100T MSD Varian, Walnut Creek, California was used for the GC/MS analysis. One microliter samples of reactants were injected into the injection port heated at 300 which was flushed with helium at a rate of 20 ml/ min for the first two minutes. Separation was carried out on a HP1 100 dimethylpolysiloxane capillary column possessing a 12.5 m length, 0.22 mm internal diameter as well as a 0.33 μm film thickness Hewett Packard, Palo Alto, California, using helium as the carrier gas. Initial temperature of the column was set at 45 for 3 min before being raised to 150 at a rate of 10 /min. The tempera- Table 2 Standards employed for the identification of chloropropanol palmitates. Commercial Abbreviations 1,2-di palmitoyl-3-chloropropanediol (3-CPDP) 1,2- dipalmitoyl-3-chloropropanediol- 13 C 3 (3-CP- 13 C 3 -DP) 1,2- dipalmitoyl-3-chloropropanediol-d 5 (3-CP-d 5 -DP) 1,3- dipalmitoyl-2-chloropropanediol (2-CPDP) 1,3- dipalmitoyl-2-chloropropanediol-d 5 (2-CP-d 5 -DP) 1-palmitoyl-3-monochloropropanol (3-CP-1-P) 2-palmitoyl-3-monochloropropanol (3-CP-2-P) 3-palmitoyl-2-monochloropropanol (2-CP-P) Glycidyl palmitate 1-myristoyl-3-monochloropropanol 1-palmitoyl-2-stearoyl-3-monochloropropanol Tripalmitin Dipalmitin Synthesized dipalmitoyl-chloropropanediol a (CPDP) (1, 1-13 C)-dipalmitoyl-chloropropanediol b (CP-1,1-13 C-DP) a - 2,3-Dichloropropanol + Palmitic acid + AlCl 3 b - 2,3-Dichloropropanol + [ 13 C 1 ]Palmitic acid + AlCl 3 1047

Anja K. K. Rahn and Varoujan A. Yaylayan Fig. 2 Comparison of electron ionization mass spectra of (a) 3-CP dipalmitate (commercial standard) (b) synthetic sample (c) 3-CP di[ 13 C-1]palmitate. ture was then immediately raised to 300, at a rate of 20 /min, and held there for 5 min before raising to 350, again at a rate of 20 /min, which was held for another 5 mins. A constant flow of 1 ml/min was held throughout separation. Compounds separated were detected using an ionization voltage of 70 ev, an EMV of 1500 V, along with a scanning range m/z 40-650. The MS transfer line was set at 275. The identity and purity of the chromatographic peaks were determined by using NIST AMDIS version 2.1 http://chemdata.nist.gov/mass-spc/amdis/. The identity of CP palmitate esters was verified by comparison of their retention times with that of commercial standards Table 1048

Diagnostic ions of chloropropanol esters 2 and through isotope labeling technique. Ion abundances reported in the tables represent the average of two measurements. Due to the experimental conditions the masses of ions containing chlorine atom were rounded to the nearest integer. 3 RESULTS AND DISCUSSION Various CP esters were generated as described in the experimental section by reacting propionic, butyric, valeric, hexanoic, decanoic and palmitic acid anhydrides independently with 3-CP, 2-CP, 1,3-DCP and 2,3-DCP as illustrated in Fig. 1 and Table 1. Table 2 lists the selected commercial standards used to verify the retention times and EI mass spectra of the synthesized CP palmitates. As in the case of CP acetates 12, isotope labeling was used to confirm the proposed fragmentation pathways of CP palmitates. 3.1 Characterization and confirmation of EIMS fragmentation pathways of CP palmitates using isotope labeling approach The previously studied 12 mass spectral fragmentations of CP acetates were used as a guide to predict the general behavior of CP palmitates and other esters under electron ionization conditions. This study allowed the identification of characteristic fragmentation patterns of the CP esters which can generate the most abundant peaks in their mass spectra. Analysis of the data revealed the existence of five fragmentation pathways some of which could be considered as specific to CP esters and others as general to the esters of fatty acids. Four of the five pathways are shown in Figs. 3 and 4 where a generalized 3-monochloropropanediol 3-CP diester is used as an example, since such structures exhibited all the four pathways characteristic of isomers 3, 4, 5, 6 and 7 shown in Fig. 1 and termed pathways a, m, r and c. The remaining pathway termed Chl was characteristic of only dichlorinated isomers 1 and 2 shown in Fig. 5 for isomer 2. Isomers 1 and 2 also exhibited fragmentation pathways a, m and c common to the remaining Fig. 3 Proposed EI mass spectral fragmentation patterns of 3-CP dipalmitate (isomer 6) (see also Table 3). 1049

Anja K. K. Rahn and Varoujan A. Yaylayan Fig. 4 Comparison of EI mass spectral fragmentation patterns of 3-CP and 2-CP dipalmitates (isomers 6 and 7) (see Table 3). isomers as shown in Fig. 5. All the CP esters studied also exhibited ions due to the neutral loss of HCl molecule however in low intensities. 3.2 The α-cleavage of ester groups generating acylium ions pathway a The fragmentation through α-cleavage of the ester groups was termed pathway a and is shown in Figs. 3 and 5, generating the acylium ion series A at m/z 28.00 M R where M R is a saturated alkyl side chain. This series can result from the α-cleavage of any ester group. Table 3 lists important members of this ion series arising from the various isomers of CP palmitates listed in Table 2. Isotope labeling studies demonstrated the absence of chlorine, carbon and hydrogen atoms originating from the glycerol backbone in these ions and at the same time incorporating a single 13 C 1 atom from palmitic acid. This ion can be used to identify the nature of the fatty acid residues in the CP esters. 3.3 Formation of chlorinated cyclic acyloxonium ions pathway c Originating from the molecular ion M. or from the McLafferty rearrangement product, the ions in series C at m/z 120.53 M R or at m/z 135.53 R methyl group are generated under electron ionization conditions through a displacement rearrangement rd mechanism, resulting in the loss of a single fatty acid moiety and formation of the ion M-RCO 2 whose cyclic nature was confirmed recently 12 see Fig. 3. The same ion series has also been observed under various soft ionization conditions 1, 12 and has been referred to in the literature as diacylglyceride ions. This ion series could have great diagnostic utility during the analysis of CP esters 12. Furthermore, isotope labeling studies 14 using CP acetates R CH 3 have confirmed the elemental composition of this ion. In the EI mass spectrum of the 3-CP dipalmitate, this ion at m/z 332 constitutes the base peak see Table 3 thus serving as a better indicator for the identity of the fatty acid side chain than the acylium 1050

Diagnostic ions of chloropropanol esters from glycerol backbone and one C-1 atom from palmitic acid see Table 3. This ion however was absent in the spectra of monoesters as shown in Table 3. Similar cyclic acyloxonium ion formation was observed originating in a post McLafferty rearrangement step see below and Fig. 3. Ions in series M can undergo pathway c to produce mainly the cyclic acyloxonium ion at m/z 135.53 through the displacement of the ester group at sn-1 position. The loss of the ester group at sn-1 positions 15 rather than at sn-2 supports a formation mechanism 12 involving the preferential displacement of the ester group from the less hindered position through an S N 2 mechanism. Furthermore, these studies have also demonstrated the following leaving group priority under electron ionization conditions; esters halides alcohols. The order of leaving group priority might be different under standard reaction conditions 16. Consequently, it is expected that ions in series M to preferentially form the cyclic acyloxonium ion at m/z 135.6 rather than generating ion series C as shown in Fig. 3. Fig. 5 Proposed EI mass spectral fragmentation patterns of dichloropropanol monopalmitates (isomers 1 and 2) (see Tables 4 and 5). ion generated through pathway a. Isotope labeling experiments further confirmed the elemental composition of this peak m/z 332 by observing the incorporation of one chlorine atom, three carbon atoms and five hydrogen atoms 3.4 McLafferty rearrangement pathway m McLafferty rearrangement pathway m in Fig. 3 occurs when γ-h of the fatty acid rearranges with simultaneous β-cleavage of the ester bond resulting in the formation of the charge retention product of a methyl ester regardless of the length of the original fatty acid see Fig. 3. This process effectively generates a new CP diester with one of the fatty acids being converted into an acetate group. Cyclization of this newly formed diester through pathway c can either generate the ion series C or the specific cyclic oxonium ion at m/z 135.53. The identity and the structure of this ion originating from 3-CP diacetate has been con- Table 3 Selected ions from series a A, C, C I and C II originating from chloropropanol palmitate standards b. m/z (abundance) Standards A C C I C II 239 43 332 135 147 330 508 368 3-CPDP 239 (15) 43 (38) 332 (100) 135 (11) 147 (17) 330 ( 5) 508 (35) 368 ( 34) 3-CP- 13 C 3 -DP 239 (12) 43 (50) 335 (100) 138 (15) 150 (15) 333 ( 8) 511 (30) 371 ( 37) 3-CP-d 5 -DP 239 (12) 43 (45) 337 (100) 140 (13) 152 (14) 335 ( 9) 513 (30) 373 ( 42) 2-CPDP 239 (10) 43 (35) 332 ( 30) 135 ( 8) 147 (15) 330 ( 9) 508 (40) 368 (100) 2-CP-d 5 -DP 239 (15) 43 (50) 337 ( 40) 140 (11) 152 (15) 335 ( 9) 513 (40) 373 (100) 3-CP-1-P 239 (11) 43 (64) 135 ( 8) 147 (20) 3-CP-2-P 239 (12) 43 (65) 135 ( 6) 147 (24) 2-CP-P 239 (10) 43 (49) 135 (12) 147 (10) Glycidyl Palmitate 239 ( 5) 43 (63) CPDP 239 (12) 43 (60) 332 (100) 135 (15) 147 (22) 330 (10) 508 (20) 368 ( 35) CP-(1,1-13 C)-DP 240 (15) 43 (50) 333 (100) 136 (20) 148 (20) 331 (12) 510 (20) 370 ( 40) Chlorine Content 0 0 1 1 1 1 0 0 a see Figs. 3 and 4 and Table 2; b see supporting information for more details 1051

Anja K. K. Rahn and Varoujan A. Yaylayan firmed 14 using stable isotope labeling technique that indicated the participation of the carbon atoms of the glycerol backbone as well as the presence of the C1 and C2 atoms of acetic acid 14. This ion has been proposed 14 as a universal marker for the presence of CP diacetates when samples are analyzed under electron ionization conditions. CP dipalmitates also generated the ion at m/z 135.53 through the McLafferty rearrangement. Isotope labeling experiments demonstrated that in addition to single chlorine atom, three carbon and five hydrogen atoms in m/z 135 originated from the glycerol backbone of CP dipalmitate see Table 3. Furthermore, the data also indicated the incorporation of a single 13 C 1 atom from palmitic acid moiety. 3.5 The α-h rearrangement pathway r followed by formation of monounsaturated cyclic acyloxonium ion ion series C This study also revealed a fragmentation pattern related to ion series C through the loss of two successive hydrogen atoms termed ion series C see Fig. 3. As shown in Fig. 3, this series can generate two types of monounsaturated cyclic acyloxonium ions, one type can retain the chlorine atom and was termed type I C I, whereas the other loses the chlorine atom and was termed type II C II. These ions are proposed to originate from the parent radical cation which can undergo α-hydrogen rearrangement designated as pathway r from one of the α-carbons to the radical cation site on the ester carbonyl oxygen. The new radical cation can undergo cyclization pathway c either through the loss of an ester group to form a cyclic acyloxonium radical cation of type I or through neutral loss of HCl to form a cyclic acyloxonium radical cation of type II. Both radical cations can stabilize themselves through the loss of a hydrogen atom to form new series of unsaturated cyclic oxonium ions termed ion series C I and C II. Related radical cyclic acyloxonium ions have also been observed under TOF-ESI/MS conditions during analysis of heated oils 17. As anticipated, the ion series C I and C II show the expected label incorporation pattern consistent with the proposed structure and the formation pathways see Table 3. Ions generated from C I series are not all of high intensities with m/z 147.6 being the most prominent ion within the series see Fig. 3 and Table 3. However, ion series C II give rise to the base peak in 2-chloropropanediol-dipalmitate Table 3. Figure 4 compares pathways of cyclic acyloxonium ion formation in 3-CP and 2-CP dipalmitates. The 3-CP diplamitate prefers to undergo ionic S N 2 type cyclization due to the presence of the leaving group at a primary carbon and forms the stable five-membered cyclic acyloxonium ion as the base peak at m/z 332. On the other hand, the leaving group able to generate a stable five-membered ring at m/z 552 in 2-CP dipalmitate is located at the hindered secondary carbon atom; consequently, it prefers to either displace the ester from a primary carbon atom and form the relatively less stable six-membered cyclic acyl oxonium ion at m/z 332 or undergo hydrogen atom rearrangement and free-radical mediated cyclization through the loss of a chlorine atom from C-2 and form a stable secondary radical as an intermediate that can be converted into a stable fivemembered acyloxonium ion C II at m/z 368 as shown in Fig. 4. On the other hand, the 3-CP dipalmitate prefers pathway c to form m/z 332 as the base peak and form the ion at m/z 368 only in 34 relative intensity. 3.6 Chloronium ion formation through the loss of fatty Table 4 Mass spectral information and selected ions from series a A, C and Chl generated from 1,3-dichloropropanol (DCP) esters c. R b m/z (abundance) M A C Chl [M-HCl] + Base Peak C 2 H 5 185 (8) 57 (100) 112 ( 1), 110 ( 1) 149 ( 2) 57 C 3 H 7 199 (5) 71 (100) 135 (1) 112 ( 3), 110 ( 4) 163 ( 3) 71 C 4 H 9 213 (6) 85 (100) 135 (1) 112 ( 5), 110 ( 6) 177 ( 4) 85 C 5 H 11 227 (7) 99 (100) 135 (1) 112 ( 7), 110 (12) 191 ( 5) 99 C 9 H 19 283 (8) 155 ( 50) 135 (4) 112 ( 8), 110 (11) 247 ( 7) 61 C 15 H 31 367 (5) 239 ( 25) 135 (9) 112 (10), 110 (15) 331 (12) 61 a see Fig. 5, b R= fatty acid side chain; c see supporting information for more details 1052

Diagnostic ions of chloropropanol esters acid radical pathway Chl This pathway is characteristic of dichlorinated CPs see Fig. 5 and Tables 4 and 5 and results from free radical cyclization initiated by the chlorine free radical through the loss of a fatty acid radical. This ion series is characterized by the presence of two chlorine atoms in their structures. Although dichlorinated CPs can also undergo the characteristic pathways of other CP esters such as c, a, and m, however, their mass spectra are dominated by intense peaks arising from fatty acid fragmentations thus obscuring the ions relevant to CP esters. 3.7 Generality of the proposed fragmentation pathways In addition to the palmitate esters listed in Table 2, various other short chain CP esters numbering more than twenty Table 1 were also analyzed to confirm the generality of the five fragmentation pathways discussed above: α-cleavage of ester groups pathway a, formation of chlorinated cyclic acyloxonium ions pathway c, McLafferty rearrangement pathway m, α-h rearrangement pathway r and chloronium ion formation pathway Chl shown in Figs. 3 to 5. Complete isotopic labeling analysis of the EI fragmentation pathways of the CP acetates has been previ- Table 5 Selected ions from series a A, C and Chl generated from 2,3-dichloropropanol (DCP) esters b. R c m/z (abundance) M A C Chl [M-HCl] + Base Peak C 2 H 5 185 ( 7) 57 (100) 112 ( 1), 110 ( 2) 149 ( 2) 57 C 3 H 7 199 ( 7) 71 (100) 112 ( 4), 110 ( 7) 163 ( 3) 71 C 4 H 9 213 ( 7) 85 (100) 135 ( 1) 112 (10), 110 (13) 177 ( 3) 85 C 5 H 11 227 ( 9) 99 (100) 135 ( 2) 112 (13), 110 (20) 191 ( 3) 99 C 9 H 19 283 (17) 155 ( 45) 135 ( 8) 112 (21), 110 (33) 247 (12) 61 C 15 H 31 367 (19) 239 ( 33) 135 (14) 112 (23), 110 (35) 331 (11) 55 a see Fig. 5, b see supporting information for more details; c R = fatty acid side chain Table 6 Selected ions a from series b A, C, C I and C II originating from CP diesters c. R d m/z (abundance) A C C m e C I C II Base peak C 2 H 5 57 (100) 149 ( 10) 135 ( 2) 57 43 ( 63), C 3 H 7 71 (100) 43 ( 20), C 4 H 9 57 ( 76), 85 (100) 43 ( 80), C 5 H 11 71 ( 87), 99 (100) 43( 45), C 9 H 19 81 ( 55), 155 ( 74) 163 ( 19) 135 ( 6) 71 177 ( 22) 135 (10) 147 (3) 85 191 ( 37) 135 (15) 147 (5) 99 247 (100) 135 (19) 147 (15), 204 (14) 283 (2) 247 a for details see Table S7 in the supplementary material; b see Figs. 3 and 5; c see supporting information for more details; d R = fatty acid side chain; e ion series C originating from McLafferty rearrangement product 1053

Anja K. K. Rahn and Varoujan A. Yaylayan ously published 14. Acylium ions in series A at m/z 28.00 M R was also observed in all the short chain CP esters studied Tables 4 to 6. Most base peaks of short chain fatty acids were originated from acylium ions. Similarly, ion series C at m/z 120.53 M R were observed in all the CP esters Tables 4 to 6 with a distinct trend of increasing intensity with increasing fatty acid chain length generating base peaks when R C 9 H 19 and C 15 H 31 of CP diesters. A similar trend of increasing intensity with the increasing chain length of fatty acids was also observed in the formation of cyclic acyloxonium ions originating in a post-mclafferty rearrangement process Table 6. The intensities of these ions encountered in various CP palmitates shown in Table 3 suggest that the degree of chlorination and the number of ester groups di- or mono-ester have less of an influence on their intensities whereas the fatty acid chain length is directly proportional to their intensities C m in Table 6. On the other hand, α-h rearrangement pathway r followed by formation of unsaturated cyclic acyloxonium ions was more characteristic of longer chain fatty acids specifically for C II ions which were observed only when R was C 9 H 19 and C 15 H 31 see Tables 3 and 6. Finally, the characteristic chloronium ions for the dichlorinated CPs at m/z 112 and m/z 110 were also observed in all the dichlorinated CP esters studied exhibiting increasing trend in intensity with increasing chain lengths Tables 4 and 5. 4 CONCLUSIONS Ions that were characterized in this study and were generated as part of the ion series that retain the chlorine atom in their structures such as series C and C II could be used as mass spectrometric markers for the presence of 3-CP and 2-CP dipalmitates, and the specific ions at m/z 135.5 and 147.6 could be used as markers not only for dipalmitates but also for monopalmitates. In addition, due to their low intensities, ions at m/z 112 and 110 could be utilized only to a limited extend as markers for dichlorinated CPs. In general, the intensities of these marker ions increase with increasing fatty acid chain lengths, considering the majority of fatty acids found in food possess chain lengths equal to or greater than palmitic acid C 16 -C 22 it is expected therefore that the intensities of the marker ions generated from the various CP esters in food to remain high, thus providing a great potential to be utilized as diagnostic markers under EI conditions. REFERENCES 1 Destaillats, F.; Craft, B. D.; Sandoz, L.; Nagy, K. Formation mechanisms of monochloropropanediol MCPD fatty acid diesters in refined palm Elaeis guineensis oil and related fractions. Part I: Formation mechanism. Food Addit. Contam. A 29, 29-37 2012. 2 Nagy, K.; Sandoz, L.; Craft, B. D.; Destaillats, F. Massdefect filtering of isotope signatures to reveal the source of chlorinated palm oil contaminants. Food Addit. Contam. 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