A Simple Procedure To Detect Lipid-Derived Carbonyl-Phenol Adducts

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Chapter 6

A Simple Procedure To Detect Lipid-Derived Carbonyl-Phenol Adducts Rosario Zamora and Francisco J. Hidalgo* Instituto de la Grasa, Consejo Superior de Investigaciones Científicas, Carretera de Utrera km 1, Campus Universitario – Edificio 46, 41013-Seville, Spain *E-mail: [email protected]

Carbonyl-phenol adducts produced in the reactions of (E)-2-alkenals and (E,E)-2,4-alkadienals with phenolics were derivatized with methyl chloroformate and studied by gas chromatography–mass spectrometry to create a database that could be employed for their detection in complex mixtures of lipids and phenolics, in particular reactions involving lipid hydroperoxides. The obtained results showed that hydroperoxides produced adducts derived from acrolein, crotonaldehyde, and 2-pentenal when the 13-hydroperoxide of methyl linolenate was used, and acrolein and 2-octenal when the 13-hydroperoxide of methyl linoleate was employed. On the contrary, adducts derived from (E,E)-2,4-alkadienals could not be detected in these mixtures. Although only two types of lipid-derived aldehydes have been studied, the obtained results support that this methodology can be extended to other lipid oxidation products and phenolics and employed to quantitatively determine carbonyl-phenol adduct content. Therefore, this methodology may constitute a powerful tool to investigate the formation of carbonyl-phenol adducts in complex systems.

© 2019 American Chemical Society Granvogl and MacMahon; Food-Borne Toxicants: Formation, Analysis, and Toxicology ACS Symposium Series; American Chemical Society: Washington, DC, 2019.

Introduction Lipid oxidation is a major source of food-borne toxicants (1). Thus, degradation of food lipids generates potentially toxic products that have been shown to correlate with inflammatory diseases, cancer, atherosclerosis, aging, etc. (2). As an alternative to prevent the formation of lipid oxidation products, or their consequences, the use of phenolic antioxidants is quite extended. Their action is still under discussion, although a combined effect of chelating (to avoid formation of free radicals), free radical scavenging (to break free radical chains), and carbonyl trapping properties (to avoid spreading of the lipid oxidative damage to other food macromolecules) has been suggested (3). Of these, the last function is the least known, although recent studies have shown that phenolics act as an alternative sink in the removal of lipid oxidation products (4). In fact, some carbonyl-phenol adducts have been shown to be produced under usual cooking conditions and be present in fried foods (5). Lipid oxidation produces a wide array of reactive carbonyls with a considerable diversity of structures, including alkanals, 2-alkenals, 2,4alkadienals, 4-hydroxy-2-alkenals, 4-oxo-2-alkenals, and 4,5-epoxy-2-alkenals, among others. Because of this diversity, different carbonyl-phenol adducts, also with a wide range of structures and stabilities, are produced (6–10). In addition, each adduct is produced as a series of diastereomers because new chiral centers are created (8–10). Some of the produced adducts are unstable and suffer further reactions, including polymerization reactions. For these reasons, carbonyl-phenol reactions are very complex and studied with difficulty, and stabilization of produced adducts is frequently required. In the past, this stabilization has often been achieved by means of acetylation (8–10), a slow process that cannot be carried out in aqueous solutions and complicates the analysis of the obtained mixtures. In an attempt to find a simpler procedure to analyze carbonyl-phenol adducts, this study explores the possibility of stabilizing and separating these adducts after derivatization with methyl chloroformate. Derivatives obtained after treatment with methyl chloroformate have been used for the separation and quantification of numerous metabolites (11), including phenolic compounds (12), although their use for the separation and identification of carbonyl-phenol adducts remains to be investigated. This study describes the development of a reference library of methyl chloroformate–derivatized carbonyl-phenol adducts with their mass spectrometric and retention index information, which can be employed for their detection. As an initial step, this library includes the adducts produced with (E)-2-alkenals and (E,E)-2,4-alkadienals as lipid carbonyls, and resorcinol, 2-methylresorcinol, orcinol (5-methylresorcinol), and 2,5-dimethylresorcinol as phenolic compounds. In addition, the developed library has been employed to study the formation of carbonyl-phenol adducts in the reaction between lipid hydroperoxides and phenolic compounds.

92 Granvogl and MacMahon; Food-Borne Toxicants: Formation, Analysis, and Toxicology ACS Symposium Series; American Chemical Society: Washington, DC, 2019.

Materials and Methods Chemicals Different hydroperoxides and lipid-derived reactive carbonyls were employed in these studies. Lipid hydroperoxides were prepared by oxidation of the corresponding fatty acids with lipoxygenase and later esterification with diazomethane following previously described procedures (13, 14). (E)-2-Alkenals, (E,E)-2,4-alkadienals, phenolics, and all other chemicals were purchased from Sigma-Aldrich (St. Louis, MO) or Merck (Darmstadt, Germany) and were of the highest available grade.

Reactive Carbonyl/Phenolic Reaction Mixtures Mixtures of the hydroperoxides or the lipid-derived reactive carbonyls (30 μmol in 30 μL of methanol) and phenolics (30 μmol in 170 μL of water) were mixed with 300 μL of 0.3 M sodium phosphate solution, pH 8, and heated overnight at 60 °C under nitrogen. At the end of the heating time, samples were cooled (10 min at room temperature) and derivatized with methyl chloroformate.

Derivatization with Methyl Chloroformate Cooled samples were treated successively with methanol (100 μL), 1 M NaOH (100 μL), pyridine (35 μL), and methyl chloroformate (20 μL). Samples were then stirred and, after 20 s, methyl chloroformate (20 μL) was added. Samples were stirred again and, after 2 min, 0.1 M sodium bicarbonate solution (400 μL) was added. Samples were stirred again and extracted with dichloromethane (700 μL). Finally, samples were centrifuged at 2000 g for 5 min, and the organic layer was separated and analyzed by gas chromatography–mass spectrometry (GC–MS).

GC–MS Analyses GC–MS analyses were conducted with an Agilent 7820A gas chromatograph (Agilent Technologies, Santa Clara, CA) coupled with an Agilent 5977 mass selective detector (quadrupole type). A fused-silica HP-5MS ultra inert (UI) capillary column (30 m × 0.25 mm inner diameter, with a coating thickness of 0.25 μm) (Agilent Technologies, Santa Clara, CA) was used, and 1 μL of sample was injected in the pulsed splitless mode. Working conditions were as follows: carrier gas, helium (1 mL/min at constant flow); injector, 250 °C; oven temperature program, from 100 °C (1 min) to 300 °C at 15 °C/min and then held at 300 °C for 5 min; transfer line to mass selective detector, 280 °C; electron ionization, 70 eV; ion source temperature, 230 °C; and mass range, 28–550 amu.


Results Reaction of (E)-2-Alkenals with Phenolics The reaction of (E)-2-alkenals with phenolics produces two types of carbonylphenol adducts: chromane-2,7-diols (1) and 2H-chromen-7-ols (2) (Figure 1) (7). After derivatization with methyl chloroformate, adducts 1 were converted into adducts 3 and adducts 2 were converted into adducts 4. Figure 2A–C shows the extracted ion chromatograms obtained for a mixture of 2-methylresorcinol with acrolein (Figure 2A), crotonaldehyde (Figure 2B), or (E)-2-pentenal (Figure 2C), respectively, after derivatization with methyl chloroformate. The peak corresponding to adduct 3 was the main reaction product in all assayed reactions and always appeared as a single peak (Table 1). In addition, the chromatograms usually showed some smaller peaks that corresponded to adducts 4. Adducts 4 usually appeared as two peaks, although a single peak was always observed with acrolein and three peaks appeared in mixtures involving orcinol (Table 2). Although adducts 1 and 2 were derivatized with methyl chloroformate, only the phenolic group was protected. Therefore, methyl chloroformate did not react with the hemiacetalic hydroxyl group of chromane-2,7-diols (1). Different reactions involving a variety of (E)-2-alkenals (acrolein [2-propenal], crotonaldehyde [(E)-2-butenal], (E)-2-pentenal, and (E)-2-octenal) and phenolic compounds (resorcinol, 2-methylresorcinol, orcinol, and 2,5-dimethylresorcinol) were studied. Retention indices and mass spectra of produced adducts 3 are collected in Table 1. In addition, changes in retention indices as a function of (E)-2-alkenal chain length is shown in Figure 3. Retention indices increased as a function of chain length; this increase was linear (r > 0.98, p < 0.1) for the aldehydes having between 3 and 5 carbon atoms (Figure 3A). Slopes of the obtained lines depended on the involved phenolics and decreased in the order resorcinol > 2-methylresorcinol > orcinol > 2,5-dimethylresorcinol. Mass spectra also depended on the aldehyde chain length, but all isolated adducts exhibited the same fragmentation pattern. These fragments corresponded to the molecular ion, the loss of the alkyl R residue from adduct 3, the loss of the group CH2CHO from adduct 5, and the loss of the alkenal from adduct 5. These last two losses could be produced because the hemiacetal ring was opened to produce adduct 5 and the carbonyl group was recovered. In relation to the intensity of the fragments, adducts derived from acrolein or crotonaldehyde usually exhibited a behavior different to that of adducts derived from (E)-2-pentenal and (E)-2octenal. Thus, the base peak of the shorter aldehydes was more related to the opening of the hemiacetal form and the loss of the aldehyde group. On the contrary, the base peak of adducts obtained with (E)-2-pentenal or (E)-2-octenal was always the loss of either the ethyl or the pentyl group, respectively, in the cyclic structure 3 (the R group in Figure 1). This loss was also important in adducts derived from crotonaldehyde, but it was absent in adducts derived from acrolein.


Figure 1. Carbonyl-phenol adducts formed in the reaction of (E)-2-alkenals and phenolics and major fragments of produced adducts observed by mass spectrometry.


Figure 2. Extracted ion chromatograms of the reactions of 2-methylresorcinol with (A) acrolein (m/z 161), (B) crotonaldehyde (m/z 175), (C) (E)-2-pentenal (m/z 175), (D) (E,E)-2,4-heptadienal (m/z 173), and (E) 13-hydroperoxide of methyl linolenate (traces at m/z 161, m/z 175, m/z 175, and m/z 173 for adducts derived from acrolein, crotonaldehyde, (E)-2-pentenal, and (E)-2-octenal, respectively). Structures of adducts 3 and 4 are given in Figure 1. Structure of adduct 10 is given in Figure 4. Compounds in layer E were identified on the basis of their retention indices and mass spectra.


Table 1. Retention Indices and Mass Spectra of Adducts of Chromane-2,7-diols (3) Produced in the Reaction between (E)-2-Alkenals and Phenolics MS (m/z, %) alk

phen

RI

M+

M+ – Ra

M+ – F1b

M+ – alk

acr

res

1929

224 (59)

223 (0)

181 (18)

168 (54)

acr

mres

1983

238 (83)

237 (0)

195 (21)

182 (58)

acr

orc

2074

238 (72)

237 (0)

19 (26)

182 (100)

acr

dmr

2111

252 (81)

251 (0)

209 (27)

196 (100)

crot

res

1987

238 (78)

223 (41)

195 (100)

168 (38)

crot

mres

2033

252 (81)

237 (32)

209 (100)

182 (35)

crot

orc

2107

252 (68)

237 (38)

209 (60)

182 (100)

crot

dmr

2134

266 (37)

251 (30)

223 (59)

196 (100)

pent

res

2086

252 (32)

223 (100)

209 (19)

168 (5)

pent

mres

2121

266 (37)

237 (100)

223 (17)

182 (5)

pent

orc

2181

266 (31)

237 (100)

223 (8)

182 (13)

pent

dmr

2202

280 (35)

251 (100)

237 (8)

196 (16)

oct

res

2360

294 (19)

223 (100)

251 (3)

168 (4)

oct

mres

2383

308 (18)

237 (100)

265 (2)

182 (4)

oct

orc

2431

208 (15)

237 (100)

265 (0.4)

182 (10)

oct

dmr

2442

322 (19)

251 (100)

279 (1)

196 (13)

Abbreviations: acr = acrolein; alk = (E)-2-alkenal; crot = crotonaldehyde; dmr = 2,5dimethylresorcinol; mres = 2-methylresorcinol; MS = mass spectra; oct = (E)-2-octenal; orc = orcinol; pent = (E)-2-pentenal; phen = phenolic; res = resorcinol; RI = retention indices Chemical structures are collected in Figure 1. Retention indices were determined in a fused-silica HP-5MS UI capillary column. a R corresponds to the alkyl residue shown in Figure 1. b F1 corresponds to the fragment CH2CHO.


Figure 3. Change of retention index of carbonyl-phenol adducts 3 (A) and 4 (B) as a function of the chain length of the (E)-2-alkenals. Studied phenolics were resorcinol (□), 2-methylresorcinol (○), orcinol (4), and 2,5-dimethylresorcinol (5). Retention indices were determined in a fused-silica HP-5MS UI capillary column. Several symbols with different retention indices appear at each aldehyde chain length in panel B because several adducts 4 were produced for each aldehyde (Table 2).

Retention indices and mass spectra of produced adducts 4 are collected in Table 2. These adducts had a lower molecular weight than adducts 3, and the free hydroxyl group of adducts 3 was absent. Therefore, they had lower retention indices than their homologous adducts 3.

Table 2. Retention Indices and Mass Spectra of Adducts of 2H-chromen-7-ols (4) Produced in the Reaction between (E)-2-Alkenals and Phenolics MS (m/z, %) alk

phen

RI

M+

M+

6–R

7–R

8

205 (31)

161 (100)

147 (23)

146 (14)

220 (81)

219 (43)

175 (100)

161 (42)

160 (29)

1809

220 (87)

219 (58)

175 (100)

161 (64)

160 (8)

dmr

1890

234 (100)

233 (70)

189 (80)

175 (79)

174 (22)

res

1668

220 (10)

205 (100)

161 (60)

147 (4)

146 (24)

acr

res

1642

206 (62)

acr

mres

1723

acr

orc

acr crot

–R

Continued on next page.


Table 2. (Continued). Retention Indices and Mass Spectra of Adducts of 2H-chromen-7-ols (4) Produced in the Reaction between (E)-2-Alkenals and Phenolics MS (m/z, %) alk

phen

RI

M+

M+

–R

6–R

7–R

220 (32)

205 (100)

161 (78)

147 (4)

146 (20)

1741

234 (10)

219 (100)

175 (45)

161 (6)

160 (44)

mres

1756

234 (33)

219 (100)

175 (55)

161 (5)

160 (35)

crot

orc

1755

234 (26)

219 (100)

175 (20)

161 (7)

160 (51)

crot

orc

1802

234 (7)

219 (100)

175 (61)

161 (2)

160 (11)

crot

orc

1848

234 (31)

219 (100)

175 (71)

161 (2)

160 (8)

crot

dmr

1859

248 (8)

233 (100)

189 (39)

175 (7)

174 (28)

crot

dmr

1890

248 (30)

233 (100)

189 (47)

175 (5)

174 (22)

pent

res

1775

234 (2)

205 (100)

161 (54)

147 (6)

146 (24)

pent

res

1819

234 (13)

205 (100)

161 (60)

147 (6)

146 (23)

pent

mres

1838

248 (2)

219 (100)

175 (36)

161 (8)

160 (38)

pent

mres

1873

248 (13)

219 (100)

175 (39)

161 (7)

160 (36)

pent

orc

1863

248 (10)

219 (100)

175 (9)

161 (8)

160 (51)

pent

orc

1892

248 (1)

219 (100)

175 (49)

161 (4)

160 (10)

pent

orc

1954

248 (13)

219 (100)

175 (55)

161 (3)

160 (9)

pent

dmr

1939

262 (1)

233 (100)

189 (31)

175 (8)

174 (25)

pent

dmr

1997

262 (14)

233 (100)

189 (35)

175 (5)

174 (23)

oct

res

2136

276 (6)

205 (100)

161 (40)

147 (5)

146 (12)

oct

mres

2178

290 (6)

219 (100)

175 (26)

161 (7)

160 (21)

oct

orc

2167

290 (5)

219 (100)

175 (6)

161 (9)

160 (29)

oct

orc

2173

290 (1)

219 (100)

175 (33)

161 (8)

160 (8)

oct

orc

2251

290 (7)

219 (100)

175 (32)

161 (23)

160 (7)

oct

dmr

2205

304 (1)

233 (100)

189 (20)

175 (10)

174 (16)

crot

res

1705

crot

mres

crot

8

Abbreviations: acr = acrolein; alk = (E)-2-alkenal; crot = crotonaldehyde; dmr = 2,5dimethylresorcinol; mres = 2-methylresorcinol; MS = mass spectra; oct = (E)-2-octenal; orc = orcinol; pent = (E)-2-pentenal; phen = phenolic; res = resorcinol; RI = retention indices Chemical structures are collected in Figure 1. Retention indices were determined in a fused-silica HP-5MS UI capillary column.

Analogously to the above described for adducts 3, retention indices of adducts 4 increased as a function of chain length; this increase was linear (r > 0.98, p < 0.001) for aldehydes having between 4 and 8 carbons (Figure 3B). Slopes of the obtained lines also depended on the involved phenolics and decreased in the order 99 Granvogl and MacMahon; Food-Borne Toxicants: Formation, Analysis, and Toxicology ACS Symposium Series; American Chemical Society: Washington, DC, 2019.

resorcinol > 2-methylresorcinol > orcinol > 2,5-dimethylresorcinol similarly to the above described for adducts 3. Although this order was identical for adducts 3 and 4, there was not a clear relationship between the slopes determined for adducts 3 and those determined for adducts 4. Mass spectra also depended on the aldehyde chain length. However, all adducts 4 exhibited a similar fragmentation pattern. These fragments derived from four ions with an even number of hydrogen atoms: the molecular ion 4, the adduct 6 formed after a loss of CO2, the adduct 7 produced by loss of the protecting group, and the quinone 8. In relation to the intensity of the fragments, the base peak of all adducts, except those derived from acrolein, corresponded to the loss of the alkyl side chain (R in Figure 1) from the molecular ion (4). Acrolein was an exception most likely because this loss corresponded to the loss of a hydrogen atom. Base ions in adducts 4 derived from acrolein corresponded to either the molecular ion or the ions formed after the loss of CO2 (6).

Reaction of (E,E)-2,4-Alkadienals with Phenolics The reaction of (E,E)-2,4-alkadienals with phenolics produces alk-1-en-1ylchromane-2,7-diols (9) (Figure 4) (8). Because of the creation of chiral centers, several diastereomers were observed in the total ion chromatogram obtained for a mixture of 2-methylresorcinol and 2,4-heptadienal (Figure 2D). One of them was usually produced to a higher extent than the others. Adducts were derivatized in the presence of methyl chloroformate. However, and analogously to the adducts 1 derived from 2-alkenals, only the phenolic group, and not the hemiacetalic hydroxyl group, was protected. Different reactions involving a variety of (E,E)-2,4-alkadienals [(E,E)2,4-hexadienal (hxd); (E,E)-2,4-heptadienal (hd); (E,E)-2,4-octadienal (od); (E,E)-2,4-nonadienal (nd); and (E,E)-2,4-decadienal (dd)] and phenolic compounds (resorcinol, 2-methylresorcinol, 2,5-dimethylresorcinol, and orcinol) were studied. Retention indices and mass spectra of main adducts 10 are collected in Table 3. As observed for adducts derived from (E)-2-alkenals, retention indices increased as a function of chain length; this increase was linear (r > 0.999, p < 0.0001) for the five (E,E)-2,4-alkadienals studied (Figure 5). Also, the slopes of the obtained lines depended on the involved phenolics and decreased in the order resorcinol > 2-methylresorcinol > orcinol > 2,5-dimethylresorcinol. As expected, mass spectra depended on the aldehyde chain length, but all adducts exhibited the same fragmentation pattern. These fragments were derived from four ions with an even number of hydrogen atoms: the molecular ion (10), the molecular ion with a free carbonyl group (11), the dehydrated molecular ion (12), and the ion (13) produced after a loss of ketene (Figure 4). In relation to the intensity of the fragments, the base peak of adducts derived from longer aldehydes (C7–C10) was derived from the dehydrated ion 12. On the contrary, the base peak of most adducts derived from 2,4-hexadienal mostly derived from the molecular ion 10. In addition, the molecular ion was more abundant when shorter-chain aldehydes and less substituted phenolics were involved (Table 3). 100 Granvogl and MacMahon; Food-Borne Toxicants: Formation, Analysis, and Toxicology ACS Symposium Series; American Chemical Society: Washington, DC, 2019.

Figure 4. Carbonyl-phenol adducts formed in the reaction of (E,E)-2,4-alkadienals and phenolics and major fragments of produced adducts observed by mass spectrometry.


Table 3. Retention Indices and Mass Spectra of Adducts of Alk-1-en-1-ylchromane-2,7-diols (10) Produced in the Reaction between (E,E)-2,4-Alkadienals and Phenolics MS (m/z, %) M+

12 – R

11 – F1a

12 – F2b

12 – F3c

102

akd

phen

RI

hxd

res

2157

264 (100)

246 (59)

231 (42)

222 (10)

221 (74)

205 (52)

187 (42)

168 (9)

hxd

mres

2193

278 (100)

260 (45)

245 (32)

236 (7)

235 (45)

219 (85)

201 (35)

182 (8)

hxd

orc

2230

278 (89)

260 (13)

245 (38)

236 (15)

235 (100)

219 (52)

201 (17)

182 (40)

hxd

dmr

2249

292 (92)

274 (11)

259 (29)

250 (11)

249 (64)

233 (100)

215 (16)

196 (36)

hd

res

2240

278 (100)

260 (40)

231 (60)

236 (15)

235 (82)

205 (92)

201 (20)

168 (11)

hd

mres

2275

292 (87)

274 (25)

245 (37)

250 (8)

249 (39)

219 (100)

215 (15)

182 (10)

hd

orc

2303

292 (84)

274 (9)

245 (45)

250 (15)

249 (78)

219 (100)

215 (5)

182 (47)

hd

dmr

2319

306 (82)

288 (10)

259 (40)

264 (11)

263 (57)

233 (100)

229 (6)

196 (43)

od

res

2335

292 (50)

274 (15)

231 (6)

250 (16)

249 (100)

205 (56)

215 (5)

168 (7)

od

mres

2363

306 (73)

288 (13)

245 (38)

264 (15)

263 (76)

219 (100)

229 (6)

182 (10)

od

orc

2388

306 (81)

288 (10)

245 (47)

264 (18)

263 (100)

219 (72)

229 (3)

182 (55)

od

dmr

2400

320 (64)

302 (7)

259 (31)

278 (10)

277 (54)

233 (100)

243 (3)

196 (40)

nd

res

2425

306 (76)

288 (23)

231 (87)

264 (11)

263 (68)

205 (100)

229 (8)

168 (12)

nd

mres

2451

320 (60)

302 (10)

245 (55)

278 (9)

277 (32)

219 (100)

243 (6)

182 (15)

nd

orc

2469

320 (93)

302 (8)

245 (73)

278 (17)

277 (91)

219 (100)

243 (3)

182 (76)

nd

dmr

2479

334 (44)

316 (3)

259 (25)

292 (6)

291 (30)

233 (100)

257 (1)

196 (28)

12

13

Granvogl and MacMahon; Food-Borne Toxicants: Formation, Analysis, and Toxicology ACS Symposium Series; American Chemical Society: Washington, DC, 2019.

M+ – akd

MS (m/z, %) M+

12 – R

11 – F1a

12 – F2b

12 – F3c

akd

phen

RI

dd

res

2485

320 (94)

302 (30)

231 (89)

278 (12)

277 (85)

205 (100)

243 (1)

168 (29)

dd

mres

2536

334 (52)

316 (17)

245 (37)

292 (2)

291 (26)

219 (100)

257 (14)

182 (21)

dd

orc

2559

334 (77)

316 (13)

245 (57)

292 (17)

291 (63)

219 (100)

257 (1)

182 (65)

dd

dmr

2565

348 (41)

330 (3)

259 (21)

306 (6)

305 (30)

233 (100)

271 (1)

196 (32)

12

13

M+ – akd

Abbreviations: akd = alkadienal; dmr = 2,5-dimethylresorcinol; mres = 2-methylresorcinol; MS = mass spectra; orc = orcinol; phen = phenolic; res = resorcinol; RI = retention indices Chemical structures are collected in Figure 4. a F1 corresponds to the fragment CH2CHO. b F2 corresponds to the fragment RCHCH. c F3 corresponds to the fragment CH3OCO.


Figure 5. Change of retention index of adducts as a function of the chain length of the (E,E)-2,4-alkadienal. Studied phenolics were resorcinol (□), 2-methylresorcinol (○), orcinol (4), and 2,5-dimethylresorcinol (5). Retention indices were determined in a fused-silica HP-5MS UI capillary column.

Reaction of Lipid Hydroperoxides with Phenolics The created database with all previous retention indices and mass spectra was employed to investigate the formation of short-chain aldehyde/phenolic adducts in mixtures of lipid hydroperoxides and phenolics. As an example, Figure 2E shows the extracted ion chromatogram obtained from a mixture of the 13-hydroperoxide of methyl linolenate and 2-methylresorcinol. The main carbonyl-phenol adducts observed were the chromane-2,7-diols 3a (from acrolein), 3b (from crotonaldehyde), and 3c (from (E)-2-pentenal). In addition, the 2H-chromen-7-ols 4b and 4c (from crotonaldehyde), and 4d and 4e (from (E)-2-pentenal) were also detected in trace amounts. On the other hand, neither the 2H-chromen-7-ol 4a derived from acrolein, nor the alk-1-en-1-ylchromane-2,7-diol 10 derived from (E,E)-2,4-heptadienal could be detected. In addition, the adducts between 2-methylresorcinol and 2-octenal were not found. Analogously, in reactions involving the 13-hydroperoxide of methyl linoleate and 2-methylresorcinol (data not shown), chromane-2,7-diols derived from (E)-2-octenal could be detected, but not those derived from (E)-2-pentenal. In these last reactions, the chromane-2,7-diol 3a derived from acrolein was the main carbonyl-phenol adduct found (data not shown).

Discussion A recent study showed that toxicologically relevant aldehydes produced during the frying process are trapped by food phenolics (5). Thus, chromane2,7-diols (1) and 2H-chromen-7-ols (2) derived from quercetin and acrolein, crotonaldehyde, and (E)-2-pentenal were detected in fried onions. However, it is unclear whether these were the only carbonyl-phenol adducts formed in fried onions. To advance our present knowledge of the formation of these adducts in food products, this investigation proposes a novel analytical procedure 104 Granvogl and MacMahon; Food-Borne Toxicants: Formation, Analysis, and Toxicology ACS Symposium Series; American Chemical Society: Washington, DC, 2019.

for the study of carbonyl-phenol adducts. The procedure is quite simple, and carbonyl-phenol adducts can be derivatized in an aqueous system without the need of water removal. The reaction is very fast and adducts can be concentrated by extraction. The results obtained in this study show that the methodology can be applied easily to detect adducts derived from (E)-2-alkenals and (E,E)-2,4-alkadienals. The formed adducts have been characterized, and they can be easily identified by means of their retention indices and mass spectra. Furthermore, the obtained data have been applied to the identification of carbonyl-phenol adducts in hydroperoxide/phenolic reaction mixtures. As shown in Figure 2E, diverse carbonyl-phenol adducts were identified in a mixture of the 13-hydroperoxide of methyl linolenate and 2-methylresorcinol. These adducts are derived from the reaction of generated acrolein, crotonaldehyde, and (E)-2-pentenal with the phenolic. As expected, adducts derived from (E)-2-octenal were not detected because (E)-2-octenal is the product of ω6 fatty acyl chains and methyl linolenate is a ω3 fatty acyl ester. On the contrary, when the reaction between 13-hydroperoxide of methyl linoleate (a ω6 fatty acyl ester) and 2-methylresorcinol was studied, the adduct detected was that derived from (E)-2-octenal and not that derived from (E)-2-pentenal. In both cases, the main adduct produced in the reaction was that derived from acrolein. This is not surprising because acrolein is known to be a major product of lipid oxidation (15, 16). As opposed to the adducts derived from (E)-2-alkenals, adducts derived from (E,E)-2,4-alkadienals were not detected in any of the studied systems involving hydroperoxides. This can be a consequence of both the lower yield in which these carbonyl compounds are produced and their higher instability. Thus, when Suh et al. (15) studied the degradation ω3 fatty acids, they found that the yield of acrolein was about 0.2%, the yield of (E)-2-pentenal was 0.03%, and the yield of (E,E)-2,4-heptadienal was 0.01%. In addition, the carbonyl-phenol reaction for (E,E)-2,4-alkadienals has a low reaction rate (8), and (E,E)-2,4-alkadienals are thermally degraded to produce alkanals and (E)-2-alkenals (17). Although only two series of short-chain aldehydes have been studied in this work, it is expected that the same methodology can be applied to the carbonyl-phenol adducts derived from other short-chain aldehydes produced in the course of lipid oxidation. Furthermore, the sensitivity of the technique and its discriminating power can be improved if a more sensitive detector with an increased discriminating power, like a TOF-MS, is employed. Moreover, this methodology should also be valid for carbonyl-phenol adducts derived from more complex phenolics. However, these adducts are less volatile. Therefore, a liquid chromatography–mass spectrometry procedure would likely be more appropriate for the study of these adducts. Additional studies involving these less volatile and more common phenolics in foods will complete the database now started and contribute to a better understanding of the presence and the role of carbonyl-phenol adducts in foods. In addition, all of these methodologies should allow the quantitative determination of the formed adducts, as already shown for other compounds produced by alkyl chloroformate derivatization (11). 105 Granvogl and MacMahon; Food-Borne Toxicants: Formation, Analysis, and Toxicology ACS Symposium Series; American Chemical Society: Washington, DC, 2019.

Acknowledgments We are indebted to José L. Navarro for technical assistance. This study was supported in part by the European Union (FEDER funds) and the Plan Nacional de I + D of the Ministerio de Economía y Competitividad of Spain (Project AGL201568186-R).

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A Simple Procedure To Detect Lipid-Derived Carbonyl-Phenol Adducts

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