Differentiation of Whole Grain from Refined Wheat (T. aestivum) Flour

Article pubs.acs.org/JAFC

Differentiation of Whole Grain from Refined Wheat (T. aestivum) Flour Using Lipid Profile of Wheat Bran, Germ, and Endosperm with UHPLC-HRAM Mass Spectrometry Ping Geng, James M. Harnly, and Pei Chen* Food Composition and Methods Development Laboratory, Beltsville Human Nutrition Research Center, Agricultural Research Service, U.S. Department of Agriculture, Beltsville, Maryland 20705, United States S Supporting Information *

ABSTRACT: A comprehensive analysis of wheat lipids from milling fractions of bran, germ, and endosperm was performed using ultrahigh-performance liquid chromatography−high-resolution accurate-mass multistage mass spectrometry (UHPLCHRAM-MSn) with electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) in both positive and negative modes. About 155 lipid compounds, including free fatty acids (FA), oxylipins, alk(en)ylresorcinols (ARs), γ-oryzanol, sphingolipids, triglycerides (TGs), diglycerides (DGs), phospholipids, and galactolipids were characterized from the three milling fractions. Galactolipids and phospholipids were proposed to be potential discriminatory compounds for refined flour, whereas γ-oryzanols, ARs, TGs, and DGs could distinguish whole wheat flour from a refined one based on principal component analysis (PCA). KEYWORDS: whole grain, UHPLC, APCI, ESI, PCA, phospholipid, galactolipid, triglyceride, alk(en)ylresorcinols, γ-oryzanol, sphingolipid

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INTRODUCTION Wheat flours can be divided into two subgroups, whole wheat flour (WF) and refined wheat flour (RF). WF contains the entire grain kernel―the bran, germ, and endosperm. The bran and germ fractions are removed from the milling process for RF, leaving endosperm only. The germ contains many essential nutrients and antioxidants; it also contains natural oil (wheat germ oil). Once the germ is crushed and exposed to the air, the oil begins to oxidize and, in time, becomes rancid. Removal of the germ increases the shelf life of flour considerably and avoids the possibility of flour getting a strong flavor from rancid oils. However, WF’s health benefits cannot be overlooked. Epidemiological studies find that whole-grain intake is protective against cancer, cardiovascular disease, diabetes, and obesity.1−4 With the rise of consumer awareness, WF has become increasingly popular. The trend demands new methods for the analysis of whole-grain foods and further understanding of the differences in the chemical components between WF and RF. In the whole wheat kernel, lipids form 8−15% of the germ, about 6% of the bran, and 1−2% of the starchy endosperm.5 Although lipids in wheat or wheat flour are minor constituents, their composition is very complex,6,7 and they play important roles in wheat production, storage, processing, products, nutrition, and consumer acceptance of finished goods.8 Early publications on the analysis of wheat flour lipids often emphasized one or a few lipid classes.9−11 A recent study of lipophilic phytochemicals in wheat bran using gas chromatography−mass spectrometry (GC-MS) profiled alkylresorcinols, steroids, and triglycerides, but lacked polar lipids, such as phospholipids and galactolipids, due to the limitation of GC and the milling fraction selection of bran only.12 A comprehensive analysis of wheat lipids on milling fractions has not been reported. Such an analysis is This article not subject to U.S. Copyright. Published XXXX by the American Chemical Society

important as the lipid profiles are quite different in wheat structural parts. Germ lipids are predominantly nonpolar in character (77−85%), and polar lipids make up a minor proportion (13−17%), whereas the profile of endosperm lipids is distinctly different from that of germ and bran lipids. Endosperm lipids provide the only substantial source of galactolipids (predominantly monogalactosyl diglyceride (MGDG) and digalactosyl diglyceride (DGDG)) and phospholipids (predominantly phosphatidylcholine (PC), lysophosphatidylcholine (LPC), phosphatidylethanolamine (PE), and lysophosphatidylethanolamine (LPE)) among the various whole-grain components.8 Due to their remarkable structural diversity, lipid classes differ in their ionization capacity and produce polarity-dependent forms of molecular cations and anions. Therefore, in a comprehensive analysis using mass spectrometry (MS), both positive and negative ion modes and different ionization techniques must be used. APCI-MS has proven to be a valuable technique for the analysis of lipids from a variety of classes. This instrumental method readily produces useful ions by gentle fragmentation of large neutral molecules such as TGs, which are often difficult to analyze using other techniques, such as ESI. Molecules that are easily ionized, such as phospholipids, are better suited for ESI MS with collision-induced dissociation (CID). In the present work, we have performed a detailed and comprehensive characterization of the lipids present in wheat bran, germ, and endosperm using UHPLC-HRAM-MSn with ESI and APCI in both positive and negative modes. The approach Received: March 30, 2015 Revised: June 10, 2015 Accepted: June 17, 2015

A

DOI: 10.1021/acs.jafc.5b01599 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry

Article

Table 1. Major Fatty Acids Identified in Germ

a

error (ppm)

tentative identification

abbreviation

3.5 MS2 [279.2]: 261.2 (100),a 243.2 (58), 223.1 (27), 209.2 (30), 195.1 (21), C18H30O2 137.2 (31), 123.2(42), 109.2(35), 95.2 (38); MS3 [279.2→261.2]: 243.3 (100), 173.2 (13), 135.2 (13); MS4 [279.2→261.2→243.3]: 215.2 (12), 201.2 (44), 187.3 (61), 173.2 (100), 161.2 (28), 159.2 (45), 147.3 (22), 145.2 (50), 133.2 (20), 131.3 (26) 4.5 MS2 [277.2]: 259.4 (41), 257.3 (7), 233.3 (100), 205.3 (4), 179.3 (5)

α-linolenic acid

C18:3

2.5 MS2 [281.2]: 263.2 (100), 245.2 (19); MS3 [281.2→263.2]: 245.2 (100); MS4 [281.2→263.2→245.2]: 217.1 (26), 203.2 (43), 189.2 (93), 175.2 (96), 163.3 (39), 161.2 (100), 149.2 (37), 147.1 (71), 135.1 (29), 133.2 (55), 121.2 (28), 119.1 (26), 109.2 (13), 107.2 (11), 105.2(16), 95.2 (17), 81.2 (13) 3.5 MS2 [279.2]: 261.3 (100), 259.3 (5), 243.3 (5); MS3 [279.2→261.2]: 243.3 (100), 233.5 (16), 219.1 (22), 205.4 (16), 109.2 (25), 107.3 (17)

C18H32O2

linoleic acid

C18:2

0.5 MS2 [257.2]: 239.3 (100), 225.2 (24), 187.2 (22), 183.2 (17), 173.1 (20), 169.2 (17), 103.2 (20), 89.0 (28) 1.5 MS2 [255.2]: 237.3 (100), 236.7 (11), 211.2 (21), 193.3 (12)

C16H32O2

palmitic acid

C16:0

C18H34O2

oleic acid

C18:1

−0.724

1.5 MS2 [283.3]: 265.3 (100), 247.3 (27); MS3 [283.3→265.3]: 247.3 (100); MS4 [283.3→265.3→247.3]: 205.2 (26), 191.2 (65), 177.3 (97), 165.2 (19), 163.2 (100), 151.2 (17), 149.1 (98), 137.1 (19), 135.1 (73), 123.2 (19), 121.2 (48), 109.2 (30), 107.2 (19), 95.2 (34), 93.2 (17), 81.2 (24) 2.5 MS2 [281.2]: 263.3 (100)

[M + H]+ [M − H]−

−0.725 −1.248

0.5 MS2 [285.2]: 271.0 (23), 267.3 (35), 257.2 (20), 253.1 (100) 1.5 MS2 [283.3]: 265.3 (100), 251.2 (11), 239.5 (20), 221.3 (26)

C18H36O2

stearic acid

C18:0

10.36

[M + H]+

0.138

C20H38O2

gadoleic acid

C20:1

10.45

[M − H]−

−0.982

1.5 MS2 [311.3]: 293.2 (100), 275.3 (24); MS3 [311.3→293.2]: 275.4 (100); MS4 [311.3→293.2→275.4]: 219.3 (60), 205.2 (70), 191.3 (80), 177.2 (72), 163.2 (70), 149.2 (100), 135.2 (66), 121.2 (59), 109.2 (24), 107.2 (29), 93.2 (22) 2.5 MS2 [309.3]: 291.4 (100), 265.4 (54), 209.2 (67)

ion m/z

RT

adduct

279.2320

4.57

[M + H]+

0.513

277.2172

4.63

[M − H]−

−0.373

281.2473

5.93

[M + H]+

−0.735

279.2329

5.95

[M − H]−

−0.192

257.2475

6.87

[M + H]+

−0.026

255.2330

6.91

[M − H]−

0.182

283.2630

7.43

[M + H]+

−0.554

281.2484

7.48

[M − H]−

285.2786 283.2639

9.76 9.82

311.2945

309.2796

RDB

MS2 data

formula

Characteristic ions for identification are in bold. Same as other tables.

Figure 1. Extracted ion chromatograms of fatty acid ions at m/z 277.21−277.23, 279.23−279.24, and 281.24−281.25 in ESI negative mode. B



Article

Table 2. Oxylipins Putatively Identified in Germ ion m/z

RT

error (ppm)

adduct −

formula

RDB

MS2 data


abbreviation

329.2334

1.05

[M − H]

309.2071

1.16

[M − H]− −0.106 C18H30O4

4.5 MS2 [309.2]: 291.3 (100), 273.3 (4), 265.3 (5), 251.4 (4), 221.3 (8), 209.3 (9), 195.3 (20), 185.2 (7), 171.2 (8)

311.2228

1.32

[M − H]−

0.055 C18H32O4

3.5 MS2 [311.2]: 293.4 (97), 275.4 (8), 267.3 (20), 249.2 (9), 9-hydroxy-13-oxo-10-octade241.4 (6), 223.3 (11), 211.3 (11), 184.1 (24), 183.2 (100), cenoic acid; 13-hydroxy-9171.2 (6) oxo-10-octadecenoic acid

13-oxo-9-HODE; 9-oxo-13-HODE

313.2384

1.41

[M − H]− −0.105 C18H34O4

2.5 MS2 [313.2]: 295.3 (100), 277.4 (19), 213.2 (4), 201.2 (7), 12,13-dihydroxy-9-octadece195.3 (13), 183.3 (60), 129.3 (4); MS3 [313.2→295.3]: noic acid 277.3 (100), 251.4 (24), 211.3 (10), 195.3 (56), 181.3 (12), 179.3 (44), 171.2 (12), 113.2 (7)

12,13-DiHOME(9)

293.2122

1.65

[M − H]− −0.062 C18H30O3

4.5 MS2 [293.2]: 275.4 (100), 265.4 (6), 249.3 (7), 235.3 (20), (9Z,11E)-13-keto-9,11-octade- 13-KODE 224.2 (6), 205.4 (9), 195.2 (18), 179.2 (7); MS3 cadienoic acid [293.2→275.4]: 257.3 (10), 231.3 (100), 177.3 (9)

295.2276

1.68

[M − H]− −0.908 C18H32O3

3.5 MS2 [295.2]: 277.4 (100), 267.5 (3), 259.3 (2), 251.3 (47), 13-hydroxy-9,11-octadecadie223.3 (5), 213.2 (5), 197.1 (9), 195.3 (13), 179.2 (18), noic acid 171.2 (16), 169.2 (9), 167.3 (2), 153.2 (11), 151.2 (20), 141.3 (29)

13-HODE

295.2276

1.96

[M − H]− −0.908 C18H32O3

3.5 MS2 [295.2]: 277.3 (100), 251.4 (7), 195.2 (23), 179.2 (14), 9-hydroxy-10,12-octadecadie171.2 (34) noic acid

9-HODE

293.2122

2.06

[M − H]− −0.062 C18H30O4

4.5 MS2 [293.2]: 275.2 (67), 249.3 (47), 235.3 (17), 209.3 (7), (10E,12Z)-9-keto-10,12-octa- 9-KODE 197.3 (36), 185.3 (100), 179.2 (9), 167.2 (10), 149.2 (7), decadienoic acid 141.3 (10), 125.3 (8), 113.2 (26)

297.2434

2.19

[M − H]− −0.398 C18H34O3

2.5 MS2 [297.2]: 279.3 (100), 253.5 (9), 251.4 (17), 235.3 (10), 9,10-epoxyoctadecanoic acid 198.2 (5), 197.2 (14), 184.2(9), 183.2 (24), 171.3 (45), 157.3 (12), 155.2 (6)

9,10-EODA

297.2434

5.11

[M − H]− −0.398 C18H34O3

2.5 MS2 [297.2]: 297.5 (24), 281.1 (6), 279.2 (100), 253.4 (85), 9,10-epoxyoctadecanoic acid 251.3 (49), 240.1 (7), 235.2 (15), 183.1 (92)

9,10-EODA

0.160 C18H34O5

2.5 MS2 [329.2]: 314.1 (10), 311.3 (100), 293.4 (48), 291.2 (7), 9,12,13-trihydroxy-10-octade- 9,12,13-THODE 275.3 (31), 229.3 (35), 211.2 (34), 201.2 (61), 199.3 (29), cenoic acid 197.2 (8), 183.3 (7), 181.2 (17), 171.3 (73), 169.2 (12)

(Heraeus, South Plainfield, NJ, USA) at 4 °C. The supernatant was filtered through a 17 mm (0.20 μm) PVDF syringe filter (VWR Scientific, Seattle, WA, USA) and stored at −80 °C until analysis. For FCMS analysis, whole wheat flour and all purpose unbleached flour from three different brands were used as WF and RF, respectively. Each of the six samples was extracted in triplicate. The injection volume for all samples was 2 μL. UHPLC-HRAM-MSn Conditions. The UHPLC-HRAM-MSn system consisted of an LTQ Orbitrap XL MS with an Accela 1250 binary pump, a PAL HTC Accela TMO autosampler, an Accela PDA detector (Thermo Fisher Scientific, San Jose, CA, USA), and a G1316A column compartment (Agilent, Santa Clara, CA, USA). APCI and ESI detection were investigated in both the positive and negative ion modes. The ESI conditions were set as follows: sheath gas at 80 (arbitrary units), auxiliary and sweep gas at 10 (arbitrary units), spray voltage at −4.0 kV for negative mode and at 4.5 kV for positive mode; capillary temperature at 300 °C; capillary voltage at −50 V for negative mode and at 50 V for positive mode; and tube lens offset at −120 V for negative mode and at 120 V for positive mode. APCI source was operated at 450 °C, the heated capillary temperature was 275 °C, the corona discharge current was set to 5.0 μA, and sheath gas (N2) and auxiliary gas (N2) were at 60 and 5 arbitrary units, respectively. The scan event cycle used a FT full scan mass spectrum at a resolving power of 30000 and three data-dependent MS2, MS3, MS4 events acquired by LTQ triggered by the most intensive ion from each previous scan event. The automated gain control (AGC) target value was set to 200,000 for FT full scan and to 10,000 for IT MSn scan, respectively. MSn activation parameters used an isolation width of 1 amu, maximum ion injection time of 200 ms, normalization collision energy at 35%, and activation time of 30 ms.

enabled the analysis of a wide range of compounds from fatty acids to intact high molecular weight lipids such as sterol esters and TGs. UHPLC-HRAM-MSn has been used to analyze polyphenols, glucosinolates, flavonoids, etc., in this laboratory.13−17 The fuzzy chromatography mass spectrometric (FCMS) fingerprinting combined PCA was used to visualize the chemical difference between WF and RF. In comparison with full LC separation, FCMS aims to obtain an adequate separation one class of compounds from another as fast as possible on a relatively short (≤50 mm) column. Although optimal separation cannot be achieved on each individual compound, FCMS can avoid ion suppression effects to some extent and speed up high-throughput analysis.

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9-hydroperoxy-10,12,15-octadecatrienoic acid

EXPERIMENTAL PROCEDURES

Chemicals. HPLC grade methanol, formic acid, and n-butanol were purchased from VWR International, Inc. (Clarksburg, MD, USA). HPLC water was prepared from distilled water using a Milli-Q system (Millipore Laboratories, Bedford, MA, USA). Materials and Sample Preparation. The bran, germ, and endosperm of the same brand were purchased from local grocery stores. Bran and germ flakes were ground into fine powder and then passed through a 60 mesh sieve. One hundred milligrams of each sample was extracted with 4.0 mL of water-saturated n-butanol5,6,11,18 using a sonicator (Advanced Sonic Processing Systems, Oxford, CT, USA) at 16 kHz with 300 W power for 40 min. The slurry mixture was centrifuged at 13000g for 15 min in a Contifuge 28RS centrifuge C



Article

Figure 2. Structures of 5-n-alk(en)ylresorcinols present in wheat.

Table 3. Alk(en)ylresorcinols Identified from Bran ion m/z

RT

error (ppm)

formula

RDB

345.2788

5.83

−0.020

C23H36O2

5.5

MS2 [345.3]: 327.6 (21), 275.5 (12), 261.5 (17), 177.0 (21), 163.1 (63), 123.1 (100)

AR-C17:2

371.2946

6.81

0.385

C25H38O2

6.5

MS2 [371.3]: 355.2 (36), 301.3 (32), 275.2 (38), 259.1 (72), 240.9 (36), 177.2 (53), 163.2 (100), 147.1 (37), 137.2 (46), 129.1 (60), 123.1 (99)

AR-C19:3

347.2945

7.32

0.124

C23H38O2

4.5

MS2 [347.3]: 123.1 (100); MS3 [347.3→123.1]: 95.0 (100), 65.1 (11)

AR-C17:1

391.3207

7.69

0.072

C25H42O3

4.5

MS2 [391.3]: 373.4 (62), 348.2 (10), 233.2 (12), 149.1 (100), 139.1 (21), 123.0 (10)

AR-C19:oxo

373.3101

8.14

−0.019

C25H40O2

5.5

MS2 [373.3]: 357.1 (52), 341.2 (28), 289.2 (16), 275.3 (21), 261.3 (27), 163.2 (56), 149.3 (18), 137.2 (19), 123.2 (100)

AR-C19:2

349.3104

9.56

0.839

C23H40O2

3.5

MS2 [349.3]: 334.4 (2), 123.2 (4), 111.0 (100); MS3 [349.3→111.0]: 93.2 (100)

AR-C17:0

375.3259

10.09

0.381

C25H42O2

4.5

MS2 [375.3]: 123.1 (100); MS3 [375.3→123.1]: 95.1 (100), 77.0 (5), 67.1 (17)

AR-C19:1

419.3519

10.66

−0.072

C27H46O3

4.5

MS2 [419.4]: 401.4 (100), 177.1 (14), 163.1 (35), 149.2 (35), 123.1 (29); MS3 [419.4→401.4]: 319.4 (14), 177.1 (20), 163.2 (76), 149.1 (100), 123.2 (58)

AR-C21:oxo

375.3259

10.25

0.381

C25H42O2

4.5

MS2 [375.3]: 123.1 (100); MS3 [375.3→123.1]: 95.1 (100), 67.2 (23)

AR-C19:1

377.3416

12.78

0.511

C25H44O2

3.5

MS2 [377.3]: 127.0 (2), 123.1 (2), 111.2 (100); MS3 [377.3→111.2]: 93.2 (100)

AR-C19:0

403.3572

13.24

0.354

C27H46O2

4.5

MS2 [403.4]: 123.1 (100); MS3 [403.4→123.1]: 95.2 (100), 57.1 (14)

AR-C21:1

447.3835

13.86

0.228

C29H50O3

4.5

MS2 [447.4]: 429.4 (100), 177.3 (12), 163.1 (39), 149.1 (30); MS3 [447.4→429.4]: 347.3 (10), 333.4 (9), 177.2 (14), 163.2 (85), 149.1 (100), 123.1 (34)

AR-C23:oxo

429.3729

14.31

0.193

C29H48O2

5.5

MS2 [429.4]: 411.5 (24), 401.5 (24), 341.2 (18), 219.2 (13), 205.3 (26), 191.3 (14), 177.3 (20), 175.1 (6), 165.2 (100), 163.2 (17), 149.2 (6); MS3 [429.4→165.1]: 137.2 (100), 119.3 (11), 109.3 (20), 67.2 (4)

AR-C23:2

MS2 data

D

abbreviation



Article

Table 3. continued ion m/z

RT

405.3730

15.86

431.3884

error (ppm)

MS2 data

formula

RDB

0.293

C27H48O2

3.5

MS2 [405.4]: 387.4 (40), 251.2 (28), 169.1 (27), 140.8 (79), 127.2 (75), 124.8 (67), 123.3 (100)

AR-C21:0

abbreviation

16.42

0.099

C29H50O2

4.5

MS2 [431.4]: 413.6 (8), 361.5 (8), 347.3 (9), 333.4 (17), 319.1 (12), 305.3 (8), 291.4 (5), 277.2 (7), 137.2 (5), 123.1 (100)

AR-C23:1

433.4042

18.91

0.193

C29H52O2

3.5

MS2 [433.4]: 433.8 (78), 415.7 (72), 405.5 (70), 279.4 (50), 265.1 (41), 251.4 (49), 237.5 (41), 197.2 (61), 182.7 (41), 169.0 (52), 167.4 (56), 155.2 (100), 138.1 (41), 123.2 (59)

AR-C23:0

461.4355

21.92

0.192

C31H56O2

3.5

MS2 [461.4]: 111.2 (100)

AR-C25:0

Figure 3. Proposed characteristic ions from CID spectra of ARs in positive mode.

Figure 4. Structures of γ-oryzanol identified in wheat bran: 1, campesteryl ferulate; 2, sitosteryl ferulate; 3, campestanyl ferulate; 4, sitostanyl ferulate. Data Processing for PCA. Each FCMS fingerprint consisted of a one-dimensional matrix (ion counts versus mass for m/z 100−1300 in nominal resolution), obtained by averaging spectra acquired from 0.1 to 5.0 min. The nominal masses of each FCMS spectrum were exported to Excel (Microsoft, Inc., Bellingham, WA, USA) from Xcalibur for data preprocessing. The preprocessing in Excel involved combining MS fingerprints of all the samples into one matrix, sorting the data by sample names, and aligning the masses (each spectrum had a different number of masses because the m/z below the detection threshold would not be exported into the mass list from the spectrum). A zero was inserted for each missing m/z, using an Excel macro written in-house.

The LC separation was carried out on a Poroshell 120EC-C18 column (2.1 × 150 mm, 2.7 μm) (Agilent, Santa Clara, CA, USA) with a flow rate of 0.4 mL/min. The mobile phase consisted of a combination of A (0.1% formic acid in water) and B (0.1% formic acid in methanol). A gradient elution program was employed as follows: 85% B at 0−3.0 min, 90% B at 5.0−7.0 min, 95−100% B over 15.0−20.0 min, and held at 100% B until 100.0 min. The column temperature was set at 40 °C. A Poroshell 120EC-C18 column (3.0 × 50 mm, 2.7 μm) was used for FCMS fingerprinting with the same flow rate and temperature. The flowing elution gradient was 85% B at 0−1.0 min, 100% B at 1.1−3.7 min, and 85% B at 3.8−5.0 min. E



Article

Table 4. γ-Oryzanols Identified from Bran ion m/z

RT

adduct −

MS2 data

error (ppm)

formula

RDB

−1.105

C38H54O4

12.5 MS2 [573.4]: 558.4 (100)

24-methylenecholesterol ferulate

tentative identification

573.3943

21.73

[M − H]

573.3943

21.96

[M-H]−

−1.105

C38H54O4

12.5 MS2 [573.4]: 558.5 (100), 193.2 (4)

24-methylenecholesterol ferulate

601.4261

23.33

[M − H]−

−0.222

C40H58O4

12.5 MS2 [601.4]: 586.6 (100), 193.1 (1)

cycloartenyl ferulate

575.4105

24.30

[M − H]−

−0.145

C38H56O4

11.5 MS2 [575.4]: 560.5 (100), 193.2 (3); MS3 [575.4→560.5]: 545.5 (100); MS4 [575.4→560.5→545.5]: 501.5 (7), 179.1 (100)

campesteryl ferulate

601.4261

24.30

[M − H]−

−0.222

C40H58O4

12.5 MS2 [601.4]: 586.6 (100), 193.1 (1)

cycloartenyl ferulate

603.4417

25.17

[M − H]−

−0.304

C40H60O4

11.5 MS2 [603.4]: 588.5 (100), 193.2 (2)

cycloartanyl ferulate

589.4261

25.39

[M − H]−

−0.227

C39H58O4

11.5 MS2 [589.4]: 574.5 (100), 193.1 (3); MS3 [589.4→574.5]: 559.5 (100); MS4 [589.4→574.5→559.5]: 179.0 (100)

sitosteryl ferulate

577.4261

25.41

[M − H]−

−0.231

C38H58O4

10.5 MS2 [577.4]: 562.5 (100), 193.1 (3); MS3[577.4→562.5]: 547.5 (5), campestanyl ferulate 534.5 (21), 518.6 (95), 518.0 (16), 178.2 (12), 177.1 (100)

591.4413

26.58

[M − H]−

−0.987

C39H60O4

10.5 MS2 [591.4]: 576.5 (100); MS3 [591.4→576.5]: 561.7 (4), 548.5 (18), 532.6 (100), 532.0 (15), 178.2 (9), 177.1 (93)

sitostanyl ferulate

Figure 5. Structures of wheat sphingolipids. One data matrix for each acquisition mode was obtained: APCI +, APCI−, ESI+, and ESI−. The data matrices were then imported to software SIMCA-P (version 13.0 Umetrics, Umea, Sweden) for PCA.

20−30 min, ARs were found mainly prior to DGs, and the majority of the peaks eluted after 40 min were identified as TGs. Identification of the lipid compounds was based on chromatographic behavior, accurate mass measurements, consecutive MS2−MS4 analyses, ring-plus-double-bond equivalent (RDB), and comparison with data in the literature. A total of 155 lipid compounds were tentatively identified from germ, bran, and endosperm. The MSn data listed in the tables were from the milling fraction with the most reliable spectra in terms of peak intensity. Identification of Free Fatty Acids. The FAs of wheat and wheat products have been studied by many investigators in the form of methyl derivatives by gas chromatography (GC).9 When liquid chromatography is used, the FAs can be analyzed directly without being converted to their methyl esters. The characteristic fragmentation of free FA in APCI negative and positive

■

RESULTS AND DISCUSSION ESI- and APCI-HRAM-MSn detection in both positive and negative ionization modes were used to obtain complementary information on the structural features and the conjugated forms of lipids. The total ion chromatograms (TICs) of germ acquired in APCI(±) and ESI(±) are shown in the Supporting Information (Figure S1). From first glance, most of the peaks appearing in the negative spectra were eluted before 40 min; those were identified as free FA, oxylipins, γ-oryzanol, phospholipids, sphingolipids, galactolipids, etc., whereas for positive spectra, DGs were identified from peaks eluting around F



Article

modes is demonstrated in Figure S2. The putative identification of wheat free FAs and APCI(±) data are listed in Table 1. The distributions of the three most abundant free FAs across endosperm, bran, and germ are compared in Figure 1 with the germ fraction giving the highest peaks. Identification of Oxylipins. Oxylipins are a diverse family of secondary metabolites that are produced by oxidative metabolism of polyunsaturated fatty acids (PUFA), such as linolenic acid and linoleic acid.19−22 In plants, oxylipins serve as signal molecules in developmental processes (e.g., pollen formation) and in defense mechanisms (e.g., pathogenesis, wounding, and herbivores).23 Milling, baking, and storage are known to trigger autoxidation, leading to the formation of additional oxylipins. A whole-grain wheat feeding study on pigs24 also found that oxylipins in the plasma of pigs correlated with WF and RF bread consumed, with higher oxylipin concentrations found after consumption of WF. It is not clear whether oxylipins may have health detrimental effects or may just be considered as markers for the shelf life of flour. The oxylipins in wheat are tentatively identified in Table 2 using APCI negative MS, MS2, and MS3 spectra and literature research.19−22,25−28 The characteristic MS2 and MS3 spectra in APCI negative mode are shown in Figure S3. Identification of Alk(en)ylresorcinols. Alkylresorcinols and alkenylresorcinols are amphiphilic derivatives of 1,3dihydroxybenzene with an odd-numbered alkyl or alkenyl chain at position 5 of the benzene ring (Figure 2). ARs have been extensively evaluated as biomarkers for the intake of whole-grain and bran products of wheat and rye.29,30 Among commonly consumed foods, ARs have been reported to be found mainly in the bran fraction of cereal grains,31,32 where homologues with alkyl chains in the range of C15−C25 dominate, although derivatives with unsaturated and oxygenated side chains also exist. The MS/MS spectra of three ARs,

Table 5. Characteristic Anion of 2-Hydroxy Fatty Acids in Sphingolipids

1

Not observed in this work.

Table 6. Ceramides and Cerebrosides Identified from Wheat ion m/z

RT

error (ppm)

formula

RDB

−

−0.008

C40H75O9N

4.5

MS2 [712.5]: 550.5 (45), 532.5 (100), 271.3 (10); MS3 [712.5→532.5]: 514.5 (26), 340.4 (51), 324.4 (90), 306.4 (100), 278.2 (59), 225.4 (53)

GlcCer(d18:24,8/16:0(2OH))

adduct

MS2 data


712.5369

15.40

[M − H]

714.5525

16.37

[M − H]−

−0.078

C40H77O9N

3.5

MS2 [714.5]: 552.6 (56), 534.6 (100), 271.3 (6); MS3 [714.5→534.6]: 516.6 (13), 296.4 (15), 280.3 (63), 253.3 (26), 225.3 (100)

GlcCer(d18:18/16:0(2OH))

716.5682

18.07

[M − H]−

−0.009

C40H79O9N

2.5

MS2 [716.6]: 554.8 (58), 536.5 (100), 271.3 (10)

GlcCer(d18:0/16:0(2OH))

740.5680

18.10

[M − H]−

−0.278

C42H79O9N

4.5

MS2 [740.6]: 578.6 (46), 560.6 (100), 299.4 (14); MS2 [740.6]: 684.0 (9), 678.9 (10), 672.0 (5), 578.5 (35), 560.5 (100), 299.4 (5), 279.0 (14)

GlcCer(d18:24,8/18:0(2OH))

742.5839

19.17

[M − H]−

0.059

C42H81O9N

3.5

MS2 [742.6]: 712.1 (5), 710.8 (5), 699.8 (9), 625.9 (7), 580.7 (33), 562.7 (100), 503.8 (20), 299.5 (6)

GlcCer(d18:18/18:0(2OH))

768.5994

20.98

[M − H]−

−0.138

C44H83O9N

4.5

MS2 [768.6]: 606.6 (48), 588.6 (100), 570.6 (3), 327.4 (7), GlcCer(d18:24,8/20:0(2OH)) 281.3 (2)

770.6151

22.01

[M − H]−

−0.073

C44H85O9N

3.5

MS2 [770.6]: 608.6 (54), 590.7 (100), 489.4 (5), 415.3 (4), GlcCer(d18:18/20:0(2OH)) 327.4 (9), 279.3 (54); MS3 [770.6→590.7]: 572.5 (6), 562.7 (10), 352.4 (17), 309.4 (13), 281.4 (100)

840.6570

22.76

[M − H]−

−0.025

C48H91O10N

4.5

MS2 [840.7]: 678.8 (25), 660.5 (8), 436.5 (100), 381.4 (17), GlcCer(t18:18/24:1(2OH)) 335.5 (10)

822.6460

23.90

[M − H]−

−0.555

C48H89O9N

5.5

MS2 [822.7]: 754.1 (10), 660.7 (54), 642.6 (100), 381.4 (9), GlcCer(d18:24,8/24:1(2OH)) 335.4 (3) G



Article


RT

error (ppm)

formula

RDB

−

−0.678

C48H93O10N

3.5

MS2 [842.7]: 773.7 (8), 680.7 (19), 662.6 (12), 438.5 (100), GlcCer(t18:18/24:0(2OH)) 383.6 (16), 337.4 (13)

adduct

MS2 data


842.6721

24.95

[M − H]

654.6041

25.19

[M − H]−

−0.149

C40H81O5N

1.5

MS2 [654.6]: 410.4 (24), 398.4 (23), 355.5 (100), 309.5 (31)

680.6194

25.52

[M − H]−

−0.658

C42H83O5N

2.5

MS2 [680.6]: 662.6 (11), 612.3 (6), 436.5 (24), 424.5 (30), Cer(t18:0/24:1(2OH)) 381.4 (100), 335.5 (23), 267.5 (6)

824.6620

26.20

[M − H]−

−0.129

C48H91O9N

4.5

MS2 [824.7]: 794.3 (57), 662.7 (37), 644.6 (100), 383.5 (17), 337.6 (4)

668.6197

26.61

[M − H]−

−0.221

C41H83O5N

1.5

MS2 [668.6]: 650.7 (8), 424.5 (26), 412.5 (11), 369.4 (100), Cer(t18:0/23:0(2OH)) 323.4 (25), 267.3 (8), 255.3 (6)

680.6198

26.72

[M − H]−

−0.070

C42H83O5N

2.5

MS2 [680.6]: 662.6 (10), 612.0 (7), 438.4 (18), 426.5 (12), Cer(t18:18/24:0(2OH)) 383.4 (100), 337.5 (14)

826.6777

27.20

[M − H]−

−0.068

C48H93O9N

3.5

MS2 [826.7]: 780.6 (50), 758.9 (56), 664.6 (50), 646.6 (100), 383.3 (12)

GlcCer(d18:18/24:0(2OH))

682.6353

28.05

[M − H]−

−0.290

C42H85O5N

1.5

MS2 [682.6]: 438.5 (19), 426.5 (18), 383.4 (100), 337.5 (20); MS3 [682.6→383.4]: 337.4 (100)

Cer(t18:0/24:0(2OH))

696.6511

29.59

[M − H]−

−0.069

C43H87O5N

1.5

MS2 [696.7]: 452.5 (22), 440.6 (21), 397.6 (100), 351.5 (21)

Cer(t18:0/25:0(2OH))

710.6663

31.23

[M − H]−

−0.701

C44H89O5N

1.5

MS2 [710.7]: 466.5 (18), 454.5 (20), 411.4 (100), 365.5 (25)

Cer(t18:0/26:0(2OH))

Cer(t18:0/22:0(2OH))

GlcCer(d18:24,8/24:0(2OH))

Table 7. Abbreviations of Acyls and Characteristic Fragment Ions of TGs/DGs

a

name

symbol

CN:DBa

ion B

ion B − H2O

ion C

ion C − H2O

myristic palmitoleic palmitic linolenic linoleic oleic stearic gadoleic arachidic erucic

M Po P Ln L O S G A E

C14:0 C16:1 C16:0 C18:3 C18:2 C18:1 C18:0 C20:1 C20:0 C22:1

285 311 313 335 337 339 341 367 369 395

267 293 295 317 319 321 323 349 351 377

211 237 239 261 263 265 267 293 295 321

193 219 221 243 245 247 249 275 277 303

CN, carbon number; DB, double bond.

Identification of γ-Oryzanols. γ-Oryzanol is a mixture of ferulic acid esters of sterols and triterpene alcohols (Figure 4), which is generally found in rice, particularly in the bran fraction.34−36 It has also been identified in wheat,37,38 but in low concentrations, 6.3 mg/100 g, as compared with those in brown rice, 60.2−65.6 mg/100 g.35 The molecular mass of γ-oryzanol could be resolved by APCI in negative mode on the basis of the formation of the deprotonated anion [M − H]−. The positive mode spectra generated the cation of the intact triterpene alcohol or sterol moieties ([M + H − 194]+ from neutral loss of ferulic acid36,38 as major peak and protonated ion [M + H]+ in low abundance. CID spectra of deprotonated molecular ions showed abundant ions of [M − H − CH3]•− resulting from the loss of a methyl group in the ferulic acid moiety. Another anion, [M − H − 2CH3]•−, could be yielded for sterol ferulates with a double bond between

Figure 6. Structures and notation of the fragment ions of TGs/DGs and other acylglycerols used in this work.

representing unsaturated, oxygenated, and saturated species, are shown in Figure S4. Seventeen ARs were tentatively identified from the bran sample in Table 3 using APCI positive MS, MS2, and MS3 spectra and literature research (Figure 3).32,33 H



Article

Figure 7. Extracted ion chromatograms of A ions of DGs/TGs at mass range 551.50−551.52, 575.50−575.51, 577.51−577.53, 579.53−579.54, 599.50− 599.51, 601.51−601.23, 603.53−603.54, or 629.54−629.56 in APCI positive mode.

Table 8. Diglycerides (DGs) and Triglycerides (TGs) Tentatively Identified from Germ ion m/z

RT

error (ppm)b

formulaa

RDB

MS2 data

+

−0.573

C39H64O5

7.5

MS2 [613.5]: 595.5 (100), 594.9 (10), 539.5 (23), 521.4 (29), 503.4 (17); MS3 [613.5→595.5]: 577.6 (53), 559.5 (31), 539.6 (100), 521.4 (63), 503.6 (45), 425.4 (26), 411.5 (21), 397.3 (29), 351.4 (25)

adducta

abbreviation

613.4823

19.18

[M + H]

595.4720

19.18

[M + H − H2O]+

−0.146

C39H63O4+

8.5

MS2 [595.5]: 539.6 (100), 521.6 (47), 317.3 (70), 261.3 (18), 211.3 (22)

615.4982

20.85

[M + H]+

−0.165

C39H66O5

6.5

MS2 [615.5]: 597.6 (100), 517.5 (8), 503.5 (6), 491.6 (5), 337.4 (6), 317.4 (4); MS3 [615.5→597.6]: 579.6 (47), 561.5 (20), 541.5 (81), 523.4 (100), 505.6 (35), 317.4 (27); MS4 [615.5→597.6→523.4]: 505.4 (100)

597.4880

20.85

[M + H − H2O]+

C39H65O4+

7.5

MS2 [597.5]: 541.4 (100), 523.4 (63), 505.5 (25), 337.4 (18), 319.3 (19), 317.3 (34), 263.2 (19), 261.3 (22), 245.3 (11), 243.2 (16)

591.4980

22.10

[M + H]+

C37H66O5

4.5

MS2 [591.5]: 573.4 (100), 335.4 (36), 317.4 (19), 313.3 (48), 261.3 (24), 243.3 (6); MS3 [591.5→573.4]: 517.5 (18), 499.5 (19), 317.3 (100), 313.3 (21), 261.3 (39), 243.3 (9)

573.4882

22.10

[M + H − H2O]+

C37H65O4+

5.5

MS2 [573.5]: 499.5 (16), 317.4 (100), 313.3 (54), 261.3 (56), 243.2 (33), 239.2 (15)

617.5133

22.46

[M + H]+

−1.055

C39H68O5

5.5

MS2 [617.5]: 599.5 (100); MS3 [617.5→599.5]: 581.6 (16), 543.5 (100), 525.7 (55), 507.7 (14), 337.4 (23), 319.2 (31), 263.3 (33), 245.3 (14)

599.5032

22.46

[M + H − H2O]+

−0.312

C39H67O4+

6.5

MS2 [599.5]: 581.6 (16), 543.5 (100), 525.7 (55), 507.7 (14), 337.4 (23), 319.2 (31), 263.3 (33), 245.3 (14)

593.5135

23.72

[M + H]+

−0.452

C37H68O5

3.5

MS2 [593.5]: 575.7 (79), 574.9 (26), 501.4 (5), 353.4 (4), 337.4 (89), 319.3 (5), 313.3 (100), 279.3 (5), 263.3 (17), 257.2 (7), 245.3 (14), 239.2 (6)

575.5039

23.72

[M + H − H2O]+

C37H67O4+

4.5

MS2 [575.5]: 501.5 (11), 319.4 (60), 313.3 (100), 263.3 (51), 245.4 (31), 239.1 (13), 221.3 (5), 175.3 (11)

0.440

−0.510

0.807

0.891

I

LnLn

LLn

PLn

LL

PL



Article


RT

adducta +

error (ppm)b

formulaa

RDB

MS2 data

−0.648

C39H70O5

4.5

MS2 [619.5]: 601.5 (100), 545.5 (22), 527.5 (37), 339.4 (4), 337.3 (5); MS3 [619.5→601.5]: 583.6 (19), 545.4 (47), 527.5 (100), 317.4 (4); MS4 [619.5→601.5→527.5]: 509.7 (100), 499.7 (12), 491.6 (5), 457.6 (11), 445.4 (10), 443.6 (11), 429.5 (20), 317.6 (6), 245.3 (5)

abbreviation

619.5292

24.26

[M + H]

601.5195

24.26

[M + H − H2O]+

0.770

C39H69O4+

5.5

MS2 [601.5]: 583.6 (14), 545.4 (100), 527.5 (71), 509.5 (31), 339.5 (28), 319.4 (29), 265.3 (26), 263.4 (25), 247.3 (13), 245.4 (19)

551.5039

24.99

[M + H − H2O]+

0.930

C35H67O4+

2.5

MS2 [551.5]: 313.2 (4), 297.3 (3), 295.4 (5), 257.4 (3), 239.1 (100), 221.1 (12)

PP

595.5290

25.57

[M + H]+

C37H70O5

2.5

MS2 [595.5]: 577.6 (100), 339.4 (100), 313.4 (100), 283.3 (12), 265.4 (25), 257.4 (17), 247.3 (11), 239.2 (14)

PO

577.5191

25.57

[M + H − H2O]+

C37H69O4+

3.5

MS2 [577.5]: 503.5 (8), 321.4 (8), 313.3 (28), 303.4 (4), 265.4 (100), 247.4 (39), 239.2 (27), 221.2 (4)

621.5449

26.15

[M + H]+

C39H72O5

3.5

MS2 [621.5]: 603.7 (100), 547.5 (12), 529.5 (39), 511.6 (8), 339.3 (74), 321.2 (4), 265.4 (22), 247.3 (10)

603.5350

26.15

[M + H − H2O]+

C39H71O4+

4.5

MS2 [603.5]: 529.6 (19), 511.6 (6), 339.3 (24), 321.4 (12), 265.3 (100), 247.3 (31); MS3 [603.5→265.3]: 247.4 (100)

621.5449

26.54

[M + H]+

C39H72O5

3.5

NA

603.5350

26.54

[M + H − H2O]+

C39H71O4+

4.5

MS2 [603.5]: 341.4 (100), 319.3 (57), 263.4 (50), 245.3 (28)

647.5604

26.98

[M + H]+

C41H74O5

4.5

MS2 [647.6]: 629.6 (100), 573.5 (29), 555.6 (34), 537.6 (6), 367.4 (6), 337.3 (6)

629.5508

26.98

[M + H − H2O]+

0.735

C41H73O4+

5.5

MS2 [629.5]: 611.6 (30), 573.6 (100), 555.5 (66), 537.6 (18), 461.4 (9), 367.5 (9), 349.4 (17), 293.3 (12), 263.3 (15), 245.2 (5)

579.5348

27.99

[M + H − H2O]+

0.195

C37H71O4+

2.5

MS2 [579.5]: 323.4 (22), 267.3 (37), 249.3 (16), 239.0 (100)

PS

623.5605

28.53

[M + H]+

C39H74O5

2.5

MS2 [623.6]: 605.4 (100), 367.3 (27), 341.4 (38), 339.2 (29), 313.0 (11)

mix of PG and SO

605.5504

28.53

[M + H − H2O]+

C39H73O4+

3.5

MS2 [605.5]: 531.5 (14), 349.4 (16), 341.4 (40), 321.4 (9), 313.3 (12), 293.4 (100), 275.4 (31), 267.1 (18), 265.3 (99), 249.3 (9), 247.3 (27), 239.0 (14), 179.3 (8)

649.5762

29.07

[M + H]+

C41H76O5

3.5

MS2 [649.6]: 631.8 (100), 631.1 (25), 575.6 (25), 557.5 (59), 367.4 (54), 339.5 (58), 293.4 (18), 265.5 (13)

631.5660

29.07

[M + H − H2O]+

C41H75O4+

4.5

MS2 [631.6]: 613.6 (8), 575.6 (8), 574.5 (25), 557.6 (43), 556.5 (16), 539.6 (15), 367.4 (22), 349.4 (19), 339.4 (13), 323.3 (8), 321.1 (13), 293.3 (100), 275.3 (28), 265.4 (77), 247.3 (29)

649.5762

29.64

[M + H]+

C41H76O5

3.5

NA

631.5660

29.64

[M + H − H2O]+

C41H75O4+

4.5

MS2 [631.6]: 574.5 (63), 557.3 (28), 556.4 (34), 369.5 (100), 367.4 (17), 319.4 (64), 293.3 (40), 265.3 (21), 263.3 (48), 245.4 (25), 203.3 (14)

675.5920

30.05

[M + H]+

−0.299

C43H78O5

4.5

MS2 [675.6]: 657.7 (100), 601.7 (32), 583.6 (38)

657.5816

30.05

[M + H − H2O]+

−0.057

C43H77O4+

5.5

MS2 [657.6]: 639.7 (22), 601.6 (100), 583.7 (60), 565.6 (29), 395.4 (15), 377.5 (14), 321.4 (29), 319.2 (18), 303.4 (14), 263.3 (21), 245.4 (15)

873.6973

39.38

[M + H]+

C57H92O6

11.5

MS2 [873.7]: 855.7 (36), 803.7 (15), 595.5 (100), 593.5 (60), 335.5 (4); MS3 [873.7→595.5]: 577.8 (50), 539.5 (59), 521.5 (52), 317.2 (100), 243.2 (43)

890.7234

39.38

[M + NH4]+

875.7130

42.98

[M + H]+

−1.010

0.109

−0.566

0.518

−0.566 0.518 −0.775

−0.644

0.104

−0.542

0.020

−0.542 0.020

0.723

C57H92O6 0.780

C57H94O6

OL

OO

SL

GL

GO

AL

EL

LnLnLn

MS2 [890.7]: 873.8 (100), 595.5 (55) 10.5

MS2 [875.7]: 857.6 (49), 597.5 (66), 595.6 (100), 337.4 (7); MS3 [875.7→597.5]: 579.6 (30), 541.5 (100), 523.5 (72), 337.4 (29), 335.2 (24), 319.4 (40), 317.2 (98), 263.3 (35), 261.1 (47), 243.3 (23) J

LnLnL



Article


RT

adducta

error (ppm)b

formulaa

0.919

C57H94O6 C55H94O6

+

892.7388 851.7131

42.98 46.74

[M + NH4] [M + H]+

868.7384

46.74

[M + NH4]+

877.7284

47.47

[M + H]+

894.7516

47.47

[M + NH4]+

827.7122

49.04

[M + H]+

844.7385

49.04

[M + NH4]+

853.7281

52.06

[M + H]+

870.7534

52.06

[M + NH4]+

879.7435

52.88

[M + H]+

896.7672

52.88

[M + NH4]+

879.7435

53.60

[M + H]+

896.7679

53.60

[M + NH4]+

855.7438

57.15

[M + H]+

872.7682

57.15

[M + NH4]+

829.7290

57.64

[M + H]+

846.7551

57.64

[M + NH4]+

855.7438

58.54

[M + H]+

872.7682

58.54

[M + NH4]+

881.7601

60.54

[M + H]+

898.7858

60.54

[M + NH4]+

881.7601

61.49

[M + H]+

898.7858

61.49

[M + NH4]+

8.5

C55H94O6

0.493

C57H96O6

C53H94O6

9.5

C55H96O6

6.5

C57H98O6

7.5

C57H98O6

8.5

C55H98O6

8.5

6.5

C55H98O6 1.245

C53H96O6

C55H98O6

5.5

C57H100O6

6.5

C57H100O6

C57H100O6

PLnL

MS2[879.8]: 861.7 (44), 599.6 (100); MS3 [879.7→599.5]: 581.6 (14), 543.4 (100), 525.6 (57), 507.6 (23), 337.2 (41), 319.3 (52), 263.3 (59), 245.3 (29)

LLL

MS2 [879.8]: 861.7 (33), 599.6 (28), 597.6 (100), 541.5 (11), 523.5 (13), 339.4 (4) MS2 [896.8]: 879.6 (100), 601.5 (22), 599.6 (29), 597.6 (40); MS3 [896.8→879.7]: 861.8 (36), 599.5 (31), 597.6 (100) MS2 [855.7]: 837.8 (100), 757.7 (15), 601.5 (92), 573.5 (69), 545.4 (8), 527.6 (16), 517.3 (10), 339.3 (8)

LLnO

PoLO

MS2 [829.7]: 811.6 (67), 573.4 (100), 551.5 (40), 499.5 (7), 317.3 (38), 313.4 (6), 261.3 (25); MS3 [829.7→[573.5]: 317.4 (100), 313.3 (35), 261.3 (45), 243.3 (21)

PLnP

MS2 [855.8]: 837.7 (22), 599.6 (100); MS3 [855.8→599.5]: 581.6 (16), 543.5 (100), 525.5 (51), 507.6 (21), 337.4 (11), 319.4 (12), 263.3 (13), 245.3 (8); MS3 [855.8→575.5]: 319.4 (67), 313.3 (91), 263.3 (100), 245.3 (52), 239.1 (28)

PLL

MS2 [872.8]: 855.6 (100), 599.5 (52), 575.5 (47); MS3 [872.8→855.6]: 837.8 (19), 757.5 (6), 599.5 (100), 543.6 (8), 525.5 (13) 7.5

C57H100O6 0.944

MS2 [853.7]: 835.6 (20), 597.5 (100); MS3 [853.7→597.5]: 579.5 (16), 541.4 (100), 523.5 (58), 505.6 (19), 337.4 (9), 319.4 (9), 317.3 (25), 261.3 (11), 243.2 (8)

MS2 [846.8]: 829.7 (30), 811.7 (21), 573.5 (100), 551.5 (40), 317.3 (10), 261.2 (6)

C55H98O6

0.944

MLL

MS2 [872.8]: 855.7 (72), 837.8 (16), 601.5 (100), 575.6 (54), 573.5 (95)

C53H96O6

0.214

MS2 [827.7]: 599.5 (100); MS3 [827.7→599.5]: 543.4 (100), 525.5 (52), 507.6 (20), 337.3 (17), 319.4 (24), 263.4 (16); MS3 [827.7→547.5]: 319.5 (98), 285.3 (43), 263.4 (100), 245.3 (32), 211.1 (54)

MS2 [896.8]: 879.6 (100), 599.5 (60); MS3 [896.8→879.6]: 861.7 (39), 599.6 (100), 597.5 (40), 525.5 (13)

C57H98O6 0.214

LLnL

MS2 [870.8]: 853.8 (100), 597.5 (61), 575.6 (28), 573.4 (22)

C57H98O6

−0.133

MS2 [877.7]: 859.8 (46), 599.5 (31), 597.6 (100); MS3 [877.7→597.5]: 579.7 (26), 541.6 (100), 523.5 (77), 505.6 (28), 337.4 (27), 335.3 (18), 319.3 (33), 317.4 (91), 263.3 (32), 261.3 (40), 245.3 (15), 243.3 (21)

MS2 [844.7]: 827.7 (100), 599.6 (36), 547.6 (23)

C55H96O6 −0.133

LnLnP

MS2 [894.8]: 877.6 (100), 599.5 (23), 597.6 (42)

C53H94O6 0.156

abbreviation

MS2 [892.7]: 875.6 (100), 597.5 (46), 595.5 (34) MS2 [851.7]: 833.6 (19), 595.5 (100), 539.5 (12), 521.6 (17) MS2 [868.7]: 851.6 (100), 595.5 (60), 573.5 (47); MS3 [868.7→573.5]: 499.6 (17), 317.3 (100), 313.4 (32), 261.3 (52), 243.2 (15)

C57H96O6 −0.141

MS2 data

RDB

MS2 [881.8]: 863.7 (30), 601.6 (10), 599.5 (100); MS3 [881.8→601.5]: 545.6 (70), 544.5 (100), 527.7 (67), 526.6 (53), 339.4 (39), 337.4 (32), 319.3 (69), 265.4 (63), 263.3 (63), 247.4 (17), 245.3 (40)

LLO

MS2 [898.8]: 881.7 (100), 601.5 (55), 599.6 (47) 7.5

MS2 [881.8]: 603.5 (41), 599.5 (100); MS3 [881.8→599.5]: 543.5 (100), 525.5 (62), 507.6 (18), 339.4 (28), 317.3 (67), 265.3 (20), 261.3 (34), 243.4 (14); MS3 [881.8→603.5]: 339.4 (25), 321.3 (11), 265.2 (100), 247.2 (23)

OLnO

NA K



Article


RT

adducta +

error (ppm)b

formulaa

RDB

0.214

C55H98O6

6.5

MS2 data

abbreviation

MS2 [855.8]: 837.7 (22), 599.6 (100), 575.6 (2), 313.4 (4); MS3 [855.8→599.6]: 581.5 (86), 563.6 (46), 543.5 (100), 525.5 (73), 507.7 (33), 339.4 (28), 317.1 (15), 261.2 (9), 243.2 (6)

855.7438

61.53

[M + H]

872.7682

61.53

[M + NH4]+

881.7601

62.92

[M + H]+

898.7858

62.92

[M+NH4]+

907.7752

64.66

[M + H]+

0.311

924.8021 831.7441

64.66 65.49

[M + NH4]+ [M + H]+

0.581

848.7703

65.49

[M + NH4]+

857.7603

67.80

[M + H]+

874.7852

67.80

[M+NH4]+

883.7760

70.32

[M + H]+

900.8003

70.32

[M + NH4]+

883.7762

72.08

[M + H]+

900.8003

72.08

[M + NH4]+

C57H102O6

MS2 [900.8]: 883.6 (100), 603.6 (57), 599.4 (61)

551.5034 824.7717

74.07 74.07

ion A [M + NH4]+

C51H98O6 C51H98O6

MS2 [551.5]: 313.3 (6), 295.1 (5), 239.2 (100), 221.2 (24) MS2 [824.8]: 551.5 (100); MS3 [824.8→551.5]: 295.3 (33), 239.3 (100), 221.3 (16)

PPP

909.7910

74.22

[M + H]+

MS2 [909.8]: 891.7 (43), 629.7 (12), 599.5 (100); MS3 [909.8→629.5]: 573.7 (100), 555.7 (72), 367.4 (44), 349.3 (17), 319.4 (65), 293.4 (78), 275.5 (44), 263.2 (64), 245.4 (26); MS3 [909.8→599.5]: 543.5 (100), 525.5 (52), 507.6 (20), 337.4 (15), 319.4 (18), 263.4 (23), 245.3 (13)

LLG

926.8160

74.22

[M + NH4]+

833.7601

76.66

[M + H]+

850.7860

76.66

[M + NH4]+

859.7755

79.64

[M + H]+

876.8011

79.64

[M + NH4]+

859.7755

81.75

[M + H]+

C55H98O6 0.944

C57H100O6

MS2 [872.8]: 855.6 (100), 837.7 (25), 599.5 (69), 577.6 (43), 573.5 (40) 7.5

MS2 [881.8]: 863.9 (23), 597.6 (100); MS3 [881.8→597.6]: 579.5 (68), 561.5 (46), 541.5 (96), 523.5 (100), 521.5 (24), 505.5 (52), 317.3 (25) MS2 [898.8]: 881.7 (100), 603.5 (37), 601.6 (32), 597.5 (50), 337.6 (7)

LLnS

C59H102O6

8.5

MS2 [907.8]: 889.7 (41), 627.6 (30), 597.5 (100); MS3 [907.8→627.5]: 571.4 (34), 317.4 (100), 293.3 (40), 261.1 (33)

LLnG

C59H102O6 C53H98O6

4.5

C57H100O6

C53H98O6 1.204

C55H100O6

C57H102O6

5.5

0.476

C57H102O6

C59H104O6

6.5

C53H100O6

6.5

7.5

C55H102O6

3.5

C55H102O6

MS2 [883.8]: 865.7 (46), 601.6 (100); MS3 [883.8→601.6]: 583.5 (21), 545.5 (100), 527.5 (75), 509.5 (29), 319.4 (6), 265.4 (4), 263.3 (4), 245.2 (6)

OLO

MS2 [883.8]: 865.7 (24), 599.5 (100); MS3 [883.8→603.5]: 529.6 (22), 511.4 (23), 341.5 (100), 319.4 (80), 263.3 (91), 245.3(46); MS3 [883.8→599.5]: 581.6 (12), 543.5 (100), 525.5 (43), 507.5 (16), 337.3 (14), 319.4 (15), 263.3 (15), 245.2 (10)

LLS

MS2 [833.8]: 577.7 (100), 551.5 (75); MS3 [833.8→551.5]: 239.2 (100), 221.3 (13); MS3 [833.8→577.5]: 321.3 (14), 313.4 (32), 265.3 (100), 247.3 (28), 239.1 (24)

POP

MS2 [850.8]: 577.6 (100), 551.5 (26) 4.5

C55H102O6 0.678

PLO

MS2 [926.8]: 909.7 (100), 629.6 (58), 599.6 (42)

C53H100O6 0.678

MS2 [857.8]: 839.7 (25), 601.6 (100), 577.6 (4), 575.6 (4); MS3 [857.8→575.5]: 501.6 (26), 319.3 (68), 313.4 (100), 263.3 (100), 245.3 (66), 239.1 (24); MS3 [857.8→601.5]: 545.4 (100), 527.5 (73), 509.6 (26), 339.4 (18), 319.4 (14), 265.3 (15), 263.4 (17); MS3 [857.8→577.5]: 313.3 (59), 265.3 (100), 247.4 (41), 239.2 (43)

MS2 [900.8]: 883.8 (39), 603.5 (40), 601.6 (100); MS3 [900.8→601.6]: 545.6 (100), 527.3 (67), 339.5 (68), 319.3 (69), 265.3 (36), 263.3 (49), 245.3 (35)

C59H104O6 0.999

PLP

MS2 [874.8]: 857.6 (39), 601.5 (100), 577.5 (56), 575.5 (58)

C57H102O6

1.225

MS2 [924.8]: 907.7 (100), 629.7 (15), 627.6 (25), 597.5 (52) MS2 [831.7]: 813.6 (14), 575.4 (100), 551.5 (51); MS3 [831.7→575.5]: 501.6 (15), 319.3 (82), 313.3 (100), 263.4 (73), 245.3 (40), 239.2 (14); MS3 [831.7→551.5]: 239.3 (100), 221.3 (16) MS2 [848.8]: 831.8 (2), 575.5 (100), 551.4 (41)

C55H100O6 1.225

PLnO

MS2 [859.8]: 841.7 (18), 603.5 (100), 577.5 (79); MS3 [859.8→577.5]: 321.3 (12), 313.3 (32), 265.4 (100), 247.4 (31), 239.2 (28); MS3 [859.8→603.5]: 529.5 (36), 339.3 (47), 321.4 (15), 265.3 (100), 247.4 (31)

POO

MS2 [876.8]: 603.6 (56), 577.5 (100) 4.5

MS2 [859.8]: 841.6 (35), 603.6 (75), 579.5 (100), 575.5 (72) L

PLS



Article


RT

adducta

error (ppm)b

+

formulaa

876.8011

81.75

[M + NH4]

C55H102O6

885.7906

82.70

[M + H]+

0.037

C57H104O6

5.5

902.8176 885.7906

82.70 84.31

[M + NH4]+ [M + H]+

0.037

C57H104O6 C57H104O6

5.5

902.8176

84.31

[M + NH4]+

911.8065

87.78

[M + H]+

928.8334

87.78

[M + NH4]+

911.8065

91.05

[M + H]+

928.8334

91.05

[M+NH4]+

937.8220 954.8480

93.40 93.40

[M + H]+ [M + NH4]+

861.7905

96.91

[M + H]+

878.8170

96.91

[M + NH4]+

887.8062

100.28

[M + H]+

904.8326

100.28

[M + NH4]+

913.8221

104.34

[M + H]+

930.8481

104.34

[M + NH4]+

913.8221

107.23

[M + H]+

930.8481

107.23

[M + NH4]+

C59H106O6

C59H106O6

MS2 [885.8]: 867.9 (15), 603.5 (100), 529.6 (11); MS3 [885.8→603.5]: 529.6 (28), 339.3 (50), 321.2 (13), 265.3 (100), 247.3 (35) MS2 [902.8]: 885.6 (20), 603.4 (100) MS2 [885.8]: 867.9 (18), 629.5 (100); MS3 [885.8→629.5]: 611.6 (18), 573.6 (100), 555.5 (69), 537.6 (18), 367.5 (12), 319.3 (12), 293.3 (13), 263.3 (14), 245.3 (7); MS3 [885.8→605.5]: 349.4 (25), 341.4 (29), 313.4 (26), 293.4 (100), 275.3 (33), 239.2 (27); MS3 [885.8→575.5]: 501.5 (23), 319.3 (74), 313.3 (57), 263.3 (100), 245.3 (64), 239.1 (25)

OOO

PLG

MS2 [902.8]: 885.6 (53), 629.5 (100), 605.5 (49), 575.5 (53) 6.5

C59H106O6

0.310

abbreviation

MS2 [876.8]: 859.5 (11), 603.6 (83), 579.6 (70), 575.5 (100)

C57H104O6 0.310

MS2 data

RDB

MS2 [911.8]: 893.7 (87), 629.5 (100), 601.5 (59); MS3 [911.8→629.5]: 611.6 (17), 573.6 (100), 555.5 (73), 367.4 (20), 293.4 (20), 263.3 (20)

OLG

MS2 [928.8]: 911.4 (63), 631.7 (78), 629.5 (100), 601.6 (81); MS3 [928.8→631.6]: 573.8 (14), 557.6 (51), 367.4 (31), 349.4 (21), 339.4 (16), 293.4 (100), 275.4 (31), 265.4 (65), 247.3 (13) 6.5

C59H106O6

MS2 [911.8]: 893.7 (21), 599.5 (100); MS3 [911.8→599.5]: 543.6 (100), 525.6 (39), 507.6 (21), 319.3 (12), 263.3 (15); MS3 [911.8→631.6]: 369.2 (46), 319.2 (100), 263.4 (23), 245.4 (31)

LLA

MS2 [928.8]: 911.8 (100), 631.6 (69), 599.4 (55)

0.141

C61H108O6 C61H108O6

7.5

MS2 [937.8]: 919.9 (43), 657.7 (13), 599.6 (100), 543.6 (15), 525.6 (13) MS2 [954.8]: 937.8 (100), 657.6 (50), 599.6 (46); MS3 [954.8→599.5]: 581.6 (15), 543.6 (100), 525.5 (49), 507.5 (21), 337.4 (23), 319.3 (15), 263.3 (15), 245.3 (11); MS3 [954.8→657.6]: 601.6 (58), 395.5 (41), 377.6 (18), 321.4 (100), 319.6 (25), 263.2 (34), 245.3 (14)

LLE

−0.078

C55H104O6

3.5

MS2 [861.8]: 605.4 (100), 579.6 (83), 577.7 (71); MS3 [861.8→605.5]: 531.2 (14), 341.4 (17), 321.4 (13), 265.2 (100), 247.2 (21); MS3 [861.8→577.5]: 313.3 (26), 265.3 (100), 247.4 (21), 239.2 (27)

POS

C55H104O6 −0.020

C57H106O6

MS2 [878.8]: 861.7 (13), 605.5 (100), 579.6 (54), 577.6 (89) 4.5

C57H106O6 0.254

C59H108O6

C59H108O6

C59H108O6

POG

MS2 [904.8]: 887.8 (8), 631.6 (100), 605.6 (86), 577.5 (74) 5.5

C59H108O6 0.254

MS2 [887.8]: 869.7 (26), 631.6 (100), 605.6 (52), 577.5 (42); MS3 [887.8→577.5]: 321.3 (18), 313.2 (17), 265.3 (100), 247.2 (29), 239.1 (36); MS3 [887.8→631.6]: 557.5 (66), 367.4 (23), 349.2 (25), 339.6 (34), 321.2 (22), 293.4 (100), 275.4 (25), 265.4 (73), 247.4 (19)

MS2 [913.8]: 895.7 (23), 631.6 (100), 603.5 (44); MS3 [913.8→603.5]: 339.4 (71), 265.3 (100), 247.4 (27); MS3 [913.8→631.6]: 557.6 (58), 367.4 (30), 349.3 (32), 339.4 (32), 293.3 (100), 275.2 (44), 265.2 (85), 247.3 (38)

OOG

MS2 [930.8]: 913.8 (43), 631.5 (100), 603.5 (85) 5.5

MS2 [913.8]: 895.7 (17), 657.6 (100), 601.6 (21), 583.5 (14); MS2 [913.8→657.6]: 639.5 (31), 601.6 (100), 583.7 (73), 565.5 (21), 321.5 (16), 263.4 (17)

PLE

MS2 [930.8]: 913.9 (39), 657.5 (100), 633.6 (31), 575.7 (15); MS3 [930.8→657.6]: 639.6 (18), 601.6 (100), 583.7 (69), 321.4 (20), 303.3 (17), 263.4 (14), 245.3 (7); MS3 [930.8→633.6]: 614.1 (14), 559.7 (24), 559.1 (15), 451.9 (22), 377.5 (31), 321.5 (100), 303.5 (33), 239.1 (13); MS3 [930.8→575.5]: 319.3 (99), 313.3 (73), 263.3 (100), 245.4 (50), 239.1 (21)

For [M + H − H2O]+, formula of cation is listed; for other ions, formula of neutral molecules is listed. bError of adduct ions or fragment ions was not reported. a

M



Article

Figure 8. APCI+ MS2 spectra of [M + H]+ ions from TG isomers at m/z 855.7.

Figure 9. APCI+ MS3 spectra of A ions from TG isomers [855.7→599.6].

Identification of Sphingolipids. Sphingolipids constitute a class of distinct compounds that are both endogenous to mammalian cells and available exogenously through dietary consumption. These unique lipids are composed of a sphingoid long-chain base, a fatty acid tethered to the amino group of the sphingosine, and a variable polar headgroup (Figure 5A).

C-5 and C-6. Characteristic ions also include deprotonated ferulic acid at m/z 193 as well as a fragment at m/z 177 derived from deprotonated ferulic acid.36 The APCI(±) MSn spectra of campestanyl ferulate re shown in Figure S5. The major γ-oryzanol components were deduced by combining APCI positive and negative data, listed in Table 4. N



Article

milling fractions were about the same on the basis of the peak areas in their extracted ion chromatography. Identification of Triglycerides and Diglycerides. According to the literature,18 TGs in whole wheat contain mainly linoleic acid (59%), palmitic acid (16.7%), and oleic acid (16.5%) with lower concentrations of some other fatty acids, such as linolenic acid (4.3%), arachidic acid (1.9%), palmitoleic acid (0.7%), and stearic acid (0.3%). The APCI full scan spectra of TGs showed primarily two types of structurally important ions, “molecular ions” ([M + H]+ and [M + NH4]+) and “diglycerides” [M − RCOO]+ ions, the fragment ions formed by the loss of one fatty acid molecule from the [M + H]+ ion of TGs (Figure 6, A ions). In the ESI full scan spectra, adduct ions [M + NH4]+ and [M + Na]+ were observed instead of protonated molecules [M + H]+, and no obvious fragment ions were produced. Therefore, the molecular masses of the individual TGs can be readily determined from the peaks of [M + H]+ and [M + NH4]+ in APCI spectra, or [M + NH4]+ and [M + Na]+ in ESI spectra, respectively. DGs formed A ions by losing a molecule of water from their [M + H]+ ion. Another abundant fragment ion in the MS/MS spectra of TGs is formed by subsequent losses of a fatty acid molecule and an acyl RCO (B ions). Furthermore, dehydrated ion B, acyl ions (C ions), and dehydrated C ions were observed as well. The structure of the fragment ions observed in both the APCI and ESI MS/MS spectra are shown in Figure 6. The masses of acyls and characteristic fragment ions of TGs/DGs are listed in Table 7. Molecular weight can also be determined from ions A or C using the following equations:

Figure 10. Structures and abbreviations of common wheat phospholipids.

The sphingosine base and the fatty acid constitute ceramide. There is considerable structural variation among the sphingolipids of different organisms with respect to the types of polar head groups and ceramide backbones. The principal molecular species of sphingolipids reported in wheat include the dihydroxy longchain base sphinganine (d18:0) and the trihydroxy long-chain base 4-hydroxysphinganine (t18:0) for ceramide (Figure 5B) AND 4,8-sphingadienine (d18:24,8), 8-sphingenine (d18:18), and 4-hydroxy-8-sphingenine (t18:18) for glucosylceramide (Figure 5C). The major fatty acids in wheat sphingolipids are 2-hydroxy fatty acids, and the major sugar in wheat monoglycosyl ceramide (cerebroside) is glucose.39 In APCI/ESI negative mode, ceramide and cerebroside compounds mainly yielded [M − H]− and [M + FA − H]− ions, due to the formation of adducts with formic acid in the mobile phase. The acid adducts were useful diagnostic ions as sphingolipid selective markers. All cerebrosides gave product ions [M − H − 162]− and [M − H − 180]− in their MS2 spectra (Table 6; Figure S6). Anions of 2-hydroxy fatty acids, which can be used to determine the length of the fatty acyl chain moiety, are listed in Table 5.40,41 Twelve cerebrosides and seven ceramides were tentatively identified (Table 6). Their distributions in three

[M + H]+ = C1 + C2 + C3 + 90 A12 = C1 + C2 + 73

C1 = [M + H]+ − A 23 − 17 C2 = [M + H]+ − A13 − 17

C3 = [M + H]+ − A12 − 17

The above equations are very helpful in elucidating composition of saturated TGs, which showed no or extremely low intensity of [M + H]+ ions in APCI spectra. Extracted ion chromatograms of A ions could be reconstructed to deconvolute the overlapping peaks, facilitating the identification of TGs/DGs with the same A ion (Figure 7). Every TG molecule contains three distinct attachment sites for fatty acids.

Table 9. Characteristic Ions of Phospholipids ESI positive compound molecular ions [M + H] [M + H]+

[M + H − 141] m/z 184

PA

[M + NH4]+

[M + H − 98]+

PI a

fragment ions

PE PC PG PS

+

negative fragment ions

sn-2/sn-1a

R1COO , R2COO [M − 15]− R1COO−, R2COO− [M − H − 87]−, [M − H − 87 − R2COOH]−, [M − H − 87 − R1COOH]−, R1COO−, R2COO− R1COO−, R2COO−, [M − H − R2COOH]−, [M − H − R1COOH]−, [M − R2CO]− m/z 241, R1COO−, R2COO−, [M − H − R2COOH]−, [M − H − R1COOH]−, [M − R2CO]−

>1 >1 >1

Differentiation of Whole Grain from Refined Wheat (T. aestivum) Flour

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