MS of New Carotenoid Esters in Mango and

Article pubs.acs.org/JAFC

Composition by LC-MS/MS of New Carotenoid Esters in Mango and Citrus Fabiane C. Petry and Adriana Z. Mercadante* Food Research Center (FoRC), Department of Food Science, Faculty of Food Engineering, University of Campinas (UNICAMP), 13083-862 Campinas, SP, Brazil ABSTRACT: Interest in the composition of carotenoid esters of fruits is growing because esterification may affect their bioavailability. Thus, the aim was to provide a detailed identification of carotenoid esters in citrus and mango. Orange cv. ‘Valencia’ and cv. ‘Pera’ presented 9 free carotenoids, 38 monoesters, and 60 diesters. Violaxanthin and luteoxanthin derivatives were the major ones, followed by antheraxanthin, lutein, zeaxanthin, β-cryptoxanthin, and zeinoxanthin esters, many of them reported for the first time in orange pulp. The carotenoid ester composition of tangor cv. ‘Murcott’, reported for the first time, showed 8 free carotenoids, 34 monoesters, and 33 diesters, with β-cryptoxanthin esters as major compounds, followed by violaxanthin and zeaxanthin esters. In citrus, carotenoids were acylated mainly with capric, lauric, myristic, myristoleic, palmitic, palmitoleic, and oleic acids. In mango, 5 free carotenoids, 2 monoesters, and 19 diesters were identified, from which many violaxanthin and neoxanthin esters were reported for the first time. KEYWORDS: carotenoid esters, Citrus sinensis L. Osbeck, Citrus reticulata L. Blanco X Citrus sinensis L. Osbeck, HPLC-DAD-(APCI)MSn, identification, Mangifera indica L., xanthophylls, mango, orange, mandarin, LC-MS

■

INTRODUCTION Carotenoids are a widespread group of fat-soluble natural pigments biosynthesized by higher plants, algae, and some microorganisms. Carotenoids are associated with reduced risk of developing cancer, cardiovascular disorders, age-related macular degeneration, and cataract formation.1−3 Animals and humans are able to convert some carotenoids into vitamin A (retinol), a nutrient related to vision, maintenance of epithelial surfaces, immune response, reproduction, and embryonic growth and development.4 Hydroxylated carotenoids can be present in fruits in the free form and also acylated with fatty acids (FA), forming esters. In plant tissues, esterification facilitates carotenoid storage5 and has also been related to a biological mechanism to protect sensitive molecules from photo-oxidation.6 Acylation modifies chemical properties, such as water solubility, which is lower in esterified xanthophylls than in the corresponding free xanthophylls and even than in carotenes,7 which could affect the bioavailability of such nutrients. In fact, esterification did not impair lutein bioavailability in humans, although it depended not only on the presence or absence of esterified fatty acids but also on factors related to dissolution of the carotenoid.8 From this standpoint, characterization of the native carotenoid composition of fruits and vegetables is mandatory for subsequent studies on human bioavailability. In addition, β-cryptoxanthin esters have been shown to be more stable against heat than free β-cryptoxanthin, a property that can be explored by the food industry.9 Therefore, increasing interest about the carotenoid ester composition of fruits has arisen in the past years. Esterification increases possible structure natural variation, increasing the complexity of carotenoid analysis. A single xanthophyll can be acylated with different FAs, but the chromophore remains unchanged, turning identification into © 2016 American Chemical Society

a challenging step. Therefore, information about the carotenoid ester composition of foods is frequently not available, because a saponification step is usually applied to make carotenoid analysis simpler by releasing the FA bound to carotenoids and eliminating interfering compounds such as chlorophylls and triacylglycerols. Oranges (Citrus sinensis) and mandarins (Citrus reticulata) are the most representative species from the Citrus genus in terms of worldwide production and consumption.10 Considering the composition of the nonsaponified carotenoid extract from orange, only a few studies are found.11−15 However, some of them studied only esters of one xanthophyll or group of xanthophylls. For example, Breithaupt and co-workers11,12 studied the β-cryptoxanthin esters in orange juice, whereas Dugo and co-workers14 investigated the epoxycarotenoid esters. On the other hand, when the complete native composition was studied, only a few carotenoid esters were identified, despite the large diversity of possible esters that can be found in orange and orange juice. For example, Dugo and co-workers13 identified only 10 esters among the 62 peaks separated, whereas Giuffrida and co-workers15 identified 18 esters among the 25 carotenoids reported. From all of the above studies, a total of 22 carotenoid monoesters and 20 diesters from oranges were described. Belonging to the mandarin group, common mandarin (C. reticulata Blanco) and mandarin hybrids, including tangor (C. reticulata × C. sinensis) and tangelo (C. reticulata × Citrus paradisi), can be found.16 Mandarins have a simpler carotenoid ester composition compared to oranges, although studies about the composition of the nonsaponified carotenoid extract of Received: Revised: Accepted: Published: 8207

July 19, 2016 October 4, 2016 October 7, 2016 October 7, 2016 DOI: 10.1021/acs.jafc.6b03226 J. Agric. Food Chem. 2016, 64, 8207−8224

(all-E)-lutein (13Z)- or (15Z)-violaxanthin dibutyrate (9Z)-violaxanthin butyrate

(all-E)-zeaxanthin (all-E)-violaxanthin dibutyrate

(9Z)- or (9′Z)- antheraxanthin (9′Z)-neoxanthin dibutyrate

6b; V, P 7a; M

8; V, P 9; M

10; P 11; M

8208

unidentified apocarotenoid derivative (9Z)-violaxanthin dibutyrate

14; T

(9Z)- or (9′Z)-luteoxanthin dibutyrate (9Z)-violaxanthin butyrate-caproate

18; M

(all-E)-violaxanthin laurate

(9Z)-violaxanthin caprate

(all-E)-antheraxanthin dibutyrate

(all-E)-luteoxanthin laurate

(all-E)-violaxanthin myristate

20; V, P, T

21; V

22; M

23; V, P, T

24a; V, P, T

19; M

17; V

(13Z)- or (15Z)-violaxanthin laurate 2 not identified

16; V, P

15; M

13; M

(13Z)- or (15Z)-violaxanthin laurate 1 (all-E)-violaxanthin butyrate-caproate

12; V, P

7b; M

5; V, P 6a; M

(all-E)-violaxanthin (all-E)-luteoxanthin (all-E)-antheraxanthin (9Z)-violaxanthin (all-E)-violaxanthin butyrate or neoxanthin butyrate mutatoxanthin (all-E)-neoxanthin dibutyrate

carotenoid

1; V, P, M 2; V, P 3a; V, P 3b; V, P, M 4; M

peaka

21.4−21.8

20.5−21.1

20.0−20.1

19.0−19.5

18.5−19.0

17.8−18.0

17.2−17.3

16.9−17.2

16.5−16.6

15.7−15.8

15.5

15.1−15.2

14.8−15.0

13.4 13.7−13.8

12.9−13.0 13.1−13.2

12.1−12.2

11.2−11.3 12.1−12.2

10.2−10.4 11.2

6.5 7.4−7.5 8.7−9.0 8.7−9.0 9.2

tRb (min)

439, 469

439, 468 421, 448 444, 472

469

435,

468 436,

477 469

435,

472 435,

nd

d

400, 422, 447

329, 417, 436, 463 421, 445, 473

418, 439, 465

312, 396, 417, 444 329, 412, 436, 465

328, 428

328, 412, 435, 464 329, 435

390, 413, 435

422, 440, 329, 413, 465 328, 418, 462 417, 440,

416, 444, 330, 412, 463 330, 412, 463 423, 450, 416, 440,

400, 426, 450 416, 440, 469

416, 398, 420, ndd 417,

λmaxc (nm)

nc

e

49

nc

0

0

f

ncf

nce 44

0

15

16

39

39

9

0

0

24

16 16

nce

71

93

0

0

84

0

79

15

44 80

0 0

ncf

nce 20 94

0 36

ncf 0

nce 81 44 69

0 0 0 ncf 0

% AB/AII

84 89 44 nce 87

% III/II

811.6

783.6

725.6

755.6

783.7

769.5

741.5

783.6

783.7

741.5

ndg

769.5

783.8

585.6 741.6

569.4 741.6

671.5

ndg 741.5

585.6 741.6

601.5 601.5 585.6 601.6 671.5

[M + H]+ (m/z)

723.5 [M + H − 18]+, 653.5 [M + H − 4:0]+, 649.4 [M + H − 92]+, 635.4 [M + H − 4:0 − 18]+, 631.4 [M + H − 92 − 18]+, 565.5 [M + H − 4:0 − 4:0]+, 547.4 [M + H − 4:0 − 4:0 − 18]+ 765.6 [M + H − 18]+, 747.6 [M + H − 18 − 18]+, 691.5 [M + H − 92]+, 673.5 [M + H − 92 − 18]+, 583.4 [M + H − 12:0]+, 565.4 [M + H − 12:0 − 18]+, 547.5 [M + H − 12:0 − 18 − 18] + 765.6 [M + H − 18]+, 747.5 [M + H − 18 − 18]+, 691.5 [M + H − 92]+, 673.5 [M + H − 92 − 18]+, 583.4 [M + H − 12:0]+, 565.4 [M + H − 12:0 − 18]+, 547.4 [M + H −12:0 − 18 − 18] + 723.5 [M + H − 18]+, 653.5 [M + H − 4:0]+, 649.4 [M + H − 92]+, 635.4 [M + H − 4:0 − 18]+, 631.4 [M + H − 92 − 18]+, 565.5 [M + H − 4:0 − 4:0]+, 547.4 [M + H − 4:0 − 4:0 − 18]+ 751.6 [M + H − 18]+, 681.5 [M + H − 4:0]+, 677.4 [M + H − 92]+, 663.5 [M + H − 4:0 − 18]+, 659.5 [M + H − 92 − 18]+, 653.4 [M + H − 6:0]+, 635.5 [M + H − 6:0 − 18]+, 565.4 [M + H − 4:0 − 6:0]+, 547.4 [M + H − 4:0 − 6:0 − 18]+ 765.6 [M + H − 18]+, 747.6 [M + H − 18 − 18]+, 691.5 [M + H − 92]+, 673.6 [M + H − 92 − 18]+, 583.4 [M + H − 12:0]+, 565.4 [M + H − 12:0 − 18]+, 547.5 [M + H − 12:0 − 18 − 18] + 737.6 [M + H − 18]+, 719.6 [M + H − 18 − 18]+, 663.5 [M + H − 92]+, 645.5 [M + H − 92 − 18]+, 583.4 [M + H − 10:0]+, 565.4 [M + H − 10:0 − 18]+, 547.5 [M + H − 10:0 − 18 − 18]+, 221.1 707.5 [M + H − 18]+, 637.5 [M + H − 04:0]+, 619.5 [M + H − 04:0 − 18]+, 549.4 [M + H − 04:0 − 04:0]+, 531.4 [M + H − 04:0 − 04:0 − 18]+ 765.7 [M + H − 18]+, 747.6 [M + H − 18 − 18]+, 691.5 [M + H − 92]+, 673.6 [M + H − 92 − 18]+, 583.4 [M + H − 12:0]+, 565.4 [M + H − 12:0 − 18]+, 547.5 [M + H − 12:0 − 18 − 18]+ 793.7 [M + H − 18]+, 775.6 [MH − 18 − 18]+, 719.5 [M + H − 92]+, 701.5 [M + H − 92 − 18]+, 583.4 [M + H − 14:0]+, 565.4 [M + H − 14:0 − 18]+, 547.5 [M + H − 14:0 − 18 − 18]+

583.5 [M + H − 18], 565.5 [M + H − 18 − 18], 509.4 [M + H − 92], 491.4 [M + H − 92 − 18], 221.1 583.5 [M + H − 18], 565.5 [M + H − 18 − 18], 509.5 [M + H − 92], 491.4 [M + H − 92 − 18], 221.1 567.5 [M + H − 18]+, 549.5 [M + H − 18−18]+, 493.4 [M + H − 92]+, 475.4 [M + H − 92 − 18]+, 221.1 583.6 [M + H − 18]+, 565.5 [M + H − 18−18]+, 509.5 [M + H − 92] +, 491.5 [M + H − 92 − 18] +, 221.0 653.4 [M + H − 18]+, 635.4 [M + H − 18 − 18]+, 583.4 [M + H − 4:0]+, 579.4 [M + H − 92]+, 565.4 [M + H − 4:0 − 18]+, 561.4 [M + H − 92 − 18]+ 567.5 [M + H − 18]+, 549.6 [M + H − 18 − 18]+, 493.4 [M + H − 92]+, 221.0 723.5 [M + H − 18]+, 653.5 [M + H − 04:0]+, 649.4 [M + H − 92]+, 635.4 [M + H − 4:0 − 18]+, 631.4 [M + H − 92 − 18]+, 565.5 [M + H − 4:0 − 4:0]+, 547.4 [M + H − 4:0 − 4:0 − 18]+ 551.5 [M + H − 18]+ → 533.4 [M + H − 18 − 18]+, 495.3 [M + H − 56 − 18]+ 723.5 [M + H − 18]+, 653.5 [M + H − 4:0]+, 649.4 [M + H − 92]+, 635.4 [M + H − 4:0 − 18]+, 631.4 [M + H − 92 − 18]+, 565.5 [M + H − 4:0 − 4:0]+, 547.4 [M + H − 4:0 − 4:0 − 18]+ 653.4 [M + H − 18]+, 635.4 [M + H − 18 − 18]+, 583.4 [M + H − 4:0]+, 579.4 [M + H − 92]+, 565.4 [M + H − 4:0 − 18]+, 561.4 [M + H − 92 − 18]+ 551.4 [M + H − 18]+, 533.4 [M + H − 18 − 18]+, 477.4 [M + H − 92]+ 723.5 [M + H − 18]+, 653.5 [M + H − 4:0]+, 649.4 [M + H − 92]+, 635.4 [M + H − 4:0 − 18]+, 631.4 [M + H − 92−18]+, 565.5 [M + H −4:0 − 4:0]+, 547.4 [M + H − 4:0 − 4:0 − 18]+ 567.5 [M + H − 18]+, 549.6 [M + H − 18 − 18]+, 493.4 [M + H − 92]+, 221.0 723.5 [M + H − 18]+, 653.5 [M + H − 4:0]+, 649.4 [M + H − 92]+, 635.4 [M + H − 4:0 − 18]+, 631.4 [M + H − 92 − 18]+, 565.5 [M + H − 4:0 − 4:0]+, 547.4 [M + H − 4:0 − 4:0 − 18]+ 765.8 [M + H − 18]+, 747.7 [M + H − 18−18]+, 691.7 [M + H − 92]+, 673.2 [M + H − 92 − 18]+, 583.5 [M + H − 12:0]+, 565.5 [M + H − 12:0 − 18]+, 547.5 [M + H − 12:0 − 18 − 18]+ 751.6 [M + H − 18]+, 681.5 [M + H − 04:0]+, 677.4 [M + H − 92]+, 663.5 [M + H − 4:0 − 18]+, 659.5 [M + H − 92 − 18]+, 653.4 [M + H − 6:0]+, 635.5 [M + H − 6:0 − 18]+, 565.4 [M + H − 4:0 − 6:0]+, 547.4 [M + H − 4:0 − 6:0 − 18]+ ndg

fragment ions (m/z)

Table 1. Chromatographic, UV/Vis, and Mass Spectroscopy Characteristics, Obtained by HPLC-DAD-APCI-MS/MS, of Carotenoids from Orange cv. ‘Valencia’, Orange cv. ‘Pera’, Tangor cv. ‘Murcott’, and Mango cv. ‘Tommy Atkins’

Journal of Agricultural and Food Chemistry Article

DOI: 10.1021/acs.jafc.6b03226 J. Agric. Food Chem. 2016, 64, 8207−8224

(all-E)-phytoene (all-E)-β-cryptoxanthin (9Z)- or (9′Z)-luteoxanthin laurate

auroxanthin laurate

(all-E)-neoxanthin butyrate-myristate (all-E)-violaxanthin butyrate-laurate

(all-E)-luteoxanthin myristate

phytofluene 1 phytofluene 2 (all-E)-violaxanthin palmitoleate

(9Z)-or (9′Z)-luteoxanthin myristate (9Z)-violaxanthin butyrate-laurate

(all-E)-violaxanthin palmitate

(all-E)-violaxanthin butyrate-myristate

(9Z)- or (9′Z)-antheraxanthin laurate (9Z)- or (9′Z)-luteoxanthin oleate

(all-E)-luteoxanthin palmitate

(9Z)- or (9′Z)-luteoxanthin myristate (Z)-ζ-carotene (13Z)- or (15Z)-β-carotene

(9Z)-violaxanthin palmitate

mutatoxanthin laurate 1

(9Z)-violaxanthin butyrate-myristate

(9Z)- or (9′Z)-luteoxanthin palmitate

25a; T 25b; T 25c; V, P, T

26; V, P, T

27; M

29; V, P

30; T 31a; T 31b; V, P, T

32; V, P, T

34; V, P

35a; M

35b; V, P, T

8209

35d; P

35e; P

36; V, P, T 37; M

38a; V

38b; V, P

39a; M

39b; V, P, T

35c; V, P

33; M

28; M

(9Z)-violaxanthin laurate

carotenoid

24b; V, P, T

peaka

Table 1. continued

30.9−31.5

31.2−31.4

30.3−30.8

30.1

29.7−30.0 29.9−30.2

28.4−29.0

28.4−29.0

28.4−29.0

28.4−29.0

28.7−28.9

27.9−28.3

27.1−27.3

26.2−26.7

24.8 25.3 25.3−25.7

24.5−25.0

24.4−24.6

24.1−24.3

23.8−24.3

22.7 22.7 22.3−22.9

21.4−21.8

tRb (min)

425

417,

298

435,

445

436,

419 443,

418,

313, 390, 417, 442

329, 413, 436, 465

312, 395, 441 376, 398, 339, 420, 465 330, 416, 464 400, 424,

330, 418, 440, 464 312, 397, 418, 443 ndd

417, 440, 470

412, 439, 469

312, 388, 417, 443 330, 414, 437, 466

330, 348, 368 330, 348, 368 417, 440, 464

400, 421, 447

417, 440, 470

417, 441, 470

328, 414, 467 274, 285, ndd 315, 397, 443 380, 401,

λmaxc (nm)

ncf

nce

ncf

ncf

nce

nce

ncf

nce

10

541.5 537.5

ncf 39

90 8

83

811.7

ncf

nce

839.7

881.7

767.6

839.7

839.7

ncf

nce

865.7

767.6

21

ncf

nce

881.7

839.7

853.7

100

0

ncf

nce 89

16

70

ncf

nce 811.7

543.5 543.5 837.7

ncf ncf ncf

80 nce nce

853.7

811.7

0

ndg

783.6

545.5 553.5 783.6

783.7

[M + H]+ (m/z)

0

100

88

0

ncf ncf 11

nce nce 100

83

11

% AB/AII

56

% III/II

fragment ions (m/z)

821.7 [M + H − 18]+, 803.6 [M + H − 18 − 18]+, 747.6 [M + H − 92]+, 729.6 [M + H − 92 − 18]+, 583.4 [M + H − 16:0]+, 565.4 [M + H − 16:0 − 18]+, 547.4 [M + H − 16:0 − 18 − 18]+ 749.6 [M + H − 18]+, 731.6 [M + H − 18 − 18]+, 675.6 [M + H − 92]+, 567.4 [M + H − 12:0]+, 549.4 [M + H − 12:0 − 18]+, 531.4 [M + H − 12:0 − 18 − 18]+ 863.7 [M + H − 18]+, 793.7 [M + H − 4:0]+, 789.7 [M + H − 92]+, 775.6 [M + H − 4:0 − 18]+, 771.8 [M + H − 92 − 18]+, 653.5 [M + H − 14:0]+, 635.5 [M + H − 14:0 − 18]+, 565.5 [M + H − 4:0 − 14:0]+, 547.4 [M + H − 4:0 − 14:0 − 18]+ 821.7 [M + H − 18]+, 803.8 [M + H − 18 − 18]+, 747.6 [M + H − 92]+, 729.6 [M + H − 92 − 18]+, 583.4 [M + H − 16:0]+, 565.5 [M + H − 16:0 − 18]+, 547.4 [M + H − 16:0 − 18 − 18]+

765.6 [M + H − 18]+, 747.6 [M + H − 18 − 18]+, 691.6 [M + H − 92]+, 673.6 [M + H − 92 − 18]+, 583.5 [M + H − 12:0]+, 565.4 [M + H − 12:0 − 18]+, 547.5 [M + H − 12:0 − 18 − 18]+ 489.4 [M + H − 56]+, 475.4 [M + H − 70]+, 463.3 [M + H − 82]+, 421.4, 395.3 535.5 [M + H − 18]+, 461.4 [M + H − 92]+ 765.6 [M + H − 18]+, 747.6 [M + H − 18 − 18]+, 691.5 [M + H − 92]+, 673.6 [M + H − 92 − 18]+, 583.4 [M + H − 12:0]+, 565.4 [M + H − 12:0 − 18]+, 547.4 [M + H − 12:0 − 18 − 18]+ 765.6 [M + H − 18]+, 747.6 [M + H − 18 − 18]+, 691.5 [M + H − 92]+, 673.5 [M + H − 92 − 18]+, 583.4 [M + H − 12:0]+, 565.5 [M + H − 12:0 − 18]+, 547.4 [M + H − 12:0 − 18 − 18]+ 863.7 [M + H − 18]+ → 775.6 [M + H − 04:0 − 18]+, 771.8 [M + H − 92 − 18]+, 635.5 [M + H − 14:0 − 18]+, 547.4 [M + H − 04:0 − 14:0 − 18]+ 835.7 [M + H − 18]+, 765.6 [M + H − 04:0]+, 761.6 [M + H − 92]+, 747.6 [M + H − 4:0 − 18]+, 743.6 [M + H − 92 − 18]+, 653.4 [M + H − 12:0]+, 635.4 [M + H − 12:0 − 18]+, 565.4 [M + H − 4:0 − 12:0]+, 547.4 [M + H − 4:0 − 12:0 − 18]+ 793.6 [M + H − 18]+, 777.8 [M + H − 18 − 18]+, 719.6 [M + H − 92]+, 701.6 [M + H − 92 − 18]+, 583.4 [M + H − 14:0]+, 565.4 [M + H − 14:0 − 18]+, 547.4 [M + H − 14:0 − 18 − 18]+ 487.6 [M + H − 56]+, 461.4 [M + H − 82], 393.4, 337.2 487.6 [M + H − 56]+, 461.4 [M + H − 82], 393.4, 337.2 819.7 [M + H − 18]+, 801.6 [M + H − 18 − 18]+, 745.6 [M + H − 92]+, 727.6 [M + H − 92 − 18]+, 583.4 [M + H − 16:1]+, 565.3 [M + H − 16:1 − 18]+, 547.5 [M + H − 16:1 − 18 − 18]+ 793.7 [M + H − 18]+, 775.6 [M + H − 18 − 18]+, 719.5 [M + H − 92]+, 701.6 [M + H − 92 − 18]+, 583.5 [M + H − 14:0]+, 565.4 [M + H − 14:0 − 18]+, 547.3 [M + H − 14:0 − 18 − 18]+ 835.7 [M + H − 18]+, 765.6 [M + H − 4:0]+, 761.6 [M + H − 92]+, 747.6 [M + H − 4:0 − 18]+, 743.6 [M + H − 92 − 18]+, 653.4 [M + H − 12:0]+, 635.4 [M + H − 12:0 − 18]+, 565.4 [M + H − 4:0 − 12:0]+, 547.4 [M + H − 4:0 − 12:0 − 18]+ 821.7 [M + H − 18]+, 803.6 [M + H − 18 − 18]+, 747.6 [M + H − 92]+, 729.6 [M + H − 92 − 18]+, 583.4 [M + H − 16:0]+, 565.4 [M + H − 16:0 − 18]+, 547.4 [M + H − 16:0 − 18 − 18]+ 863.7 [M + H − 18]+, 793.7 [M + H − 4:0]+, 789.7 [M + H − 92]+, 775.6 [M + H − 4:0 − 18]+, 771.8 [M + H − 92 − 18]+, 653.5 [M + H − 14:0]+, 635.5 [M + H − 14:0 − 18]+, 565.5 [M + H − 4:0 − 14:0]+, 547.4 [M + H − 4:0 − 14:0 − 18]+ 749.7 [M + H − 18]+, 731.5 [M + H − 18 − 18]+, 675.6 [M + H − 92]+, 567.5 [M + H − 12:0]+, 549.4 [M + H − 12:0 − 18]+, 531.4 [M + H − 12:0 − 18 − 18]+ 847.7 [M + H − 18]+, 829.8 [M + H − 18 − 18]+, 773.6 [M + H − 92]+, 755.6 [M + H − 92 − 18]+, 583.4 [M + H − 18:1]+, 565.4 [M + H − 18:1 − 18]+, 547.4 [M + H − 18:1 − 18 − 18]+ 821.7 [M + H − 18]+, 803.6 [M + H − 18 − 18]+, 747.6 [M + H − 92]+, 729.6 [M + H − 92 − 18]+, 583.4 [M + H − 16:0]+, 565.4 [M + H − 16:0 − 18]+, 547.4 [M + H − 16:0 − 18 − 18]+ 793.7 [M + H − 18]+, 775.6 [M + H − 18 − 18]+, 719.5 [M + H − 92]+, 701.6 [M + H − 92 − 18]+, 583.5 [M + H − 14:0]+, 565.4 [M + H − 14:0 − 18]+, 547.3 [M + H − 14:0 − 18 − 18]+ 486.4 [M − 56]+, 459.4 [M + H − 82]+, 391.3 ndg

Journal of Agricultural and Food Chemistry Article

DOI: 10.1021/acs.jafc.6b03226 J. Agric. Food Chem. 2016, 64, 8207−8224

mutatoxanthin laurate 2

(all-E)-violaxanthin caprylate-laurate

(9Z)- or (9′Z)-luteoxanthin palmitate (Z)-zeaxanthin caprate

40b; V, P, T

40c; V

40d; P

(all-E)-ζ-carotene mutatoxanthin myristate

antheraxanthin oleate or mutatoxanthin oleate violaxanthin caprate-laurate

42a; T 42b; V, P

42c; V, P

8210

(all-E)-β-carotene (all-E)-zeaxanthin myristoleate (9Z)-violaxanthin butyrate-palmitate

(9Z)- or (9′Z)-antheraxanthin myristate (9Z)-β-carotene

(9Z)-ζ-carotene

(13Z)- or (15Z)-zeaxanthin myristate 1 zeaxanthin laurate (all-E)-antheraxanthin palmitate

43c; T, M 43d; T 44a; M

44b; V, P

45b; T

45c; T

(13Z)- or (15Z)-violaxanthin laurate-myristoleate

48b; T

48a; V, P

(9Z)- or (9′Z)-antheraxanthin oleate (9Z)-violaxanthin caprate-laurate

47b; V, P

46; T 47a; V, P

45a; M

violaxanthin dilaurate

43b; V, P, T

43a; V, P, T

(all-E)-violaxanthin butyrate-palmitate

41; M

40e; T

lutein 3-O-laurate (all-E)-antheraxanthin myristate

carotenoid

39c; P 40a; V, P, T

peaka

Table 1. continued

40.2−41.0

40.2−41.0

38.6−39.5

38.4 38.6−39.5

37.7

37.7

37.6−37.9

36.7−37.2

35.8−36.3 36.3 36.7

35.9−36.6

35.9−36.6

34.8−35.4

35.1 34.8−35.4

33.9−34.2

32.6

32.8

32.2

32.2−32.8

31.5 32.2−32.8

tRb (min)

ncf ncf

nce nce

ndd

ncf ncf

nce nce

330, 416, 437, 464

50

23

ncf ncf

nce nce

ndd 426, 443, 468 330, 420, 440, 467 328, 418, 436, 464

ncf

nce

991.8

937.7

849.7

751.6 823.7

779.7

541.5

ncf

100

795.7

537.5 777.7 909.7

965.8

537.5

14

0 0 ncf

nd

937.8

849.7

541.5 795.6

0

31

21

33 13 60

73

nd

ncf

0 ncf

909.7

723.7

839.8

909.7

767.6

ndg 795.6

[M + H]+ (m/z)

330, 415, 440, 467 330, 420, 447, 472 ndd, 380, 401, 425 ndd

422, 451, 468 422, 451, 468 330, 415, 437, 466

409, 437, 464

73

nce

ndd 409, 437, 464

100 nce

380, 401, 425 403, 427, 448

330, 418, 442, 469 417, 440, 470 0

ncf

nce

420, 439, 465

90

ncf

nce

395, 424, 442

ncf 0

nce 29

ndd 417, 444, 470

% AB/AII

% III/II

λmaxc (nm) fragment ions (m/z)

733.6 [M + H − 18]+, 659.7 [M + H − 92]+, 551.5 [M + H − 12:0]+, 533.4 [M + H − 12:0 − 18]+ 805.7 [M + H − 18]+, 787.8 [M + H − 18 − 18]+, 731.7 [M + H − 92]+, 567.4 [M + H − 16:0]+, 549.4 [M + H − 16:0 − 18]+, 531 [M + H −16:0 − 18 − 18]+ 831.7 [M + H − 18]+, 813.6 [M + H − 18 − 18]+, 757.7 [M + H − 92]+, 739.5 [M + H − 92 − 18]+, 567.4 [M + H − 18:1]+, 549.4 [M + H − 18:1 − 18]+ 919.8 [M + H − 18]+, 845.6 [M + H − 92]+, 827.7 [M + H − 92 − 18]+, 765.6 [M + H − 10:0]+, 747.6 [M + H − 10:0 − 18]+, 737.6 [M + H − 12:0]+, 719.5 [M + H− 12:0 − 18]+, 565.4 [M + H − 10:0 − 12:0]+, 547.4 [M + H − 10:0 − 12:0 − 18]+ 973.8 [M + H − 18]+, 899.8 [M + H − 92]+, 881.7 [M + H − 92 − 18]+, 791.6 [M + H − 12:0]+, 773.7 [M + H − 12:0 − 18]+, 765.6 [M + H − 14:1]+, 747.6 [M + H − 14:1 − 18]+, 565.4 [M + H − 12:0 − 14:1]+, 547.4 [M + H − 12:0 − 14:1 − 18]+

761.8 [M + H − 18]+, 687.5 [M + H − 92]+, 551.4 [M + H − 14:0]+, 533.4 [M + H − 14:0 − 18]+

485.4 [M + H − 56]+, 471.4, 459.4 [M + H − 82], 391.4

891.7 [M + H − 18]+, 821.6 [M + H − 4:0]+, 817.6 [M + H − 92]+, 803.8 [M + H − 4:0 − 18]+, 799.6 [M + H − 92 − 18]+, 653.5 [M + H − 16:0]+, 635.4 [M + H − 16:0 − 18]+, 565.4 [M + H − 4:0 − 16:0]+, 547.4 [M + H − 4:0 − 16:0 − 18]+ 485.4 [M + H − 56]+, 471.4, 459.4 [M + H − 82], 391.4 777.7 [M + H − 18]+, 759.8 [M + H − 18 − 18]+, 703.5 [M + H − 92]+, 567.4 [M + H − 14:0]+, 549.4 [M + H − 14:0 − 18]+, 531.4 [M + H − 14:0 − 18 − 18]+ 831.7 [M + H − 18]+, 813.6 [M + H − 18 − 18]+, 757.7 [M + H − 92]+, 739.5 [M + H − 92 − 18]+, 567.4 [M + H − 18:1]+, 549.4 [M + H − 18:1 − 18]+ 919.8 [M + H − 18]+, 845.8 [M + H − 92]+, 827.7 [M + H − 92 − 18]+, 765.6 [M + H − 10:0]+, 747.6 [M + H − 10:0 − 18]+, 737.5 [M + H − 12:0]+, 719.5 [M + H − 12:0 − 18]+, 565.5 [M + H − 10:0 − 12:0]+, 547.4 [M + H − 10:0 − 12:0 − 18]+ 947.8 [M + H − 18]+, 873.6 [M + H − 92]+, 855.7 [M + H − 92 − 18]+, 765.6 [M + H − 12:0]+, 747.6 [M + H − 12:0 − 18]+, 565.4 [M + H − 12:0 − 12:0]+, 547.4 [M + H − 12:0 − 12:0 − 18]+ 481.4 [M + H − 56]+, 445.4 [M + H − 92]+ 759.8 [M + H − 18]+, 685.5 [M + H − 92]+, 551.4 [M + H − 14:1]+, 533.4 [M + H − 14:1 − 18]+ 891.7 [M + H − 18]+, 821.6 [M + H − 4:0]+, 817.6 [M + H − 92]+, 803.8 [M + H − 4:0 − 18]+, 799.6 [M + H − 92 − 18]+, 653.5 [M + H − 16:0]+, 635.4 [M + H − 16:0 − 18]+, 565.4 [M + H − 4:0 − 16:0]+, 547.4 [M + H − 4:0 − 16:0 − 18]+ 777.7 [M + H − 18]+, 759.6 [M + H − 18 − 18]+, 703.6 [M + H − 92]+, 567.5 [M + H − 14:0]+, 549.4 [M + H − 14:0 − 18]+, 531.5 [M + H − 14:0 − 18 − 18]+ nd

733.6 [M + H − 18]+ → 533.5 [M + H − 12:0 − 18]+ 777.7 [M + H − 18]+, 759.8 [M + H − 18 − 18]+, 703.5 [M + H − 92]+, 567.4 [M + H − 14:0]+, 549.4 [M + H − 14:0 − 18]+, 531.4 [M + H − 14:0 − 18 − 18]+ 749.6 [M + H − 18]+, 731.6 [M + H − 18 − 18]+, 675.5 [M + H − 92]+, 567.4 [M + H − 12:0]+, 549.4 [M + H − 12:0 − 18]+, 531.4 [M + H − 12:0 − 18 − 18]+ 891.7 [M + H − 18]+, 817.8 [M + H − 92]+, 799.7 [M + H − 92 − 18]+, 765.6 [M + H − 8:0]+, 747.6 [M + H − 8:0 − 18]+, 709.5 [M + H − 12:0]+, 691.5 [M + H − 12:0 − 18]+, 565.4 [M + H − 8:0 − 12:0]+, 547.4 [M + H − 8:0 − 12:0 − 18]+ 821.7 [M + H − 18]+, 803.7 [M + H − 18 − 18]+, 747.6 [M + H − 92]+, 729.7 [M + H − 92 − 18]+, 583.4 [M + H − 16:0]+, 565.4 [M + H − 16:0 − 18]+, 547.4 [M + H− 16:0 − 18 − 18]+ 705.6 [M + H − 18]+, 551.4 [M + H − 10:0]+, 533.4 [M + H − 10:0 − 18]+

Journal of Agricultural and Food Chemistry Article

DOI: 10.1021/acs.jafc.6b03226 J. Agric. Food Chem. 2016, 64, 8207−8224

(13Z)- or (15Z)-violaxanthin laurate-myristate

(9Z)-violaxanthin caprate-myristate

(13Z)- or (15Z)-mutatoxanthin palmitate (13Z)- or (15Z)-zeaxanthin myristate 2 (9Z)- or (9′Z)-antheraxanthin palmitate (all-E)-violaxanthin laurate-myristate or (all-E)-luteoxanthin laurate-myristate (9Z)- or (9′Z)-luteoxanthin dilaurate (Z)-zeinoxanthin laurate

49a; P, T

49b; V, T

50a; V, P

8211

46.6−47.2

(13Z)- or (15Z)-violaxanthin laurate-palmitate

(9Z)-zeaxanthin myristate (all-E)-violaxanthin dimyristate

violaxanthin laurate-oleate or luteoxanthin laurate-oleate

55b; V, P

55c; T 56a; V, P, T, M 56b; V, P, T

56c; V, P, T

55a; V, P, T

(9Z)- or (9′Z)-luteoxanthin lauratemyristate

46.1 46.6−47.2

(13Z)- or (15Z)-violaxanthin dimyristate violaxanthin caprate-palmitate or luteoxanthin caprate-palmitate

54b; V, P, T

54a; V, P, T

53b; T

53a; T

46.6−47.2

45.7−46.3

45.7−46.3

44.5−45.2

44.5−45.2

44.4

44.2

43.3−44.1

(9Z)- or (9′Z)-luteoxanthin dilaurate β-cryptoxanthin caprate or zeinoxanthin caprate (13Z)- or (15Z)-β-cryptoxanthin laurate (9Z)-violaxanthin laurate-myristate

52b; V, P, T

43.3−44.1

violaxanthin laurate-palmitoleate or luteoxanthin laurate-palmitoleate

43.0

42.5−43.2

42.5−43.2

42.5−43.2

41.9

41.4−41.9

41.6

41.6

40.2−41.0

tRb (min)

52a; V, P, T

51d; T

51c; V, P, T

51b; V, P, T

51a; V, P

50b; T

(9Z)-violaxanthin dilaurate

carotenoid

48c; V, P, T

peaka

Table 1. continued

436,

444,

442,

418,

312, 392, 418, 443

nd

330, 446 416, 439, 466

nd

329, 415, 437, 463 nd

313, 395, 441 330, 418, 462 337, 420, 470 328, 413, 465

312, 395, 417, 443 330, 417, 441, 469 ndd

330, 400, 426, 449 ndd, 423, 444, 470 330, 417, 440, 469 ndd

329, 413, 435, 464

328, 413, 436, 464 328, 412, 434, 463

λmaxc (nm)

17 0 ncf ncf

nce nce

nce

nce

0 nce

nc

19

10

nc

0

80

28

17

nce 0

ncf

nce

ncf

nce

ncf

nce 24

ncf

nce

44

13

50

nc

f

nc

19

e

16

19

13

% AB/AII

25

61

30

78

% III/II

993.9

1047.9

779.7 1021.8

1021.9

993.9

1021.9

993.8

735.7

707.6

965.8

1019.9

735.6

965.8

993.8

823.7

779.7

823.7

965.9

993.8

965.8

[M + H]+ (m/z) fragment ions (m/z)

975.8 [M + H − 18]+, 901.9 [M + H − 92]+, 883.8 [M + H − 92 − 18]+, 793.6 [M + H − 12:0]+, 775.6 [M + H − 12:0 − 18]+, 765.7 [M + H − 14:0]+, 747.6 [M + H − 14:0 − 18]+, 565.4 [M + H − 12:0 − 14:0]+, 547.4 [M + H − 12:0 − 14:0 − 18]+ 1003.9 [M + H − 18]+, 929.8 [M + H − 92]+, 911.8 [M + H − 92 − 18]+, 793.7 [M + H − 14:0]+, 775.6 [M + H − 14:0 − 18]+, 565.4 [M + H − 14:0 − 14:0]+, 547.4 [M + H − 14:0 − 14:0 − 18]+ 975.9 [M + H − 18]+, 901.8 [M + H − 92]+, 883.8 [M + H − 92 − 18]+, 821.7 [M + H − 10:0]+, 803.8 [M + H − 10:0 − 18]+, 737.5 [M + H − 16:0]+, 719.6 [M + H − 16:0 − 18]+, 565.4 [M + H − 10:0 − 16:0]+, 547.4 [M + H − 10:0 − 16:0 − 18]+ 1003.9 [M + H − 18]+, 929.9 [M + H − 92]+, 911.8 [M + H − 92 − 18]+, 821.7 [M + H − 12:0]+, 803.8 [M + H − 12:0 − 18]+, 765.6 [M + H − 16:0]+, 747.6 [M + H − 16:0 − 18]+, 565.4 [M + H − 12:0 − 16:0]+, 547.4 [M + H − 12:0 − 16:0 − 18]+ 761.7 [M + H − 18]+, 669.6 [M + H − 92 − 18]+, 551.4 [M + H − 14:0]+, 533.4 [M + H − 14:0 − 18]+ 1003.9 [M + H − 18]+, 929.8 [M + H − 92]+, 911.8 [M + H − 92 − 18]+, 793.7 [M + H − 14:0]+, 775.6 [M + H − 14:0 − 18]+, 565.5 [M + H − 14:0 − 14:0]+, 547.4 [M + H − 14:0 − 14:0 − 18]+ 1029.9 [M + H − 18]+, 955.9 [M + H − 92]+, 937.8 [M + H − 92 − 18]+, 847.7 [M + H − 12:0]+, 829.8 [M + H − 12:0 − 18]+, 765.6 [M + H − 18:1]+, 747.6 [M + H − 18:1 − 18]+, 565.4 [M + H − 12:0 − 18:1]+, 547.4 [M + H − 12:0 − 18:1 − 18]+ 975.8 [M + H − 18]+, 901.8 [M + H − 92]+, 883.8 [M + H − 92 − 18]+, 793.6 [M + H − 12:0]+, 775.6 [M + H − 12:0 − 18]+, 765.5 [M + H − 14:0]+, 747.8 [M + H − 14:0 − 18]+, 565.5 [M + H − 12:0 − 14:0]+, 547.4 [M + H − 12:0 − 14:0 − 18]+

642.6 [M − 92]+, 535.4 [M + H − 12:0]+

1001.9 [M + H − 18]+, 927.8 [M + H − 92]+, 909.8 [M + H − 92 − 18]+, 819.7 [M + H − 12:0]+, 801.7 [M + H − 12:0 − 18]+, 765.6 [M + H − 16:1]+, 747.6 [M + H − 16:1 − 18]+, 565.4.4 [M + H − 12:0 − 16:1]+, 547 [M + H − 12:0 − 16:1 − 18]+ 947.8 [M + H − 18]+, 873.6 [M + H − 92]+, 855.7 [M + H − 92 − 18]+, 765.6 [M + H − 12:0]+, 775.6 [M + H − 12:0 − 18]+, 565.4 [M + H − 12:0 − 12:0]+, 547.4 [M + H − 12:0 − 12:0 − 18]+ 614.5 [M − 92]+, 535.4 [M + H − 10:0]+

805.7 [M + H − 18]+, 787.5 [M + H − 18 − 18]+, 731.5 [M + H − 92]+, 713.4 [M + H − 92 − 18]+, 567.4 [M + H − 16:0]+, 549.4 [M + H − 16:0 − 18]+, 531.4 [M + H − 16:0 − 18 − 18]+ 975.8 [M + H − 18]+, 901.9 [M + H − 92]+, 883.8 [M + H − 92 − 18]+, 793.6 [M + H − 12:0]+, 775.6 [M + H − 12:0 − 18]+, 765.6 [M + H − 14:0]+, 747.5 [M + H − 14:0 − 18]+, 565.4 [M + H − 12:0 − 14:0]+, 547.4 [M + H − 12:0 − 14:0 − 18]+ 947.8 [M + H − 18]+, 873.6 [M + H − 92]+, 855.7 [M + H − 92 − 18]+, 765.6 [M + H − 12:0]+, 775.6 [M + H − 12:0 − 18]+, 565.4 [M + H − 12:0 − 12:0]+, 547.4 [M + H − 12:0 − 12:0 − 18]+ 642.6 [M + H − 92]+, 535.4 [M + H − 12:0]+

947.8 [M + H − 18]+, 873.8 [M + H − 92]+, 855.7 [M + H − 92 − 18]+, 765.6 [M + H − 12:0]+, 747.6 [M + H − 12:0 − 18]+, 565.4 [M + H − 12:0 − 12:0]+, 547.4 [M + H − 12:0 − 12:0 − 18]+ 975.9 [M + H − 18]+, 901.9 [M + H − 92]+, 883.7 [M + H − 92 − 18]+, 793.6 [M + H − 12:0]+, 775.6 [M + H − 12:0 − 18]+, 765.6 [M + H − 14:0]+, 747.6 [M + H − 14:0 − 18]+, 565.4 [M + H − 12:0 − 14:0]+, 547.4 [M + H − 12:0 − 14:0 − 18]+ 947.8 [M + H − 18]+, 873.8 [M + H − 92]+, 855.7 [M + H − 92 − 18]+, 793.6 [M + H − 10:0]+, 775.6 [M + H − 10:0 − 18]+, 737.6 [M + H − 14:0]+, 719.7 [M + H − 14:0 − 18]+, 565.4 [M + H − 10:0 − 14:0]+, 547.4 [M + H − 10:0 − 14:0 − 18]+ 805.7 [M + H − 18]+, 787.8 [M + H − 18 − 18]+, 731.7 [M + H − 92]+, 567.4 [M + H − 16:0]+, 549.4 [M + H − 16:0 − 18]+, 531 [M + H − 16:0 − 18 − 18]+ 761.7 [M + H − 18]+, 669.6 [M + H − 92 − 18]+, 551.4 [M + H − 14:0]+, 533.4 [M + H − 14:0 − 18]+

Journal of Agricultural and Food Chemistry Article

DOI: 10.1021/acs.jafc.6b03226 J. Agric. Food Chem. 2016, 64, 8207−8224

(13Z)- or (15Z)-zeinoxanthin myristate (13Z)- or (15Z)-β-cryptoxanthin myristate (9Z)-β-cryptoxanthin laurate

59a; V, P, T

59b; T

8212

(all-E)-violaxanthin myristate-oleate

(9Z)- or (9′Z)-antheraxanthin dilaurate (Z)-zeinoxanthin palmitoleate or (Z)-β-cryptoxanthin palmitoleate (13Z)- or (15Z)-β-cryptoxanthin oleate (all-E)-violaxanthin myristate-palmitate

61a; V, P, T

61b; V, P, T

violaxanthin palmitoleate-palmitate or luteoxanthin palmitoleate-palmitate β-cryptoxanthin oleate or zeinoxanthin oleate (all-E)-violaxanthin dioleate

62d; V, P

63a; V, P

62e; P

62c; V, P, T

(all-E)-antheraxanthin laurate-myristate (9Z)- or (9′Z)-luteoxanthin lauratepalmitate

62b; V, P, T

62a; V, P, M

61d; T

61c; P, T

(13Z)- or (15Z)-violaxanthin myristate-palmitate

60c; V, P

60b; T

60a; V, P

(9Z)-violaxanthin laurate-palmitate

58a; V, P, T 58b; V, P, T

(13Z)- or (15Z)-β-cryptoxanthin myristoleate or (all-E)-zeinoxanthin myristoleate zeaxanthin palmitate violaxanthin myristate-palmitoleate or luteoxanthin myristate-palmitoleate (all-E)-antheraxanthin dilaurate

carotenoid

(all-E)-violaxanthin laurate-palmitate or (all-E)-luteoxanthin laurate-palmitate (all-E)-β-cryptoxanthin laurate (9Z)-violaxanthin dimyristate

57c; V, P

57b; V, P

56e; T 57a; V, P, T

56d; T

peaka

Table 1. continued

51.8- 52.2

51.0

50.6−51.0

50.6−51.0

50.6−51.0

50.6−51.0

50.2

50.2

50.0−50.6

50.0−50.6

49.3−49.8

49.7

49.3−49.8

49.1

48.8−49.3

48.1−48.6 48.1−48.6

47.3−48.1

47.3−48.1

47.2 47.3−48.1

47.2

tRb (min)

nce

ndd

70

nce

ndd

417, 439, 465

nce

ndd

ncf

nce

0

ncf

ncf

ncf

0

12

15

13

0

10

23

33

465

446,

443,

441,

0

ncf

nce

50

17

20

30

11

0 11

nc

f

0

0

30

63

25 74

nc

419, 444, 469

330, 419, 466 330, 420, 467 330, 418, 470 419, 438,

419, 438, 465

330, 420, 446, 475 ndd

331, 420, 440, 465 329, 443

420, 451, 478 328, 414, 436, 465 328, 415, 437, 465

nd

d

e

ncf

nce

418, 443, 466

ncf ncf

nce nce

ndd ndd

ncf

nce

nd,d 419, 444, 468

% AB/AII

% III/II

λmaxc (nm)

1129.9

817.7

1075.9

1021.9

977.8

1049.9

817.8

789.7

949.8

1075.9

1049.9

735.6

763.6

763.6

1021.9

735.6 1021.9

1021.9

949.8

807.7 1047.9

761.6

[M + H]+ (m/z) fragment ions (m/z)

1111.9 [M + H − 18]+, 1037.9 [M + H − 92]+, 1019.0 [M + H − 18]+, 847.8 [M + H − 18:1]+, 829.8 [M + H − 18:1 − 18]+, 565.5 [M + H − 18:1 − 18:1]+, 547.4 [M + H − 18:1 − 18:1 − 18]+

1031.9 [M + H − 18]+, 957.9 [M + H − 92]+, 939.9 [M + H − 92 − 18], 821.7 [M + H − 14:0]+, 803.8 [M + H − 14:0 − 18]+, 793.6 [M + H − 16:0]+, 775.6 [M + H − 16:0 − 18]+, 565.5 [M + H − 14:0 − 16:0]+, 547.4 [M + H − 14:0 − 16:0 − 18]+ 959.8 [M + H − 18]+, 777.7 [M + H − 12:0]+, 759.6 [M + H − 12:0 − 18]+, 749.6 [M + H − 14:0]+, 731.6 [M + H − 14:0 − 18]+, 549.4 [M + H − 12:0 − 14:0]+, 531.4 [M + H − 12:0 − 14:0 − 18]+ 1003.9 [M + H − 18]+, 929.9 [M + H − 92]+, 911.8 [M + H − 92 − 18]+, 821.7 [M + H − 12:0]+, 803.6 [M + H − 12:0 − 18]+, 765.6 [M + H − 16:0]+, 747.5 [M + H − 16:0 − 18]+, 565.5 [M + H − 12:0 − 16:0]+, 547.4 [M + H − 12:0 − 16:0−18]+ 1057.9 [M + H − 18]+, 983.9 [M + H − 92]+, 965.9 [M + H − 92 − 18]+, 821.7 [M + H − C16:1]+, 819.9 [M + H − 16:0]+, 803.6 [M + H − C16:1 − 18]+, 801.8 [M + H − 16:0 − 18]+, 565.5 [M + H − 16:1 − 16:0]+, 547.4 [M + H − 16:1 − 16:0 − 18]+ 725.7 [M + H − 92]+, 535.4 [M + H − 18:1]+

724.7[M − 92]+, 535.4 [M + H − 18:1]+

1031.9 [M + H − 18]+, 957.8 [M + H − 92]+, 939.8 [M + H − 92 − 18]+, 821.8 [M + H − 14:0]+, 803.6 [M + H − 14:0 − 18]+, 793.7 [M + H − 16:0]+, 775.6 [M + H − 16:0 − 18]+, 565.4 [M + H − 14:0 − 16:0]+, 547.5 [M + H − 14:0 − 16:0 − 18]+ 1057.9 [M + H − 18]+, 983.9 [M + H − 92]+, 965.9 [M + H − 92 − 18]+, 847.7 [M + H − 14:0]+, 829.6 [M + H − 14:0 − 18]+, 793.7 [M + H − 18:1]+, 775.6 [M + H − 18:1 − 18]+, 565.5 [M + H − 14:0 − 18:1]+, 547.5 [M + H − 14:0 − 18:1 − 18]+ 931.9 [M + H − 18]+, 749.6 [M + H − 12:0]+, 731.6 [M + H − 12:0 − 18]+, 549.4 [M + H − 12:0 − 12:0]+, 531.4 [M + H − 12:0 − 12:0 − 18]+ 696.7 [M − 92]+, 535.4 [M + H − 16:1]+

643.5 [M + H − 92]+, 535.4 [M + H − 12:0]+

671.6 [M + H − 92]+, 535.4 [M + H − 14:0]+

789.8 [M + H − 18]+, 697.9 [M + H − 92 − 18]+, 551.4 [M + H − 16:0]+ 1029.9 [M + H − 18]+, 955.8 [M + H − 92]+, 937.8 [M + H − 92 − 18]+, 819.6 [M + H − 14:0]+, 801.6 [M + H − 14:0 − 18]+, 793.6 [M + H − 16:1]+, 775.5 [M + H − 16:1 − 18]+, 565.4 [M + H − 14:0 − 16:1]+, 547.4 [M + H − 14:0 − 16:1 − 18]+ 931.8 [M + H − 18]+, 857.7 [M + H − 92]+, 749.6 [M + H − 12:0]+, 731.6 [M + H − 12:0 − 18]+, 549.4 [M + H − 12:0 − 12:0]+, 531.4 [M + H − 12:0 − 12:0 − 18]+ 1003.9 [M + H − 18]+, 929.9 [M + H − 92]+, 911.8 [M + H − 92 − 18]+, 821.7 [M + H − 12:0]+, 803.8 [M + H − 12:0 − 18]+, 765.6 [M + H − 16:0]+, 747.6 [M + H − 16:0 − 18]+, 565.4 [M + H − 12:0 − 16:0]+, 547.4 [M + H − 12:0 − 16:0 − 18]+ 643.5 [M + H − 92]+, 535.4 [M + H − 12:0]+ 1003.9 [M + H − 18]+, 929.9 [M + H − 92]+, 911.8 [M + H − 92 − 18]+, 793.6 [M + H − 14:0]+, 775.8 [M + H − 14:0 − 18]+, 565.4 [M + H − 14:0 − 14:0]+, 547.4 [M + H − 14:0 − 14:0 − 18]+ 1003.9 [M + H − 18]+, 929.9 [M + H − 92]+, 911.8 [M + H − 92 − 18]+, 821.6 [M + H − 12:0]+, 803.7 [M + H − 12:0 − 18]+, 765.7 [M + H − 16:0]+, 747.5 [M + H − 16:0 − 18]+, 565.5 [M + H − 12:0 − 16:0]+, 547.4 [M + H − 12:0 − 16:0 − 18]+ 671.7 [M + H − 92]+, 535.4 [M + H − 14:0]+

668.6 [M − 92]+, 535.5 [M + H − 14:1]+

Journal of Agricultural and Food Chemistry Article

DOI: 10.1021/acs.jafc.6b03226 J. Agric. Food Chem. 2016, 64, 8207−8224

(9Z)-violaxanthin myristate-palmitate or luteoxanthin myristatepalmitate 1 (all-E)-β-cryptoxanthin myristate (all-E)-lutein 3-O-caprate-3′-O-laurate (9Z)-β-cryptoxanthin oleate

(all-E)-violaxanthin palmitate-oleate

(9Z)- or (9′Z)-antheraxanthin laurate-myristate (9Z)-zeinoxanthin myristate

(all-E)-lutein dilaurate (13Z)- or (15Z)-β-cryptoxanthin palmitate or zeinoxanthin palmitate (9Z)-violaxanthin myristate-palmitate or luteoxanthin myristatepalmitate 2 (all-E)-antheraxanthin dimyristate

(all-E)-violaxanthin dipalmitate

(9Z)- or (9′Z)-antheraxanthin laurate-oleate zeaxanthin dilaurate (9Z)-violaxanthin dipalmitate or luteoxanhin dipalmitate (all-E)-lutein 3-O-myristate-3′-Olaurate (all-E)-lutein 3-O-laurate-3′-O-myristate (9Z)- or (9′Z)-antheraxanthin dimyristate or mutatoxanthin dimyristate 1 (all-E)-β-cryptoxanthin palmitate (all-E)-antheraxanthin myristateoleate (9Z)- or (9′Z)-antheraxanthin dimyristate or mutatoxanthin dimyristate 2 (all-E)-lutein 3-O-palmitate-3′-Olaurate (9Z)-β-cryptoxanthin palmitate or (all-E)-zeinoxanthin palmitate (all-E)-zeaxanthin laurate-myristate

63b; V, P, T

64b; V, P

64c; V, P, T

65a; V, P 65b; P, T

65d; V, P, T

65e; V, P, M

66a; V, P

8213

70a; V, P

69; T

68c; T

68b; V, P, T

67b; V, P, T 68a; V, P

67a; V, P

66e; T

66d; T

66b; V, P, T 66c; V, P

65c; V, P

64d; T

64a; V, P, T

63c; V, P, T 63d; P, T

carotenoid

peaka

Table 1. continued

58.1−58.5

57.9

57.2

57.1−57.6

56.1−56.5 57.1−57.6

56.1−56.5

55.2

55.2

55.0−55.4 55.0−55.4

55.0−55.4

53.8−54.2

53.8−54.2

53.8−54.2

53.8−54.2 53.8−54.2

53.6

53.2−53.7

53.2−53.7

53.2−53.7

51.8−52.2 52.1

51.8−52.2

tRb (min)

ncf ncf

nce nce

ncf ncf

nce nce nce

412, 444, 466 ndd

0 0 ncf ncf ncf ncf

10 29 nce nce nce nce

417, 444, 467 nd,d 416, 444, 470 420, 448, 474

421, 450, 474 330, 420, 443, 470 ndd

ncf

ncf ncf

nce nce

412, 444, 466

9

43

330, 412, 440, 465 ndd ndd

417, 440, 470

421, 445, 472

ncf

nce

ndd

nc ncf

f

nc nce

16

e

15

0

15

0 ncf

ncf

% AB/AII

31

36

40

0

9 nce

nce

% III/II

329, 417, 440, 465 331, 419, 443, 468 428, 444, 470 ndd

330, 420, 447, 472 412, 438, 465

420, 451, 476 420, 444, 470

ndd

λmaxc (nm)

961.8

791.7

ndg

1005.9

791.7 1059.9

1005.9

ndg

ndg

933.8 1077.9

1031.9

1077.9

1005.9

1049.9

ndg 791.8

763.7

977.8

1103.9

817.7

763.7 ndg

1049.9

[M + H]+ (m/z) fragment ions (m/z)

869.8 [M + H − 92]+, 761.7 [M + H − 12:0]+, 733.6 [M + H − 14:0]+, 669.5 [M + H − 12:0 − 92]+, 641.6 [M + H − 14:0 − 92]+, 533.4 [M + H − 12:0 − 14:0]+

789.6 [M + H − 12:0]+ → 533.4 [M + H − 12:0 − 16:0]+; 733.5 [M + H − 16:0]+ → 533.4 [M + H − 16:0 − 12:0]+ 699.6 [M + H − 92]+, 535.4 [M + H − 16:0]+

699.6 [M + H − 92]+, 535.4 [M + H − 16:0]+ 1041.9 [M + H − 18]+, 967.9 [M + H − 92]+, 831.7 [M + H − 14:0]+, 813.6 [M + H − 14:0 − 18]+, 777.7 [M + H − 18:1]+, 759.6 [M + H − 18:1 − 18]+, 549.4 [M + H − 14:0 − 18:1]+, 531.4 [M + H − 14:0 − 18:1 − 18]+ 987.9 [M + H − 18]+, 913.8 [M + H − 92]+, 777.7 [M + H − 14:0]+, 759.6 [M + H − 14:0 − 18]+, 549.4 [M + H − 14:0 − 14:0]+, 531.4 [M + H − 14:0 − 14:0 − 18]+

1031.9 [M + H − 18]+, 957.8 [M + H − 92]+, 939.8 [M + H − 92 − 18]+, 821.7 [M + H − 14:0]+, 803.8 [M + H − 14:0 − 18]+, 793.6 [M + H − 16:0]+, 775.6 [M + H − 16:0 − 18]+, 565.5 [M + H − 14:0 − 16:0]+, 547.4 [M + H − 14:0 − 16:0 − 18]+ 987.8 [M + H − 18]+, 777.6 [M + H − 14:0]+, 759.5 [M + H − 14:0 − 18]+, 549.4 [M + H − 14:0 − 14:0]+, 531.4 [M + H − 14:0 − 14:0 − 18]+ 1059.9 [M + H − 18]+, 985.8 [M + H − 92]+, 967.9 [M + H − 92 − 18]+, 821.7 [M + H − 16:0]+, 803.6 [M + H − 16:0 − 18]+, 565.4 [M + H − 16:0 − 16:0]+, 547.3 [M + H − 16:0 − 16:0 − 18]+ 1013.9 [M + H − 18]+, 939.9 [M + H − 92]+, 831.8 [M + H − 12:0]+, 813.7 [M + H − 12:0 − 18]+ 749.7 [M + H − 18:1]+, 731.7 [M + H − 18:1 − 18]+, 549.4 [M + H − 12:0 − 18:1]+, 531.4 [M + H − 12:0 − 18:1 − 18]+ 733.6 [M + H − 12:0]+, 641.5 [M + H − 12:0 − 92]+, 533.4 [M + H − 12:0 − 12:0]+ 1059.9 [M + H − 18]+, 985.9 [M + H − 92]+, 967.9 [M − 92 − 18]+, 821.7 [M + H − 16:0]+, 803.6 [M + H − 16:0 − 18]+, 565.4 [M + H − 16:0 − 16:0]+, 547.4 [M + H − 16:0 − 16:0 − 18]+ 761 [M + H − 12:0]+ → 669.7 [M + H − 92]+, 533 [M + H − 12:0 − 14:0]+; 733 [M + H − 14:0]+ → 641.5 [M + H − 92]+, 533 [M + H − 14:0 − 12:0]+ 761 [M + H − 12:0]+ → 669.7 [M + H − 92]+, 533 [M + H − 12:0 − 14:0]+; 733 [M + H − 14:0]+ → 641.5 [M + H − 92]+, 533 [M + H − 14:0 − 12:0]+ 987.9 [M + H − 18]+, 913.8 [M + H − 92]+, 777.7 [M + H − 14:0]+, 759.6 [M + H − 14:0 − 18]+, 549.4 [M + H − 14:0 − 14:0]+, 531.4 [M + H − 14:0 − 14:0 − 18]+

733 [M + H − 12:0]+ → 641.5 [M + H − 92]+, 533 [M + H − 12:0 − 12:0]+ 698.7 [M − 92]+, 535.4 [M + H − 16:0]+

1085.9 [M + H − 18]+, 1011.9 [M + H − 92]+, 993.9 [M + H − 92 − 18]+, 847.6 [M + H − 16:0], 829.6 [M + H − 16:0 − 18], 821.7 [M + H − 18:1]+, 803.6 [M + H − 18:1 − 18]+, 565.4 [M + H − 16:0 − 18:1]+, 547.4 [M + H − 16:0 − 18:1 − 18]+ 959.8 [M + H − 18]+, 777.6 [M + H − 12:0]+, 759.6 [M + H − 12:0 − 18]+, 749.6 [M + H − 14:0]+, 731.6 [M + H − 14:0 − 18]+, 549.4 [M + H − 12:0 − 14:0]+, 531.5 [M + H − 12:0 − 14:0 − 18]+ 671.5 [M + H − 92]+, 535.4 [M + H − 14:0]+

1031.9 [M + H − 18]+, 957.9 [M + H − 92]+, 939.9 [M + H − 92 − 18]+, 821.8 [M + H − 14:0]+, 803.6 [M + H − 14:0 − 18]+, 793.6 [M + H − 16:0]+, 775.6 [M + H − 16:0 − 18]+, 565.4 [M + H − 14:0 − 16:0]+, 547.4 [M + H − 14:0 − 16:0 − 18]+ 671.5[M + H − 92]+, 535.4 [M + H − 14:0]+ 733.6 [M + H − 10:0]+ → 533.4 [M + H − 10:0 − 12:0]+; 705.6 [M + H − 12:0]+ → 533.4 [M + H − 12:0 − 10:0]+ 725.7 [M + H − 92]+, 535.4 [M + H − 18:1]+

Journal of Agricultural and Food Chemistry Article

DOI: 10.1021/acs.jafc.6b03226 J. Agric. Food Chem. 2016, 64, 8207−8224

lutein dipalmitate (all-E)-zeaxanthin dipalmitate

79b; V, P 80; V, P

8214

65.8−66.1 67.7−68.4

65.8−66.1

63.6−64.0 65.2−65.5

63.6−64.0

62.4−62.8

62.4−62.8

61.7−62.1 61.7−62.1

60.6−61.2

60.4−60.8

59.0−59.7 59.8−60.2

58.1−58.5

tRb (min)

33 14 nce nce 22

ndd ndd 421, 451, 478

nce

ndd 420, 443, 470 425, 450, 474

ncf

nce

416, 446, 469

ncf 0

ncf

0 0

ncf

0

ncf ncf

33

422, 449, 475

nce nce

ndd ndd

nc

f

nc

12

e

0 0

0

% AB/AII

31

38 35

31

% III/II

330, 416, 441, 469 418, 443, 468

420, 444, 471 420, 444, 471

420, 446, 471

λmaxc (nm)

ndg 1045.9

1071.9

ndg 1017.9

1061.9

ndg

989.9

989.9 1061.9

1087.9

1033.9

ndg ndg

1033.9

[M + H]+ (m/z) fragment ions (m/z)

1015.9 [M + H − 18]+, 941.8 [M + H − 92]+, 805.6 [M + H − 14:0]+, 787.6 [M + H − 14:0 − 18]+, 777.7 [M + H − 16:0]+, 759.6 [M + H − 16:0 − 18]+, 549.4 [M + H − 14:0 − 16:0]+, 531.4 [M + H − 14:0 − 16:0 − 18]+ 761.7 [M + H − 14:0]+ → 669.6 [M + H − 92]+, 533.5 [M + H − 14:0 − 14:0]+ 815.7 [M + H − 16:0]+ → 533.4 [M + H − 16:0 − 18:1]+; 789.7 [M + H − 18:1]+ → 533.4 [M + H − 18:1 − 16:0]+ 1015.9 [M + H − 18]+, 941.8 [M + H − 92]+, 805.7 [M + H − 14:0]+, 787.6 [M + H − 14:0 − 18]+, 777.6 [M + H − 16:0]+, 759.5 [M + H − 16:0 − 18]+, 549.4 [M + H − 14:0 − 16:0]+, 531.4 [M + H − 14:0 − 16:0 − 18]+ 1069.9 [M + H − 18]+, 995.9 [M + H − 92]+, 831.7 [M + H − 16:0]+, 813.6 [M + H − 16:0 − 18]+, 805.7 [M + H − 18:1]+, 787.6 [M + H − 18:1 − 18]+, 549.4 [M + H − 16:0 − 18:1]+, 531.4 [M + H − 16:0 − 18:1 − 18]+ 897.7 [M + H − 18]+, 761.6 [M + H − 14:0]+, 669.5 [M + H − 14:0 − 18]+, 533.4 [M + H − 14:0 − 14:0]+ 1043.9 [M + H − 18]+, 969.9 [M + H − 92]+, 805.7 [M + H − 16:0]+, 787.6 [M + H − 16:0 − 18]+, 549.4 [M + H − 16:0 − 16:0]+, 531.4 [M + H − 16:0 − 16:0 − 18]+ 897.9 [M + H − 18]+, 789.7 [M + H − 12:0]+, 733.6 [M + H − 16:0]+, 697.6 [M + H − 12:0 − 92]+, 641.6 [M + H − 16:0 − 92]+, 533.4 [M + H − 12:0 − 16:0]+ 789.8 [M + H − 14:0]+ → 533.5 [M + H − 14:0 − 16:0]+; 761.8 [M + H − 16:0]+ → 533.5 [M + H − 16:0 − 14:0]+ 1044.0 [M + H − 18]+, 969.8 [M + H − 92]+, 805.7 [M + H − 16:0]+, 787.6 [M + H − 16:0 − 18]+, 549.4 [M + H − 16:0 − 16:0]+, 531.4 [M + H − 16:0 − 16:0 − 18]+ 815.7 [M + H − 18:1]+ → 533.4 [M + H − 18:1 − 18:1]+ 925.8 [M + H-92]+, 789.7 [M + H − 14:0]+, 761.6 [M + H − 16:0]+, 697.6 [M + H − 14:0 − 92]+, 669.5 [M + H − 16:0−92]+, 533.4 [M + H − 14:0 − 16:0]+ 979.0 [M + H − 92]+, 815.9 [M + H − 16:0]+, 789.8 [M + H − 18:1]+, 723.6 [M + H − 16:0 − 92]+, 697.8 [M + H − 18:1 − 92]+, 533.5 [M + H − 16:0 − 18:1]+ 789.7 [M + H − 16:0]+ → 697.6 [M + H − 16:0 − 92]+, 533.5 [M + H − 16:0 − 16:0]+ 953.8 [M + H − 92]+, 789.7 [M + H − 16:0]+, 697.6 [M + H − 16:0 − 92]+, 533.4 [M + H − 16:0 − 16:0]+

a

Numbered according to the chromatograms shown in Figures 3−6. V, orange cv. ‘Valencia’; P, orange cv. ‘Pera’; T, tangor cv. ‘Murcott’; M, mango cv. ‘Tommy Atkins’. bRetention time on C30 column (see Material and Methods for chromatographic conditions). cLinear gradient containing MeOH, MTBE, and H2O mixtures. dUV/vis spectrum was not clearly detected because of coelution. e% III/II was not calculated because of poor definition of the UV/vis spectrum or because it was not detected. f% Ab/AII was not calculated because of poor definition of the UV/vis spectrum or because it was not detected. g[M + H]+ or MS/MS fragments were not detected. Underlined fragments are the most abundant in the MS/MS spectrum. Bold face indicates MS in-source fragment ions. Identification followed by Arabic numerals (1, 2, ...) refer to respective isomers, when the cis−trans configuration or position of the cis double bond was not assigned.

79a; V, P

77b; P 78; V, P

77a; V, P

(all-E)-lutein 3-O-palmitate-3′-Omyristate antheraxanthin dipalmitate or mutatoxanthin dipalmitate (all-E)-lutein dioleate (all-E)-zeaxanthin myristate-palmitate zeaxanthin palmitate-oleate

(all-E)- antheraxanthin myristatepalmitate (all-E)-lutein dimyristate (all-E)-lutein 3-O-palmitate-3′-Ooleate (9Z)- or (9′Z)-antheraxanthin myristate-palmitate (all-E)-antheraxanthin palmitateoleate zeaxanthin dimyristate antheraxanthin dipalmitate or mutatoxanthin dipalmitate (all-E)-zeaxanthin laurate-palmitate

carotenoid

76b; V, P

76a; V, P

75a; V, P 75b; V, P

74; V, P

73; V, P, T

71; V, P 72; V, P

70b; V, P

peaka

Table 1. continued

Journal of Agricultural and Food Chemistry Article

DOI: 10.1021/acs.jafc.6b03226 J. Agric. Food Chem. 2016, 64, 8207−8224

Article

Journal of Agricultural and Food Chemistry mandarin pulp or juice are also scarce.17,18 Mainly esters of βcryptoxanthin were reported in those studies.17,18 With regard to the nonsaponified carotenoid extract, mangoes are characterized by two major esters, namely, (allE)-violaxanthin and (9Z)-violaxanthin dibutyrates,19 with further details on the composition of mango esters given by Ornelas-Paz and co-workers.20 Although interest in the natural composition of carotenoids of fruits is growing, information about the carotenoid ester composition of citrus and mango is still lacking. Therefore, the aim of this study was to identify, by HPLC-DAD-(APCI)-MS/MS, carotenoids and carotenoid esters present in oranges, mandarin, and mango pulps, including minor ones. Furthermore, these fruits were chosen because some carotenoids, such as violaxanthin, are present in all three matrices and therefore could help in the identification of its esters.

■

Corp., Miliford, MA, USA). Several combinations of different ratios and linear gradients of MeOH, MTBE, and ultrapure water (H2O) were tested as mobile phase for the separation of carotenoids and their esters (results not shown). The best separation was obtained using the following conditions: mobile phases A (MeOH/MTBE/H2O, 81:15:4) and phase B (MeOH/MTBE/H2O, 16:80.4:3.6), in a linear gradient changing from 99 to 66% A in 30 min, maintaining this condition for 5 min, changing from 66 to 44% A in 15 min, keeping this condition for 5 min, changing from 44 to 22% A in 15 min and from 22 to 0% A in 5 min, returning to the initial conditions (99% A) in 5 min, and keeping this condition for 5 min. The flow rate was set at 0.9 mL/min, the column temperature was maintained at 35 °C, the UV/vis spectra were recorded between 250 and 600 nm, and the chromatograms were processed at 286, 348, and 450 nm. The MS parameters were the same as previously described by Chiste and Mercadante,22 except for the dry gas flow set at 5 L/min and nebulizer set at 60 psi. The mass spectra were acquired with scan range from m/ z 100 to 1200. Carotenoids were identified according to the following combined information: elution order on C30 column, cochromatography with authentic standards, UV/vis spectrum features ((λmax), spectral fine structure (% III/II) and cis-peak intensity (% AB/AII)), mass spectrum, and comparison with data available in the literature.12,19,21−31

MATERIALS AND METHODS

Materials. Reagents and solvents of analytical grade (Synth, São Paulo, Brazil) were used for carotenoid extraction. HPLC mobile phases were constituted by methyl tert-butyl ether (MTBE) (Tedia, Fairfield, OH, USA), methanol (MeOH) (J. T. Baker, Phillipsburg, NJ, USA), both of chromatographic grade, and ultrapure water (Millipore, Billerica, MA, USA). Samples and solvents were filtered before HPLC analysis, using, respectively, membranes of 0.22 and 0.45 μm (Millipore). The following carotenoid standards were used: (all-E)lutein, (all-E)-zeaxanthin, (all-E)-β-cryptoxanthin, (all-E)-α-carotene, (all-E)-β-carotene (DSM Nutritional Products, Basel, Switzerland), (all-E)-violaxanthin, (all-E)-antheraxanthin, and (9′Z)-neoxanthin (CaroteNature, Lupsingen, Switzerland). Samples. Approximately 50 fruits of each citrus cultivar, orange (C. sinensis L. Osbeck) cv. ‘Valencia’ and cv. ‘Pera’ and tangor (C. reticulata L. Blanco × C. sinensis L. Osbeck) cv. ‘Murcott’, were harvested at full-ripening stage, whereas mango (5 kg) (Mangifera indica L. cv. ‘Tommy Atkins’) was purchased in a local market in Campinas, Brazil. All of the citrus samples were provided by the Centro APTA Citros Sylvio Moreira from the Agronomic Institute of Campinas (IAC), Brazil. All of the fruits were washed and manually peeled, the seeds were removed, and the pulp was homogenized, maintaining the juice vesicles as intact as possible in the citrus samples, and immediately frozen in liquid nitrogen. The frozen pulp was lyophilized at −60 °C below 40 μHg (Liobras, São Paulo, Brazil) until constant weight. The freeze-dried pulp was ground into powder, homogenized, vacuum packaged in polyethylene bags, and stored in the dark at −80 °C. Carotenoid Extraction. Lyophilized pulp (1.00 g) was extracted with ethyl acetate, and an equal amount of Na2CO3 was added prior to extraction. The mixture was submitted to magnetic stirring for 5 min, followed by filtration under vacuum, and further extraction was carried out with methanol (MeOH). Diethyl ether/petroleum ether (1:1) and a NaCl (10%, p/v) aqueous solution were added to the combined carotenoid extracts, and phase separation was achieved by centrifugation (16900g for 10 min at 20 °C). Anhydrous Na2SO4 was added to remove water traces from the extract, which was concentrated to dryness in a rotatory evaporator (t < 35 °C), and the dry extract was stored under nitrogen, in the dark at −36 °C. For orange cv. ‘Valencia’ extract, to remove triacylglycerols (TAGs) that interfere with the MS signals, an additional two-step cleanup procedure was carried out.21 HPLC-DAD-MS/MS Analysis. Carotenoid extracts were analyzed in a HPLC (Shimadzu, Kyoto, Japan), equipped with an LC-20AD quaternary pump, a DGU-20A5 online degasser, and a manual injection valve (Rheodyne, Rohnert Park, CA, USA) with a 20 μL loop. The equipment was connected in series with a SPD-M20A diode array detector (DAD) (Shimadzu) and an AmaZon speed ETD mass spectrometer, with an ion-trap analyzer and APCI ionization source from Bruker Daltonics (Bremen, Germany). Separation was carried out on a 250 mm × 4.6 mm i.d., 5 μm, YMC C30 column (Waters

■

RESULTS AND DISCUSSION

MS Fragmentation Pattern of Carotenoid Esters. Some features of the in-source fragmentation pattern of violaxanthin esters were previously described, for example, detection of protonated molecule ([M + H]+) and losses of water, FAs, or both together.31 We confirmed by APCI(+)-MS/MS the detection of [M + H]+ of all violaxanthin esters. MS/MS spectra showed that the most intense fragment of both monoand diesters of violaxanthin was the neutral loss of one water molecule from the protonated molecule ([M + H − 18]+), for example, peaks 20 and 62a (Table 1). In monoesters, an additional fragment corresponding to the loss of two water molecules from the protonated molecules ([M + H − 18 − 18]+) was also detected at very low intensity (e.g., peak 20). Loss of a C7H8 fragment (e.g., toluene), originated from the polyene chain, alone [M + H − 92]+ or along with one water molecule [M + H − 92 − 18]+ was always detected in both mono- and diesters, whereas [M + H − 92]+ was detected at low intensity. For monoesters, fragments at m/z 583 and 565 were detected with various intensities, corresponding, respectively, to elimination of the FA moiety ([M + H − FA]+) alone and also together with water ([M + H − FA − 18]+), for example, peaks 20 and 34 (Table 1). In violaxanthin diesters, when the same FA is esterified on both sides (homodiesters), Figure 1, fragments representing the loss of each FA moiety ([M + H − FA]+) alone and together with one water molecule ([M + H − FA − 18]+) were detected. In the case of heterodiesters (e.g., peak 62a, Figure 1), when different FAs are esterified in each side of the carotenoid, two fragments ([M + H − FA1]+ and [M + H − FA2]+) corresponding to the loss of each FA moiety were eliminated from [M + H]+, as well as their respective elimination together with water ([M + H − FA1 − 18]+ and [M + H − FA2 − 18]+). In addition, after the loss of two FA moieties, a fragment representing the carotenoid backbone at m/z 565 ([M + H − 2FA]+ or [M + H − FA1 − FA2]+) was always detected, along with a fragment at m/z 547 due to the FA plus water elimination ([M + H − 2FA − 18]+ or [M + H − FA1 − FA2 − 18]+). The MS spectra illustrating these losses are shown in Figure 2. As expected, all (Z)-isomer esters of violaxanthin presented MS fragmentation patterns identical to that of the (all-E)-, and 8215

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group on the other side. MS and MS/MS spectra of luteoxanthin esters were virtually the same of those of violaxanthin. Neoxanthin is a structural isomer of violaxanthin as well and contains an allenic group on one side of the molecule and one epoxy group on the other side, instead of the two epoxy groups of violaxanthin. Neoxanthin esters showed fragmentation patterns close to those of violaxanthin esters, including detectable [M + H]+, and fragments representing losses of water [M + H − 18]+, C7H8 [M + H − 92]+, C7H8 plus water [M + H − 92 − 18]+, FA(s), and FA(s) plus water. The fragmentation pattern of antheraxanthin esters was characterized by detectable [M + H]+, and MS/MS fragments consisting in neutral loss of one water molecule from the protonated molecule ([M + H − 18]+), but it was not the most intense fragment, unlike that occurring for violaxanthin esters. Both mono- and diesters showed fragments indicating loss of C7H8 (toluene) [M + H − 92]+ and also [M + H − 92 − 18]+, for example, peaks 40a and 57b. Only for antheraxanthin monoesters (see peak 40a) was an additional fragment with very low intensity indicating loss of two water molecules ([M + H − 18 − 18]+) also detected. In addition, fragments at m/z 567 and 549 were also detected, representing, respectively, the losses of FA ([M + H − FA]+) and FA plus water ([M + − FA − 18]+). These later fragments dominated the MS/MS spectra of antheraxanthin monoesters, with varying intensities between them. In antheraxanthin diesters, MS/MS fragments representing loss of FAs ([M + H − FA]+ for homodiesters or [M + H − FA1]+ and [M + H − FA2]+ for heterodiesters) dominated the MS/MS spectra, with intensities varying between them. As a consequence, after the loss of two FA moieties, the fragment representing the carotenoid backbone at m/z 549 ([M + H − 2FA]+ or [M + H − FA1 − FA2]+) is always registered, as well as at m/z 531 due to the water elimination together with FAs ([M + H − 2FA − 18]+ or [M + H − FA1 − FA2 − 18]+). Mutatoxanthin is a structural isomer of antheraxanthin, with a 5,8-furanoid group instead of an epoxide group at position 5,6- observed in antheraxanthin. Thus, the fragmentation pattern of mutatoxanthin esters is essentially the same as that observed for antheraxanthins. The fragmentation pattern in APCI(+)-MS of esters of both β-cryptoxanthin and zeinoxanthin is essentially the same, characterized by detectable [M + H]+ and fragments representing loss of toluene ([M + H − 92]+) and at m/z 535 due to FA moiety loss [M + H − FA]+. The fragmentation pattern of zeaxanthin monoesters consists in detectable [M + H]+ and the most common MS/MS fragments representing losses of free hydroxyl group [M + H − 18]+, toluene [M + H − 92]+, FA [M + H − FA]+, resulting in a fragment at m/z 551, and the loss of [M + H − FA − 18]+, resulting in the backbone at m/z 533, for example, peaks 43d and 55c. For diesters, the MS/MS fragments represent losses of each FA alone and together with toluene and also loss of the two FA acids together, resulting in a fragment at m/z 533, for example, peaks 70a and 80. Considerations for the Identification of Carotenoid Esters. We tested many linear gradients of combinations of MeOH, MTBE, and H2O for separation of carotenoid esters of orange cv. ‘Valencia’, which has one of the most complex patterns among the samples studied herein, and the best separation condition was chosen (Figure 3) and applied to the other fruits (Figures 4−6). However, both UV/vis and MS spectra indicated that two or more compounds coeluted in

Figure 1. Chemical structures of monoester and diesters from (all-E)violaxanthin found in orange.

therefore differentiation was only possible considering UV/vis spectrum characteristics and elution order. Lutein presents one β- and one ε-ring, leading to an asymmetric structure. Therefore, the presence of cis double bonds and monoacylation with one FA or diacylation with two different FAs results in the formation of regioisomers. In APCI(+)-MS conditions, the in-source cation formed by cleavage at the ε-ring is allylic to the double bond, providing more stability when compared to the cation produced by cleavage at the β-ring.32 This fact produces characteristic MS fragmentation patterns that are very useful to unequivocally assign the FA moiety position of the two possible regioisomeric forms of lutein.21,32,33 The most abundant in-source fragment ion results from neutral loss of the corresponding moiety (hydroxyl group or FA) at position 3′ of lutein (e.g., peaks 66d and 66e). Additionally, [M + H]+ of all the peaks related to lutein was not detected under the MS conditions used in the present study. Luteoxanthin, neoxanthin, antheraxanthin, and mutatoxanthin are also asymmetric xanthophylls detected in the ester form in our work. However, different from lutein, no perceptible differences in MS and MS/MS spectra between regioisomeric forms of these carotenoids were observed in the conditions applied, and therefore assignment of the position of the fatty acid in these carotenoids was not possible (e.g., peaks 23, 27, and 45c). Luteoxanthin is a structural isomer of violaxanthin, with one epoxy group at one side of the molecule and one furanoid 8216

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Figure 2. MS and MS/MS spectra of (A) (all-E)-violaxanthin myristate (peak 24a) and (B) (all-E)-violaxanthin myristate-palmitate (peak 62a).

with the data processing software. Unfortunately, using this approach some spectroscopic features, such as fine structure and cis-peak intensity, were lost. In other cases, information about the UV/vis spectrum remained unclear even after application of the approach described above, and therefore only the assignment of the molecular weight (MW) of carotenoids and their esters was possible (peaks 24a, 25b, 35d, 39c, 40d, 42c, 45c, 46, 51b, 52a, 55a,b, 56b, 56e, 57a, 57c, 60c, 62c−e, 63b, 65bc, 66b-c, 67a, 68b, 75a,b, 77a, and 79a,b). Because there are many carotenoids with the same MW in

many peaks, especially in citrus samples. The combined information given by DAD and MS detectors is the minimum requirement to tentatively identify carotenoid esters. In our study, due to coelution, the UV/vis spectra of some peaks were often deformed or even not available, hampering the identification of carotenoid esters based on this information. A similar situation was previously reported for nonsaponified orange juice extracts.12 On the other hand, we observed UV/vis spectra resembling the pure carotenoids that are coeluting just by moving the cursor throughout the chromatographic peak 8217

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Figure 3. Chromatogram from orange cv. ‘Valencia’, obtained by HPLC-DAD, processed at 450 nm. (Inset) Chromatogram zoom, ranging from 45 to 53 min.

Figure 4. Chromatogram from orange cv. ‘Pera’, obtained by HPLC-DAD, processed at 450 nm. (Inset) Chromatogram zoom, ranging from 46 to 53 min.

these samples, this information alone was not sufficient to affirm peak assignments. Chromatographic and spectroscopic characteristics of carotenoids and carotenoid esters separated in all analyzed fruits

are shown in Table 1. As expected, the typical elution behavior of free carotenoids was maintained for carotenoid esters. The elution order of (Z)- and (all-E)-carotenoids on the C30 column is well-known24,26,29 and confirmed by our results. 8218

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Figure 5. Chromatograms from tangor cv. ‘Murcott’, obtained by HPLC-DAD, processed at 450 nm (A) and at 286 and 348 nm (B).

UV/vis spectrum with a cis-peak at 329 nm and maximum absorption wavelengths (λmax) with 3−4 nm bathochromic shift compared to that from (all-E)-neoxanthin, as well as elution after (all-E)-neoxanthin dibutyrate (peak 6a). Moreover, the nearness of the Z double bond to the allenic group in the (9Z)neoxanthin leads to a higher steric hindrance than in the (9′Z)stereoisomer, and therefore the latter is the most common form naturally found in fruits.27 Further confirmation was given by coelution of free forms of (all-E)-violaxanthin and (9′Z)neoxanthin standards (data not shown), and this premise was used to identify their acylated forms in the cases of peaks 9 and 11. Peaks 43a and 43b coeluted and were identified, respectively, as violaxanthin caprate-laurate and violaxanthin dilaurate. It was not possible to assign the cis−trans configuration of these carotenoids, because λmax at 437 nm is either too low for the (all-E)-isomer (typical λmax at 438−440 nm) or too high for (Z)-violaxanthin isomers, with typical λmax at 434−436 nm. Peak 54b also showed λmax at 437 nm, with loss of fine structure and the presence of a cis-peak, and considering its elution

(9Z)-Isomers eluted after the corresponding (all-E)-isomers, for example, peaks 1 and 3b (free carotenoids) as well as peaks 9 and 15 (esterified carotenoids). Moreover, (13Z)- or (15Z)isomers (e.g., peaks 12 and 16) eluted before the corresponding (all-E)-isomers (e.g., peak 20). Another feature of carotenoid elution on the C30 column is that carotenoids possessing a 5,8furanoid ring usually show longer retention times compared to their corresponding 5,6-epoxycarotenoid.26,29 This behavior was also observed here for free carotenoids (e.g., peaks 1 and 2), as well as for carotenoid esters (e.g., peaks 15 and 18). Therefore, these characteristics of elution order on the C30 column were considered very useful for tentative identification of carotenoid esters in the samples. Due to similar MS spectra, differentiation between violaxanthin esters and neoxanthin esters is only possible considering the earlier elution of neoxanthin derivatives compared to the respective violaxanthins. Using this strategy, peaks 6a, 11, and 27 were identified as neoxanthin derivatives, whereas peak 4 could not be unequivocally assigned. Peak 11 was assigned as (9′Z)-neoxanthin dibutyrate on the basis of the 8219

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Figure 6. Chromatogram from mango cv. ‘Tommy Atkins’, obtained by HPLC-DAD, processed at 450 nm.

and were tentatively assigned as (9Z)- or (9′Z)-antheraxanthin dimyristate or mutatoxanthin dimyristate. Another pair of carotenoids with the same MW, βcryptoxanthin and zeinoxanthin, was found in the saponified extract of pulp from both orange varieties (data not shown), but they were not detected in free form in nonsaponified extracts. Although previous studies reported only the presence of β-cryptoxanthin esters in orange juice,11−15 we expected to find here zeinoxanthin esters as well. In fact, we have detected several esters of β-cryptoxanthin and/or zeinoxanthin in citrus cultivars; however, all of these peaks coeluted with esters of other carotenoids, impairing assessment of the UV/vis spectrum, essential information for differentiation between these carotenoids, considering that mass spectra of their esters are not different from each other (e.g., peaks 53a and 62e, generically assigned as β-cryptoxanthin or zeinoxanthin esters). Considering these facts, undoubtedly designation of the monoester in peaks 53a, 56d, 61c, 62e, 65b and 69 was not possible, and the tentative assignment was based on elution order. Peaks 51d, 59b, and 64d were assigned as zeinoxanthin esters by their UV/vis spectral features coherent with those of zeinoxanthin derivatives, especially higher fine structure than that of β-cryptoxanthin. Peaks 53b, 60a, 60b, 61d, and 64a were identified as (Z)-β-cryptoxanthin esters, on the basis of UV/vis features, and the position of the double bond was attributed considering elution order of the compound relative to the corresponding (all-E)-β-cryptoxanthin ester (peak 58a, 63c, or 67b). The elution order of carotenoid esters with respect to their acylated FA can be logically deduced. Considering esters from the same carotenoid, the retention time on reversed-phase columns generally increases as the MW of the FA also increases, except for carotenoid acylated with unsaturated fatty acids that elutes earlier than the corresponding saturated FA. Indeed, the presence of a double bond in FA is approximately equivalent in reversed-phase HPLC to shortening the FA chain

before the corresponding (all-E)- and (9Z)-correspondents (peaks 56a and 58b, respectively), peak 54b was assigned as (13Z)- or (15Z)-violaxanthin dimyristate. Peak 59a also showed λmax at 437 nm, but its identification as (9Z)violaxanthin laurate-palmitate was supported by its elution after (all-E)-violaxanthin laurate-palmitate or (all-E)-luteoxanthin laurate-palmitate (peak 57c). This reasoning was applied to tentatively identify peak 60c as (13Z)- or (15Z)violaxanthin myristate-palmitate and peak 24b as (9Z)violaxanthin laurate, although their UV/vis spectra were not detected. A similar situation occurred for peaks 79a and 79b, with λmax at 447 nm, an intermediate value between that of lutein (λmax ∼ 445 nm) and zeaxanthin (λmax ∼ 450 nm), and with MS features revealing coelution of diester of zeaxanthin with a diester of lutein. Three peaks with [M + H]+ at m/z 779 and fragmentation pattern characteristic of zeaxanthin myristate (peaks 45c, 50b, and 55c) were detected in tangor cv. ‘Murcott’. Peak 55c was readily identified as (9Z)-zeaxanthin myristate on the basis of its λmax similar to that of (9Z)-β-carotene29 and elution after the other two peaks. Peak 50b with λmax at 444 nm was tentatively identified as (13Z)- or (15Z)-zeaxanthin myristate, whereas peak 45c was tentatively identified as another (13Z)- or (15Z)zeaxanthin myristate isomer because of its earlier elution. On the other hand, some peaks with the same pronated molecule and MS features but with UV/vis spectra not detected were tentatively identified either as violaxanthin or luteoxanthin derivatives; for example, peaks 63b and 65c refer to (9Z)violaxanthin myristate palmitate or (all-E)- or (9Z)-luteoxanthin myristate-palmitate, respectively. Similarly, peak 51b was tentatively identified as (all-E)-violaxanthin lauratemyristate or (all-E)-luteoxanthin laurate-myristate, and peak 66c was assigned as (9Z)-violaxanthin dipalmitate or luteoxanhin dipalmitate. Following this logic, two peaks (peak 67a and 68b) with similar MS spectra as that of (all-E)antheraxanthin dimyristate (peak 65d) eluted after this peak 8220

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Journal of Agricultural and Food Chemistry length by two carbons.34 For example, considering two (all-E)violaxanthin esters, peak 63a with [M + H]+ at m/z 1129 eluted before peak 64b with [M + H]+ at m/z 1103. This occurred because in peak 63a both FAs acylated to violaxanthin are unsaturated (18:1), whereas in peak 64b, only one of the FAs is unsaturated (18:1) and the other FA has a shorter saturated chain (16:0). This fact was considered for tentative identification of peak 42c (undetected UV/vis spectrum) as antheraxanthin or mutatoxanthin oleate. Additionally, we observed that homodiesters elute before heterodiesters with the same MW. For example, (9Z)violaxanthin dimyristate, esterified with 14:0 at both ends (peak 58b), elutes earlier than (9Z)-violaxanthin capratemyristate (peak 59a), acylated with 12:0 and 16:0. Considering this behavior, peak 55b, with no detected UV/vis spectrum, was assigned as (13Z)- or (15Z)-violaxanthin laurate-palmitate because of its elution after (13Z)- or (15Z)-violaxanthin dimyristate (peak 54b) and before (all-E)-violaxanthin dimyristate (peak 56a). Similarly, peak 57c was assigned as (all-E)-violaxanthin laurate-palmitate or (all-E)-luteoxanthin laurate-palmitate. (All-E)-violaxanthin laurate (peak 20) and (9Z)-violaxanthin caprate (peak 21) have a difference of two carbons in the acylating FA and eluted very close to each other, with respect to the typical behavior of (all-E)- and (9Z)-isomers on the C30 column. Thus, although with no detected UV/vis spectrum, peak 24a was tentatively identified as (all-E)-violaxanthin myristate, due to the elution close to (9Z)-violaxanthin laurate (peak 24b). In other cases of coelution, two different λmax values could be obtained throughout the peak, but with a slight increase or decrease in typical λmax values, as observed in peak 70a (λmax at 448 nm) identified as (all-E)-zeaxanthin lauratemyristate, and peak 70b (λmax at 446 nm) identified as (all-E)antheraxanthin myristate-palmitate (typical λmax at 444−445 nm). Carotenoid and Carotenoid Ester Composition of Mango and Citrus. Mango and tangor cv. ‘Murcott’ were characterized by the presence of xanthophylls preferentially in the totally esterified form, whereas orange extracts presented peaks of similar intensities throughout all of the chromatogram, indicating the occurrence of free and partially and totally esterified carotenoids. In the initial 16 min of the chromatographic run of orange cv. ‘Valencia’ extract, mass spectra showed interfering compounds with fragment patterns different from those of carotenoids. In this region, only three (peaks 3a and 3b, 6b, and 8) of seven peaks had the identity confirmed by MS analysis. The additional two-step cleanup procedure applied to eliminate potential interferences enabled the detection of [M + H]+ of peaks 1, 2, 5, and 13. However, several artifacts were formed, probably because this cleanup procedure increases the time of analysis, exposing epoxycarotenoids to isomerization and rearrangement reactions. This suggest that the procedure should be applied with caution for the analysis of carotenoids from orange. The cleanup procedure was not applied for orange cv. ‘Pera’ because its carotenoid profile was quite similar to that of cv. ‘Valencia’, especially in the elution region of interfering compounds, and it was not necessary for tangor cv. ‘Murcott’ and mango. Free Xanthophylls in Mango and Citrus. Free xanthophylls accounted for approximately 8.5% in oranges, 3.8% in tangor cv. ‘Murcott’, and 1.2% in mango (% of total area). (All-E)-violaxanthin (peak 1), (all-E)-luteoxanthin (peak 2), (all-E)-antheraxanthin (peak 3a), (9Z)-violaxanthin (peak

3b), mutatoxanthin (peaks 5), (all-E)-lutein (peak 6b), (all-E)zeaxanthin (peak 8), and (9Z)- or (9′Z)-antheraxanthin (peak 10) were found in free form only in orange varieties, except for (all-E)-violaxanthin and (9Z)-violaxanthin that were also present in mango. Free (all-E)-β-cryptoxanthin (peak 25b) was detected only in tangor cv. ‘Murcott’. The identification of such peaks was carried out according to criteria previously reported;22,26,28−30 therefore, only some important considerations are made hereafter. In citrus samples, peak 3 was a mixture of (all-E)antheraxanthin and (9Z)-violaxanthin because both [M + H]+ were detected, respectively, at m/z 585 and 601, as well as their characteristic MS/MS fragments. Interestingly, the UV/vis spectrum of (all-E)-antheraxanthin was not influenced by coelution with (9Z)-violaxanthin, probably because the amount of free antheraxanhin is much higher when compared to that of violaxanthin. These carotenoids are very difficult to separate, even on C30 column with gradient elution, because both also coeluted under different chromatographic conditions.25 Peak 5 was generically assigned as mutatoxanthin, because some features of its UV/vis spectrum were not detected, impairing the assessment of cis−trans configuration. On the other hand, this peak probably corresponds to (9′Z)- or (9Z)mutatoxanthin isomers because it eluted close to (all-E)-lutein on the C30 column, as observed in other studies.25,35 The MS spectrum of free (all-E)-β-cryptoxanthin (peak 25a) was detected only in tangor cv. ‘Murcott’ sample, although its pure UV/vis spectrum was not detected because of coelution. In fact, injection of (all-E)-β-cryptoxanthin standard as well as the chromatogram of a saponified extract of orange cv. ‘Valencia’ (data not shown) shows a peak at this retention time with UV/vis characteristic of (all-E)-β-cryptoxanthin, undoubtedly confirming its identification. Carotenes. Carotenes represented 24.2% of the carotenoids in mango and 9.3% in tangor cv. ‘Murcott’ (% of total area), whereas in oranges the estimation could not be accomplished due to peak coelution. The linear carotenes phytoene (peak 25a) and phytofluene (peaks 30 and 31a), as well as (all-E)-ζcarotene (peak 42a) and (9Z)-ζ-carotene (45b), were detected only in tangor cv. ‘Murcott’. On the other hand, peak 36, assigned as (Z)-ζ-carotene was found in all of the citrus samples. (All-E)-β-carotene (peak 43c) is one of the major carotenoids in mango, and therefore UV/vis and MS spectra resembling the pure carotenoid were obtained in the mango chromatogram (Figure 6) and supported the identification of this carotene in tangor cv. ‘Murcott’. In mango, we also detected the presence of (13Z)- or (15Z)-β-carotene (peak 37) and (9Z)-β-carotene (peak 45a). In all nonsaponified orange samples, (all-E)-β-carotene was not detected because in the elution region of this carotene several peaks of esters also eluted with no baseline separation. However, (all-E)-β-carotene was detected when extracts were saponified. As a matter of fact, (all-E)-β-carotene was also found in low amounts in the saponified extract of orange juice in other works.25 Carotenoid Esters. Monoesters comprised 28.6−30.8% in oranges, 47.3% in tangor cv. ‘Murcott’, and around 1% in mango. In addition, diesters represented 53.7−56.3% of carotenoids in orange varieties, 22.6% in tangor cv. ‘Murcott’, and 46.4% in mango (% of total area). On the other hand, peak coelution impaired even a rough estimation of percentages of the major carotenoid esters in oranges. Orange cv. ‘Valencia’ and cv. ‘Pera’ showed quite similar qualitative profiles. In fact, 8221

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(∼20.3%), followed by (all-E)-violaxanthin dibutyrate (∼13.5%) and (9Z)-violaxanthin dibutyrate (∼7.7%). These results are in agreement with previously published works.19,20 We also found (all-E)-violaxanthin butyrate (∼1.1%, peak 4), (9Z)-violaxanthin butyrate (0.9%, peak 7b), and (all-E)violaxanthin butyrate-caproate (1.2%, peak 13), also previously reported.20 Additionally, we report for the first time the presence of (9Z)-violaxanthin butyrate-caproate (0.7%, peak 19), as well as the following (all-E)- and (9Z)-violaxanthin diesters: butyrate-laurate, butyrate-myristate, caprylate-laurate, butyrate-palmitate, dimyristate, and myristate-palmitate, which together represent about 18% of the mango carotenoids. The presence of neoxanthin esters in mango, namely, (all-E)and (9′Z)-neoxanthin dibutyrates (peaks 6a and 11, respectively), as well as (all-E)-neoxanthin butyrate-myristate (peak 27) was reported here for the first time, supported by previous results36 where free neoxanthin was found. On the other hand, neoxanthin was not detected in any of the citrus varieties analyzed here. Indeed, previous evidence27 indicated that neoxanthin is not present in detectable levels in a wide range of orange juices from Spain. Although neoxanthin is a major carotenoid in citrus green stages, it disappears progressively during fruit development until being undetectable even in early chromoplastic tissues.37,38 In summary, we provided a detailed identification, by HPLCDAD-MS/MS, of carotenoids and their esters in citrus and mango, including most of the minor compounds. The major esters of oranges cv. ‘Valencia’ and cv. ‘Pera’ were derived from violaxanthin and luteoxanthin, and 52 of them were identified in our study. We reported for the first time the presence of 19 antheraxanthin esters, 11 lutein esters, and 7 zeaxanthin diesters, as well as some of the violaxanthin esters in orange pulps. In tangor, β-cryptoxanthin esters were the major compounds, followed by violaxanthin mono- and diesters, the latter being reported for the first time in mandarin varieties. The fatty acids more frequently esterified with citrus carotenoids were 12:0, 14:0, 16:1, 16:0, and 18:1, whereas in mango several short-chain acylating fatty acids such as 4:0 and 6:0 were found. In mango, besides the well-known predominance of violaxanthin dibutyrates, we reported for the first time the identification of neoxanthin esters as well as 10 (all-E)- and (9Z)-violaxanthin diesters. The current study demonstrates substantial differences in the carotenoid composition between nonsaponified and saponified extracts of citrus and mango. Although it is not completely understood how esterification modifies carotenoid bioavailability and functions, it is clear that esterification of carotenoids has a high impact on their solubility. In addition, with regard to carotenoid esters, some MS features were deduced: (1) the protonated molecule was always detected, with the exception of lutein derivatives; (2) characteristic backbone and losses of C7H8 (92u), characteristic of polyene chain, were always detected by MS/MS; (3) losses of one and both fatty acids were always observed by MS/MS; (4) regioisomeric forms of lutein esters were distinguished considering that the most abundant in-source fragment ion was produced from neutral loss of the corresponding moiety (hydroxyl group or FA) at position 3′ of lutein ; (5) in diesters, loss of water from the protonated molecule and losses of fatty acids with water were detected only for epoxy-xanthophylls by MS/MS.

the differences observed between the orange cultivars are probably due to the high number of coelutions and possible quantitative differences between these cultivars. Some esters were detected in cv. ‘Valencia’ (peaks 21, 38a, 40c, and 49b) but were not detected in cv. ‘Pera’ variety, which in turn also had some esters (peaks 35d, 35e, 39c, 40d, 49a, 61c, 62e, 63d, 65b, and 77b) not detected in cv. ‘Valencia’. Information available in the literature for orange esters makes comparison difficult because either cis−trans configuration or position of the cis double bond was omitted in previous studies. In a generic comparison, 17 monoesters of violaxanthin and luteoxanthin were identified in the present study in cv. ‘Valencia’ and cv. ‘Pera’ against only 8 previously reported in orange juices from eight varieties.13−15 Moreover, we found 30 diesters of violaxanthin and luteoxanthin, whereas 20 diesters of these xanthophylls were previously reported.13−15 Antheraxanthin diesters in orange pulp were identified for the first time here. We identified (all-E)-antheraxanthin dilaurate (peak 57b), laurate-myristate (peak 62b), dimyristate (peak 65d), myristate-oleate (peak 68a), myristate-palmitate (peak 70b), and palmitate-oleate (peak 74), as well as (9Z)- or (9′Z)-antheraxanthin dilaurate (peak 61b), laurate-myristate (peak 64c), laurate-oleate (peak 66a), myristate-palmitate (peak 73). In previous studies, only antheraxanthin monoacylated with palmitic acid (16:0) was found.13,15 Besides (all-E)antheraxanthin monoacylated with 16:0, we further identified this xanthophyll monoacylated with 14:0, as well as (9Z)antheraxanthin monoesterified with 12:0, 14:0, 16:0, and 18:1. In our study, we detected mutatoxanthin esterified with 12:0 (peaks 38b and 40b) as well as with 14:0 (peak 42b), whereas Giuffrida and co-workers15 reported mutatoxanthin monoesterified with 14:0 and 16:0. No information about the presence of lutein and zeaxanthin esters in orange pulp or juice is reported in the literature of orange or orange juices.13−15 In our study, one lutein monoester (peak 39c) and several lutein and zeaxanthin diesters were identified in pulp of orange varieties, whereas zeaxanthin monoesters were not found in the present study. The carotenoid pattern of tangor cv. ‘Murcott’ was dominated by two major peaks consisting of (all-E)-βcryptoxanthin laurate (∼15.0%) and (all-E)-β-cryptoxanthin myristate (∼10.9%), followed by (all-E)-β-cryptoxanthin palmitate (∼4.1%), as previously reported.17 The presence of violaxanthin mono- and diesters (e.g., peaks 24a, 24b, and 48c) in mandarin samples is reported for the first time in our work. Whereas in oranges only zeaxanthin diesters were found, in tangor we found several zeaxanthin monoesters, for example, acylated with 10:0, 12:0, 14:1, 14:0, and 16:0 (peaks 40e, 43d, 45c, 46, 50b, 55c, and 56e), also not previously described in the literature. Peak 14, detected only in tangor pulp, showed UV/vis features characteristic of an apocarotenoid, particularly close to apo-12′-violaxanthal.23 Moreover, we expect that the free form of such an apocarotenoid should elute earlier than it actually did, so peak 14 probably refers to an acylated form of apo-12′violaxanthal. However, assignment confirmation was not possible, because the [M + H]+ was not detected in the MS spectrum, and therefore this peak remained unidentified. Mango has a particular carotenoid ester composition, with several short-chain acylating FAs. As can be noted in Figure 6 and Table 1, mango presents several violaxanthin and neoxanthin esters, as well as β-carotene. The major constituent of the nonsaponified extract of mango was (all-E)-β-carotene 8222

DOI: 10.1021/acs.jafc.6b03226 J. Agric. Food Chem. 2016, 64, 8207−8224

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AUTHOR INFORMATION

Corresponding Author

*(A.Z.M.) Phone: +55-19-35212163. Fax: +55-19-35212153. E-mail: [email protected], [email protected]. Funding

We thank Sao Paulo Research Foundation (FAPESP) (Grants 2013/07914-8 and 2013/08904-5) and National Counsel of Technological and Scientific Development (CNPq Grant 308484/2014-2) for their financial support. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Agronomic Institute of Campinas for providing the citrus samples. REFERENCES

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DOI: 10.1021/acs.jafc.6b03226 J. Agric. Food Chem. 2016, 64, 8207−8224