Generic Characterization of Apolar Metabolites in Red Chili Peppers

Article pubs.acs.org/JAFC

Generic Characterization of Apolar Metabolites in Red Chili Peppers (Capsicum frutescens L.) by Orbitrap Mass Spectrometry. Sebastiaan Bijttebier,*,†,§ Kaouther Zhani,‡,§ Els D’Hondt,† Bart Noten,† Nina Hermans,§ Sandra Apers,§ and Stefan Voorspoels† †

Business Unit Separation and Conversion Technology (SCT), Flemish Institute for Technological Research (VITO), Boeretang 200, 2400 Mol, Belgium ‡ Department of Horticulture and Landscape, Sousse University, Higher Institute of Agronomy, 4042 Chott Mariem, Tunisia § NatuRA, University of Antwerp, Universiteitsplein 1, 2610 Antwerp, Belgium S Supporting Information *

ABSTRACT: The aim of the present study was to develop a generic analytical method for the identification and quantitation of apolar plant metabolites in biomass using liquid chromatography−photodiode array−accurate mass mass spectrometry (LCPDA-amMS). During this study, a single generic sample preparation protocol was applied to extract apolar plant metabolites. Compound identification was performed using a single generic screening method for apolar compounds without the need for dedicated fractionation. Such a generic approach renders vast amounts of information and is virtually limited by only the solubility and detector response of the metabolites of interest. Method validation confirmed that this approach is applicable for quantitative purposes. Furthermore, an identification−quantitation strategy based on amMS and molar extinction coefficients was used for carotenoids, eliminating the need for reference standards for each carotenoid. To challenge the validated method, chili peppers (Capsicum frutescens L.) were analyzed to unravel their complex phytochemical composition (carotenoids, glycolipids, glycerolipids, capsaicinoids, lipid-soluble vitamins). KEYWORDS: LC-PDA-amMS, quantitation without reference standards, extinction coefficients, generic analysis of apolar plant metabolites, biomass composition, Capsicum frutescens L., carotenoids

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INTRODUCTION Biomass such as algae and vegetables often contains natural components of high value including the so-called phytochemicals, such as antioxidants and essential oils, that are barely utilized from these resources today.1,2 These natural products are thought to provide beneficial health effects and have an increasing sales market in the pharmaceutical, cosmetics, and food supplements industry.3 Knowing the composition of these resources is thus an essential first step in a potential valorization process. Because of the complexity of compound mixtures in biological samples, the usage of simple analytical instrumentation such as liquid chromatography−ultraviolet detection (LCUV) has shifted toward more selective and more complex separation and detection systems such as ultrahigh-performance liquid chromatography−photodiode array−accurate mass mass spectrometry (UHPLC-PDA-amMS) instrumentation to achieve more definitive compound identification.4 Accurate mass MS detectors allow the tentative identification of compounds without the use of analytical standards.5 Because of the limited availability of analytical standards for plant metabolites, this utility has shown to be essential in natural products exploration.6 Furthermore, modern high-end analytical instrumentation allows the detection of multiple compound classes, such as carotenoids, capsaicinoids, and glycolipids, in a single analysis, resulting in enormous time and consumables savings. To avoid compound discrimination during extraction, no cleanup steps should be used and the © 2014 American Chemical Society

number of sample preparation steps should be limited as much as possible. Such a generic approach is virtually limited only by the solubility (extractability, chromatographic retention) and detector response (UV absorbance and ionization efficiency) of the compounds of interest. Furthermore, the use of a generic analytical screening application allows the setup of a compound database with specific chromatographic and spectrometric data. This database enables an effective dereplication during structure elucidation of the extract composition.7 The aim of the present study was to develop, optimize, and validate a generic analytical method for the identification and quantitation of apolar plant metabolites in biological matrices using UHPLC-PDA-amMS. Authentic standards of multiple phytochemical classes and various biological matrices were used to confirm and emphasize the generic character of the method. To challenge the validated method, the widely studied chili pepper matrix (Capsicum frutescens L.) was selected as it is known for its complex phytochemical composition (carotenoids, glycolipids, glycerolipids, capsaicinoids, lipid-soluble vitamins).8−13 Finally, an identification−quantitation strategy based on the use of amMS data and extinction coefficients was applied for carotenoids, eliminating the need for reference standards for each carotenoid. Received: Revised: Accepted: Published: 4812

January 17, 2014 April 23, 2014 April 24, 2014 April 24, 2014 dx.doi.org/10.1021/jf500285g | J. Agric. Food Chem. 2014, 62, 4812−4831

Journal of Agricultural and Food Chemistry

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Article

more with 15 mL of hexane. The pooled hexane fractions were evaporated, dissolved in 10 mL of dichloromethane + 0.1% butylated hydroxytoluene, an aliquot was used for analysis, and the remaining extract was stored in the dark under nitrogen at −25 °C until further treatment. For saponification 2 mL of 10% sodium hydroxide (in methanol) was added to 2 mL of dichloromethane extract and was consecutively shaken for 6 h at room temperature in the dark under an inert atmosphere. Afterward, the solution was washed five times with water to remove the alkali.14 An aliquot of the saponified dichloromethane extract was used for analysis. Instrumental Analysis. The analytical method was previously described4 and is briefly summarized as follows. For analysis, 1.25 μL of extract was injected with a CTC PAL autosampler (CTC Analytics, Zwingen, Switzerland) on a 100 mm × 2.1 mm, 1.8 μm, Acquity UPLC HSS C18 SB column (Waters, Milford, MA, USA) and thermostatically (35 °C) eluted with an Accela quaternary solvent manager and a Hot Pocket column oven (Thermo Fisher Scientific, Bremen, Germany) with a chromatographic gradient. The mobile phase solvents consisted of 50:22.5:22.5:5 (v/v/v/v) water + 5 mM ammonium acetate/methanol/acetonitrile/ethyl acetate (A) and 50:50 (v/v) acetonitrile/ethyl acetate (B), and the gradient was set as follows (min/% A): 0.0/90, 0.1/90, 0.8/70, 20.0/9, 20.1/0, 20.4/0, 20.5/90, 23.0/90. For detection, an Exactive amMS (Thermo Fisher Scientific) was used in positive atmospheric pressure chemical ionization (APCI) mode. Full scan and all-ion fragmentation data were acquired with a scanning range of m/z 100−1400 at a resolution of 50000 full width at half-maximum (fwhm). The Accela PDA detector (Thermo Fisher Scientific) was set to scan from 190 to 800 nm.

MATERIALS AND METHODS

Chemicals. LC-MS grade methanol, acetonitrile, and ethyl acetate were purchased from Biosolve (Valkenswaard, The Netherlands). Ultrapure water with a resistivity of 18.2 MΩ·cm at 25 °C was generated with a Millipore system. Dichloromethane for gas chromatography, n-hexane for gas chromatography, acetone for gas chromatography, sodium hydrogen carbonate, sodium hydroxide, and sodium chloride for analysis were purchased from Merck (Darmstadt, Germany). Ammonium acetate, (D-Ala2)-leucine enkephalin, sand (quartz), and butylated hydroxytoluene were purchased from SigmaAldrich (Bornem, Belgium). Certified reference material BCR 485 consisting of freeze-dried mixed vegetables was bought from IRMM (Geel, Belgium). Commercially available mixtures to calibrate the mass spectrometer, that is, MSCAL5-1EA (caffeine, tetrapeptide “Met-ArgPhe-Ala”, Ultramark) for positive ion mode and MSCAL6-1EA (sodium dodecyl sulfate, taurocholic acid sodium salt, Ultramark) for negative ion mode, were purchased from Supelco (Bellefonte, PA, USA). Lycopene, α-carotene, phytoene, lutein, zeaxanthin, tunaxanthin, astaxanthin dipalmitate, phytofluene, antheraxanthin, violaxanthin, canthaxanthin, and astaxanthin were purchased from Carotenature (Ostermundigen, Switzerland). β-Carotene, trans-β-apo-8′-carotenal, campesterol, stigmasterol, β-sitosterol, glyceryl trioleate, glyceryl dioleate, γ-tocopherol, α-tocopherol, phylloquinone, ergocalciferol, and cholecalciferol were purchased from Sigma-Aldrich. Glycerol dioleate and glycerol trioleate were obtained from Chem Service (West Chester, PA, USA). Monogalactosyldiacylglycerol with average MW 752.369 and digalactosyldiacylglycerol with average MW 926.767 were purchased from Avanti (Alabaster, AL. USA). Preparation of Standard Solutions. Standard stock solutions and working solutions were prepared of each analyte separately at a concentration of approximately 200 μg/mL. Stock solutions of glycoand glycerolipids were prepared in methanol. The stock solutions of sterols and lipid-soluble vitamins were prepared in methanol + 0.1% butylated hydroxytoluene. Stock solutions of carotenoids were prepared in dichloromethane + 0.1% butylated hydroxytoluene. Standard stock and working solutions were stored at −25 °C in the dark under an inert atmosphere (nitrogen). Dilutions of the stock solutions were prepared in dichloromethane + 0.1% butylated hydroxytoluene for analysis. Sample Preparation. Red bell peppers (Capsicum annuum) and apples (Malus domestica) were bought in a local store. After removal of the stems and kernels, they were cut into pieces, frozen with liquid nitrogen, and immediately freeze-dried. After drying, they were milled and homogenized with a Grindomix GM 200 from Retsch (Haan, Germany) at 10000 rpm and stored in the dark under nitrogen at −25 °C. Fruits of five chili pepper accessions (Capsicum frutescens L.), namely, ‘Tebourba’, ‘Korba’, ‘Somâa’, ‘Awlad Haffouzz’, and ‘Souk jedid’, were collected from plants cultivated in the experimental station of the Higher Institute of Agriculture, Chott Mariem, Tunisia. The peduncles of the peppers were removed, and the fruits were freezedried, milled, and stored at −25 °C until analysis. Before sample preparation, the samples were allowed to equilibrate to room temperature. One gram of sample was mixed with 1 g of sodium hydrogen carbonate, spiked with trans-β-apo-8′-carotenal (internal standard), and mixed with sand. Ultrapure water was added until the sample was visually completely hydrated (approximately 3 mL, depending on the matrix). The mixture was kept in the dark under N2 to allow swelling of the matrix for better analyte extraction. Afterward, the sample was mixed again with sand and loaded into a 33 mL accelerated solvent extraction (ASE) cell (Thermo Fisher Scientific, Bremen, Germany). The mixture was extracted three times (5 min static extraction) with 70:30 acetone/ methanol + 0.1% butylated hydroxytoluene at 40 °C and 1050 psi, resulting in approximately 120 mL of sample extract. The three extracts were combined in a separation funnel, and 100 mL of 10% sodium chloride (aqueous) and 15 mL of hexane were added. The hexane phase was retained, and the polar phase was extracted twice

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RESULTS AND DISCUSSION Method Validation. Carotenoids were chosen as model compounds for the optimization of a generic sample preparation and screening method for apolar plant metabolites. Carotenoids are apolar plant metabolites that can easily degrade or isomerize under the influence of light, oxygen, enzymes, heat, oxidants, and acid or alkaline conditions.15 It can hence be assumed that if no isomerization or degradation occurs for carotenoids during sample preparation, most other apolar compounds should also be extracted without any artifact formation. The chromatographic gradient and MS settings were optimized on an UHPLC-PDA-amMS for a set of carotenoids representing the wide polarity range of carotenoids found in nature.4 A validation study of the optimized method was performed for a range of compounds from several apolar metabolite classes (12 carotenoids, 5 lipid-soluble vitamins, 1 phytosterol) and different matrices. Furthermore, the extraction efficiency of other apolar plant metabolites (glycolipids, glycerolipids, steryl derivatives, chlorophyll) for which no commercial reference standards were available was investigated to confirm its generic character. Dynamic Range and Sensitivity. High dynamic ranges (up to 4 orders of magnitude) with good linear fits were obtained in MS for most compound calibration curves. Because of the lower sensitivity of carotenoids in UV detection and the high cost of carotenoid standards, the dynamic ranges of the carotenoids were not tested to their full extent with PDA detection. However, it could be observed that smaller dynamic ranges were acquired for the very apolar carotenes in both MS and PDA, suggesting lower solubility to be the cause. Detection and quantitation limits are of less importance for valorization objectives as predominantly compounds with high concentrations are targeted. Precision. The repeatability (intraday RSD) was obtained from the relative standard deviations (RSDs) of the recoveries from the spiking experiments. Because of the high price of 4813

dx.doi.org/10.1021/jf500285g | J. Agric. Food Chem. 2014, 62, 4812−4831


Article

Figure 1. Representative structures of the different compound groups detected in chili peppers during this study.

analytical standards, it was chosen to obtain the intermediate precision (interday RSD) of the method with compounds natively present in red bell pepper (Capsicum variety). Five aliquots of freeze-dried pepper were extracted and analyzed the same day. This experiment was repeated for 3 days in total. Intraday RSDs ranged between 0.5 and 8.4% and interday RSDs between 0.0 and 6.0%. Trueness. Certified reference material (CRM) is commercially available for only a very limited set of plant metabolites. Furthermore, reference standards are often not commercially available or very expensive for plant metabolites. Therefore, the trueness could only be assessed for a select set of compounds. A CRM of mixed vegetables, BCR 485 (consisting of sweet corn, tomatoes, and carrots), with certified concentration values for all-trans-α- and β-carotene, lutein, and lutein + zeaxanthin was extracted in triplicate. Values of 110 ± 8, 105 ± 3, 100.5 ± 2.7, and 94.5 ± 1.4% of the certified concentration values were found with MS, respectively, and 110.5 ± 2.5, 106.2 ± 1.1, 105 ± 4, and 101 ± 5% with PDA, respectively, with the uncertainties being the RSD of the three replicates. These results show good accuracy of the method for the quantitative determination of carotenoids natively present in biological matrices. Spiking experiments were performed in triplicate on aliquots of freeze-dried apple (M. domestica) using an analytical standard mixture with 11 carotenoids, 6 lipid-soluble vitamins, and 1 phytosterol (concentrations ranging between 5.3 and 140 μg/g dw). The recoveries (R (%)) for all spiked compounds except violaxanthin ranged between 86 and 115% with MS detection, showing good accuracy. The slightly lower recoveries (74−87% in MS) found for violaxanthin can be attributed to epoxide rearrangements (which lead to large hypsochromic shifts in UV

absorbance), catalyzed by the acids naturally present in the apple matrix.16 Because of the low UV absorbance of phytosterols and lipid-soluble vitamins, only the recoveries of most of the spiked carotenoids could be calculated with PDA detection, showing similar values compared to the results calculated with MS. Extraction Efficiency. Because of the lack of commercially available CRMs and reference standards, the conventional approach of method validation could be performed for only a select set of apolar plant metabolites. To investigate the extraction efficiency for a larger range of apolar plant metabolites, the two most critical steps in sample preparation were tested by exhaustive extraction of compounds naturally present in peppers. In a first experiment, an aliquot of freezedried red bell pepper was extracted five times with ASE followed by separate preparation of the obtained extracts. Assuming 100% extraction, the relative abundances of the compounds were calculated in the respective extracts. In a second experiment, the transfer of compounds to the hexane phase during liquid−liquid extraction was investigated by backextracting the polar phase six times with hexane. After the first three hexane extracts had been pooled, an extra 240 mL of 10% sodium chloride solution was added to the polar phase. The polar phase was extracted three more times. The first two apolar extracts resulting from this second series of extractions were pooled. Subsequently, the three remaining extracts (consisting of three pooled, two pooled, and a single extract) were prepared separately for analysis. Again assuming 100% extraction, the relative abundances of the compounds were calculated in the respective extracts. The results showed that during the two most critical steps in the extraction procedure, 4814


4815

C54H82O5

C52H78O4

C52H76O4

capsorubin-myristate (C14:0)

all-trans-capsanthin-laurate (C12:0)

-

13

14

C40H56O

β-cryptoxanthin

9

12

C40H54O2

nigroxanthin

8

C52H80O5

C30H40O

all-trans-apo-carotenal

7

karpoxanthin-laurate (C12:0)

C40H56O2

all-trans-zeaxanthin

6

11

C40H56O3

cis-antheraxanthin

5

C52H78O5

C40H56O3

all-trans-capsanthin

4

capsorubin-laurate (C12:0)

C40H56O3

cis-capsanthin

3

10

C40H56O4

cycloviolaxanthin

2

15.38

15.01

15.01

14.77

13.85

13.61

12.62

10.38

10.12

8.67

7.93

7.77

7.29

6.77

C40H56O3

retention time (min)

mol formula

cucurbitaxanthin A

identity

1

compd

435, 465, 498

475

424, 448, 477

479

456, 483

448, 478

465

455, 481

450, 472

474

467

418, 443, 472

426, 449, 476

max absorbance (nm)

pos neg

pos neg

pos neg

pos neg

pos neg

pos neg

pos neg

pos neg

pos neg

pos neg

pos neg

pos neg

pos neg

pos neg

MS mode

765.58206 764.57505

767.59687 766.59080

811.62242 810.61682

785.60732 784.60170

783.59123 782.58620

553.44074 552.43436

567.41995 -

417.31602 416.30875

569.43546 568.42884

585.43029 584.42385

585.43027 584.42352

585.43029 584.42392

601.42497 -

585.43068 -

accurate mass of precursor ion

583.414, 565.405 749.587, 567.420 -

−1.33 0.06 −0.55 0.31

565.404 672.513, 658.497, 564.397

767.597, 585.431, 567.420, 549.410, 475.357 -

−0.67 0.74

0.55 0.18

583.414, 565.405 -

535.43 -

-

-

551.425 -

567.419, 549.409, 493.352, 475.357 35 -

567.419, 549.409, 493.352, 475.35735, 461.342 -

567.419 -

-

567.420 -

in-source fragments

−1.24 0.93

0.63 1.27

0.51 -

1.99 0.70

0.26 0.46

0.12 0.62

0.09 0.05

0.12 0.74

−0.28 -

0.79 -

mass deviation (ppm)

Table 1. Values for Maximum Absorbance, Precursor and Product Ions, and Retention Times of the Carotenoids Present in the Five Chili Pepper Accessions

-

c, e

b, e

a, b,f

b,e

a, b, c, e

a, b

d

d

a

d

c

a, b

a, b

identification type

Journal of Agricultural and Food Chemistry Article


4816

C52H78O3

C56H86O4

C52H78O3

all-trans-zeaxanthin-laurate (C12:0)

cis-capsanthin-palmitate (C16:0)

cis-zeaxanthin-laurate (C12:0)

26

27

28

C54H82O4

all-trans-capsanthin-myristate (C14:0)

23

C40H64

C40H62

all-trans-phytofluene

22

all-trans-phytoene

C52H78O4

cucurbitaxanthin A-laurate (C12:0)

21

25

C52H78O4

mutatoxanthin-laurate (C12:0)

20

C54H82O4

C40H56

all-trans-β-carotene

19

cis-antheraxanthin-myristate (C14:0)

16.45

C54H82O4

all-trans-capsanthin-myristate (C14:0)

18

24

16.34

C52H78O4

cis-antheraxanthin-laurate (C12:0)

17

17.52

17.21

17.16

17.08

17.05

16.83

16.66

16.54

16.15

16.03

15.85

C52H78O4

cis-capsanthin-laurate (C12:0)

16

15.72


C52H78O4

mol formula

all-trans-capsanthin-laurate (C12:0)

identity

15

compd

Table 1. continued

424, 447, 474

463

455, 483

276, 288, 297

450, 477

473

334, 349, 369

426, 451, 474

405, 428, 457

455, 482

474

448, 478

463

474

max absorbance (nm)

pos neg

pos neg

pos neg

pos neg

pos neg

pos neg

pos neg

pos neg

pos neg

pos neg

pos neg

pos neg

pos neg

pos neg

MS mode

751.60229 750.59536

823.65929 822.65320

751.60171 750.59539

545.50774 -

795.62827 794.62168

795.62760 794.62178

543.49255 -

767.59682 766.59099

767.59653 766.59097

537.44556 536.43894

795.62818 794.62201

767.59672 766.59051

767.59704 766.59063

767.59639 766.59059


749.587, 567.420, 549.409 777.617, 567.420, 549.409 566.413, 227.202

−0.74 −0.07 −0.52 0.19

777.617, 567.420, 549.409 566.413, 227.202 777.617, 567.420, 549.409 463.430 551.425 805.650, 567.420, 549.409 566.413, 255.232 -

−1.24 −0.10 −0.40 −0.23 −0.62 −0.88 −0.33 −0.73 0.05 −0.11 −0.37

-

749.587, 567.420, 549.409 -

−0.61 0.56 0.22 -

567.420 -

−0.99 0.53

457.384, 445.384 -

749.587, 567.420 -

−0.33 0.09

0.15 0.35

749.587, 567.420, 549.409 674.528, 660.513, 566.413

in-source fragments

−1.17 0.04


-

-

c

d

c

c, e

d

a, b

b

d

c, e

c

c

c, e

identification type



18.44

18.58

C54H82O3

C54H82O3

C54H82O3

-

C58H88O4

C52H78O2

C64H100O5

C64H100O5

-

C54H82O2

C66H104O5

C66H104O5

cis-zeaxanthin-myristate (C14:0)

all-trans-zeaxanthin-myristate (C14:0)

all-trans-zeaxanthin-myristate (C14:0)

-

-

β-cryptoxanthin-laurate (C12:0)

all-trans-capsanthin-dilaurate (C12:0, C12:0)

cis-capsanthin-dilaurate (C12:0, C12:0)

-

β-cryptoxanthin-myristate (C14:0)

all-trans-capsanthin-laurate-myristate (C12:0, C14:0)

cis-capsanthin-laurate-myristate (C12:0, C14:0)

31

32

33

34

35

36

4817

37

38

39

40

41

42

19.97

19.78

19.56

19.50

19.32

19.12

18.8

18.37

18.16

18.00

17.83

C56H86O4

all-trans-capsanthin-palmitate (C16:0)

30

17.54

C54H82O4


mol formula

mutatoxanthin-myristate (C14:0)

identity

29

compd

Table 1. continued

463

474

455, 483

474

463

476

456, 482

434, 456

473

455, 479

454, 481

423, 447, 477

472

405, 430, 456

max absorbance (nm)

pos neg

pos neg

pos neg

pos neg

pos neg

pos neg

pos neg

pos neg

pos neg

pos neg

pos neg

pos neg

pos neg

pos neg

MS mode

976.78790

977.79388 976.78806

762.63197

-

948.75618

949.76483 948.75682

735.60815 734.60119

848.66846

-

778.62666

779.63263 778.62677

779.63226 778.62652

823.65985 822.65301

794.62109


551.425 761.622, 551.425 550.418

−1.81 −0.54 −1.33 −0.22

749.587, 549.409 -

749.587, 549.409 -

−1.81 −0.88 −1.04

535.430 -

-

749.587, 549.409 -

749.587, 549.409 -

535.430 -

-

804.642 44

−0.08

-

−1.52

0.51 −0.84

0.94 0.63

0.41

-

-

805.650, 567.420, 549.409 -

−0.05 −0.18

−0.36

777.617, 567.420, 549.409 -

in-source fragments

−0.97


-

c, e

c, e

-

-

e

c, e

-

-

c, e

c, e

c

e

b

identification type



4818

C66H104O5

C56H86O2

C68H108O5

C68H108O5

C68H108O5

C70H112O5

C70H112O5

C70H112O5

C72H116O5

C66H104O4

C68H108O4

cis-capsanthin-laurate-myristate (C12:0, C14:0)

β-cryptoxanthin-palmitate (C16:0)

all-trans-capsanthin-dimyristate (C14:0, C14:0)

all-trans-capsanthin-laurate-palmitate (C12:0, C16:0)

cis-capsanthin-dimyristate (C14:0, C14:0)

all-trans-capsanthin-myristate-palmitate (C14:0, C16:0)

cis-capsanthin-myristate-palmitate (C14:0, C16:0)

cis-capsanthin-myristate-palmitate (C14:0, C16:0)

all-trans-capsanthin-dipalmitate (C16:0, C16:0)

all-trans-zeaxanthin-laurate-myristate (C12:0- C14:0)

all-trans-zeaxanthin-dimyristate (C14:0C14:0)

43

44

45

46

47

48

49

50

51

52

53

21.37

21.37

21.33

21.19

21.10

20.93

20.72

20.57

20.36

20.25

20.13


453, 480

453, 480

473

463

464

475

464

473

473

457, 482

464

max absorbance (nm)

pos neg

pos neg

pos neg

pos neg

pos neg

pos neg

pos neg

pos neg

pos neg

pos neg

pos neg

MS mode

988.82436

960.79331

1060.88213

1032.85057

1032.84970

1032.85050

1.004.81860

1.004.81839

1.005.82526 1.004.81983

790.66218

976.78853


−0.96

−0.73

−0.65

−0.92

−1.76

−0.99

−1.61

761.622, 533.414 760.614

761.622, 733.591, 533.414 760.614, 732.584

805.648, 549.409 804.640, 255.233

805.648, 777.617, 549.409 776.611, 548.4

805.648, 777.617 776.611

805.648, 777.617, 549.409 776.611, 548.4

777.617, 549.409 776.611

805.650, 749.588, 549.409 776.611

777.617 776.611, 227.202

−1.68 −0.39 −1.82

-

749.587, 549.409 -

in-source fragments

−1.45

−0.40


c, e

c, e

c, e

-

-

-

-

c, e

c, e

c, e

-

identification type

a Dictionary of Natural Products:18 present in Capsicum annuum. bCarotenoids Handbook: present in Capsicum annuum and match with UV reference spectrum. cIdentified by Giuffrida et al..13 dMatch with analytical standard. eIdentified by Schweiggert et al.;10 fIdentified by Deli et al.8

mol formula

identity

compd

Table 1. continued




Article

Figure 2. Chromatogram of the PDA signal at 470 nm depicts the elution profiles of the carotenoids present in the Korba chili pepper accession.

the greater part of compounds is extracted during the first three extractions (>90%). Measurement Uncertainty. The results of the spiking experiments, CRM analysis, and intermediate precision were used to estimate the total expanded measurement uncertainty (U; k = 2). As not all compounds included during the spiking experiments were available for intermediate precision experiments, the intra- and interday RSDs of compounds of the same compound group were used to calculate U (e.g., for PDA analysis, the RSDs of β-carotene were applied for the calculation of U for α-carotene and lycopene). The obtained U values ranged between 10 and 26% (MS) and between 14 and 21% (PDA) for all compounds except violaxanthin, for which the U values were higher (44 and 59% for MS and PDA, respectively) as the bias of the lower R (%) obtained for violaxanthin was included in the calculation. Identification Strategy. Identification of compounds is relatively easy when analytical standards are available. However, in natural products research, analytical standards are often very expensive or not commercially available. Therefore, identification is often based upon the available chromatographic and spectrometric information.8−13 Carotenoids absorb UV light in a high-wavelength area (typical absorbance maxima at 400−500 nm) because of their conjugated system in the polyene backbone (e.g., molecular structures 4 and 45 in Figure 1).17 PDA detectors are therefore very selective for the detection of carotenoids. However, the applicability of PDA detectors is limited as they are only useful for compounds that absorb UV or visible light and provide only very little structural information, which makes it impossible to identify unknown compounds with PDA spectra.10 MS detectors are much more selective detectors than PDA detectors and can therefore facilitate the identification of plant metabolites. Orbitrap MS detectors can routinely generate mass spectra with a resolving power up to 140,000 at fwhm and obtain mass accuracies within 2 ppm (ppm), which enables the calculation of the most probable molecular formulas of the generated ions and fragments.5 However, to date, even the most state-of-the-art MS detectors are often not capable of distinguishing between isomers, whereas PDA detectors in many cases are.4,15 Furthermore, chromatography can be used to separate isomers before detection. The combination of orthogonal analytical techniques therefore provides a powerful tool for the identification of unknown compounds. During this study carotenoids, steryl derivatives, glycolipids, glycerolipids, capsaicinoids, and lipid-soluble vitamins were

tentatively identified in chili pepper samples. The information provided by the analysis alone is, however, not always enough for peak identification at an acceptable confidence level. Therefore, other sources of information such as in-house and commercial compound databases and peer-reviewed publications can give additional information for successful dereplication.7 During this study, the Dictionary of Natural Products18 and the Carotenoids Handbook19 were consulted as commercial databases. Both of these databases give information on the occurrence of plant metabolites in nature. The Carotenoids Handbook also lists the UV absorbance spectra of most of the carotenoids known to exist in nature. These were used as reference spectra for comparison of experimental UV spectra if identification by amMS spectra resulted in several possible structures. In the present study structures were assigned to unknown peaks only when both the mass/charge (m/z) ratios and molecular formulas of the ions were in agreement. PDA spectra often provided confirmation for the proposed structures when the reference information was available. Furthermore, the assigned identities often matched with their occurrence in chili peppers as described in the literature and databases. Figure 1 depicts the molecular structures of the identified plant metabolite classes. Tables 1 and 4 show the diagnostic amMS and PDA data used for compound identification in the chili pepper samples. These tables also specify the literature and databases used for confirmation of compound identity. Carotenoids in Chili Peppers. Identification of Carotenoids. A wavelength of 470 nm is very specific to measure carotenoid absorbance. This wavelength was used to depict the carotenoids present in the ‘Awlad Haffouzz’ accession (Figure 2). The PDA chromatograms of the other four accessions appeared identical (not shown). All of the peaks in Figure 2 could be tentatively attributed to carotenoids as no chlorophyll peaks were detected (derived from the PDA profiles of the peaks). The greater part of PDA peaks in the chromatograms contained the same absorption profiles. As described previously,10 carotenoid esterification with fatty acids does not affect the polyene backbone, and therefore esterification has virtually no effect on the absorption spectrum. The characteristic UV profiles of the nonesterified carotenoids, such as capsanthin, antheraxanthin, and zeaxanthin, can therefore also be used to locate their esters. Complementary to PDA detection, fatty acid esters of carotenoids easily fragment during MS ionization, and therefore information on the type of fatty acid moieties (number of carbons, number of double 4819



Article

Figure 3. Chromatogram of the PDA signal at 470 nm depicts the elution profiles of the carotenoids present in the Korba chili pepper accession extract after 1 year of storage, after saponification. Peaks: 1, capsorubin; 2, cucurbitaxanthin A; 3, cis-capsanthin; 4, cis-capsanthin; 5, trans-capsanthin; 6, cis-antheraxanthin; 7, mutatoxanthin; 8, karpoxanthin; 9, cis-zeaxanthin; 10, trans-zeaxanthin; 11, cis-β-apo-8′-carotenal; 12, trans-β-apo-8′carotenal; 13, β-cryptoxanthin; 14, trans-β-carotene; 15, cis-β-carotene.

Figure 4. Correlation of the retention times and m/z values of the compounds identified in the chili peppers with the analytical screening method. The data labels match the compound numbers in Tables 1 and 4

capsanthin, in agreement with previuos findings.10 Other studies10,17,20 have shown that the cis isomers of carotenoids can be identified on the basis of a small hypsochromic shift (shorter wavelength) of the maximum absorbance values and a

bonds) can be obtained from full scan amMS spectra, as described earlier.4,10,13 Most of the carotenoids in the chili pepper samples were esterified with laurate, myristate, and palmitate, and the major carotenoid present in the sample was 4820



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Table 2. Carotenoid Concentrations in the Five Chili Pepper Cultivars Calculated with Analytical Standards and Molar Extinction Coefficients concentration (μg/g dw) compd

identity

4 5 6 19 22 25 3 12 + 13 15 16 17 18 23 24 26 + 27

all-trans-capsanthina cis-antheraxanthina,e all-trans-zeaxanthina β-carotenea all-trans-phytofluenea all-trans-phytoenea cis-capsanthinf capsorubin-myristate + capsanthin-laurateb,g,h,i all-trans-capsanthin-laurateb,g cis-capsanthin-lauratef cis-antheraxanthin-laurateb,e,j all-trans-capsanthin-myristateb,g all-trans-capsanthin-myristateb,g cis-antheraxanthin-myristateb,e,j all-trans-zeaxanthin-laurate + cis-capsanthinpalmitateb,g,h,i all-trans-capsanthin-palmitateb,g cis-zeaxanthin-myristateb,k all-trans-zeaxanthin-myristateb,k all-trans-zeaxanthin-myristateb,k all-trans-capsanthin-dilaurateb,g cis-capsanthin-dilaurateb,g all-trans-capsanthin-laurate-myristateb,g cis-capsanthin-laurate-myristateb,g cis-capsanthin-laurate-myristateb,g all-trans-capsanthin-dimyristateb,g all-trans-capsanthin-laurate-palmitateb,g cis-capsanthin-dimyristateb,g all-trans-capsanthin-myristate-palmitateb,g cis-capsanthin-myristate-palmitateb,g cis-capsanthin-myristate-palmitateb,g all-trans-capsanthin-dipalmitateb,g all-trans-zeaxanthin-dimyristate + all-trans-zeaxanthinlaurate-myristateb,k β-cryptoxanthinc,k capsorubin-lauratec,g β-cryptoxanthin-lauratec,k β-cryptoxanthin-myristatec,k β-cryptoxanthin-palmitatec,k cucurbitaxanthin Ad,k cycloviolaxanthind,l nigroxanthind,k karpoxanthin-laurated,l mutatoxanthin-laurate + cucurbitaxanthin A -laurated,h,k cis-zeaxanthin-laurate + mutatoxanthin-myristated,h,k

30 31 32 33 37 38 41 42 43 45 46 47 48 49 50 51 52 + 53 9 10 36 40 44 1 2 8 11 20 + 21 28 + 29


‘Awlad Haffouzz’

7.93 8.67 10.12 16.34 16.66 17.08 7.77 15.01 15.72 15.85 16.03 16.15 16.83 17.05 17.16 + 17.21

130 27 25 130 27 90

± ± ± ± ± ±

20 5 5 20 6 10

260 ± 50 70 ± 20

‘Tebourba’ 70 18 13.0 120 22 68

± ± ± ± ± ±

10 3 2.5 20 5 9

220 ± 50 80 ± 20

‘Korba’ 100 22 25 80 17 48

± ± ± ± ± ±

20 4 5 10 4 7

180 ± 40 70 ± 10

‘Somâa’ 100 26 9.0 100 21 70

± ± ± ± ± ±

20 5 1.8 20 4 10

310 ± 60 90 ± 20

‘Souk jedid’ 110 24 12.0 110 20 58

± ± ± ± ± ±

20 4 2.3 20 4 8

190 ± 40 50 ± 10

70 430 120 80 160

± ± ± ± ±

20 90 30 20 30

70 390 150 80 190

± ± ± ± ±

20 80 30 20 40

70 320 120 70 160

± ± ± ± ±

20 70 30 10 30

100 540 140 80 190

± ± ± ± ±

20 110 30 20 40

60 450 110 60 200

± ± ± ± ±

10 90 20 10 40

17.83 18 18.16 18.37 19.12 19.32 19.78 19.97 20.13 20.36 20.57 20.72 20.93 21.1 21.19 21.33 21.37 + 21.37

60 11.3 80 39 380 18 700 37 120 700 30 120 370 17 70

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

10 2.3 20 7 80 4 200 8 30 100 6 20 80 4 10

80 19 70 50 420 13.8 800 27 110 800 25 140 500 11.1 90

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

20 4 10 10 90 2.9 200 6 20 200 5 30 100 2.3 20

50 14 90 50 330 20 700 44 120 700 36 130 400 12.6 80

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

10 2.8 20 10 70 4 100 9 20 100 8 30 80 2.7 20

70 13.4 50 70 430 19 900 50 150 800 42 160 370 16 80

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

10 2.7 10 10 90 4 200 10 30 200 9 30 80 3 20

60 14.6 37 28 210 11.2 500 30 80 600 39 110 330 21 80

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

10 2.9 7 5 40 2.4 100 10 20 100 8 20 70 4 20

13.61 13.85 18.8 19.56 20.25 6.77 7.29 12.62 14.77 16.45 + 16.54

30 23 28 20 24 14 11 7.4 30 21

17.52 + 17.54

23 ± 5

80 ± 20 ± ± ± ± ± ± ± ± ± ±

10 7 9 6 8 5 4 2.4 10 7

110 ± 20 29 22 28 28 36 8.4 12 5.9 30 20

± ± ± ± ± ± ± ± ± ±

9 7 9 9 11 2.8 4 1.9 10 7

24 ± 5

120 ± 20 24 15 24 23 28 11 6.4 4.7 30 20

± ± ± ± ± ± ± ± ± ±

7 5 8 7 9 4 2.1 1.6 10 7

29 ± 6

80 ± 10 26 30 27 18 31 14 15 7.4 40 22

± ± ± ± ± ± ± ± ± ±

8 10 9 6 9 5 5 2.4 10 7

27 ± 5

60 ± 10 19 14 12 8 29 11 10 3 20 15

± ± ± ± ± ± ± ± ± ±

6 4 4 2 9 4 3 1 10 5

25 ± 5

a

Carotenoid concentrations calculated with analytical standards and LC-amMS. bQuantitation with standards containing the same polyene backbone (the U (%) of zeaxanthin and capsanthin estimated during validation were used as uncertainties on the measurements of zeaxanthin and capsanthin esters, respectively). cQuantitation with the extinction coefficients (εmol) from the literature.17 dQuantitation with the εmol from a compound with a similar polyene backbone. eBecause antheraxanthin was not available as a reference standard at the time of validation, the average U (%) obtained from validation data was used as uncertainty for the measurement of antheraxanthin derivatives. fToo low intensity for quantitation. gQuantitated with capsanthin standard. hPeak overlap. iPredominantly capsanthin is present. jQuantitated with antheraxanthin standard. kQuantitated with zeaxanthin standard. lQuantitated with violaxantin standard.

depending on the diluent.15 These shifts can be similar to or

characteristic peak around 330−350 nm. Several cis-carotenoids could be detected in the chili pepper samples, although in lesser amounts compared to their trans counterparts. It is generally known that absorbance maxima of carotenoids can shift

even greater than the shifts in maximum absorbances that occur during trans to cis isomerization of carotenoids.15 The correct 4821



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isomer form, cis or all-trans, was therefore attributed only when a reference standard of the compound of interest was available. Table 1 shows the diagnostic amMS and PDA data used for carotenoid identification in the chili pepper samples. During positive APCI, carotenoids predominantly ionized to generate protonated molecules [M + H]+, whereas predominantly radical molecular ions (M•−) were formed during negative APCI. A substantial amount of carotenoid product ions was detected due to in-source fragmentation, thereby providing additional structural information. Five of the 53 detected carotenoid peaks in the chili pepper accessions could be identified with reference standards. Forty-four unknown carotenoids could be tentatively identified on the basis of spectrophotometric and mass spectrometric data, chromatography, databases, and the literature. As the carotenoid content of different Capsicum varieties has been frequently reported, confirmation of identity was often obtained from comparison with the findings of other studies.8,10,13 Four minor unknown carotenoids could not be assigned a tentative structure. These data show that the applied generic apolar screening method performs in the separation and identification of carotenoids as well as earlier reported methods that were optimized for the dedicated identification of carotenoids in chili peppers.10,13 To confirm the identity of the esterified carotenoids, the sample extracts were saponified. Due to circumstances, the saponification procedure was performed after 1 year of storage of the extracts in the dark, at −25 °C. Analysis of the extract before saponification revealed that no significant change had occurred in the carotenoid content during storage. The chromatogram of the saponified extract (Figure 3) shows the profile of the hydrolyzed carotenoids. The peaks of cucurbitaxanthin A (6.77 min), two cis-capsanthins (7.38 and 7.62 min), all-trans-capsanthin (7.78 min), cis-antheraxanthin (8.55 min), all-trans-zeaxanthin (10.05 min), and β-cryptoxanthin (13.61 min) augmented in the chromatogram of the saponified extract, which confirmed the presence of their esterified derivatives in the untreated extract (Figure 2). Furthermore, new peaks appeared in the saponified extract at 5.67, 8.26, 9.25, 9.82, 10.23, 13.61, and 16.88 min that could be identified as capsorubin, mutatoxanthin, karpoxanthin, cis-zeaxanthin, cis-βapo-8′-carotenal, β-cryptoxanthin, and cis-β-carotene, respectively. Their free form was not detected before saponification, which confirms their presence as carotenoid esters in the untreated chili pepper extracts. The three main maximum absorbance wavelengths of the isomers all-trans-lutein and cis-zeaxanthin are in close agreement and are only distinguishable in UV absorbance by a characteristic cis peak of cis-zeaxanthin. A cis peak was clearly detected in the PDA spectrum of peak 9 in the chromatograms of the saponified extracts (Figure 3), indicating the presence of cis-zeaxanthin. It has also been reported that zeaxanthin and lutein are distinguishable by in-source fragmentation in positive APCI mode by the relative heights of the [M + H]+ and [M + H − H2O]+ peaks.4 Comparison of the MS spectrum of peak 9 in Figure 3 showed close resemblance with that of a transzeaxanthin reference standard and differed from the spectrum of a trans-lutein reference standard. Peak 9 was therefore designated cis-zeaxanthin. An overview of the chromatographic separation versus molecular mass of all the compounds identified in the chili pepper extracts during this study is given in Figure 4. Although most of the compounds were separated by chromatography, orthogonal detector selectivity remains essential for compound

differentiation. This can in many cases not be achieved by PDA detection alone and highlights the added value of high-end instrumentation in this research field. Quantitation of Carotenoids with Analytical Reference Standards. The concentrations of carotenoids for which an analytical standard was available were calculated with amMS (Table 2). The concentrations calculated with reference standards represent only a small fraction of the total number of carotenoids in the samples. Most of the xanthophylls in the samples appeared esterified, for which analytical standards are not commercially available. Quantitation of Carotenoids with Extinction Coefficients. (a) Quantitation of Carotenoids Using Standards with the Same Polyene Backbone. In UV analysis, carotenoids can be quantitated by using reference standards having the same polyene backbone and thus extinction coefficient.15 However, molar extinction coefficients (εmol) can be used for the calculation of the concentration expressed in weight units (e.g., μg/g) only when the molecular mass is known. To our knowledge earlier publications never used this quantification strategy in combination with amMS. Accurate mass MS enables the tentative identification of unknown carotenoids and therefore the designation of its polyene backbone and molecular weight. An LC-PDA-amMS configuration combined with the use of extinction coefficients should therefore allow unparalleled speed in the identification and quantification of unknown carotenoids. During this study, the majority of carotenoid esters, predominantly capsanthin esters, were quantitated with this procedure, that is, with a reference standard of free capsanthin (Table 2). The uncertainties represent the U of capsanthin estimated during method validation. Because UV absorbance profiles of carotenoids generally overlap, coeluting carotenoids could not be quantitated separately. In the few cases that coelution happened, the sum of both compounds was quantitated with the standard linked to the carotenoid with the highest contribution to the signal, as was judged from the PDA and MS spectra (Table 2). To check the validity of quantitating carotenoid esters with standards containing the same polyene backbone, the total capsanthin content in the extracts without saponification (2100−3300 mg/kg dw) was compared to the free capsanthin concentration in the saponified extracts (2600−3800 mg/kg dw). The total capsanthin content in the extracts without saponification was calculated by conversion of the ester concentrations to free capsanthin concentrations followed by summation. The recoveries of the total capsanthin content in the extracts without saponification with respect to the capsanthin concentrations in the saponified extracts ranged between 83 and 87%, within the U of capsanthin (21%). These results demonstrate the suitability of using standards having the same polyene backbone as the analytes of interest for the quantitation of compounds for which no reference standards are available. The slightly lower concentrations obtained for the samples without saponification can be explained by the high density of peaks in the chromatograms, whereby the baseline is artificially elevated and integration is slightly biased. Care should, however, be taken when all-trans-carotenoids are used to quantitate cis-carotenoids, as extinction coefficients of ciscarotenoids are generally smaller.21 Only small amounts of cis isomers were present in the chili pepper samples (4.5−6.0% for capsanthin). Higher percentages of cis isomers were detected (7.5−19% for capsanthin) in the saponified extracts. It is 4822



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Table 3. Concentrations of Fat-Soluble Vitamins and Free Phytosterols in the Chili Pepper Accessions, Calculated with Reference Standards concentration (μg/g dw)

a

compd

identity


54 55 56 60 61 62

γ-tocopherola α-tocopherol phylloquinone stigmasterol campesterolb β-sitosterolb

10.98 11.87 13.37 14.55 14.59 15.19

‘Awlad Haffouzz’

Generic Characterization of Apolar Metabolites in Red Chili Peppers

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