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HPLC-DAD-APCI-MSn analysis of the genuine carotenoid pattern of pineapple (Ananas comosus [L.] Merr.) infructescence
Journal Pre-proofs HPLC-DAD-APCI-MS n analysis of the genuine carotenoid pattern of pineapple (Ananas comosus [L.] Merr.) infructescence Christof B. Steingass, Kathrin Vollmer, Peter E. Lux, Carolin Dell, Reinhold Carle, Ralf M. Schweiggert PII: DOI: Reference:
S0963-9969(19)30595-2 https://doi.org/10.1016/j.foodres.2019.108709 FRIN 108709
To appear in:
Food Research International
Received Date: Revised Date: Accepted Date:
7 June 2019 17 September 2019 21 September 2019
Please cite this article as: Steingass, C.B., Vollmer, K., Lux, P.E., Dell, C., Carle, R., Schweiggert, R.M., HPLCDAD-APCI-MS n analysis of the genuine carotenoid pattern of pineapple (Ananas comosus [L.] Merr.) infructescence, Food Research International (2019), doi: https://doi.org/10.1016/j.foodres.2019.108709
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© 2019 Published by Elsevier Ltd.
HPLC-DAD-APCI-MSn analysis of the genuine carotenoid pattern of pineapple (Ananas comosus [L.] Merr.) infructescence
CHRISTOF B. STEINGASS1,2*, KATHRIN VOLLMER2,3, PETER E. LUX2,3, CAROLIN DELL2, REINHOLD CARLE2,4, RALF M. SCHWEIGGERT1
1
Department of Beverage Research, Chair Analysis and Technology of Plant-based Foods, Geisenheim
University, Von-Lade-Strasse 1, 65366 Geisenheim, Germany 2
Institute of Food Science and Biotechnology, Chair Plant Foodstuff Technology and Analysis, University
of Hohenheim, Garbenstrasse 25, 70599 Stuttgart, Germany 3
Institute of Nutritional Sciences, Chair Food Biofunctionality, University of Hohenheim, Garbenstrasse
28, 70599 Stuttgart, Germany 4
Biological Science Department, Faculty of Science, King Abdulaziz University, P.O. Box 80257, Jeddah
21589, Saudi Arabia
Corresponding author. Phone: +49 6722 502 314. E-mail: [email protected]
*
Abstract The genuine carotenoid pattern of pineapple infructescence was assessed by HPLC-DAD-APCI-MSn analysis. Prevailing pigments in the shell of ‘MD2’ (syn. “Extra Sweet”) fruits were (all-E)-lutein and (all-E)-β-carotene, in addition to chlorophylls a and b. The edible flesh contained (all-E)-violaxanthin, (all-E)-β-carotene and diverse esters of (9Z)violaxanthin with caprylic, capric, lauric, and myristic acid. The latter esters have been reported for the first time as pineapple constituents. Total carotenoid concentrations in the edible fractions of the four varieties ‘Sugar Loaf’, ‘Smooth Cayenne’, ‘MD2’, and ‘Queen Victoria’ cultivated in Ghana ranged between 29 and 565 µg/100 g of fresh weight (FW). Total carotenoids in the flesh of fully ripe ‘MD2’ fruits exported by air freight amounted to 302 µg/100 g of FW, those in green ripe samples dispatched by sea freight to 359–432 µg/100 g of FW. All yellow fleshed cultivars exhibited a highly similar qualitative carotenoid profile.
Keywords: carotenoids; xanthophyll esters; tropical fruit
Abbreviations: APCI, atmospheric pressure chemical ionisation; BHA, butylated hydroxyanisole; BHT, butylated hydroxytoluene; CID, collision induced dissociation; dah, days after harvest; FW, fresh weight; HPLC-DAD, high performance liquid chromatography-diode array detection; LP, light petroleum; MSn, multiple-stage mass spectrometry; RAE, retinol activity equivalent; tBME, tert-butyl methyl ether; tR, retention time.
1
Introduction
After banana and citrus, pineapples (Ananas comosus [L.] Merr.) belong to the most important tropical fruits in the international trade (Lobo & Paull, 2017). Total exports of fresh pineapples from their tropical production countries to the EU and the US markets were estimated at 3.35 million tonnes in 2018. The most important supplier was Costa Rica (Altendorf, 2018). Apart from the overall appearance, odour (i.e., aroma, pineapple flavour), texture, juiciness, and sweetness, the colour of the flesh has been found to be among the most important attributes determining consumer acceptance of fresh pineapples (Ramsaroop & Saulo, 2007). Consequently, one of the targets of past pineapple breeding programs was to develop intensely coloured yellow to orange fleshed varieties (Lobo & Paull, 2017). The hybrid ‘MD2’, often marketed as “Extra Sweet” or “Golden Ripe”, was launched by Del Monte Fresh Produce Hawaii Inc. and represents one of the most important varieties for fresh fruit export (Bartholomew et al., 2003). Another newly bread cultivar termed ‘FLHORAN41’ with a deep yellow flesh and an orange-red to scarlet coloured shell resulted from a breeding program of CIRAD (Brat et al., 2004). Of the more than 100 pineapple varieties, merely 6 to 8 are cultivated commercially (Lobo & Paull, 2017). In particular, large-scale companies exporting sea freighted pineapples produce the yellow fleshed ‘MD2’, reaching market shares of approx. 90 and 85% in the US and the EU, respectively (Loeillet, 2014). ‘MD2’ represents approx. 95% of the pineapples grown in Ghana. ‘Smooth Cayenne’ (4%) and ‘Sugar Loaf’ (1%) are offered on the European niche market of air freighted premium fruits (Loeillet & Paqui, 2010). Air freighted ‘Queen Victoria’ (or ‘Queen’) pineapples available in Europe originate from South Africa, Mauritius, Reunion, and Ghana (CBI Ministry of Foreign Affairs, 2014). The total carotenoids in the edible flesh of fully ripe ‘Smooth Cayenne’ and ‘FLHORAN41’ pineapples were reported to amount to 246 and 580 μg/100 g of fresh weight (FW), respectively. Prevailing pigment in both genotypes was β-carotene, accounting for 31–38% of the total carotenoids. Significantly elevated total carotenoids of 2,545 and 2,915 μg/100 g of FW, respectively, were found in the shell of the aforementioned two pineapple varieties. However, carotenoid pigments other than β-carotene have not been identified (Brat et al., 2004). Similarly, merely β-carotene has been assigned to one of 14 different carotenoids found in ‘Tropical Gold’ pineapple flesh (Gil et al., 2006). Yano et al. (2005) have reported total carotenoid concentrations of ~200 μg/100 g of FW in the flesh of an unspecified pineapple genotype. Three major pigments in the alkaline saponified extract were assigned to (9Z)-violaxanthin (50% of the total concentration), (all-E)-violaxanthin
(28%), and (all-E)-β-carotene (22%). However, detailed studies of the carotenoid profiles are virtually unavailable and, particularly, the genuine carotenoid pattern has been disregarded in previous studies. Therefore, the present contribution targeted at the in-depth characterisation of carotenoids in the shell and pulp of the pineapple infructescence applying HPLC-DAD-APCI-MSn. In addition, the carotenoids in the edible fraction of ‘Queen Victoria’, ‘Sugar Loaf’, ‘Smooth Cayenne’, and ‘MD2’ (syn. “Extra Sweet”) pineapples harvested at full maturity were quantitated by HPLC-DAD. Fruits of ‘MD2’ were additionally harvested at a green ripe stage, exported by sea freight and analysed upon their arrival in Europe to explore possible influences of postharvest procedures on the carotenoid patterns.
2 2.1
Materials and methods Pineapple samples
All studied pineapples were imported from Ghana and provided by the fruit distributer Schumacher (Filderstadt-Bernhausen, Germany). Fully ripe ‘Queen Victoria’, ‘Sugar Loaf’, ‘Smooth Cayenne’, and ‘MD2’ (syn. “Extra Sweet”) fruits were imported via air freight and analysed approx. 2 days after harvest (dah). In addition, ‘MD2’ pineapples were harvested at a premature green ripe stage and brought to Germany by sea freight. The latter samples were analysed upon their arrival (14 dah) and after a further week of storage at room temperature (21 dah). A detailed description of the harvest and postharvest procedures for sea and air freighted pineapples has been provided elsewhere (Steingass et al., 2014). Five individual fruits (n = 5) of each variety or ripening stage were randomly selected and analysed in analytical duplicates.
2.2
Carotenoid analyses
Sample preparation Carotenoids were extracted from fresh pineapple peel and pulp as described previously (Schweiggert et al., 2011) including the following modifications. For immediate neutralisation of genuine fruit acids, 200 g of the respective, carefully and minimally cut pineapple tissue was homogenised with the admixture of 4.0 g calcium carbonate and 2.0 g sodium hydrogen carbonate using a laboratory blender (Grindomix GM 200, Retsch, Haan, Germany). An aliquot of 2.0 ± 0.2 g of the obtained puree was immediately mixed with 3 mL of the extraction solution (methanol/ethyl acetate/light petroleum (LP), 1/1/1, v/v/v) containing each 0.1 g/L 2,6-di-tert-butyl-p-cresol (BHT) and 3-tert-butyl-4-hydroxyanisole (BHA). The sample was homogenised using an Ultra-Turrax® homogeniser (type 18/10, IKA®-Werke, Staufen, Germany) and
centrifuged for 3 min at 2,500 rpm, equalling 671 × g (Labofuge 200, Heraeus Material Technology, Hanau, Germany). The upper organic layer was recovered and the remainder was re-extracted twice with each 3 mL of the aforementioned extraction mixture. The combined organic phase was subsequently washed twice with each 3 mL ultrapure H2O, dried with anhydrous sodium sulphate, and evaporated to dryness under reduced pressure at 30 °C. Carotenoid extracts were redissolved in 0.3 mL tBME/methanol (1/1, v/v) and filtered over a 0.45 μm polytetrafluoroethylene filter (Chromafil® O45/15 MS, Macherey-Nagel, Düren, Germany) into amber glass vials prior to HPLC analyses.
Alkaline hydrolysis For cleavage of xanthophyll esters, the above-mentioned carotenoid extract was dissolved in 2 mL LP containing each 0.1 g/L BHA and BHT. After adding 2 mL of 15% w/w methanolic potassium hydroxide, the sample headspace was flushed with nitrogen and the extract stirred for 3 h at room temperature. Following the admixture of 5 mL H2O, the carotenoids were extracted with 2 mL diethyl ether/n-hexane (1/1, v/v). The organic layer was collected and the aqueous remainder was re-extracted twice with each 2 mL diethyl ether. The combined organic phases were washed twice with each 2 mL H2O. After evaporation to dryness, the carotenoids were dissolved in 0.3 mL tBME/MeOH (1/1, v/v) and membrane filtered as detailed above.
HPLC-DAD analyses HPLC-DAD analyses were performed using an HPLC system consisting of a separation module type 2695 equipped with a diode array detector type 2996 (both Waters, Eschborn, Germany) and a column oven thermostat Jetstream II plus (Thermotechnic Products, Langenzersdorf, Austria). Control of the system and data evaluation was performed using Millenium 32® Chromatography Manager software (Waters). Carotenoids were separated using a C30 reversed phase column (150 × 3.0 mm i.d., particle size dp = 3 μm, YMC Europe, Dinslaken, Germany) equipped with a C30 guard column (10 × 3.0 mm i.d., dp = 3 μm, YMC Europe). Eluents were tertiary mixtures of tBME/methanol/H2O (eluent A: 5/91/4, v/v/v; eluent B: 90/8/2, v/v/v). The gradient program was as follows: 100 to 50% A (60 min), 50 to 0% A (10 min), 0 to 100% A (5 min), isocratic hold at 100% A (5 min) at a flow rate of 0.42 mL/min. Prior to each injection (10 μL), the system was re-equilibrated for 5 min (100% A). Column oven temperature was set to 25 °C. Total run time was 80 min. Detection
wavelengths for xanthophylls, β-carotene, and chlorophylls were 439, 452, and 660 nm, respectively. UV/Vis spectra were recorded in the range of 200–800 nm. Linear calibration curves of (all-E)-β-carotene and (all-E)-violaxanthin were used for quantitation. Concentrations of stock solutions were determined by spectrophotometry using the appropriate molar extinction coefficients (Britton et al., 1995). Molecular weight correction factors were applied where appropriate. Total carotenoid levels were calculated as the sum of the concentrations of violaxanthin, its esters, unidentified minor constituents (as violaxanthin equivalents), and β-carotene.
HPLC-DAD-APCI-MSn analyses For HPLC-DAD-APCI-MSn experiments, a series 1100 HPLC system with a G1315B diode array detector (both Agilent, Waldbronn, Germany) was coupled on-line to an Esquire 3000+ ion trap mass spectrometer (Bruker Daltonik, Bremen, Germany) with an atmospheric pressure chemical ionisation (APCI) source. Column and HPLC settings were as detailed above. Mass spectra were recorded in the alternating polarity mode at a scan range of m/z 100–1100. All MS-parameters were adjusted as described previously (Schweiggert et al., 2012). Control of the system and data evaluation was achieved with ChemStation for LC version A.00.03 (Agilent) and Esquire software version 5.1 (Bruker), respectively.
2.3
Reagents
Chlorophyll a, chlorophyll b, and (all-E)-β-carotene were purchased from Sigma Aldrich (Taufkirchen, Germany), (all-E)violaxanthin, (all-E)-neoxanthin, and (all-E)-lutein from CaroteNature (Ostermundingen, Switzerland). Calcium carbonate, sodium hydrogen carbonate, ethyl acetate, diethyl ether, n-hexane, and tBME were obtained from Merck (Darmstadt, Germany). Potassium hydroxide, sodium sulphate, methanol, and LP (boiling point 40–60 °C) were from VWR International (Darmstadt, Germany). BHA and BHT were purchased from Fluka Chemie (Buchs, Switzerland). An arium® 611 UV (Sartorius, Göttingen, Germany) ultrapure water system was used for preparation of ultrapure H2O.
2.4
Statistics
Statistical analyses were performed using SAS software version 9.1 (SAS Institute, Cary, NC, USA). Normality and homogeneity of variances were examined by Shapiro-Wilk’s test (p < 0.05) and Levene’s test (p < 0.05), respectively.
Significant differences (p < 0.05) of means among the individual samples were determined by one-way analysis of variance (ANOVA) and Tukey's test.
3 3.1
Results and discussion Compound assignment by HPLC-DAD-APCI-MSn
Fig. 1a and b display representative HPLC-DAD chromatograms of a pineapple peel extract recorded at 660 and 452 nm, respectively. The chromatogram of genuine flesh carotenoids at a detection wavelength of 452 nm is shown in Fig. 1c. After alkaline hydrolysis, the signal intensities of compounds 8, 10–15 and 18–21 vanished, whereas those of the five early eluting compounds 1–5 increased, thus indicating the presence of esterified xanthophylls (Fig. 1d). Retention times (tR), UV/Vis absorption, and mass spectra of the individual pigments are compiled in Table 1 and their assignments will be discussed in the following sections.
Chlorophylls Three compounds were detected at 660 nm in the peel extract (Fig. 1a). Compounds 6 and 9 exhibiting protonated molecules [M + H]+ at m/z 907 and 893 were identified as chlorophyll b and chlorophyll a, respectively. CID of both chlorophylls yielded abundant fragment ions from the elimination of the phytadiene (C20H38) moiety [M + H − 278]+ at m/z 629 and 615, respectively. In addition, both compounds displayed fragment ions from the additional losses of methanol (32 amu) at m/z 597 and 583 ([M + H − 278 − 32]+) and of carbon monoxide (28 amu) at m/z 569 and 555 ([M + H − 278 − 32 − 28]+). The sequential eliminations of methanol and carbon monoxide reflected the loss of the entire carboxymethoxy group from the ε-ring together with an H atom (60 amu), thus being in accordance with Verzegnassi et al. (2000). Retention times, UV/Vis spectra, and mass spectral data of compounds 6 and 9 matched those of the analytical chlorophyll b and a standards, respectively. Compound 17 assigned to pheophytin a displayed protonated molecules [M + H]+ at m/z 871 that were 22 amu lighter than those of compound 9 (chlorophyll a). This lower molecular weight can be attributed to the replacement of the central Mg2+ ion (24 amu) by two protons. In agreement with our observations for compound 9, the MS2 experiment on pheophytin a yielded abundant product ions at m/z 593 ([M + H − 278]+) and 533 ([M + H − 278 − 60]+) from the loss of phytadiene and
the additional elimination of the carboxymethoxy group (Verzegnassi et al., 2000; van Breemen et al., 1991; Kaiser et al., 2012) (Table 1).
Carotenes and free xanthophylls Compound 16 was detected in both peel and pulp extracts (Fig. 1) and exhibited molecular ions M−• at m/z 536 and protonated molecules [M + H]+ at m/z 537. By comparing tR (43.9 min), UV/Vis, and mass spectra to those of an authentic reference standard, 16 was identified as (all-E)-β-carotene. The product ions observed in the APCI(+)-MS2 experiment were in agreement with previous reports (van Breemen et al., 2012; Schex et al., 2018). Compound 7 assigned to (all-E)-lutein displayed an abundant signal at 452 nm in the peel extract (Fig. 1b) and molecular ions M−• at m/z 568 in the APCI(−)-MS1 spectrum. In the positive ion mode, in-source fragments from the elimination of water were detected at m/z 551 ([M + H − H2O]+). Then, the CID MS2 spectrum of this in-source fragment ion presented a base peak at m/z 533 ([M + H − 2 H2O]+). The product ions at m/z 495 ([M + H − H2O − 56]+) and 477 ([M + H − 2 H2O − 56]+) may be attributed to the retro-Diels-Alder cleavage of the ε-ring (56 amu) and water eliminations (18 amu). The fragment ions at m/z 459 ([M + H − H2O − 92]+) presumably resulted from the elimination of toluene (92 amu) from the polyene chain of the dehydrated precursor ion. The characteristic product ions at m/z 429 ([M + H − H2O − 122]+) have been attributed to the eliminations of hydroxylated ε- or β-rings. This mass fragmentation was in agreement with previous reports (Schex et al., 2018; Ziegler et al., 2015; Crupi et al., 2012). The assignment was further confirmed by comparison of tR, UV/Vis and mass spectra of compound 7 with those of an authentic reference standard. Five early eluting compounds (1–5) in the saponified pulp extract (Fig. 1d) had protonated molecules [M + H]+ at m/z 601, providing evidence of xanthophyll molecules with four oxygen atoms. CID resulted in abundant fragment ions at m/z 583 ([M + H − H2O]+) and 565 ([M + H − 2 H2O]+), likely originating from elimination of water (18 amu). In addition, the common product ions at m/z 509 [M + H − 92]+) and 491 ([M + H − H2O − 92]+) appeared in the MS2 spectra that can be attributed to the neutral loss of toluene (92 amu) as discussed above. Product ions at m/z 547 ([M + H − 3 H2O]+) and m/z 393 were exclusively detected in the MS2 experiment of compound 2 that have been attributed to the threefold elimination of water and the cleavage of the double bond in allylic position to the allenic carbon of (all-E)-neoxanthin, respectively (De Rosso & Mercadante, 2007).
Further distinctive fragment ions at m/z 221 and 181 have been attributed to the cleavage of the polyene chain between positions 10,11 (or 10´,11´) and 8,9 (or 8´,9´), respectively, of a xanthophyll with a 3-hydroxy-5,6- or 5,8-epoxidated βring (Rivera et al., 2014). Recently, cyclic oxonium ions have been proposed to generate the aforementioned fragment ions after epoxy-oxepinoid and epoxy-furanoid rearrangements of the protonated molecules under electrospray ionisation (ESI) conditions (Neto et al., 2016). On the basis of the mass fragmentation detailed above and the Vis absorption maxima (λmax) at 416, 439, and 469 nm, compound 1 was identified as (all-E)-violaxanthin. Compound 2 displayed slightly shorter Vis λmax at 413, 436, and 464 nm and the spectral shape resembled that of the structural isomer (all-E)-neoxanthin. The assignments of compounds 1 and 2 were confirmed by comparison of retention times as well as UV/Vis and mass spectral characteristics to those of authentic reference standards. Compound 3 displayed Vis λmax at 399, 422, and 448, thus being congruently ~20 nm shorter than those observed for (allE)-violaxanthin (1). The mass spectral behaviour of compounds 1 and 3, however, was virtually identical. This and particularly the hypsochromic shift indicated the rearrangement of one 5,6- to a 5,8-epoxy-group and thus, 3 was assigned to (all-E)-luteoxanthin (De Rosso & Mercadante, 2007). Compound 4 displayed a spectral shape and a DIII/DII ratio of 87% resembling those of (all-E)-violaxanthin (1). The 3–4 nm shorter Vis λmax at 412, 436, and 465 nm indicated a corresponding mono-cis-isomer (Britton et al., 1995). On the basis of the UV/Vis absorption maxima and the previously reported elution order on a C30 stationary phase, compound 4 was tentatively identified as (9Z)-violaxanthin (Meléndez-Martínez et al., 2007). Accordingly, compound 5 with Vis λmax at 397, 417, and 443 was assigned to a corresponding 5,6-, 5,8-epoxide, i.e., (9Z)- or (9´Z)-luteoxanthin. The elution order of (allE)-luteoxanthin prior to the two possible (9Z)-isomer was in accordance to literature (Petry & Mercadante, 2018). Noteworthy, (all-E)-neoxanthin (2), (all-E)-luteoxanthin (3), and (9Z)-luteoxanthin (5) were merely detected in the saponified pulp extract (Fig. 1d) and thus, may represent cleavage products of minor esters or possibly workup artefacts in case of the latter two 5,8-epoxides.
Xanthophyll esters Isobaric esters of (all-E)- and (9Z)-violaxanthin (compounds 8/12, 11/13, 15/18, and 20/21) cannot be distinguished based on their MSn fragmentations. However, the UV/Vis spectra of the xanthophyll esters resembled those of the corresponding
free xanthophylls discussed above, permitting the assignment of geometrical isomers. The violaxanthin monoesters (8, 10– 15, and 18) detected in the genuine pulp extract (Fig. 1c) were characterised by distinctive fragment ions generated by the neutral losses of the fatty acid moieties of 144, 172, 200, and 228 amu for caprylic, capric, lauric, and myristic acid, respectively, as well as by the neutral loss of toluene (92 amu) and the elimination of water (18 amu). Exemplarily, APCI(+)MS2 of the most abundant ester, assigned to (9Z)-violaxanthin laurate (13), with protonated molecules [M + H]+ at m/z 783, displayed product ions at m/z 765 ([M + H − H2O]+), 747 ([M + H − 2 H2O]+), 691 ([M + H − 92]+), 673 ([M + H − 92 − H2O]+), 583 ([M + H − 200]+), 565 ([M + H − 200 − H2O]+), and 547 ([M + H − 200 − 2 H2O]+) (Fig. 2). Moreover, as observed in the MS2 spectra of the free xanthophylls 1–5, the aforementioned fragment ions at m/z 221 were detected at a low abundance, substantiating the presence of 5,6- or 5,8-epoxidated β-rings carrying hydroxyl groups (not shown in Table 1). The minor compound 19 presented protonated molecules [M + H]+ at m/z 937. CID resulted in product ions from the eliminations of water and toluene as discussed above. The distinctive product ions at m/z 765 ([M + H − 172]+), 737 ([M + H − 200]+), and 565 ([M + H − 172 − 200]+) indicated a diester with capric (172 amu) and lauric acid (200 amu) as acyl moieties (Table 1). Accordingly, compound 19 was assigned to (9Z)-violaxanthin caprate-laurate. The compounds 20 and 21 both showed protonated molecules at m/z 965 and common product ions at m/z 947 ([M + H − H2O]+), 765 ([M + H − 200]+), 747 ([M + H − 200 − H2O]+), and 565 ([M + H − 200 − 200]+). On the basis of their mass fragmentations and UV/Vis absorption spectra that resembled those of peaks 1 and 4, they were tentatively assigned to (all-E)- and (9Z)violaxanthin dilaurate.
3.2
Quantitation of pulp carotenoids by HPLC-DAD
Total carotenoids in the edible portion of all pineapple cultivars assessed are compiled in Table 2 and ranged from 29 to 565 µg/100 g of FW. Hereby, the lowest levels were determined in the white fleshed ‘Sugar Loaf’, whilst the maximum values were found in the intensely yellow fleshed ‘Queen Victoria’. Total carotenoid levels determined among the yellow fleshed pineapples were comparable to those previously reported in ‘Smooth Cayenne’ and ‘FLHORAN41’ pineapples amounting to 246 and 580 μg/100 g of fresh weight (FW), respectively (Brat et al., 2004). In general, pineapples represent nutritional sources containing “low” (0–100 µg/100 g) to “moderate” (100–500 µg/100 g) carotenoid levels according to the classification proposed by Britton et al. (2009). Nevertheless, the high proportions of (Z)-isomers observed may indicate
a deposition of the carotenoids including the provitamin A-active β-carotene in the chromoplasts in a highly bioavailable, lipid-dissolved and/or liquid-crystalline state (Schweiggert & Carle, 2017). However, to the best of our knowledge, the structure of the pigment-bearing plastids in the pineapple infructescence as well as the bioavailability of the contained carotenoids has not been described in the literature. Among the ‘MD2’ fruits, pineapples harvested green ripe displayed highest total carotenoid levels of 432 µg/100 g of FW upon their arrival in Europe (14 dah). After postharvest storage for another seven days (21 dah), total carotenoid levels dropped by 17% to 359 µg/100 g of FW. This decline may be attributed to the isomerisation and degradation of carotenoids caused by the release of acids from bruised cells (Gortner & Singleton, 1961), but also to enzymatic reactions. The cooxidation of carotenoids by lipoxygenase or the cleavage of (9´Z)-neoxanthin and (9Z)-violaxanthin as an initial step in the biosynthesis of the stress hormone abscisic acid are worth mentioning here (Britton et al., 2008). Interestingly, ‘MD2’ pineapples allowed to ripen completely attached to the plant, i.e., being harvested at full maturity, displayed lower total carotenoid levels (302 µg/100 g of FW) than those harvested green ripe (359–432 μg/100 g of FW). The abovementioned decompartmentalisation and related effects (acids, enzymes) might have been more pronounced in the fully ripe fruits due to their softer tissue and more labile cell structures (Gortner & Singleton, 1961), resulting in lower carotenoid levels in the fully ripe fruits. This hypothesis is supported by the slight increase of the peroxidase activity reported during the last 20 days of pineapple maturation (Gortner, 1965). Such market differences have also been found in the profiles of volatiles when comparing air freighted pineapples to their sea freighted counterparts (Steingass et al., 2014; Steingass et al., 2016). Noteworthy, among all samples, (9Z)-violaxanthin laurate (C12, 13) was the prevailing xanthophyll ester. In the yellow fleshed samples, further abundant esters were (9Z)-violaxanthin caprate (C10, 12), myristate (C14, 14), and palmitate (C16, 18), in addition to the less abundant (9Z)-violaxanthin caprylate (C8, 10) and further esters. Thus, the determined pineapplespecific pattern of acyl moieties was mainly based on C10 to C16 fatty acids as previously found in numerous other fruits (Hornero-Méndez, 2019). Merely one non-oxygenated carotenoid (carotene) was found, namely (all-E)-β-carotene (16). This provitamin A carotenoid contributed to approx. 15% of the total carotenoids in fully ripe ‘Queen Victoria’, ‘Sugar Loaf’, and ‘Smooth Cayenne’ flesh and 7–13% in the ‘MD2’ samples assessed. Elevated proportions of β-carotene of 20 and 31–38%, respectively, have been previously reported in the literature (Brat et al., 2004; Yano et al., 2005). The retinol activity equivalents (RAE) as calculated by the guidelines of the U.S. Institute of Medicine (2010) of our samples ranged
from 0.3 to 7.3 µg RAE/100 g. This range was similar to the RAEs of oranges (4 µg RAE/100 g), but substantially lower than those of carrot roots (1000 RAE/100 g), leafy vegetables (343 µg RAE/100 g), and apricots (125 µg RAE/100 g) as reported by Solomons & Orozco (2003). In brief, the colour of yellow fleshed varieties may particularly be determined by (9Z)-violaxanthin esters, whereas the corresponding (all-E)-isomers (8, 11, 15, and 20) merely occurred as minor constituents.
3.3
Conclusions
To the best of our knowledge, the present contribution is the first detailed study into the genuine carotenoid patterns of pineapples. We found diverse esters of violaxanthin with caprylic, capric, lauric, and myristic acid as pigments determining the colour of the pineapple flesh. Among the esterified xanthophylls, monoesters prevailed, whereas diesters were only detected as minor constituents. Interestingly, the pineapple shell displayed a chloroplast-specific pigment profile comprising (all-E)-violaxanthin, (all-E)neoxanthin, (all-E)-lutein, and (all-E)-β-carotene. By contrast, esterified (9Z)-violaxanthin prevailed in the edible flesh. The provitamin A value of pineapple fruit was shown to be moderate. Besides elucidating the deposition form and bioavailability of pineapple carotenoids, future studies may explore the carotenoid composition of additional pineapple varieties considering a larger sample set and fruits from different provenances as well as the influence of processing (juice extraction and pasteurisation), on the carotenoid pattern of pineapple juice. In addition, the pineapple shell as a valuable source for the recovery of functional compounds such as polyphenols and carotenoids merits further investigation.
Acknowledgements We cordially thank Frank Oberschilp (Peelco, Accra, Ghana) and Fritz Schumacher (Schumacher, Filderstadt-Bernhausen, Germany) for kindly providing the pineapple fruits.
Dedication In grateful remembrance of Priv.-Doz. Dr. habil. Hans-Georg Schmarr (1961–2019), Neustadt an der Weinstraße, Germany.
4
References
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Hornero-Méndez, D. (2019). Chapter 7. Occurrence of carotenoid esters in foods. In A. Z. Mercadante (Ed.), Carotenoid Esters in Foods (pp. 182–284). Cambridge: Royal Society of Chemistry. Kaiser, A., Brinkmann, M., Carle, R., & Kammerer, D. R. (2012). Influence of thermal treatment on color, enzyme activities, and antioxidant capacity of innovative pastelike parsley products. Journal of Agricultural and Food Chemistry, 60, 3291–3301. Lobo, M. G., & Paull, R. E. (Eds.) (2017). Handbook of pineapple technology: Production, postharvest science, processing and nutrition. Chichester, UK, Hoboken, NJ: John Wiley & Sons. Loeillet, D. (2014). World pineapple market. Fruitrop, 228, 18–29. Loeillet, D., & Paqui, T. (2010). Pineapple. Fruitrop, 176, 23–50. Meléndez-Martínez, A. J., Vicario, I. M., & Heredia, F. J. (2007). Geometrical isomers of violaxanthin in orange juice. Food Chemistry, 104, 169–175. Neto, F. C., Guaratini, T., Costa-Lotufo, L., Colepicolo, P., Gates, P. J., & Lopes, N. P. (2016). Re-investigation of the fragmentation of protonated carotenoids by electrospray ionization and nanospray tandem mass spectrometry. Rapid Communications in Mass Spectrometry, 30, 1540–1548. Petry, F., & Mercadante, A. (2018). New method for carotenoid extraction and analysis by HPLC-DAD-MS/MS in freeze-dried citrus and mango pulps. Journal of the Brazilian Chemical Society, 29, 205–215. Ramsaroop, R. E. S., & Saulo, A. A. (2007). Comparative consumer and physicochemical analysis of Del Monte Hawai'i Gold and Smooth Cayenne pineapple cultivars. Journal of Food Quality, 30, 135–159. Rivera, S. M., Christou, P., & Canela-Garayoa, R. (2014). Identification of carotenoids using mass spectrometry. Mass Spectrometry Reviews, 33, 353–372. Schex, R., Lieb, V. M., Jiménez, V. M., Esquivel, P., Schweiggert, R. M., Carle, R., & Steingass, C. B. (2018). HPLCDAD-APCI/ESI-MSn analysis of carotenoids and α-tocopherol in Costa Rican Acrocomia aculeata fruits of varying maturity stages. Food Research International, 105, 645-653. Schweiggert, R. M., & Carle, R. (2017). Carotenoid deposition in plant and animal foods and its impact on bioavailability. CRC Critical Reviews in Food Science and Nutrition, 57, 1807-1830.
Schweiggert, R. M., Steingass, C. B., Esquivel, P., & Carle, R. (2012). Chemical and morphological characterization of Costa Rican papaya (Carica papaya L.) hybrids and lines with particular focus on their genuine carotenoid profiles. Journal of Agricultural and Food Chemistry, 60, 2577–2585. Schweiggert, R. M., Steingass, C. B., Mora, E., Esquivel, P., & Carle, R. (2011). Carotenogenesis and physico-chemical characteristics during maturation of red fleshed papaya fruit (Carica papaya L.). Food Research International, 44, 1373–1380. Solomons, N. W., & Orozco, M. (2003). Alleviation of vitamin A deficiency with palm fruit and its products. Asia Pacific Journal of Clinical Nutrition, 12, 373–384. Steingass, C. B., Dell, C., Lieb, V., Mayer-Ullmann, B., Czerny, M., & Carle, R. (2016). Assignment of distinctive volatiles, descriptive sensory analysis and consumer preference of differently ripened and post-harvest handled pineapple (Ananas comosus [L.] Merr.) fruits. European Food Research and Technology, 242, 33–43. Steingass, C. B., Grauwet, T., & Carle, R. (2014). Influence of harvest maturity and fruit logistics on pineapple (Ananas comosus [L.] Merr.) volatiles assessed by headspace solid phase microextraction and gas chromatography–mass spectrometry (HS-SPME-GC/MS). Food Chemistry, 150, 382–391. U.S. Institute of Medicine (2010). Dietary reference intakes for vitamin A, vitamin K, arsenic, boron, chromium, copper, iodine, iron, manganese, molybdenum, nickel, silicon, vanadium, and zinc: A report of the Panel on Micronutrients … and the Standing Committee on the Scientific Evaluation of Dietary Reference Intakes, Food and Nutrition Board, Institute of Medicine. Washington, D.C: National Academy Press. van Breemen, R. B., Canjura, F. L., & Schwartz, S. J. (1991). Identification of chlorophyll derivatives by mass spectrometry. Journal of Agricultural and Food Chemistry, 39, 1452–1456. van Breemen, R. B., Dong, L., & Pajkovic, N. D. (2012). Atmospheric pressure chemical ionization tandem mass spectrometry of carotenoids. International Journal of Mass Spectrometry, 312, 163–172. Verzegnassi, L., Riffé-Chalard, C., & Gülaçar, F. O. (2000). Rapid identification of Mg-chelated chlorins by on-line high performance liquid chromatography/atmospheric pressure chemical ionization mass spectrometry. Rapid Communications in Mass Spectrometry, 14, 590–594.
Yano, M., Kato, M., Ikoma, Y., Kawasaki, A., Fukazawa, Y., Sugiura, M., Matsumoto, H., Oohara, Y., Nagao, A., & Ogawa, K. (2005). Quantitation of carotenoids in raw and processed fruits in Japan. Food Science and Technology Research, 11, 13–18. Ziegler, J. U., Wahl, S., Würschum, T., Longin, C. Friedrich H., Carle, R., & Schweiggert, R. M. (2015). Lutein and lutein esters in whole grain flours made from 75 genotypes of five Triticum species grown on multiple sites. Journal of Agricultural and Food Chemistry, 63, 5061–5071.
5 5.1
Captions Figures
Fig. 1 HPLC-DAD chromatograms of ‘MD2’ (syn. “Extra Sweet”) pineapple (Ananas comosus [L.] Merr.) peel (a, b) and pulp extracts (c). The latter was additionally analysed after alkaline hydrolysis (d).
Fig. 2 APCI(+)-MS2 experiment of compound 13 assigned to (9Z)-violaxanthin laurate (a). Mass spectral data is summarised in Table 2. Proposed mass fragmentation of violaxanthin esters (b) (Britton et al., 1995; Breithaupt, 2002).
5.2
Tables
Table 1 HPLC-DAD-APCI-MSn data of lipophilic pigments isolated from the pineapple (Ananas comosus [L.] Merr.) infructescence.
Table 2 Quantitation of prevailing pulp carotenoids (µg/100 g of fresh weight) in different pineapple (Ananas comosus [L.] Merr.) varieties harvested at full and green ripe maturity, respectively. Green ripe fruits were dispatched by sea freight and analysed at 14 and 21 days after harvest (dah).
Table 1. No.
tR (min)
1
9.5
2
λmax (nm)
DB/DII a (%)
DIII/DII b (%)
[M]−• (m/z)
[M+H]+ (m/z)
APCI(+)-MSn experiment (m/z, % base peak intensity)
Proposed structure
266, 315, 327 416/439/469
-
87
-
601
[601]: 583 (100), 565 (17), 509 (17), 491 (12), 221 (7), 181 (1)
(all-E)-Violaxanthin d
10.6
265, 315, 327 413/436/464
2
83
-
601
[601]: 583 (100), 565 (20), 547 (4), 509 (15), 491 (9), 393 (8), 221 (10), 181 (2)
(all-E)-Neoxanthin d
3
11.5
250, 300, 311 399/422/448
3
95
-
601
[601]: 583 (100), 565 (15), 509 (23), 491 (14), 221 (6), 181 (1)
(all-E)-Luteoxanthin
4
14.5
267, 314, 327 412/436/465
8
87
-
601
[601]: 583 (100), 565 (21), 509 (20), 491 (12), 221 (7), 181 (1)
(9Z)-Violaxanthin
5
16.3
251, 300, 311 397/417/443
5
83
-
601
[601]: 583 (100), 565 (18), 509 (15), 491 (12), 221 (9), 181 (1)
(9Z)- or (9´Z)-Luteoxanthin
6
17.6
256, 316, 343 467/602/650
-
-
-
907
[907]: 629 (100), 597 (60), 569 (50), 541 (24)
Chlorophyll b d
7
18.2
267, 332 sh420/445/473
5
57
568
551 c
[551]: 533 (100), 495 (49), 477 (24), 459 (44), 429 (88)
(all-E)-Lutein d
8
23.4
266, 315, 327 416/439/469
-
84
-
755
[755]: 737 (100), 719 (6), 663 (16), 645 (8), 583 (35), 565 (17), 547 (7)
(all-E)-Violaxanthin caprate (C10)
9
24.6
265, 337, 383 432/619/665
-
-
-
893
[893]: 615 (85), 583 (46), 555 (100)
Chlorophyll a d
10
25.5
267, 314, 327 412/436/464
7
87
-
727
[727]: 709 (89), 691 (3), 635 (5), 617 (7), 583 (100), 565 (27), 547 (8)
(9Z)-Violaxanthin caprylate (C8)
11
28.0
266, 315, 327 416/439/469
-
83
-
783
[783]: 765 (100), 747 (8), 691 (11), 673 (12), 583 (95), 565 (38), 547 (8)
(all-E)-Violaxanthin laurate (C12)
12
29.4
267, 314, 327 412/436/464
8
88
-
755
[755]: 737 (100), 719 (7), 663 (10), 645 (10), 583 (83), 565 (26), 547 (6)
(9Z)-Violaxanthin caprate (C10)
13
33.8
267, 314, 327 412/436/465
8
88
-
783
[783]: 765 (100), 747 (6), 691 (9), 673 (12), 583 (83), 565 (29), 547 (5)
(9Z)-Violaxanthin laurate (C12)
14
38.8
267, 314, 327 412/436/465
7
79
-
811
[811]: 793 (100), 775 (8), 719 (7), 701 (14), 583 (75), 565 (28), 547 (4)
(9Z)-Violaxanthin myristate (C14)
15
40.1
266, 315, 327 416/437/467
-
80
-
839
[839]: 821 (100), 803 (10), 747 (9), 729 (11), 583 (80), 565 (25), 547 (5)
(all-E)-Violaxanthin palmitate (C16)
16
43.9
275, 330 sh426/452/477
4
24
536
537
[537]: 481 (52), 457 (16), 445 (46), 444 (100), 399 (16), 387 (39), 347 (49)
(all-E)-β-Carotene d
17
44.0
324 413/610/667
-
-
-
871
[871]: 593 (100), 533 (82)
Pheophytin a
18
44.7
267, 314, 327 412/436/465
7
84
-
839
[839]: 821 (100), 803 (13), 747 (16), 729 (16), 583 (80), 565 (25), 547 (7)
(9Z)-Violaxanthin palmitate (C16)
19
46.3
267, 315, 328 413/436/465
3
78
-
937
[937]: 919 (39), 845 (5), 827 (15), 765 (100), 747 (12), 737 (92), 565 (46), 547 (44)
(9Z)-Violaxanthin capratelaurate (C10-C12)
20
47.4
265, 315, 328 417/440/470
-
84
-
965
[965]: 947 (2), 765 (100), 747 (20), 565 (42)
(all-E)-Violaxanthin dilaurate (C12-C12)
21
50.5
267, 315, 327 6 83 965 [965]: 947 (10), 765 (100), 747 (78), 565 (41) 413/436/465 tR, retention time; λmax, UV/Vis absorption maxima; sh, shoulder. a D /D , ratio of absorption intensity at ‘cis-band’ near UV maximum (D ) to intensity at main absorption maximum (D ). B II B II b D /D , ratio of absorption intensity at longest wavelength maximum (D ) to D . III II III II c In-source elimination of water ([M + H − H O]+). 2 d Verified by an authentic reference standard.
(9Z)-Violaxanthin dilaurate (C12-C12)
Table 2. No.
Proposed structure
‘Queen Victoria’ fully ripe 2 dah
‘Sugar Loaf’ fully ripe 2 dah
‘Smooth Cayenne’ ‘MD2’ fully ripe fully ripe 2 dah 2 dah
‘MD2’ green ripe 14 dah
‘MD2’ green ripe 21 dah
1
(all-E)-Violaxanthin
9 ± 5 bc
6±1c
6±1c
13 ± 4 b
26 ± 5 a
6 ± 3 bc
10
(9Z)-Violaxanthin caprylate
35 ± 12 a
tr.
9±1c
20 ± 3 b
25 ± 3 a
22 ± 6 ab
12
(9Z)-Violaxanthin caprate
90 ± 36 a
3±1d
21 ± 3 c
47 ± 9 b
74 ± 8 a
54 ± 20 ab
13
(9Z)-Violaxanthin laurate
151 ± 60 ab
5±1d
35 ± 3 c
109 ± 18 b
161 ± 19 a
107 ± 40 b
14
(9Z)-Violaxanthin myristate
40 ± 10 ab
tr.
14 ± 1 c
31 ± 4 b
38 ± 3 a
29 ± 7 b
16
(all-E)-β-Carotene
87 ± 32 a
4±0 f
17 ± 1 e
22 ± 4 d
34 ± 4 c
45 ± 4 b
18
(9Z)-Violaxanthin palmitate
56 ± 21 a
tr.
9±1c
30 ± 6 b
41 ± 5 ab
29 ± 9 b
Total carotenoids
565 ± 193 a
29 ± 2 d
110 ± 27 c
302 ± 44 b
432 ± 48 ab
359 ± 89 b
a
*
Calculated as sum of (all-E)-β-carotene, xanthophylls, xanthophyll esters (as free xanthophylls) and unidentified minor constituents. MD2 (syn. “Extra Sweet”); dah, days after harvest; tr., trace. Different letters within a row indicate significant (p < 0.05) differences of means (n = 5 single fruits). * The standard deviation ± 0 equals a value < 0.5 μg/100 g of FW. a
Highlights Detailed HPLC-DAD-APCI-MSn analysis of pineapple carotenoids Identification of 21 lipophilic pigments in the pineapple infructescence Xanthophyll esters are reported for the first time as pineapple constituents Quantitation of carotenoids in four varieties cultivated in Ghana
S0963-9969(19)30595-2 https://doi.org/10.1016/j.foodres.2019.108709 FRIN 108709
To appear in:
Food Research International
Received Date: Revised Date: Accepted Date:
7 June 2019 17 September 2019 21 September 2019
Please cite this article as: Steingass, C.B., Vollmer, K., Lux, P.E., Dell, C., Carle, R., Schweiggert, R.M., HPLCDAD-APCI-MS n analysis of the genuine carotenoid pattern of pineapple (Ananas comosus [L.] Merr.) infructescence, Food Research International (2019), doi: https://doi.org/10.1016/j.foodres.2019.108709
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© 2019 Published by Elsevier Ltd.
HPLC-DAD-APCI-MSn analysis of the genuine carotenoid pattern of pineapple (Ananas comosus [L.] Merr.) infructescence
CHRISTOF B. STEINGASS1,2*, KATHRIN VOLLMER2,3, PETER E. LUX2,3, CAROLIN DELL2, REINHOLD CARLE2,4, RALF M. SCHWEIGGERT1
1
Department of Beverage Research, Chair Analysis and Technology of Plant-based Foods, Geisenheim
University, Von-Lade-Strasse 1, 65366 Geisenheim, Germany 2
Institute of Food Science and Biotechnology, Chair Plant Foodstuff Technology and Analysis, University
of Hohenheim, Garbenstrasse 25, 70599 Stuttgart, Germany 3
Institute of Nutritional Sciences, Chair Food Biofunctionality, University of Hohenheim, Garbenstrasse
28, 70599 Stuttgart, Germany 4
Biological Science Department, Faculty of Science, King Abdulaziz University, P.O. Box 80257, Jeddah
21589, Saudi Arabia
Corresponding author. Phone: +49 6722 502 314. E-mail: [email protected]
*
Abstract The genuine carotenoid pattern of pineapple infructescence was assessed by HPLC-DAD-APCI-MSn analysis. Prevailing pigments in the shell of ‘MD2’ (syn. “Extra Sweet”) fruits were (all-E)-lutein and (all-E)-β-carotene, in addition to chlorophylls a and b. The edible flesh contained (all-E)-violaxanthin, (all-E)-β-carotene and diverse esters of (9Z)violaxanthin with caprylic, capric, lauric, and myristic acid. The latter esters have been reported for the first time as pineapple constituents. Total carotenoid concentrations in the edible fractions of the four varieties ‘Sugar Loaf’, ‘Smooth Cayenne’, ‘MD2’, and ‘Queen Victoria’ cultivated in Ghana ranged between 29 and 565 µg/100 g of fresh weight (FW). Total carotenoids in the flesh of fully ripe ‘MD2’ fruits exported by air freight amounted to 302 µg/100 g of FW, those in green ripe samples dispatched by sea freight to 359–432 µg/100 g of FW. All yellow fleshed cultivars exhibited a highly similar qualitative carotenoid profile.
Keywords: carotenoids; xanthophyll esters; tropical fruit
Abbreviations: APCI, atmospheric pressure chemical ionisation; BHA, butylated hydroxyanisole; BHT, butylated hydroxytoluene; CID, collision induced dissociation; dah, days after harvest; FW, fresh weight; HPLC-DAD, high performance liquid chromatography-diode array detection; LP, light petroleum; MSn, multiple-stage mass spectrometry; RAE, retinol activity equivalent; tBME, tert-butyl methyl ether; tR, retention time.
1
Introduction
After banana and citrus, pineapples (Ananas comosus [L.] Merr.) belong to the most important tropical fruits in the international trade (Lobo & Paull, 2017). Total exports of fresh pineapples from their tropical production countries to the EU and the US markets were estimated at 3.35 million tonnes in 2018. The most important supplier was Costa Rica (Altendorf, 2018). Apart from the overall appearance, odour (i.e., aroma, pineapple flavour), texture, juiciness, and sweetness, the colour of the flesh has been found to be among the most important attributes determining consumer acceptance of fresh pineapples (Ramsaroop & Saulo, 2007). Consequently, one of the targets of past pineapple breeding programs was to develop intensely coloured yellow to orange fleshed varieties (Lobo & Paull, 2017). The hybrid ‘MD2’, often marketed as “Extra Sweet” or “Golden Ripe”, was launched by Del Monte Fresh Produce Hawaii Inc. and represents one of the most important varieties for fresh fruit export (Bartholomew et al., 2003). Another newly bread cultivar termed ‘FLHORAN41’ with a deep yellow flesh and an orange-red to scarlet coloured shell resulted from a breeding program of CIRAD (Brat et al., 2004). Of the more than 100 pineapple varieties, merely 6 to 8 are cultivated commercially (Lobo & Paull, 2017). In particular, large-scale companies exporting sea freighted pineapples produce the yellow fleshed ‘MD2’, reaching market shares of approx. 90 and 85% in the US and the EU, respectively (Loeillet, 2014). ‘MD2’ represents approx. 95% of the pineapples grown in Ghana. ‘Smooth Cayenne’ (4%) and ‘Sugar Loaf’ (1%) are offered on the European niche market of air freighted premium fruits (Loeillet & Paqui, 2010). Air freighted ‘Queen Victoria’ (or ‘Queen’) pineapples available in Europe originate from South Africa, Mauritius, Reunion, and Ghana (CBI Ministry of Foreign Affairs, 2014). The total carotenoids in the edible flesh of fully ripe ‘Smooth Cayenne’ and ‘FLHORAN41’ pineapples were reported to amount to 246 and 580 μg/100 g of fresh weight (FW), respectively. Prevailing pigment in both genotypes was β-carotene, accounting for 31–38% of the total carotenoids. Significantly elevated total carotenoids of 2,545 and 2,915 μg/100 g of FW, respectively, were found in the shell of the aforementioned two pineapple varieties. However, carotenoid pigments other than β-carotene have not been identified (Brat et al., 2004). Similarly, merely β-carotene has been assigned to one of 14 different carotenoids found in ‘Tropical Gold’ pineapple flesh (Gil et al., 2006). Yano et al. (2005) have reported total carotenoid concentrations of ~200 μg/100 g of FW in the flesh of an unspecified pineapple genotype. Three major pigments in the alkaline saponified extract were assigned to (9Z)-violaxanthin (50% of the total concentration), (all-E)-violaxanthin
(28%), and (all-E)-β-carotene (22%). However, detailed studies of the carotenoid profiles are virtually unavailable and, particularly, the genuine carotenoid pattern has been disregarded in previous studies. Therefore, the present contribution targeted at the in-depth characterisation of carotenoids in the shell and pulp of the pineapple infructescence applying HPLC-DAD-APCI-MSn. In addition, the carotenoids in the edible fraction of ‘Queen Victoria’, ‘Sugar Loaf’, ‘Smooth Cayenne’, and ‘MD2’ (syn. “Extra Sweet”) pineapples harvested at full maturity were quantitated by HPLC-DAD. Fruits of ‘MD2’ were additionally harvested at a green ripe stage, exported by sea freight and analysed upon their arrival in Europe to explore possible influences of postharvest procedures on the carotenoid patterns.
2 2.1
Materials and methods Pineapple samples
All studied pineapples were imported from Ghana and provided by the fruit distributer Schumacher (Filderstadt-Bernhausen, Germany). Fully ripe ‘Queen Victoria’, ‘Sugar Loaf’, ‘Smooth Cayenne’, and ‘MD2’ (syn. “Extra Sweet”) fruits were imported via air freight and analysed approx. 2 days after harvest (dah). In addition, ‘MD2’ pineapples were harvested at a premature green ripe stage and brought to Germany by sea freight. The latter samples were analysed upon their arrival (14 dah) and after a further week of storage at room temperature (21 dah). A detailed description of the harvest and postharvest procedures for sea and air freighted pineapples has been provided elsewhere (Steingass et al., 2014). Five individual fruits (n = 5) of each variety or ripening stage were randomly selected and analysed in analytical duplicates.
2.2
Carotenoid analyses
Sample preparation Carotenoids were extracted from fresh pineapple peel and pulp as described previously (Schweiggert et al., 2011) including the following modifications. For immediate neutralisation of genuine fruit acids, 200 g of the respective, carefully and minimally cut pineapple tissue was homogenised with the admixture of 4.0 g calcium carbonate and 2.0 g sodium hydrogen carbonate using a laboratory blender (Grindomix GM 200, Retsch, Haan, Germany). An aliquot of 2.0 ± 0.2 g of the obtained puree was immediately mixed with 3 mL of the extraction solution (methanol/ethyl acetate/light petroleum (LP), 1/1/1, v/v/v) containing each 0.1 g/L 2,6-di-tert-butyl-p-cresol (BHT) and 3-tert-butyl-4-hydroxyanisole (BHA). The sample was homogenised using an Ultra-Turrax® homogeniser (type 18/10, IKA®-Werke, Staufen, Germany) and
centrifuged for 3 min at 2,500 rpm, equalling 671 × g (Labofuge 200, Heraeus Material Technology, Hanau, Germany). The upper organic layer was recovered and the remainder was re-extracted twice with each 3 mL of the aforementioned extraction mixture. The combined organic phase was subsequently washed twice with each 3 mL ultrapure H2O, dried with anhydrous sodium sulphate, and evaporated to dryness under reduced pressure at 30 °C. Carotenoid extracts were redissolved in 0.3 mL tBME/methanol (1/1, v/v) and filtered over a 0.45 μm polytetrafluoroethylene filter (Chromafil® O45/15 MS, Macherey-Nagel, Düren, Germany) into amber glass vials prior to HPLC analyses.
Alkaline hydrolysis For cleavage of xanthophyll esters, the above-mentioned carotenoid extract was dissolved in 2 mL LP containing each 0.1 g/L BHA and BHT. After adding 2 mL of 15% w/w methanolic potassium hydroxide, the sample headspace was flushed with nitrogen and the extract stirred for 3 h at room temperature. Following the admixture of 5 mL H2O, the carotenoids were extracted with 2 mL diethyl ether/n-hexane (1/1, v/v). The organic layer was collected and the aqueous remainder was re-extracted twice with each 2 mL diethyl ether. The combined organic phases were washed twice with each 2 mL H2O. After evaporation to dryness, the carotenoids were dissolved in 0.3 mL tBME/MeOH (1/1, v/v) and membrane filtered as detailed above.
HPLC-DAD analyses HPLC-DAD analyses were performed using an HPLC system consisting of a separation module type 2695 equipped with a diode array detector type 2996 (both Waters, Eschborn, Germany) and a column oven thermostat Jetstream II plus (Thermotechnic Products, Langenzersdorf, Austria). Control of the system and data evaluation was performed using Millenium 32® Chromatography Manager software (Waters). Carotenoids were separated using a C30 reversed phase column (150 × 3.0 mm i.d., particle size dp = 3 μm, YMC Europe, Dinslaken, Germany) equipped with a C30 guard column (10 × 3.0 mm i.d., dp = 3 μm, YMC Europe). Eluents were tertiary mixtures of tBME/methanol/H2O (eluent A: 5/91/4, v/v/v; eluent B: 90/8/2, v/v/v). The gradient program was as follows: 100 to 50% A (60 min), 50 to 0% A (10 min), 0 to 100% A (5 min), isocratic hold at 100% A (5 min) at a flow rate of 0.42 mL/min. Prior to each injection (10 μL), the system was re-equilibrated for 5 min (100% A). Column oven temperature was set to 25 °C. Total run time was 80 min. Detection
wavelengths for xanthophylls, β-carotene, and chlorophylls were 439, 452, and 660 nm, respectively. UV/Vis spectra were recorded in the range of 200–800 nm. Linear calibration curves of (all-E)-β-carotene and (all-E)-violaxanthin were used for quantitation. Concentrations of stock solutions were determined by spectrophotometry using the appropriate molar extinction coefficients (Britton et al., 1995). Molecular weight correction factors were applied where appropriate. Total carotenoid levels were calculated as the sum of the concentrations of violaxanthin, its esters, unidentified minor constituents (as violaxanthin equivalents), and β-carotene.
HPLC-DAD-APCI-MSn analyses For HPLC-DAD-APCI-MSn experiments, a series 1100 HPLC system with a G1315B diode array detector (both Agilent, Waldbronn, Germany) was coupled on-line to an Esquire 3000+ ion trap mass spectrometer (Bruker Daltonik, Bremen, Germany) with an atmospheric pressure chemical ionisation (APCI) source. Column and HPLC settings were as detailed above. Mass spectra were recorded in the alternating polarity mode at a scan range of m/z 100–1100. All MS-parameters were adjusted as described previously (Schweiggert et al., 2012). Control of the system and data evaluation was achieved with ChemStation for LC version A.00.03 (Agilent) and Esquire software version 5.1 (Bruker), respectively.
2.3
Reagents
Chlorophyll a, chlorophyll b, and (all-E)-β-carotene were purchased from Sigma Aldrich (Taufkirchen, Germany), (all-E)violaxanthin, (all-E)-neoxanthin, and (all-E)-lutein from CaroteNature (Ostermundingen, Switzerland). Calcium carbonate, sodium hydrogen carbonate, ethyl acetate, diethyl ether, n-hexane, and tBME were obtained from Merck (Darmstadt, Germany). Potassium hydroxide, sodium sulphate, methanol, and LP (boiling point 40–60 °C) were from VWR International (Darmstadt, Germany). BHA and BHT were purchased from Fluka Chemie (Buchs, Switzerland). An arium® 611 UV (Sartorius, Göttingen, Germany) ultrapure water system was used for preparation of ultrapure H2O.
2.4
Statistics
Statistical analyses were performed using SAS software version 9.1 (SAS Institute, Cary, NC, USA). Normality and homogeneity of variances were examined by Shapiro-Wilk’s test (p < 0.05) and Levene’s test (p < 0.05), respectively.
Significant differences (p < 0.05) of means among the individual samples were determined by one-way analysis of variance (ANOVA) and Tukey's test.
3 3.1
Results and discussion Compound assignment by HPLC-DAD-APCI-MSn
Fig. 1a and b display representative HPLC-DAD chromatograms of a pineapple peel extract recorded at 660 and 452 nm, respectively. The chromatogram of genuine flesh carotenoids at a detection wavelength of 452 nm is shown in Fig. 1c. After alkaline hydrolysis, the signal intensities of compounds 8, 10–15 and 18–21 vanished, whereas those of the five early eluting compounds 1–5 increased, thus indicating the presence of esterified xanthophylls (Fig. 1d). Retention times (tR), UV/Vis absorption, and mass spectra of the individual pigments are compiled in Table 1 and their assignments will be discussed in the following sections.
Chlorophylls Three compounds were detected at 660 nm in the peel extract (Fig. 1a). Compounds 6 and 9 exhibiting protonated molecules [M + H]+ at m/z 907 and 893 were identified as chlorophyll b and chlorophyll a, respectively. CID of both chlorophylls yielded abundant fragment ions from the elimination of the phytadiene (C20H38) moiety [M + H − 278]+ at m/z 629 and 615, respectively. In addition, both compounds displayed fragment ions from the additional losses of methanol (32 amu) at m/z 597 and 583 ([M + H − 278 − 32]+) and of carbon monoxide (28 amu) at m/z 569 and 555 ([M + H − 278 − 32 − 28]+). The sequential eliminations of methanol and carbon monoxide reflected the loss of the entire carboxymethoxy group from the ε-ring together with an H atom (60 amu), thus being in accordance with Verzegnassi et al. (2000). Retention times, UV/Vis spectra, and mass spectral data of compounds 6 and 9 matched those of the analytical chlorophyll b and a standards, respectively. Compound 17 assigned to pheophytin a displayed protonated molecules [M + H]+ at m/z 871 that were 22 amu lighter than those of compound 9 (chlorophyll a). This lower molecular weight can be attributed to the replacement of the central Mg2+ ion (24 amu) by two protons. In agreement with our observations for compound 9, the MS2 experiment on pheophytin a yielded abundant product ions at m/z 593 ([M + H − 278]+) and 533 ([M + H − 278 − 60]+) from the loss of phytadiene and
the additional elimination of the carboxymethoxy group (Verzegnassi et al., 2000; van Breemen et al., 1991; Kaiser et al., 2012) (Table 1).
Carotenes and free xanthophylls Compound 16 was detected in both peel and pulp extracts (Fig. 1) and exhibited molecular ions M−• at m/z 536 and protonated molecules [M + H]+ at m/z 537. By comparing tR (43.9 min), UV/Vis, and mass spectra to those of an authentic reference standard, 16 was identified as (all-E)-β-carotene. The product ions observed in the APCI(+)-MS2 experiment were in agreement with previous reports (van Breemen et al., 2012; Schex et al., 2018). Compound 7 assigned to (all-E)-lutein displayed an abundant signal at 452 nm in the peel extract (Fig. 1b) and molecular ions M−• at m/z 568 in the APCI(−)-MS1 spectrum. In the positive ion mode, in-source fragments from the elimination of water were detected at m/z 551 ([M + H − H2O]+). Then, the CID MS2 spectrum of this in-source fragment ion presented a base peak at m/z 533 ([M + H − 2 H2O]+). The product ions at m/z 495 ([M + H − H2O − 56]+) and 477 ([M + H − 2 H2O − 56]+) may be attributed to the retro-Diels-Alder cleavage of the ε-ring (56 amu) and water eliminations (18 amu). The fragment ions at m/z 459 ([M + H − H2O − 92]+) presumably resulted from the elimination of toluene (92 amu) from the polyene chain of the dehydrated precursor ion. The characteristic product ions at m/z 429 ([M + H − H2O − 122]+) have been attributed to the eliminations of hydroxylated ε- or β-rings. This mass fragmentation was in agreement with previous reports (Schex et al., 2018; Ziegler et al., 2015; Crupi et al., 2012). The assignment was further confirmed by comparison of tR, UV/Vis and mass spectra of compound 7 with those of an authentic reference standard. Five early eluting compounds (1–5) in the saponified pulp extract (Fig. 1d) had protonated molecules [M + H]+ at m/z 601, providing evidence of xanthophyll molecules with four oxygen atoms. CID resulted in abundant fragment ions at m/z 583 ([M + H − H2O]+) and 565 ([M + H − 2 H2O]+), likely originating from elimination of water (18 amu). In addition, the common product ions at m/z 509 [M + H − 92]+) and 491 ([M + H − H2O − 92]+) appeared in the MS2 spectra that can be attributed to the neutral loss of toluene (92 amu) as discussed above. Product ions at m/z 547 ([M + H − 3 H2O]+) and m/z 393 were exclusively detected in the MS2 experiment of compound 2 that have been attributed to the threefold elimination of water and the cleavage of the double bond in allylic position to the allenic carbon of (all-E)-neoxanthin, respectively (De Rosso & Mercadante, 2007).
Further distinctive fragment ions at m/z 221 and 181 have been attributed to the cleavage of the polyene chain between positions 10,11 (or 10´,11´) and 8,9 (or 8´,9´), respectively, of a xanthophyll with a 3-hydroxy-5,6- or 5,8-epoxidated βring (Rivera et al., 2014). Recently, cyclic oxonium ions have been proposed to generate the aforementioned fragment ions after epoxy-oxepinoid and epoxy-furanoid rearrangements of the protonated molecules under electrospray ionisation (ESI) conditions (Neto et al., 2016). On the basis of the mass fragmentation detailed above and the Vis absorption maxima (λmax) at 416, 439, and 469 nm, compound 1 was identified as (all-E)-violaxanthin. Compound 2 displayed slightly shorter Vis λmax at 413, 436, and 464 nm and the spectral shape resembled that of the structural isomer (all-E)-neoxanthin. The assignments of compounds 1 and 2 were confirmed by comparison of retention times as well as UV/Vis and mass spectral characteristics to those of authentic reference standards. Compound 3 displayed Vis λmax at 399, 422, and 448, thus being congruently ~20 nm shorter than those observed for (allE)-violaxanthin (1). The mass spectral behaviour of compounds 1 and 3, however, was virtually identical. This and particularly the hypsochromic shift indicated the rearrangement of one 5,6- to a 5,8-epoxy-group and thus, 3 was assigned to (all-E)-luteoxanthin (De Rosso & Mercadante, 2007). Compound 4 displayed a spectral shape and a DIII/DII ratio of 87% resembling those of (all-E)-violaxanthin (1). The 3–4 nm shorter Vis λmax at 412, 436, and 465 nm indicated a corresponding mono-cis-isomer (Britton et al., 1995). On the basis of the UV/Vis absorption maxima and the previously reported elution order on a C30 stationary phase, compound 4 was tentatively identified as (9Z)-violaxanthin (Meléndez-Martínez et al., 2007). Accordingly, compound 5 with Vis λmax at 397, 417, and 443 was assigned to a corresponding 5,6-, 5,8-epoxide, i.e., (9Z)- or (9´Z)-luteoxanthin. The elution order of (allE)-luteoxanthin prior to the two possible (9Z)-isomer was in accordance to literature (Petry & Mercadante, 2018). Noteworthy, (all-E)-neoxanthin (2), (all-E)-luteoxanthin (3), and (9Z)-luteoxanthin (5) were merely detected in the saponified pulp extract (Fig. 1d) and thus, may represent cleavage products of minor esters or possibly workup artefacts in case of the latter two 5,8-epoxides.
Xanthophyll esters Isobaric esters of (all-E)- and (9Z)-violaxanthin (compounds 8/12, 11/13, 15/18, and 20/21) cannot be distinguished based on their MSn fragmentations. However, the UV/Vis spectra of the xanthophyll esters resembled those of the corresponding
free xanthophylls discussed above, permitting the assignment of geometrical isomers. The violaxanthin monoesters (8, 10– 15, and 18) detected in the genuine pulp extract (Fig. 1c) were characterised by distinctive fragment ions generated by the neutral losses of the fatty acid moieties of 144, 172, 200, and 228 amu for caprylic, capric, lauric, and myristic acid, respectively, as well as by the neutral loss of toluene (92 amu) and the elimination of water (18 amu). Exemplarily, APCI(+)MS2 of the most abundant ester, assigned to (9Z)-violaxanthin laurate (13), with protonated molecules [M + H]+ at m/z 783, displayed product ions at m/z 765 ([M + H − H2O]+), 747 ([M + H − 2 H2O]+), 691 ([M + H − 92]+), 673 ([M + H − 92 − H2O]+), 583 ([M + H − 200]+), 565 ([M + H − 200 − H2O]+), and 547 ([M + H − 200 − 2 H2O]+) (Fig. 2). Moreover, as observed in the MS2 spectra of the free xanthophylls 1–5, the aforementioned fragment ions at m/z 221 were detected at a low abundance, substantiating the presence of 5,6- or 5,8-epoxidated β-rings carrying hydroxyl groups (not shown in Table 1). The minor compound 19 presented protonated molecules [M + H]+ at m/z 937. CID resulted in product ions from the eliminations of water and toluene as discussed above. The distinctive product ions at m/z 765 ([M + H − 172]+), 737 ([M + H − 200]+), and 565 ([M + H − 172 − 200]+) indicated a diester with capric (172 amu) and lauric acid (200 amu) as acyl moieties (Table 1). Accordingly, compound 19 was assigned to (9Z)-violaxanthin caprate-laurate. The compounds 20 and 21 both showed protonated molecules at m/z 965 and common product ions at m/z 947 ([M + H − H2O]+), 765 ([M + H − 200]+), 747 ([M + H − 200 − H2O]+), and 565 ([M + H − 200 − 200]+). On the basis of their mass fragmentations and UV/Vis absorption spectra that resembled those of peaks 1 and 4, they were tentatively assigned to (all-E)- and (9Z)violaxanthin dilaurate.
3.2
Quantitation of pulp carotenoids by HPLC-DAD
Total carotenoids in the edible portion of all pineapple cultivars assessed are compiled in Table 2 and ranged from 29 to 565 µg/100 g of FW. Hereby, the lowest levels were determined in the white fleshed ‘Sugar Loaf’, whilst the maximum values were found in the intensely yellow fleshed ‘Queen Victoria’. Total carotenoid levels determined among the yellow fleshed pineapples were comparable to those previously reported in ‘Smooth Cayenne’ and ‘FLHORAN41’ pineapples amounting to 246 and 580 μg/100 g of fresh weight (FW), respectively (Brat et al., 2004). In general, pineapples represent nutritional sources containing “low” (0–100 µg/100 g) to “moderate” (100–500 µg/100 g) carotenoid levels according to the classification proposed by Britton et al. (2009). Nevertheless, the high proportions of (Z)-isomers observed may indicate
a deposition of the carotenoids including the provitamin A-active β-carotene in the chromoplasts in a highly bioavailable, lipid-dissolved and/or liquid-crystalline state (Schweiggert & Carle, 2017). However, to the best of our knowledge, the structure of the pigment-bearing plastids in the pineapple infructescence as well as the bioavailability of the contained carotenoids has not been described in the literature. Among the ‘MD2’ fruits, pineapples harvested green ripe displayed highest total carotenoid levels of 432 µg/100 g of FW upon their arrival in Europe (14 dah). After postharvest storage for another seven days (21 dah), total carotenoid levels dropped by 17% to 359 µg/100 g of FW. This decline may be attributed to the isomerisation and degradation of carotenoids caused by the release of acids from bruised cells (Gortner & Singleton, 1961), but also to enzymatic reactions. The cooxidation of carotenoids by lipoxygenase or the cleavage of (9´Z)-neoxanthin and (9Z)-violaxanthin as an initial step in the biosynthesis of the stress hormone abscisic acid are worth mentioning here (Britton et al., 2008). Interestingly, ‘MD2’ pineapples allowed to ripen completely attached to the plant, i.e., being harvested at full maturity, displayed lower total carotenoid levels (302 µg/100 g of FW) than those harvested green ripe (359–432 μg/100 g of FW). The abovementioned decompartmentalisation and related effects (acids, enzymes) might have been more pronounced in the fully ripe fruits due to their softer tissue and more labile cell structures (Gortner & Singleton, 1961), resulting in lower carotenoid levels in the fully ripe fruits. This hypothesis is supported by the slight increase of the peroxidase activity reported during the last 20 days of pineapple maturation (Gortner, 1965). Such market differences have also been found in the profiles of volatiles when comparing air freighted pineapples to their sea freighted counterparts (Steingass et al., 2014; Steingass et al., 2016). Noteworthy, among all samples, (9Z)-violaxanthin laurate (C12, 13) was the prevailing xanthophyll ester. In the yellow fleshed samples, further abundant esters were (9Z)-violaxanthin caprate (C10, 12), myristate (C14, 14), and palmitate (C16, 18), in addition to the less abundant (9Z)-violaxanthin caprylate (C8, 10) and further esters. Thus, the determined pineapplespecific pattern of acyl moieties was mainly based on C10 to C16 fatty acids as previously found in numerous other fruits (Hornero-Méndez, 2019). Merely one non-oxygenated carotenoid (carotene) was found, namely (all-E)-β-carotene (16). This provitamin A carotenoid contributed to approx. 15% of the total carotenoids in fully ripe ‘Queen Victoria’, ‘Sugar Loaf’, and ‘Smooth Cayenne’ flesh and 7–13% in the ‘MD2’ samples assessed. Elevated proportions of β-carotene of 20 and 31–38%, respectively, have been previously reported in the literature (Brat et al., 2004; Yano et al., 2005). The retinol activity equivalents (RAE) as calculated by the guidelines of the U.S. Institute of Medicine (2010) of our samples ranged
from 0.3 to 7.3 µg RAE/100 g. This range was similar to the RAEs of oranges (4 µg RAE/100 g), but substantially lower than those of carrot roots (1000 RAE/100 g), leafy vegetables (343 µg RAE/100 g), and apricots (125 µg RAE/100 g) as reported by Solomons & Orozco (2003). In brief, the colour of yellow fleshed varieties may particularly be determined by (9Z)-violaxanthin esters, whereas the corresponding (all-E)-isomers (8, 11, 15, and 20) merely occurred as minor constituents.
3.3
Conclusions
To the best of our knowledge, the present contribution is the first detailed study into the genuine carotenoid patterns of pineapples. We found diverse esters of violaxanthin with caprylic, capric, lauric, and myristic acid as pigments determining the colour of the pineapple flesh. Among the esterified xanthophylls, monoesters prevailed, whereas diesters were only detected as minor constituents. Interestingly, the pineapple shell displayed a chloroplast-specific pigment profile comprising (all-E)-violaxanthin, (all-E)neoxanthin, (all-E)-lutein, and (all-E)-β-carotene. By contrast, esterified (9Z)-violaxanthin prevailed in the edible flesh. The provitamin A value of pineapple fruit was shown to be moderate. Besides elucidating the deposition form and bioavailability of pineapple carotenoids, future studies may explore the carotenoid composition of additional pineapple varieties considering a larger sample set and fruits from different provenances as well as the influence of processing (juice extraction and pasteurisation), on the carotenoid pattern of pineapple juice. In addition, the pineapple shell as a valuable source for the recovery of functional compounds such as polyphenols and carotenoids merits further investigation.
Acknowledgements We cordially thank Frank Oberschilp (Peelco, Accra, Ghana) and Fritz Schumacher (Schumacher, Filderstadt-Bernhausen, Germany) for kindly providing the pineapple fruits.
Dedication In grateful remembrance of Priv.-Doz. Dr. habil. Hans-Georg Schmarr (1961–2019), Neustadt an der Weinstraße, Germany.
4
References
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5 5.1
Captions Figures
Fig. 1 HPLC-DAD chromatograms of ‘MD2’ (syn. “Extra Sweet”) pineapple (Ananas comosus [L.] Merr.) peel (a, b) and pulp extracts (c). The latter was additionally analysed after alkaline hydrolysis (d).
Fig. 2 APCI(+)-MS2 experiment of compound 13 assigned to (9Z)-violaxanthin laurate (a). Mass spectral data is summarised in Table 2. Proposed mass fragmentation of violaxanthin esters (b) (Britton et al., 1995; Breithaupt, 2002).
5.2
Tables
Table 1 HPLC-DAD-APCI-MSn data of lipophilic pigments isolated from the pineapple (Ananas comosus [L.] Merr.) infructescence.
Table 2 Quantitation of prevailing pulp carotenoids (µg/100 g of fresh weight) in different pineapple (Ananas comosus [L.] Merr.) varieties harvested at full and green ripe maturity, respectively. Green ripe fruits were dispatched by sea freight and analysed at 14 and 21 days after harvest (dah).
Table 1. No.
tR (min)
1
9.5
2
λmax (nm)
DB/DII a (%)
DIII/DII b (%)
[M]−• (m/z)
[M+H]+ (m/z)
APCI(+)-MSn experiment (m/z, % base peak intensity)
Proposed structure
266, 315, 327 416/439/469
-
87
-
601
[601]: 583 (100), 565 (17), 509 (17), 491 (12), 221 (7), 181 (1)
(all-E)-Violaxanthin d
10.6
265, 315, 327 413/436/464
2
83
-
601
[601]: 583 (100), 565 (20), 547 (4), 509 (15), 491 (9), 393 (8), 221 (10), 181 (2)
(all-E)-Neoxanthin d
3
11.5
250, 300, 311 399/422/448
3
95
-
601
[601]: 583 (100), 565 (15), 509 (23), 491 (14), 221 (6), 181 (1)
(all-E)-Luteoxanthin
4
14.5
267, 314, 327 412/436/465
8
87
-
601
[601]: 583 (100), 565 (21), 509 (20), 491 (12), 221 (7), 181 (1)
(9Z)-Violaxanthin
5
16.3
251, 300, 311 397/417/443
5
83
-
601
[601]: 583 (100), 565 (18), 509 (15), 491 (12), 221 (9), 181 (1)
(9Z)- or (9´Z)-Luteoxanthin
6
17.6
256, 316, 343 467/602/650
-
-
-
907
[907]: 629 (100), 597 (60), 569 (50), 541 (24)
Chlorophyll b d
7
18.2
267, 332 sh420/445/473
5
57
568
551 c
[551]: 533 (100), 495 (49), 477 (24), 459 (44), 429 (88)
(all-E)-Lutein d
8
23.4
266, 315, 327 416/439/469
-
84
-
755
[755]: 737 (100), 719 (6), 663 (16), 645 (8), 583 (35), 565 (17), 547 (7)
(all-E)-Violaxanthin caprate (C10)
9
24.6
265, 337, 383 432/619/665
-
-
-
893
[893]: 615 (85), 583 (46), 555 (100)
Chlorophyll a d
10
25.5
267, 314, 327 412/436/464
7
87
-
727
[727]: 709 (89), 691 (3), 635 (5), 617 (7), 583 (100), 565 (27), 547 (8)
(9Z)-Violaxanthin caprylate (C8)
11
28.0
266, 315, 327 416/439/469
-
83
-
783
[783]: 765 (100), 747 (8), 691 (11), 673 (12), 583 (95), 565 (38), 547 (8)
(all-E)-Violaxanthin laurate (C12)
12
29.4
267, 314, 327 412/436/464
8
88
-
755
[755]: 737 (100), 719 (7), 663 (10), 645 (10), 583 (83), 565 (26), 547 (6)
(9Z)-Violaxanthin caprate (C10)
13
33.8
267, 314, 327 412/436/465
8
88
-
783
[783]: 765 (100), 747 (6), 691 (9), 673 (12), 583 (83), 565 (29), 547 (5)
(9Z)-Violaxanthin laurate (C12)
14
38.8
267, 314, 327 412/436/465
7
79
-
811
[811]: 793 (100), 775 (8), 719 (7), 701 (14), 583 (75), 565 (28), 547 (4)
(9Z)-Violaxanthin myristate (C14)
15
40.1
266, 315, 327 416/437/467
-
80
-
839
[839]: 821 (100), 803 (10), 747 (9), 729 (11), 583 (80), 565 (25), 547 (5)
(all-E)-Violaxanthin palmitate (C16)
16
43.9
275, 330 sh426/452/477
4
24
536
537
[537]: 481 (52), 457 (16), 445 (46), 444 (100), 399 (16), 387 (39), 347 (49)
(all-E)-β-Carotene d
17
44.0
324 413/610/667
-
-
-
871
[871]: 593 (100), 533 (82)
Pheophytin a
18
44.7
267, 314, 327 412/436/465
7
84
-
839
[839]: 821 (100), 803 (13), 747 (16), 729 (16), 583 (80), 565 (25), 547 (7)
(9Z)-Violaxanthin palmitate (C16)
19
46.3
267, 315, 328 413/436/465
3
78
-
937
[937]: 919 (39), 845 (5), 827 (15), 765 (100), 747 (12), 737 (92), 565 (46), 547 (44)
(9Z)-Violaxanthin capratelaurate (C10-C12)
20
47.4
265, 315, 328 417/440/470
-
84
-
965
[965]: 947 (2), 765 (100), 747 (20), 565 (42)
(all-E)-Violaxanthin dilaurate (C12-C12)
21
50.5
267, 315, 327 6 83 965 [965]: 947 (10), 765 (100), 747 (78), 565 (41) 413/436/465 tR, retention time; λmax, UV/Vis absorption maxima; sh, shoulder. a D /D , ratio of absorption intensity at ‘cis-band’ near UV maximum (D ) to intensity at main absorption maximum (D ). B II B II b D /D , ratio of absorption intensity at longest wavelength maximum (D ) to D . III II III II c In-source elimination of water ([M + H − H O]+). 2 d Verified by an authentic reference standard.
(9Z)-Violaxanthin dilaurate (C12-C12)
Table 2. No.
Proposed structure
‘Queen Victoria’ fully ripe 2 dah
‘Sugar Loaf’ fully ripe 2 dah
‘Smooth Cayenne’ ‘MD2’ fully ripe fully ripe 2 dah 2 dah
‘MD2’ green ripe 14 dah
‘MD2’ green ripe 21 dah
1
(all-E)-Violaxanthin
9 ± 5 bc
6±1c
6±1c
13 ± 4 b
26 ± 5 a
6 ± 3 bc
10
(9Z)-Violaxanthin caprylate
35 ± 12 a
tr.
9±1c
20 ± 3 b
25 ± 3 a
22 ± 6 ab
12
(9Z)-Violaxanthin caprate
90 ± 36 a
3±1d
21 ± 3 c
47 ± 9 b
74 ± 8 a
54 ± 20 ab
13
(9Z)-Violaxanthin laurate
151 ± 60 ab
5±1d
35 ± 3 c
109 ± 18 b
161 ± 19 a
107 ± 40 b
14
(9Z)-Violaxanthin myristate
40 ± 10 ab
tr.
14 ± 1 c
31 ± 4 b
38 ± 3 a
29 ± 7 b
16
(all-E)-β-Carotene
87 ± 32 a
4±0 f
17 ± 1 e
22 ± 4 d
34 ± 4 c
45 ± 4 b
18
(9Z)-Violaxanthin palmitate
56 ± 21 a
tr.
9±1c
30 ± 6 b
41 ± 5 ab
29 ± 9 b
Total carotenoids
565 ± 193 a
29 ± 2 d
110 ± 27 c
302 ± 44 b
432 ± 48 ab
359 ± 89 b
a
*
Calculated as sum of (all-E)-β-carotene, xanthophylls, xanthophyll esters (as free xanthophylls) and unidentified minor constituents. MD2 (syn. “Extra Sweet”); dah, days after harvest; tr., trace. Different letters within a row indicate significant (p < 0.05) differences of means (n = 5 single fruits). * The standard deviation ± 0 equals a value < 0.5 μg/100 g of FW. a
Highlights Detailed HPLC-DAD-APCI-MSn analysis of pineapple carotenoids Identification of 21 lipophilic pigments in the pineapple infructescence Xanthophyll esters are reported for the first time as pineapple constituents Quantitation of carotenoids in four varieties cultivated in Ghana