Food Measure (2017) 11:979–986 DOI 10.1007/s11694-017-9472-y

ORIGINAL PAPER

A simple, sensitive HPLC-DAD method for simultaneous determination of , chlorophylls and α-tocopherol in leafy vegetables

Alam Zeb1

Received: 1 October 2016 / Accepted: 17 January 2017 / Published online: 6 February 2017 © Springer Science+Business Media New York 2017

Abstract Leafy vegetables are the important compo- Keywords Carotenoids · α-Tocopherol · Chlorophylls · nents of our diet and are the source of several beneficial HPLC-DAD · Leafy vegetables phytochemicals. A sensitive, simple analytical method is therefore required to precisely measure the phytochemical Abbreviations composition. A validated reversed phase high performance HPLC High performance liquid chromatography liquid chromatography with diode array detection (HPLC- DAD Diode array detector DAD) method was developed to determine simultaneously RP Reversed phase carotenoids, chlorophylls and α-tocopherol composition ACN Acetonitrite of six leafy vegetables (B. compestris, B. rapa, B. juncea, DCM Dichloromethane M. neglecta, and two spinach varieties). Carotenoids were MeOH Methanol extracted and efficiently separated using a tertiary mobile E.A Ethylacetate gradient system of methanol–water, water and MTBE in THF Tetrahydrofuran 40 min on a reversed phase C18 column. The method was MTBE Methyl tertiary butyl ether simple, precise, accurate and highly reproducible. Twelve BHT Butylated hydroxytoluene carotenoids namely and its three isomers, β-- LOQ Limit of quantification 5,6-epoxide, neoxanthin, , two cis-isomers LOD Limit of detection of , 8-apo-carotenal, all-trans-β-carotene and RSD Relative standard deviation its 13-cis-isomer; one fatty acid ester (β-cryptoxanthin ester); α-tocopherol and chlorophyll a & b were quantified in vegetable leaves. α-Tocopherol, neoxanthin, violaxan- Introduction thin, lutein, 8-apo-carotenal, chlorophyll a and all-trans-β- carotene were present in higher amounts. Significant varia- Carotenoids are a class of organic compounds soluble in tions in the major compounds were observed in the selected lipid medium and organic solvents. They are classified vegetables. It was concluded that the developed method into and . They contribute to the was highly sensitive, accurate and can be used to analyze colour, taste and acceptability of plant foods. Carotenoids carotenoids, chlorophylls and α-tocopherol simultaneously are highly valued compounds in terms of health benefits in leafy vegetables as well as in other plant leaves. such as their role as a strong antioxidant, especially for human health [1]. An existence of extremely large numbers of carotenoids in nature and their importance in our daily life had increased the attention of analytical food chemist * Alam Zeb & scientist to determine accurately the amount of carot- [email protected] enoids in the medium of choice. High performance liquid chromatography (HPLC) is the most reliable tool to deter- 1 Biochemistry Laboratory, Department of Biotechnology, Faculty of Biological Sciences, University of Malakand, mine the type and quantity of carotenoids. Reversed phase Malakand, Pakistan (RP)-HPLC method was developed to determine carotenes,

Vol.:(0123456789)1 3 980 A. Zeb lutein and in different food materials using iso- was therefore aimed to present a simple, fast and improved cratic elution of methanol:chloroform (94:6) [2]. Similary, HPLC method for the determination of carotenoids in veg- carotenoids were separated using RP-ODS-2 column with etable leaves. This paper has also reported for the first time, the help of an isocratic mixture of highly expensive chemi- the carotenoids, chlorophylls and α-tocopherol composi- cals mixture, i.e. ACN:DCN:MeOH [3]. The VYDAC C18 tion of leafy vegetables commonly consumed in Pakistan, column was used with ACN:2-propanol and water for the which will be an additional contribution to the development separation of carotenoids in the leaves, stem and flower of global carotenoids & phytochemical database. fruits. The separated compounds were β-carotene, cis-β- carotene, α-carotene, β-cryptoxanthin, α-cryptoxanthin, lutein, zeaxanthin and other xanthophylls with co-elution Materials and methods and weak separation efficiency [4]. Another method used ACN-MeOH-DCM as elution solvents at different tempera- Chemicals and reagents ture [5]. The authors showed that separation at 21 °C was better than 34 °C. However, lutein and zeaxanthin eluted Lutein, β-carotene, 8-apo-carotenal, chlorophyll a, BHT & very closely. Dachtler et al. [6] used RP C30 column and a methanol were from Sigma–Aldrich (Steinheim, Germany). binary gradient mixture of acetone and water for separation MTBE, α-tocopherol and ammonium acetate was pur- of carotenoids in spinach leaves. The use of acetone is usu- chased from Daejung Chemicals (Daejung, South Korea). ally not recommend for separation as it causes formation Ultrapure deionized double distilled water was prepared by of artefacts in column during separation. Similarly, another Labtech (Daihan Labtech Co Ltd, South Korea). All other method was reported, which uses RP-18 column and gradi- chemicals and reagents were of analytical HPLC standard ent of MeOH–water and ethylacetate for 50 min [7]. These of highest purity. authors used fluorimetric detection for identification of carotenoids. Sample collection and preparation de Sá and Rodriguez-Amaya [8] developed HPLC method for the quantitation of carotenoids in cooked green Indigenous fresh leaves of the Brassica compestris, Bras- vegetables. A C-18 monomeric column was employed sica rapa, Brassica juncea, Malva neglecta, and two species for the separation with the help of gradient elution of of Spinacia oleracea were collected from the market (2 day ACN:MeOH:E.A with 0.05% triethylamine. Another after harvesting). Samples were crushed using laboratory method was reported by Larsen and Christensen [9] for blender in to paste. The grinded materials were used for the green vegetables. The authors used MeOH:water and ethyl- extraction on the same day. acetate for xanthophylls separation, while MeOH:THF and water:ethanol:THF for carotenoids separation in 60 min. Carotenoids extraction The method was time consuming and uses costly solvents and separate analytical procedures for the selected metab- Carotenoids extraction was carried out using the method of olites. The uses of MTBE with methanol and water had Kimura and Rodriguez-Amaya [15] with some modifica- separated major carotenoids in Arabidopsis leaf [10]. How- tion. In brief, one gram of powder was weighed in to a 250 ever, co-elution and broad peaks were the major problems. mL flask. Ice cold acetone (5 mL) was added and the mix- Similar co-elution was also reported when carotenoids ture was shaken using orbital shaker at 130 rpm for 60 min were separated using C18 column with a dual gradient sys- (Witig Labortechnik, Germany). Then 10 mL of absolute tem [11]. Another method used binary gradient system of ethanol was added and vortexed for 30 min. The extractions MeOH with 0.2% ammonium acetate and MTBE on RP were repeated until the leaves residues become colour- column for the separation of pigments in grapevine leaves less. The solvent was evaporated under vacuum at 35 °C. and berries [12]. However, co-elution was the major prob- The residue was dissolved in to HPLC solvent (2 mL) and lem. The uses of methanol, water and MTBE gradient on filtered using Agilent PFTE syringe filters (0.45 µm) and C30 separated 10 carotenoids in leaves during thermal transferred into HPLC vials. processing [13]. Amorim-Carrilho et al. [14] reviewed the methods of analyses of carotenoids. Liquid chromatography Standard calibration curves on C18 column was considered as the best technique. The major concerns for an analyst in these reported methods are Standard calibration curves of α-tocopherol, β-carotene, the co-elution, peak broadening, uses of costly and toxic 8-apo-carotenal, chlorophyll a and lutein was prepared solvent, limited applicability and complex analytical proce- fresh in HPLC solvent A and injected into HPLC as a sin- dures. Thus, continuous efforts to improve the methodology gle injection of different concentrations each time. The for carotenoids determination are warranted. This method calibration curves of β-carotene was prepared in the range

1 3 A simple, sensitive HPLC-DAD method for simultaneous determination of carotenoids,… 981 of 10–100 ng, 8-apo-carotenal (2–50 ng), α-tocopherol (LODs), limits of quantitation (LOQs), precision (inter- (1–50 ng), chlorophyll a (2–50 ng) and lutein of 2–200 ng day and intra-day precision), repeatability, stability, and at five points in triplicates. In case where standards refer- accuracy. ence compounds were not available such as isomers of lutein & β-carotene, were measured with calibration curves Data analysis of the respective compounds based on similar response fac- tors. Other compounds were extracted from spinach, puri- All identified compouds were presented as mg/ 100 g on fied and quantified using spectrophotometer as proposed by fresh weight basis. Data of each identified compounds was Kimura and Rodriguez-Amaya [15]. analyzed for variations among the selected vegetable leaves using one way ANOVA followed by Tucky test at p < 0.05. Chromatography

Carotenoids, chlorophylls and α-tocopherol were deter- Results and discussion mined using a reversed phase HPLC. The HPLC system was equipped with quaternary pump, degasser, autosam- Optimization of extraction conditions pler and a diode array detector (Agilent 1260 infinity bet- ter, Agilent Technologies, Germany). The column used Extraction of carotenoids is one of the important part in was an Agilent rapid resolution column (Agilent Zorbax analyses. Extraction was carried out in daylight Eclipse C18, Agilent Technologies, Germany) with speci- and control conditions at 20 °C. The method reported by fication of 4.6 × 100 mm, 3.5 µm, which was maintained at Kimura and Rodriguez-Amaya [15] was followed, but sol- 25 °C. The tertiary gradient system consists of solvent A as vent partition had resulted extraction of some lipid com- methanol: deionized water (92: 8, v/v) with 10 mM ammo- ponents, which was found to cause changes in the peak nium acetate, solvent B was deionized water containing shape. Therefore, absolute ice cold ethanol was used for the 0.01 mM (ammonium acetate) and solvent C was MTBE extraction. The repeated extractions with ethanol was car- (100%). The flow rate was fixed at 1 mL/min and injec- ried out until discoloration, which occurred in short time tion volume was 10 µL. The efficient gradient program was (<4 min). The method also gives excellent results for other started with 80% A, 18% B and 2% C (0 min). At 3 min the plant leaves. gradient was 80% A, 12% B and 8% C, which reached 65% A, 5% B with 30% C (25 min). The gradient then finally Optimization of HPLC conditions reached 60:0:40 (A:B:C) % at 40 min with post gradient of 10 min for recovery of the initial gradient. The spectra were The HPLC conditions were optimized to obtain a maxi- recorded in the range of 190 to 750 nm and the chromato- mum possible separation efficiency and peak purity. The grams were obtained at 450 nm using OpenLab Chemsta- optimum baseline separation was achieved with a flow rate tion (Agilent Technologies, Germany). The chromatograms of 1 mL/min at 25 °C. Different mobile phases were tried as were transferred to SigmaPlot (version 12.3) as CSV file reported in the literature. Solvents such as ACN and DCM to obtain a better resolution for print. The identification of were not used because of the high cost and toxicity. From carotenoids, chlorophylls and α-tocopherol were based on the literature studies, it was observed that methanol, water either the available standards (β-carotene, 8-apo-carotenal, and MTBE are good solvents [12, 13]. The best solvent sys- chlorophyll a and lutein), their retention times, or by com- tem consists of solvent A as methanol: deionized water (92: paring the absorption spectra reported in the literature. The 8, v/v) containing 10 mM ammonium acetate as a modi- identified compounds was quantified from the peak area fier, solvent B was deionized water with 0.01 mM ammo- using respective calibrations and represented as mg/ 100 g nium acetate and solvent C was MTBE (100%). Different of fresh weight basis. Chemical tests were performed such sets of of these mobile phases were tried. When A/B/C was as acetylation with acetic anhydride for secondary hydroxy isocratically started till 3 min with 80/20/0 and reached groups of lutein, violaxanthin, and neoxanthin, and epox- 60/0/40 at 40 min, the peaks were co-eluted. At initial elu- ide-furanoid rearrangement of 5,6-epoxy groups of violax- tion stage, the addition of MTBE at 2% was fruitful, and anthin and neoxanthin using HCl [15]. the amount of void volume has decreased significantly. An efficient baseline separation was achieved when A/B/C Method validation was isocratically started with 80/18/2%. Using this gradi- ent, excellent baseline separation was obtained for several The developed method was validated according to the carotenoids including isomers of lutein. The separation effi- approved guidelines of the International Conference on ciency of carotenoids was much better than reported meth- Harmonization (ICH), for linearity, limits of detection ods [5], and for chlorophylls [11, 16]. Chromatograms were

1 3 982 A. Zeb obtained using 292, 450 and 650 nm, however, the chro- case of lutein, the LOD & LOQ were 0.05 & 1.1 ng with matogram at 450 nm has shown all of the separated com- regression equation of Y = 0.9175 X + 62.0823 and a corre- pounds, while 292 and 650 nm chromatograms were only lation coefficient of 0.9993. The present method was highly used for quantitative purposes. The spectra were recorded sensitive than previously reported methods [5, 12, 16]. The from 250 to 750 nm to observe all possible range of pig- LOD & LOQ of 8-Apo-β-carotenal were 0.08 & 0.23 ng ments present in the leaves. with linear regression equation of Y = 3.555 X + 72.3670 with ­R2 of 0.9981. Similarly, the regression equations Method validation of chlorophyll a and all-trans-β-carotene was Y = 9.6196 X + 13.922 and Y = 5.5400 X + 24.1 with correlation coef- The developed method was validated for quantitative meas- ficient of 0.9985 and 0.9978 respectively. The sensitivity of urements using standard calibration curves of five stand- the method for these two compounds were higher than the ard compounds (Fig. 1). Five calibration curves with five respected values reported by Chauveau-Duriot et al. [16] points in triplicates of different concentration ranges were and Barba et al. [17], while comparable to the results of constructed as shown in Table 1. The standard calibration Lashbrooke et al. [12]. The standard addition method was of α-tocopherol was prepared in the concentration of 1, 10, used for the recovery of α-tocopherol, which was 101.3%, 20, 30, 50 ng per injection each with a linear regression lutein (100.6%), 8-apo-β-carotenal (94.5%), chlorophyll a equation of Y = 3.5917 X + 77.6636 and a correlation coef- (95.6%) and all-trans-β-carotene had 99.1%. These results ficient of 0.9820. The LOD and LOQ were 0.04 and 1.6 ng indicate good recovery values for all the analysed standard respectively. The LOD and LOQ of the present method compounds. The method was highly precise as indicated by were better than reported HPLC & UPLC methods [16]. In the lowest % RSD values of inter-day (n = 3) and intra-day (n = 5) precision for all the standard compounds analysed. 200 Similarly, the stability of the chromatographic method was 13 9 12 assessed by repeated (n = 9) injections of each standard compound. It was found that % RSD values of the repeated 150

1 injections were lower than reported methods, which is a 15 strong evidence for sensitivity of the method. U 100 mA Identification of carotenoids 50 Figures 2 and 3 showed a total of 16 carotenoids in all vegetable leaves with detail spectral characteristics men- 0 tioned in Table 2. Carotenoids were identified using avail- 0510 15 20 25 30 35 40 Time (min) able standards, their retention time, absorption spectra or comparing absorption spectra with the one in reported literature. Peak 1 was found to be α-tocopherol, which Fig. 1 A representative HPLC-DAD chromatograms of standard ref- erence compounds. 1 α-Tocopherol, 9 lutein, 12 8-apo-carotenal, 13 eluted at 0.9 min with absorption spectra of 292 nm and chlorophyll a and 15 All-trans-β-carotene was identified from the standard compound. Compound 2

Table 1 Validation parameters of the developed method. Values are represented as mean of the replicates Compound Regression equation R2 LOD (ng) LOQ (ng) Recovery (%) Precision Stability % RSD % RSD (n = 9) Inter-day (n = 3) Intra-day (n = 5)

α-Tocopherol Y = 3.5917 0.9820 0.4 2.6 101.3 2.25 2.25 2.221 X + 77.6636 Lutein Y = 0.9175 0.9993 0.05 1.1 100.6 0.95 1.31 1.198 X + 62.0823 8-Apo-β-carotenal Y = 3.555 X + 72.3670 0.9981 0.08 0.23 94.5 2.54 2.13 1.845 Chlorophyll a Y = 9.6196 X + 13.922 0.9985 0.09 2.1 95.6 0.68 0.93 1.134 All-trans-β-carotene Y = 5.5400 X + 24.1 0.9978 0.08 2.2 99.1 1.12 1.54 1.254

Standard calibrations were prepared in respective solvents as described in the text. Recovery of each standard was based on the standard addition method. LOQ & LOD were determined from the respective standard calibration curves

1 3 A simple, sensitive HPLC-DAD method for simultaneous determination of carotenoids,… 983

Fig. 2 A representative HPLC-DAD chromatograms of Brassica leafy vegetables. a B. compestris, b B. rapa, and c B. juncea. The details identification is given at Table 2

A Representative HPLC-DAD chromatograms of three leafy was chlorophyll b with λmax of 666 & 432 nm and was Fig. 3 vegetables. a Malva neglecta, b Sindhi Spinach and c Oriental spin- identified from the work of Kupper et al. [11]. Compound ach. The details identification is given at Table 2 3 was all-trans-anhydrolutein, which may be a degradation product of lutein [18] and can be found in several birds, which eat vegetable leaves. Compound 6 was β-carotene- The separation efficiency in terms of lutein or its isomers 5,6-epoxide with λmax of 470, 442 and 418, which was were better than previously reported methods [16, 20, 22]. identified from the author recent work [19]. It was iden- Compound 12 was identified as apo-caroten-8-al by tified as a product of oxidation, which may be formed as comparing the retention time and absorption spectra with primary metabolite, for the formation of other oxygen con- standard compound. This compound was previously taining carotenoids, epecially lutein or its metabolites. All- known as product of oxidation and metabolism during trans-neoxathin (compound 5) and its isomeric compound various conditions [19]. Similarly, compound 13 was iden- all-trans-violaxanthin (compound 6) were separated effec- tified as chlorophyll a with λmax of 663 and 431, which tively with elution times of 10 and 10.7 min. These com- was identified from the standard compounds. Peak 14 pounds have λmax of 466, 436, 418 and 470, 440 and 416 was β-Cryptoxanthin ester, which was tentatively identi- and were identified by comparing the absorption spectra fied from the work of Kupper et al. [11]. However, it was reported by Azevedo-Meleiro and Rodriguez-Amaya [20]. not possible to exactly confirm the fatty acid residue in At the retention time of 12.7 and 13 min, compound 7 & 8 the ester. Compound 15 and 16 were all-trans-β-carotene were 13- and 9-cis-zeaxanthin isomers, which were iden- and its 13-cis- isomer. All-trans-β-carotene was identified tified from the work of Updike and Schwartz [21]. Simi- using standard compound, while 13-cis-isomer was identi- larly, compounds 9, 10 and 11 were lutein, 9-cis-lutein and fied from the author’s previous work [19]. There were small 9′-cis-lutein. The absorption spectra of lutein cis-isomers peaks, which were not identified due to the limitation of were compared with the one reported by Aman et al. [22]. our HPLC system, thus the uses of mass spectrometry with

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Table 2 Identification and Peak Time (min) Spectra Identity %III/II Identification reference spectral characteristics of carotenoids present in the 1 0.9 292 α-Tocopherol 0 Standard selected vegetable leaves 2 4.9 666, 432 Chlorophyll b 0 Kupper et al. [11] 3 5.2 466 All-trans-anhydrolutein III 0 McGraw et al. [18] 4 8.5 470, 442, 418 β-Carotene-5,6-epoxide 80 Zeb [19] 5 10 466, 436, 418 All-trans-Neoxanthin 86 Taylor et al. [10] 6 10.7 470, 440, 416 All-trans-violaxanthin 90 Azevedo-Meleiro and Rodriguez-Amaya [20] 7 12.7 471, 443, 422, 336 13-cis-Zeaxanthin 57 Updike and Schwartz [21] 8 13 472, 444, 424, 336 9-cis-Zeaxanthin 133 Updike and Schwartz [21] 9 15.3 474, 446, 422 Lutein 68 Standard 10 17.6 468, 442, 418, 325 9-cis-Lutein 67 Aman et al. [22] 11 18.2 466, 440, 418, 330 9′-cis-Lutein 36 Aman et al. [22] 12 23.4 462 Apo-caroten-8-al 0 Standard 13 26.3 663, 431 Chlorophyll a 0 Standard 14 29.4 474, 446, 424 β-Cryptoxanthin ester 74 Kupper et al. [11] 15 33 476, 452, 422 All-trans-β-carotene 16 Standard 16 33.4 470, 444, 332 13-cis-β-carotene 7 Zeb [19]

this method could be a plus point to determine the exact α-tocopherol was present in higher amounts in M. fatty acid moiety in the ester and other none identifiable neglecta (5.66 mg/100 g) followed by B. compestris carotenoids. (4.97 mg/100 g) and spinach leaves (2.61 mg/100 g) as shown in Table 3. Chlorophyll b was present in Composition of vegetable leaves high amounts in B. juncea (8.1 mg/100 g) followed by M. neglecta (3.96 mg/100 g) and oriental spinach Sixteen carotenoids were identified in the selected (2.96 mg/100 g). The amount of all-trans-anhydrolutein vegetable leaves as shown in Figs. 2 and 3. The III and β-carotene-5,6-epoxide were present in very small

Table 3 Phytochemicals composition of selected vegetables leaves Identity Composition (mg/100 g) B. Compestris B. rapa B. juncea M. neglecta Spinach Spinach oriental

α-Tocopherol 4.97 ± 0.12a 1.15 ± 0.02b 1.35 ± 0.01b 5.66 ± 0.02c 2.61 ± 0.01d 1.74 ± 0.11e Chlorophyll b 0.46 ± 0.05a 3.59 ± 0.11b 8.1 ± 0.21c 3.96 ± 0.03d 0.18 ± 0.01e 2.96 ± 0.05f All-trans-anhydrolutein III 0.15 ± 0.02a 0.16 ± 0.01a 0.63 ± 0.11b 0.35 ± 0.01c 0.12 ± 0.01a – β-Carotene-5,6-epoxide 0.16 ± 0.01a 0.61 ± 0.01b 1.44 ± 0.22c 0.97 ± 0.01d – 0.18 ± 0.02a All-trans-Neoxanthin 2.86 ± 0.03a 2.82 ± 0.04a 2.88 ± 0.01a 3.99 ± 0.02b 2.91 ± 0.05a 2.95 ± 0.04a All-trans-violaxanthin 7.15 ± 0.11a 7.66 ± 0.01b 7.82 ± 0.21b 11.2 ± 0.02c 10.4 ± 0.04d 10.9 ± 0.01d 13-cis-Zeaxanthin 0.34 ± 0.02a 0.36 ± 0.02a 0.29 ± 0.01a 0.65 ± 0.001b 0.94 ± 0.06c 1.12 ± 0.01c 9-cis-Zeaxanthin 0.56 ± 0.04a 0.34 ± 0.01b 0.48 ± 0.02a 0.52 ± 0.01a 0.91 ± 0.04c 0.77 ± 0.02d Lutein 19.1 ± 0.08a 18.9 ± 0.09a 18.4 ± 0.41a 24.1 ± 0.41b 20.6 ± 0.52a 18.7 ± 0.26a 9-cis-Lutein 0.59 ± 0.02a 0.69 ± 0.01b 0.68 ± 0.03b 1.11 ± 0.01c 1.72 ± 0.05d 1.75 ± 0.02d 9′-cis-Lutein 0.98 ± 0.11a 0.93 ± 0.02a 1.1 ± 0.01b 1.18 ± 0.03c 0.95 ± 0.01a 0.84 ± 0.01d Apo-caroten-8-al 17.7 ± 0.15a 17.7 ± 1.30a 16.6 ± 0.06a 15.1 ± 0.02b 16.9 ± 0.21a 16.9 ± 0.13a Chlorophyll a 26.1 ± 1.21a 25.3 ± 1.12a 22.2 ± 0.22b 20.5 ± 0.72b 24.1 ± 0.23c 22.8 ± 0.32b β-Cryptoxanthin ester 0.46 ± 0.03a 0.62 ± 0.02b 0.59 ± 0.01b 0.16 ± 0.01c 0.18 ± 0.01c 0.18 ± 0.01c All-trans-β-carotene 15.3 ± 0.51a 15.1 ± 0.44a 12.4 ± 0.62b 6.52 ± 0.81c 12.4 ± 0.13b 11.7 ± 0.24b 13-cis-β-carotene 0.73 ± 0.02a 0.72 ± 0.11a 0.67 ± 0.01a 0.28 ± 0.01b 0.69 ± 0.02a 0.48 ± 0.001c

The values are expressed as mg/100 g of fresh weight basis (n = 5) Different letters (a-f) in the same row represent significance at p < 0.05 (Tuky’s test, n = 5)

1 3 A simple, sensitive HPLC-DAD method for simultaneous determination of carotenoids,… 985 amount less than 1 and 2 mg/100 g respectively. Among Conclusions the selected vegetable leaves, a high amounts of all-trans- neoxantin was present in M. neglecta (3.99 mg/100 g), This work has reported a highly reproducible, simple followed by oriental spinach (2.95 mg/100 g). Neoxan- method for the simultaneous determination of carotenoids, thin was present in higher amounts in all selected veg- chlorophylls and α-tocopherol in leafy vegetables. The etables than the values (0.16 mg/100 g) reported previ- nutrient composition of leafy vegetables (B. compestris, ously [4]. All-trans-violaxanthin were present in higher B. rapa, B. juncea, M. neglecta, and two spinach varie- amounts in M. neglecta (11.2 mg/100 g), followed by ties) consumed in Pakistan has been reported for the first spinach varieties. The amounts were, however, higher time. Carotenoids were efficiently separated on reversed than the reported values (3.04 mg/100 g) of Müller phase C18 column and tertiary mobile gradient system of [4]. Similarly, the cis-isomer of zeaxanthin was present methanol–water, water and MTBE. The method was found in less than 1 mg/100 g except oriental spinach, which simple, precise, accurate and highly reproducible. Sixteen contains 1.12 mg/100 g. The amount of zeaxanthin iso- phytochemicals including twelve carotenoids (lutein and its mers in spinach was relatively in equals to the amounts three isomers, β-carotene-5,6-epoxide, neoxanthin, violax- reported by Müller [4] and Humphries and Khachik [23]. anthin, two cis-isomers of zeaxanthin, 8-apo-carotenal, all- Lutein was present in high amounts (24.1 mg/100 g) in trans-β-carotene and its 13-cis-isomer); one fatty acid ester M. neglecta, followed by spinach (20.6 mg/100 g) and B. (β-cryptoxanthin ester); α-tocopherol and chlorophyll a & compestris (19.1 mg/100 g). b were quantified. The α-tocopherol, neoxanthin, violaxan- The amount of lutein in spinach was higher than the val- thin, lutein, 8-apo-carotenal, chlorophyll a and all-trans-β- ues (8.4 mg/100 g and 8.5 mg/100 g) reported by Müller carotene were present in higher amounts. Significant varia- [4] and Updike and Schwartz [21] respectively. The reason tions in the major compounds were observed in the selected may be due to the difference in the varieties, climate and vegetables. It is concluded that the developed method was soil conditions and method of analysis. Krumbein et al. [24] highly sensitive and accurate and can be applid to analyse compared the carotenoid composition of different Brassica carotenoids, chlorophylls and α-tocopherol simultaneously vegetables. The authors reported a lower amount of lutein in all leafy vegetables as well as in other plant leaves. (3.4 to 8.9 mg/100 g) than our present results, which may be due to the difference in the variety and several other Acknowledgements The author is highly grateful for the financial factors. There were two cis-isomers of lutein, which were assistant by the Higher Education Commission (HEC) Pakistan under National Research Program for Universities (NRPU), Project No. present in less than 2 mg/100 g in all analysed vegetables. 2344. The amount of 8-apo-carotenal was higher in B. compes- tris, B. rapa varieties, which was followed by spinach vari- Authors Contributions The author is the sole contributor of the eties. Similarly, the highest amounts of chlorophyll a was article. present in B. compestris (26.1 mg/100 g), followed by B. Compliance with ethical standards rapa leaves (25.3 mg/100 g). The amount of chlorophyll a was lower than reported by Krumbein et al. [24], which Conflict of interest The author declare no conflict of interest to any may be due to the variations arise from the difference in body or institution. varieties, climate, soil condition and method of extraction and analyses. The third to the last carotenoid was an ester of References β-cryptoxanthin, which was present less than 1 mg/100 g in all selected vegetables. All-trans-β-carotene was fourth 1. K. Jomova, M. Valko, Health protective effects of carotenoids largest compounds in terms of amount in all selected and their interactions with other biological antioxidants. Eur. J. vegetable leaves. The highest amounts were present Med. Chem. 70, 102–110 (2013) B. compestris B. rapa 2. S. Takagi, Determination of green leaf carotenoids by HPLC. in (15.3 mg/100 g), followed by Agric. Biol. Chem. 49, 1211–1213 (1985) (15.1 mg/100 g). The lowest amounts were present in 3. S.R.A. Adewusi, J.H. Bradbury, Carotenoids in cassava: Com- M. neglecta leaves. 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