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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 carotenoids, 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

Keywords Carotenoids · α-Tocopherol · Chlorophylls · HPLC-DAD · Leafy vegetables

AbbreviationsHPLC High performance liquid chromatographyDAD Diode array detectorRP Reversed phaseACN AcetonitriteDCM DichloromethaneMeOH MethanolE.A EthylacetateTHF TetrahydrofuranMTBE Methyl tertiary butyl etherBHT Butylated hydroxytolueneLOQ Limit of quantificationLOD Limit of detectionRSD Relative standard deviation

Introduction

Carotenoids are a class of organic compounds soluble in lipid medium and organic solvents. They are classified into carotenes and xanthophylls. They contribute to the colour, taste and acceptability of plant foods. Carotenoids are highly valued compounds in terms of health benefits 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 & scientist to determine accurately the amount of carot-enoids in the medium of choice. High performance liquid chromatography (HPLC) is the most reliable tool to deter-mine the type and quantity of carotenoids. Reversed phase (RP)-HPLC method was developed to determine carotenes,

Abstract Leafy vegetables are the important compo-nents of our diet and are the source of several beneficial phytochemicals. A sensitive, simple analytical method is therefore required to precisely measure the phytochemical composition. A validated reversed phase high performance liquid chromatography with diode array detection (HPLC-DAD) method was developed to determine simultaneously carotenoids, chlorophylls and α-tocopherol composition of six leafy vegetables (B. compestris, B. rapa, B. juncea, M. neglecta, and two spinach varieties). Carotenoids were extracted and efficiently separated using a tertiary mobile gradient system of methanol–water, water and MTBE in 40 min on a reversed phase C18 column. The method was simple, precise, accurate and highly reproducible. Twelve carotenoids namely lutein and its three isomers, β-carotene-5,6-epoxide, neoxanthin, violaxanthin, two cis-isomers of zeaxanthin, 8-apo-carotenal, all-trans-β-carotene and 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-thin, lutein, 8-apo-carotenal, chlorophyll a and all-trans-β-carotene were present in higher amounts. Significant varia-tions in the major compounds were observed in the selected vegetables. It was concluded that the developed method was highly sensitive, accurate and can be used to analyze carotenoids, chlorophylls and α-tocopherol simultaneously in leafy vegetables as well as in other plant leaves.

* Alam Zeb Azebuom@gmail.com

1 Biochemistry Laboratory, Department of Biotechnology, Faculty of Biological Sciences, University of Malakand, Malakand, Pakistan

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lutein and lycopene in different food materials using iso-cratic elution of methanol:chloroform (94:6) [2]. Similary, carotenoids were separated using RP-ODS-2 column with the help of an isocratic mixture of highly expensive chemi-cals mixture, i.e. ACN:DCN:MeOH [3]. The VYDAC C18 column was used with ACN:2-propanol and water for the separation of carotenoids in the leaves, stem and flower fruits. The separated compounds were β-carotene, cis-β-carotene, α-carotene, β-cryptoxanthin, α-cryptoxanthin, lutein, zeaxanthin and other xanthophylls with co-elution and weak separation efficiency [4]. Another method used ACN-MeOH-DCM as elution solvents at different tempera-ture [5]. The authors showed that separation at 21 °C was better than 34 °C. However, lutein and zeaxanthin eluted very closely. Dachtler et al. [6] used RP C30 column and a binary gradient mixture of acetone and water for separation of carotenoids in spinach leaves. The use of acetone is usu-ally not recommend for separation as it causes formation of artefacts in column during separation. Similarly, another method was reported, which uses RP-18 column and gradi-ent of MeOH–water and ethylacetate for 50 min [7]. These authors used fluorimetric detection for identification of carotenoids.

de Sá and Rodriguez-Amaya [8] developed HPLC method for the quantitation of carotenoids in cooked green vegetables. A C-18 monomeric column was employed for the separation with the help of gradient elution of ACN:MeOH:E.A with 0.05% triethylamine. Another method was reported by Larsen and Christensen [9] for green vegetables. The authors used MeOH:water and ethyl-acetate for xanthophylls separation, while MeOH:THF and water:ethanol:THF for carotenoids separation in 60  min. The method was time consuming and uses costly solvents and separate analytical procedures for the selected metab-olites. The uses of MTBE with methanol and water had separated major carotenoids in Arabidopsis leaf [10]. How-ever, co-elution and broad peaks were the major problems. Similar co-elution was also reported when carotenoids were separated using C18 column with a dual gradient sys-tem [11]. Another method used binary gradient system of MeOH with 0.2% ammonium acetate and MTBE on RP column for the separation of pigments in grapevine leaves and berries [12]. However, co-elution was the major prob-lem. The uses of methanol, water and MTBE gradient on C30 separated 10 carotenoids in leaves during thermal processing [13]. Amorim-Carrilho et al. [14] reviewed the methods of analyses of carotenoids. Liquid chromatography on C18 column was considered as the best technique. The major concerns for an analyst in these reported methods are the co-elution, peak broadening, uses of costly and toxic solvent, limited applicability and complex analytical proce-dures. Thus, continuous efforts to improve the methodology for carotenoids determination are warranted. This method

was therefore aimed to present a simple, fast and improved HPLC method for the determination of carotenoids in veg-etable leaves. This paper has also reported for the first time, the carotenoids, chlorophylls and α-tocopherol composi-tion of leafy vegetables commonly consumed in Pakistan, which will be an additional contribution to the development of global carotenoids & phytochemical database.

Materials and methods

Chemicals and reagents

Lutein, β-carotene, 8-apo-carotenal, chlorophyll a, BHT & methanol were from Sigma–Aldrich (Steinheim, Germany). MTBE, α-tocopherol and ammonium acetate was pur-chased from Daejung Chemicals (Daejung, South Korea). Ultrapure deionized double distilled water was prepared by Labtech (Daihan Labtech Co Ltd, South Korea). All other chemicals and reagents were of analytical HPLC standard of highest purity.

Sample collection and preparation

Indigenous fresh leaves of the Brassica compestris, Bras-sica rapa, Brassica juncea, Malva neglecta, and two species of Spinacia oleracea were collected from the market (2 day after harvesting). Samples were crushed using laboratory blender in to paste. The grinded materials were used for the extraction on the same day.

Carotenoids extraction

Carotenoids extraction was carried out using the method of Kimura and Rodriguez-Amaya [15] with some modifica-tion. In brief, one gram of powder was weighed in to a 250 mL flask. Ice cold acetone (5 mL) was added and the mix-ture was shaken using orbital shaker at 130 rpm for 60 min (Witig Labortechnik, Germany). Then 10 mL of absolute ethanol was added and vortexed for 30 min. The extractions were repeated until the leaves residues become colour-less. The solvent was evaporated under vacuum at 35 °C. The residue was dissolved in to HPLC solvent (2 mL) and filtered using Agilent PFTE syringe filters (0.45  µm) and transferred into HPLC vials.

Standard calibration curves

Standard calibration curves of α-tocopherol, β-carotene, 8-apo-carotenal, chlorophyll a and lutein was prepared fresh in HPLC solvent A and injected into HPLC as a sin-gle injection of different concentrations each time. The calibration curves of β-carotene was prepared in the range

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of 10–100  ng, 8-apo-carotenal (2–50  ng), α-tocopherol (1–50 ng), chlorophyll a (2–50 ng) and lutein of 2–200 ng at five points in triplicates. In case where standards refer-ence compounds were not available such as isomers of lutein & β-carotene, were measured with calibration curves of the respective compounds based on similar response fac-tors. Other compounds were extracted from spinach, puri-fied and quantified using spectrophotometer as proposed by Kimura and Rodriguez-Amaya [15].

Chromatography

Carotenoids, chlorophylls and α-tocopherol were deter-mined using a reversed phase HPLC. The HPLC system was equipped with quaternary pump, degasser, autosam-pler and a diode array detector (Agilent 1260 infinity bet-ter, Agilent Technologies, Germany). The column used was an Agilent rapid resolution column (Agilent Zorbax Eclipse C18, Agilent Technologies, Germany) with speci-fication of 4.6 × 100 mm, 3.5 µm, which was maintained at 25 °C. The tertiary gradient system consists of solvent A as methanol: deionized water (92: 8, v/v) with 10 mM ammo-nium acetate, solvent B was deionized water containing 0.01  mM (ammonium acetate) and solvent C was MTBE (100%). The flow rate was fixed at 1 mL/min and injec-tion volume was 10 µL. The efficient gradient program was started with 80% A, 18% B and 2% C (0 min). At 3 min the gradient was 80% A, 12% B and 8% C, which reached 65% A, 5% B with 30% C (25  min). The gradient then finally 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 recorded in the range of 190 to 750 nm and the chromato-grams were obtained at 450 nm using OpenLab Chemsta-tion (Agilent Technologies, Germany). The chromatograms were transferred to SigmaPlot (version 12.3) as CSV file to obtain a better resolution for print. The identification of carotenoids, chlorophylls and α-tocopherol were based on either the available standards (β-carotene, 8-apo-carotenal, chlorophyll a and lutein), their retention times, or by com-paring the absorption spectra reported in the literature. The identified compounds was quantified from the peak area using respective calibrations and represented as mg/ 100 g of fresh weight basis. Chemical tests were performed such as acetylation with acetic anhydride for secondary hydroxy groups of lutein, violaxanthin, and neoxanthin, and epox-ide-furanoid rearrangement of 5,6-epoxy groups of violax-anthin and neoxanthin using HCl [15].

Method validation

The developed method was validated according to the approved guidelines of the International Conference on Harmonization (ICH), for linearity, limits of detection

(LODs), limits of quantitation (LOQs), precision (inter-day and intra-day precision), repeatability, stability, and accuracy.

Data analysis

All identified compouds were presented as mg/ 100  g on fresh weight basis. Data of each identified compounds was analyzed for variations among the selected vegetable leaves using one way ANOVA followed by Tucky test at p < 0.05.

Results and discussion

Optimization of extraction conditions

Extraction of carotenoids is one of the important part in carotenoid analyses. Extraction was carried out in daylight and control conditions at 20 °C. The method reported by Kimura and Rodriguez-Amaya [15] was followed, but sol-vent partition had resulted extraction of some lipid com-ponents, which was found to cause changes in the peak shape. Therefore, absolute ice cold ethanol was used for the extraction. The repeated extractions with ethanol was car-ried out until discoloration, which occurred in short time (<4 min). The method also gives excellent results for other plant leaves.

Optimization of HPLC conditions

The HPLC conditions were optimized to obtain a maxi-mum possible separation efficiency and peak purity. The optimum baseline separation was achieved with a flow rate of 1 mL/min at 25 °C. Different mobile phases were tried as reported in the literature. Solvents such as ACN and DCM were not used because of the high cost and toxicity. From the literature studies, it was observed that methanol, water and MTBE are good solvents [12, 13]. The best solvent sys-tem consists of solvent A as methanol: deionized water (92: 8, v/v) containing 10  mM ammonium acetate as a modi-fier, solvent B was deionized water with 0.01 mM ammo-nium acetate and solvent C was MTBE (100%). Different sets of of these mobile phases were tried. When A/B/C was isocratically started till 3  min with 80/20/0 and reached 60/0/40 at 40 min, the peaks were co-eluted. At initial elu-tion stage, the addition of MTBE at 2% was fruitful, and the amount of void volume has decreased significantly. An efficient baseline separation was achieved when A/B/C was isocratically started with 80/18/2%. Using this gradi-ent, excellent baseline separation was obtained for several carotenoids including isomers of lutein. The separation effi-ciency of carotenoids was much better than reported meth-ods [5], and for chlorophylls [11, 16]. Chromatograms were

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obtained using 292, 450 and 650  nm, however, the chro-matogram at 450 nm has shown all of the separated com-pounds, while 292 and 650 nm chromatograms were only used for quantitative purposes. The spectra were recorded from 250 to 750 nm to observe all possible range of pig-ments present in the leaves.

Method validation

The developed method was validated for quantitative meas-urements using standard calibration curves of five stand-ard compounds (Fig.  1). Five calibration curves with five points in triplicates of different concentration ranges were constructed as shown in Table 1. The standard calibration of α-tocopherol was prepared in the concentration of 1, 10, 20, 30, 50  ng per injection each with a linear regression equation of Y = 3.5917 X + 77.6636 and a correlation coef-ficient of 0.9820. The LOD and LOQ were 0.04 and 1.6 ng respectively. The LOD and LOQ of the present method were better than reported HPLC & UPLC methods [16]. In

case of lutein, the LOD & LOQ were 0.05 & 1.1 ng with regression equation of Y = 0.9175 X + 62.0823 and a corre-lation coefficient of 0.9993. The present method was highly sensitive than previously reported methods [5, 12, 16]. The LOD & LOQ of 8-Apo-β-carotenal were 0.08 & 0.23  ng with linear regression equation of Y = 3.555 X + 72.3670 with R2 of 0.9981. Similarly, the regression equations 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-ficient of 0.9985 and 0.9978 respectively. The sensitivity of the method for these two compounds were higher than the respected values reported by Chauveau-Duriot et  al. [16] and Barba et  al. [17], while comparable to the results of Lashbrooke et al. [12]. The standard addition method was used for the recovery of α-tocopherol, which was 101.3%, lutein (100.6%), 8-apo-β-carotenal (94.5%), chlorophyll a (95.6%) and all-trans-β-carotene had 99.1%. These results indicate good recovery values for all the analysed standard compounds. The method was highly precise as indicated by the lowest % RSD values of inter-day (n = 3) and intra-day (n = 5) precision for all the standard compounds analysed. Similarly, the stability of the chromatographic method was assessed by repeated (n = 9) injections of each standard compound. It was found that % RSD values of the repeated injections were lower than reported methods, which is a strong evidence for sensitivity of the method.

Identification of carotenoids

Figures  2 and 3 showed a total of 16 carotenoids in all vegetable leaves with detail spectral characteristics men-tioned in Table 2. Carotenoids were identified using avail-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 eluted at 0.9  min with absorption spectra of 292  nm and was identified from the standard compound. Compound 2

Time (min)0 5 10 15 20 25 30 35 40

mA

U

0

50

100

150

200

1

9 12 13

15

Fig. 1 A representative HPLC-DAD chromatograms of standard ref-erence compounds. 1 α-Tocopherol, 9 lutein, 12 8-apo-carotenal, 13 chlorophyll a and 15 All-trans-β-carotene

Table 1 Validation parameters of the developed method. Values are represented as mean of the replicates

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

Compound Regression equation R2 LOD (ng) LOQ (ng) Recovery (%) Precision% RSD

Stability% RSD (n = 9)

Inter-day (n = 3) Intra-day (n = 5)

α-Tocopherol Y = 3.5917 X + 77.6636

0.9820 0.4 2.6 101.3 2.25 2.25 2.221

Lutein Y = 0.9175 X + 62.0823

0.9993 0.05 1.1 100.6 0.95 1.31 1.198

8-Apo-β-carotenal Y = 3.555 X + 72.3670 0.9981 0.08 0.23 94.5 2.54 2.13 1.845Chlorophyll a Y = 9.6196 X + 13.922 0.9985 0.09 2.1 95.6 0.68 0.93 1.134All-trans-β-carotene Y = 5.5400 X + 24.1 0.9978 0.08 2.2 99.1 1.12 1.54 1.254

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was chlorophyll b with λmax of 666 & 432  nm and was identified from the work of Kupper et al. [11]. Compound 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-5,6-epoxide with λmax of 470, 442 and 418, which was identified from the author recent work [19]. It was iden-tified as a product of oxidation, which may be formed as primary metabolite, for the formation of other oxygen con-taining carotenoids, epecially lutein or its metabolites. All-trans-neoxathin (compound 5) and its isomeric compound all-trans-violaxanthin (compound 6) were separated effec-tively with elution times of 10 and 10.7 min. These com-pounds have λmax of 466, 436, 418 and 470, 440 and 416 and were identified by comparing the absorption spectra reported by Azevedo-Meleiro and Rodriguez-Amaya [20]. At the retention time of 12.7 and 13 min, compound 7 & 8 were 13- and 9-cis-zeaxanthin isomers, which were iden-tified from the work of Updike and Schwartz [21]. Simi-larly, compounds 9, 10 and 11 were lutein, 9-cis-lutein and 9′-cis-lutein. The absorption spectra of lutein cis-isomers were compared with the one reported by Aman et al. [22].

The separation efficiency in terms of lutein or its isomers were better than previously reported methods [16, 20, 22].

Compound 12 was identified as apo-caroten-8-al by comparing the retention time and absorption spectra with standard compound. This compound was previously known as product of oxidation and metabolism during various conditions [19]. Similarly, compound 13 was iden-tified as chlorophyll a with λmax of 663 and 431, which was identified from the standard compounds. Peak 14 was β-Cryptoxanthin ester, which was tentatively identi-fied from the work of Kupper et al. [11]. However, it was not possible to exactly confirm the fatty acid residue in the ester. Compound 15 and 16 were all-trans-β-carotene and its 13-cis- isomer. All-trans-β-carotene was identified using standard compound, while 13-cis-isomer was identi-fied from the author’s previous work [19]. There were small peaks, which were not identified due to the limitation of our HPLC system, thus the uses of mass spectrometry with

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

Fig. 3 A Representative HPLC-DAD chromatograms of three leafy vegetables. a Malva neglecta, b Sindhi Spinach and c Oriental spin-ach. The details identification is given at Table 2

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this method could be a plus point to determine the exact fatty acid moiety in the ester and other none identifiable carotenoids.

Composition of vegetable leaves

Sixteen carotenoids were identified in the selected vegetable leaves as shown in Figs.  2 and 3. The

α-tocopherol was present in higher amounts in M. neglecta (5.66  mg/100  g) followed by B. compestris (4.97  mg/100  g) and spinach leaves (2.61  mg/100  g) as shown in Table  3. Chlorophyll b was present in high amounts in B. juncea (8.1  mg/100  g) followed by M. neglecta (3.96  mg/100  g) and oriental spinach (2.96  mg/100  g). The amount of all-trans-anhydrolutein III and β-carotene-5,6-epoxide were present in very small

Table 2 Identification and spectral characteristics of carotenoids present in the selected vegetable leaves

Peak Time (min) Spectra Identity %III/II Identification reference

1 0.9 292 α-Tocopherol 0 Standard2 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 Standard10 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 Standard13 26.3 663, 431 Chlorophyll a 0 Standard14 29.4 474, 446, 424 β-Cryptoxanthin ester 74 Kupper et al. [11]15 33 476, 452, 422 All-trans-β-carotene 16 Standard16 33.4 470, 444, 332 13-cis-β-carotene 7 Zeb [19]

Table 3 Phytochemicals composition of selected vegetables leaves

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)

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 ± 0.11eChlorophyll b 0.46 ± 0.05a 3.59 ± 0.11b 8.1 ± 0.21c 3.96 ± 0.03d 0.18 ± 0.01e 2.96 ± 0.05fAll-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 ± 0.02aAll-trans-Neoxanthin 2.86 ± 0.03a 2.82 ± 0.04a 2.88 ± 0.01a 3.99 ± 0.02b 2.91 ± 0.05a 2.95 ± 0.04aAll-trans-violaxanthin 7.15 ± 0.11a 7.66 ± 0.01b 7.82 ± 0.21b 11.2 ± 0.02c 10.4 ± 0.04d 10.9 ± 0.01d13-cis-Zeaxanthin 0.34 ± 0.02a 0.36 ± 0.02a 0.29 ± 0.01a 0.65 ± 0.001b 0.94 ± 0.06c 1.12 ± 0.01c9-cis-Zeaxanthin 0.56 ± 0.04a 0.34 ± 0.01b 0.48 ± 0.02a 0.52 ± 0.01a 0.91 ± 0.04c 0.77 ± 0.02dLutein 19.1 ± 0.08a 18.9 ± 0.09a 18.4 ± 0.41a 24.1 ± 0.41b 20.6 ± 0.52a 18.7 ± 0.26a9-cis-Lutein 0.59 ± 0.02a 0.69 ± 0.01b 0.68 ± 0.03b 1.11 ± 0.01c 1.72 ± 0.05d 1.75 ± 0.02d9′-cis-Lutein 0.98 ± 0.11a 0.93 ± 0.02a 1.1 ± 0.01b 1.18 ± 0.03c 0.95 ± 0.01a 0.84 ± 0.01dApo-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 ± 0.13aChlorophyll a 26.1 ± 1.21a 25.3 ± 1.12a 22.2 ± 0.22b 20.5 ± 0.72b 24.1 ± 0.23c 22.8 ± 0.32bβ-Cryptoxanthin ester 0.46 ± 0.03a 0.62 ± 0.02b 0.59 ± 0.01b 0.16 ± 0.01c 0.18 ± 0.01c 0.18 ± 0.01cAll-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 ± 0.24b13-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

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amount less than 1 and 2 mg/100 g respectively. Among the selected vegetable leaves, a high amounts of all-trans-neoxantin was present in M. neglecta (3.99  mg/100  g), followed by oriental spinach (2.95  mg/100  g). Neoxan-thin was present in higher amounts in all selected veg-etables than the values (0.16  mg/100  g) reported previ-ously [4]. All-trans-violaxanthin were present in higher amounts in M. neglecta (11.2  mg/100  g), followed by spinach varieties. The amounts were, however, higher than the reported values (3.04  mg/100  g) of Müller [4]. Similarly, the cis-isomer of zeaxanthin was present in less than 1  mg/100  g except oriental spinach, which contains 1.12  mg/100  g. The amount of zeaxanthin iso-mers in spinach was relatively in equals to the amounts reported by Müller [4] and Humphries and Khachik [23]. Lutein was present in high amounts (24.1  mg/100  g) in M. neglecta, followed by spinach (20.6 mg/100 g) and B. compestris (19.1 mg/100 g).

The amount of lutein in spinach was higher than the val-ues (8.4 mg/100 g and 8.5 mg/100 g) reported by Müller [4] and Updike and Schwartz [21] respectively. The reason may be due to the difference in the varieties, climate and soil conditions and method of analysis. Krumbein et al. [24] compared the carotenoid composition of different Brassica vegetables. The authors reported a lower amount of lutein (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 factors. There were two cis-isomers of lutein, which were present in less than 2 mg/100 g in all analysed vegetables. The amount of 8-apo-carotenal was higher in B. compes-tris, B. rapa varieties, which was followed by spinach vari-eties. Similarly, the highest amounts of chlorophyll a was present in B. compestris (26.1  mg/100  g), followed by B. rapa leaves (25.3  mg/100  g). The amount of chlorophyll a was lower than reported by Krumbein et al. [24], which may be due to the variations arise from the difference in varieties, climate, soil condition and method of extraction and analyses.

The third to the last carotenoid was an ester of β-cryptoxanthin, which was present less than 1  mg/100  g in all selected vegetables. All-trans-β-carotene was fourth largest compounds in terms of amount in all selected vegetable leaves. The highest amounts were present in B. compestris (15.3  mg/100  g), followed by B. rapa (15.1  mg/100  g). The lowest amounts were present in M. neglecta leaves. The values of all-trans-β-carotene in spinach varieties were higher than the reported values of Müller [4] and Krumbein et  al. [24]. The 13-cis-isomer of β-carotene was present in relatively small amounts in all vegetables. These results suggest that the amounts of chlorophyll a & b, neoxanthin, violaxanthin, lutein, 8-apo-carotenal and all-trans-β-carotene can be used as finger printing different leafy vegetables.

Conclusions

This work has reported a highly reproducible, simple method for the simultaneous determination of carotenoids, chlorophylls and α-tocopherol in leafy vegetables. The nutrient composition of leafy vegetables (B. compestris, B. rapa, B. juncea, M. neglecta, and two spinach varie-ties) consumed in Pakistan has been reported for the first time. Carotenoids were efficiently separated on reversed phase C18 column and tertiary mobile gradient system of methanol–water, water and MTBE. The method was found simple, precise, accurate and highly reproducible. Sixteen phytochemicals including twelve carotenoids (lutein and its three isomers, β-carotene-5,6-epoxide, neoxanthin, violax-anthin, two cis-isomers of zeaxanthin, 8-apo-carotenal, all-trans-β-carotene and its 13-cis-isomer); one fatty acid ester (β-cryptoxanthin ester); α-tocopherol and chlorophyll a & b were quantified. The α-tocopherol, neoxanthin, violaxan-thin, lutein, 8-apo-carotenal, chlorophyll a and all-trans-β-carotene were present in higher amounts. Significant varia-tions in the major compounds were observed in the selected vegetables. It is concluded that the developed method was highly sensitive and accurate and can be applid to analyse carotenoids, chlorophylls and α-tocopherol simultaneously in all leafy vegetables as well as in other plant leaves.

Acknowledgements The author is highly grateful for the financial assistant by the Higher Education Commission (HEC) Pakistan under National Research Program for Universities (NRPU), Project No. 2344.

Authors Contributions The author is the sole contributor of the article.

Compliance with ethical standards

Conflict of interest The author declare no conflict of interest to any body or institution.

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