Carotenoids composition in Scutellaria barbataD. Don as detected by high performance liquidchromatography-diode array detection-massspectrometry-atmospheric pressure chemicalionization

Hsin-Lan Liu a,b, Bing-Huei Chen c, Tsai-Hua Kao c,Chyuan-Yuan Shiau a,*a Department of Food Science, National Taiwan Ocean University, Keelung 202, Taiwanb Department of Food and Beverage Management, Taipei College of Maritime Technology, Taipei 111, Taiwanc Department of Food Science, Fu Jen University, Taipei 242, Taiwan

A B S T R A C T

A high performance liquid chromatography-diode array detection-mass spectrometry method

with atmospheric pressure chemical ionization mode (HPLC-DAD-MS-APCI) was devel-

oped to determine carotenoids in Scutellaria barbata D. Don (Banzhilian). Results showed that

a total of 19 carotenoids including internal standard were separated within 37 min by em-

ploying a YMC C30 column and a gradient mobile phase composed of methanol–acetonitrile

(86:14, v/v) and methylene chloride. The various carotenoids in S. barbata were identified

and quantified using internal standard β-apo-8′-carotenal, in which all-trans-lutein and its

cis isomers constituted the largest portion, followed by all-trans-β-carotene and its cis isomers.

A high recovery of 93.0–100.9% and a high reproducibility were obtained with this method.

This would allow determination of carotenoids in herbal samples, and provide a basis for

possible production of functional foods with S. barbata D. Don as raw material.

© 2014 Elsevier Ltd. All rights reserved.

A R T I C L E I N F O

Article history:

Received 3 December 2013

Received in revised form 10 March

2014

Accepted 13 March 2014

Available online 1 April 2014

Keywords:

Scutellaria barbata D. Don

Carotenoids

HPLC-DAD-MS-APCI

1. Introduction

Scutellaria barbata D. Don (S. barbata), a traditional Chinese herbbelonging to genus Scutellaria, is widely consumed in Asiancountries, especially in Taiwan, China, Japan and Korea. Manystudies have demonstrated that S. barbata possesses impor-tant biological activities, including antibacterial (Yu, Lei,Yu, Cai,& Zou, 2004), anti-inflammation (Cheung,To, Ng, & Zhang, 2009),inhibition of tumor growth (Ha, Xuan,Tang,Wong, & Fung, 2010),

induction of cancer cell apoptosis (Lee et al., 2006) and regu-lation of immunity (Dai et al., 2008), all of which are believedto be associated with the presence of various bioactive com-pounds such as flavonoids, carotenoids, alkaloids, polysaccha-rides, and diterpenoids (Qiao et al., 2011; Wang et al., 2008).However, the composition and amount of carotenoids remainuncertain.

Carotenoids represent a vital class of lipid-soluble func-tional compounds and are widely distributed in plants andanimals, with colors ranging from yellow to red. It has been

* Corresponding author. Tel.: +886 2 24622192#5111; fax: +886 2 24634203.E-mail address: cyshiau@mail.ntou.edu.tw (C-Y. Shiau).

http://dx.doi.org/10.1016/j.jff.2014.03.0081756-4646/© 2014 Elsevier Ltd. All rights reserved.

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well established that carotenoids can be synthesized in plants,microorganisms and algae, but not in animals (Inbaraj et al.,2008). As animals can only metabolize carotenoids, the amountof carotenoids present in vivo is dependent upon dietary con-centration of carotenoids (Inbaraj et al., 2008). The mostcommon carotenoids in human diet include α-carotene,β-carotene, lutein, zeaxanthin, lycopene and β-cryptoxanthin,all of which are important in enhancing biological activities.For instance, β-carotene exhibits a theoretical 100% provita-min A activity as one molecule of β-carotene can be con-verted to two molecules of vitamin A in vivo, whereas both luteinand lycopene function in antioxidation, anticancer, anti-inflammation and anti-atherosclerosis (Escobedo-Avellaneda,Gutie’rrez-Uribe, Valdez-Fragoso, Torres, & Welti-Chanes, 2014;Goulas, Exarchou, Kanetis, & Gerothanassis, 2014; Maiani et al.,2009; Yang et al., 2013). In one study, Kuhnen et al. (2009) re-ported that lutein and zeaxanthin, the main carotenoids foundin maize seeds, effectively inhibited the process of vessel for-mation, suggesting a potential role in prevention of diseasesassociated with vascular dysfunction.

Accordingly, carotenoids can be divided into carotenes andxanthophylls, with the former containing only hydrocarbonslike α-carotene and β-carotene and the latter comprising oxy-genated derivatives like lutein and zeaxanthin (Kao, Chen, &Chen, 2011; Kao, Loh, Inbaraj, & Chen, 2012). As most carot-enoids are present in all-trans form in nature in plants, the for-mation of cis isomers of carotenoids can be due to the extractionor processing (Kao et al., 2011). Nevertheless, cis-carotenoidsmay also be important in elevating biological activity, as shownby a higher bioavailability of cis-lycopene when compared toits corresponding all-trans-counterpart (Boileau, Clinton, &Erdman, 2000).Thus, the variety and amount of cis-carotenoidsin S. barbata needs to be determined as well.

The analysis of carotenoids in Chinese food and herbalsamples has frequently been conducted by thin-layer chro-matography (TLC) or high-performance-liquid-chromatography-mass-spectrometry (HPLC-MS), with the former being used toprepare various carotenoids and the latter for further separa-tion, identification and quantitation (Kao et al., 2011). Alter-natively, because of the absence of commercial cis-carotenoidstandards, a photoisomerization method can also be em-ployed to obtain these standards for subsequent identifica-tion of various cis isomers of carotenoids in food or herbalsamples (Kao et al., 2012). In light of the impact of carotenoidson human health, it is pivotal to adopt an appropriate tech-nique for determination of various carotenoids in S. barbata.The objective of this study was to develop an HPLC-DAD-MS-APCI method for analysis of carotenoids in S. barbata.The resultsof this study should not only clarify the variety and amountof carotenoids in S. barbata, but also provide a basis forpossible production of functional foods with S. barbata as rawmaterial.

2. Materials and methods

2.1. Materials

A total amount of 3 kg S. barbata was procured from a localChinese drug store and subjected to cleaning, vacuum-drying,

pouring into several separate bags and was sealed undervacuum, and stored at −20 °C until use. Carotenoid stan-dards, including all-trans-zeaxanthin and all-trans-β-carotenewere purchased from Sigma (St. Louis, MO, USA), while all-trans-lutein was from Fluka Chemical Co. (Buchs, Switzer-land) and 9- or 9′-cis-neoxanthin from ChromaDex (Irvine, CA,USA). Internal standard all-trans-β-apo-8′-carotenal was alsofrom Fluka Chemical Co. The HPLC-grade solvents such asmethanol, ethanol, toluene, acetone, hexane, acetonitrile andmethylene chloride were from Lab-Scan Co. (Gliwice, Poland).Deionized water was made using a Milli-Q water purificationsystem from Millipore Co. (Bedford, MA, USA). Anhydroussodium sulfate was from Nacalai Tesque (Kyoto, Japan). Potas-sium hydroxide was from Riedel-de Haën Co. (Seelze, Germany).The silica gel 60 TLC plates (0.5 mm thickness) were from MerckCo. (Darmstadt, Germany). A YMC C30 polymeric column(250 × 4.6 mm ID, 5 μm particle size) used to separate carot-enoids was from YMC Co. (Kyoto, Japan).

The HPLC instrument was composed of Agilent G1311Apump, G1316A column temperature controller, G1315Bphotodiode-array detector and 6130 quadrupole mass spec-trometer with multimode ion source (APCI and ESI) (AgilentCo., Palo Alto, CA, USA). The Beckman DU640 spectrophometerwas from Beckman Co. (Fullerton, CA, USA). The N-1 rotaryevaporator was from Eyela Co. (Tokyo, Japan). The Sorvall RC5Chigh-speed centrifuge was from Du Pont Co. (Wilmington, DE,USA). The DC400H sonicator was from Chuan-Hua Co. (Taipei,Taiwan). The V-U shaker was from Hsiang-Tai Co. (Taipei,Taiwan).

2.2. Extraction of carotenoids

One gram of freeze-dried S. barbata powder was mixed with30 mL of hexane/ethanol/acetone/toluene (10:6:7:7, v/v/v/v) ina 100-mL volumetric flask, which was shaken at room tem-perature for 1 h. Then 2 mL of 40% methanolic KOH solutionwere added for saponification for 16 h in the dark under ni-trogen, after which 15 mL of hexane was added and shakenfor 10 min. Then 15 mL of 10% anhydrous sodium sulfate so-lution was added and shaken vigorously for 1 min.The mixturewas settled at room temperature until separation into twolayers. The upper layer containing carotenoids was collected,and the residue was repeatedly extracted with 15 mL of hexanefour times. All the supernatants were pooled, evaporated todryness, dissolved in 5 mL of methylene chloride, filteredthrough a 0.22 μm membrane filter, and 20 μL injected for HPLC-DAD-MS-APCI analysis.

2.3. HPLC-DAD-MS-APCI analysis

A method based on Inbaraj, Chien, and Chen (2006) was modi-fied to separate carotenoids in S. barbata. A YMC C30 columnand a mobile phase of (A) methanol/acetonitrile (86:14, v/v) and(B) methylene chloride with the following gradient elution wasused: 100% A was maintained for 5 min initially, decreased to95% A in 10 min, 85% A in 15 min, 78% A in 20 min, 68% A in35 min, maintained for 5 min, and returned to 100% A in 45 min.The column temperature was 25 °C and flow rate was 1 mL/min with detection at 450 nm. The peak purity was deter-mined automatically by an Agilent G2180AA Spectral Evaluation

101j o u rna l o f f un c t i ona l f o od s 8C ( 2 0 1 4 ) 1 0 0 – 1 1 0

Software System based on degree in overlapping of absorp-tion spectrum of each peak relative to standard spectrum.

The various carotenoids in S. barbata were identified by com-paring their retention times, absorption spectra and massspectra of unknown peaks with reference standards, as wellas co-chromatography with addition of known standards. Asmost cis-carotenoid standards are not commercially avail-able, the identification of cis-carotenoids was performed basedon spectral characteristics and Q-ratios as described in severalprevious studies (Kao et al., 2011, 2012). The mass spectra ofcarotenoid standards and unknown peaks were determinedusing the APCI mode with the following condition: MW scan-ning range 400–1200, nebulizer pressure 10 psi, drying gas flow7 L/min, vaporizer temperature 230 °C, dry gas temperature330 °C, charging voltage 2000 V, capillary voltage 2000 V,fragmentor voltage 200 V, corona current 4 μA.

2.4. Photoisomerization of carotenoid standards

For further identification of cis-carotenoids, a photoisomerizationmethod was also employed. Briefly, all-trans-lutein, all-trans-zeaxanthin and all-trans-β-carotene standards were each dis-solved in methylene chloride for preparation of 50, 100 and 200ppm separately.Then 1 mL of each standard was collected andmixed with 100 μL of 0.01% iodine solution (in methanol) tofacilitate isomerization, after which the solution was illumi-nated at 25 °C in an incubator with 2000–3000 lx for 1, 1 and5 h for all-trans-lutein, all-trans-zeaxanthin and all-trans-β-carotene, respectively.Then all the illuminated solutions werefiltered through a 0.22 μm membrane filter and 20 μL of eachwere collected and injected for HPLC-DAD-MS-APCI analysis.

2.5. Preparation of neoxanthin and violaxanthinstandards by TLC

A TLC method as described by Chen, Yang, and Han (1991) wasused to prepare neoxanthin and violaxanthin standards fromspinach. A 20 g of freeze-dried spinach powder was mixed with150 mL of hexane in a flask and shaken for 1 h, after which24 mL of 40% methanolic KOH solution were added for sa-ponification for 16 h in the dark under nitrogen. Then 150 mLof hexane were added and shaken for 10 min, followed byadding 30 mL of 10% anhydrous sodium sulfate solution,shaking for 1 min and standing at room temperature in thedark for 1 h until two layers were formed.The supernatant wascollected, evaporated to dryness and dissolved in 5 mL ofethanol for TLC analysis. Prior to TLC, the silica gel-coated plateswere dried in a 100 °C oven for 1 h and a developing tank con-taining 150 mL of methanol–acetone–hexane (1:29:70, v/v/v) wascovered with a lid and presaturated with solvent vapors for30 min. Next, 1 μL carotenoid extract was spotted onto the plateat about 3 cm from the bottom eight times for a total of 8 μLon one spot with a micropipette. A total of 12 spots were doneon one plate and a total of 15 plates were used.Then each platewas placed into a tank for TLC. After the solvent front reacheda distance of about 18 cm, three colored bands were scratchedseparately and dissolved in ethanol for epoxide test and HPLC-DAD-MS-APCI analysis for further identification andquantitation. The identity of the first band was 9- or 9′-cis-neoxanthin with a concentration at 3.2 μg/mL, whereas the

identity of the third band was violaxanthin with a concentra-tion of 39.6 μg/mL.

2.6. Quantitation of carotenoids

An internal standard all-trans-β-apo-8′-carotenal was used forquantitation. For preparation of standard curves, five concen-trations of 1, 5, 10, 15 and 20 μg/mL of 9-cis-neoxanthin or 9′-cis-neoxanthin were each prepared, as were five concentrationsof 1, 4, 8, 12 and 15 μg/mL for all-trans-violaxanthin. Simi-larly, five concentrations of 0.05, 0.10, 0.15, 0.18 and 2 μg/mLwere prepared for all-trans-zeaxanthin, but for all-trans-lutein and all-trans-β-carotene, both low and high concentra-tion ranges were used with 2, 4, 5, 6 and 8 μg/mL as well as40, 55, 70, 85 and 100 μg/mL for the former, 0.05, 0.10, 0.15, 0.18and 2 μg/mL as well as 2, 15, 30, 40 and 50 μg/mL for the latter.Then each standard solution was mixed with internal stan-dard at a final concentration of 10 μg/mL, and the standardcurve was prepared by plotting concentration ratio against itsarea ratio, with the correlation coefficient (r2) and regressionequation being obtained automatically. The various carot-enoids in S. barbata were quantified using a formula as de-scribed in a previous study (Kao et al., 2011):

AsAi

a b Ci V f

recovery Ws

× +⎛⎝

⎞⎠ × × ×

×

Where As, is the peak area of carotenoids; Ai, peak area ofinternal standard; a, slope of calibration curve; b, intercept ofcalibration curve; Ci, concentration of internal standard; V,volume of extract; f, dilution factor; and Ws, weight of sample(g).

2.7. Detection and quantitation limits

The reproducibility of this method was determined based ona procedure described by International Conference on Harmo-nization (1996). Two concentrations (0.025 and 0.05 μg/mL) of9-cis-neoxanthin or 9′-cis-neoxanthin, all-trans-violaxanthin, all-trans-lutein, all-trans-zeaxanthin and all-trans-β-carotene wereprepared separately, and each injected into HPLC three times.The limit of detection (LOD) was calculated based on S/N ≥ 3,while the limit of quantitation (LOQ) was based on S/N ≥ 10.

2.8. Recovery

For recovery determination, 0.1 g of S. barbata sample was eachmixed with 2 and 6 μg of all trans-violaxanthin, 1 and 3 μg of9-cis-neoxanthin or 9′-cis-neoxanthin, 10 and 30 μg of all-trans-lutein and 5 and 15 μg of all-trans-β-carotene, separately. Like-wise, 1 g of S. barbata sample was mixed with 1 and 5 μg ofall-trans-zeaxanthin. After mixing, all the samples were sub-jected to extraction and HPLC analysis as described in the pre-ceding section. After quantitation, the carotenoid recovery wasobtained based on the ratio of the amount after HPLC (spikedamount minus original amount) relative to that before HPLC(spiked amount). Because of lack of availability of commer-cial cis-carotenoid standards, the recovery of all-trans forms ofcarotenoids was used for quantitation of their corresponding

102 j o u rna l o f f un c t i ona l f o od s 8C ( 2 0 1 4 ) 1 0 0 – 1 1 0

cis isomers based on the assumption that both possess similarextinction coefficients (Kao et al., 2011).

2.9. Precision study

The intra-day variability was determined by injecting carot-enoid extract containing 10 μg/mL internal standard into HPLCthree times each in the morning, afternoon and evening for atotal of nine injections on the same day. Likewise, the inter-day variability was measured by injecting carotenoid extractcontaining 10 μg/mL internal standard three times on the sameday for 3 consecutive days. The relative standard deviation(RSD%) was then calculated.

2.10. Statistical analysis

All experiments were conducted in triplicate and the data weresubjected to ANOVA analysis and Duncan’s multiple range testfor significance (p < 0.05) in mean comparison by StatisticalAnalysis System (SAS, 2011).

3. Results and discussion

3.1. Analysis of carotenoids by HPLC-DAD-MS-APCI

It has been well documented that a C30 column is superior toC18 column in terms of simultaneous separation of both po-sitional and geometrical isomers of carotenoids because ofgreater hydrophobic interaction between carotenoid isomersand C30 stationary phase (Inbaraj et al., 2006, 2008). For in-stance, a total of 32 carotenoids, including cis isomers, wereseparated in green algae within 49 min by employing a C30

column (Inbaraj et al., 2006), as were 18 carotenoids resolvedin Lycium barbarum within 52 min (Inbaraj et al., 2008). Never-theless, the separation time could be increased substantially

with a C30 column. Thus, it is pivotal to reduce the separationtime by developing a new HPLC solvent system.

Initially a gradient mobile phase composed of (A) methanol/acetonitrile/water (82:14:4, v/v/v) and (B) methylene chloride(100%), as developed by Kao et al. (2011), was used to sepa-rate various carotenoids in S. barbata by using an Agilent HPLCsystem. The separation time was reduced to be within 40 min,apparently caused by enhancement of solvent strength ofmobile phase. However, the resolution remained inadequate,implying that the selectivity of mobile phase to sample com-ponents needs to be further improved. After adjustment ofvarious mobile phases, a total of 19 carotenoids including in-ternal standard β-apo-8′-carotenal was resolved in S. barbatawithin 37 min by using a binary mobile phase of (A) methanol/acetonitrile (86:14, v/v) and (B) methylene chloride (100%) withthe following gradient elution: 100% A in the beginning, main-tained for 5 min, decreased to 95% A in 10 min, 85% A in 15 min,78% A in 20 min, 68% A in 35 min, maintained for 5 min andreturned to 100% A in 45 min. An adequate resolution ofall-trans-carotenoids and their cis isomers in S. barbata as shownin Fig. 1 was attained. Table 1 shows the retention time, re-tention factor (k), separation factor (α), and purity of variouscarotenoids in S. barbata, which ranged from 7.2 to 36.3 min,1.10–9.68, 1.03–1.31 and 89.8–99.9%, respectively. It is worthpointing out that with the exception of violaxanthin (purity89.8%) the purities of the other carotenoids were higher than94%. For reproducibility study, the relative standard deviation(RSD) for the intra-day variability was from 1.5 to 6.1% and theinter-day variability from 0.6 to 4.8% (Table 1), revealing thata high precision of this developed method was achieved.

Compared to some other published reports, our study dem-onstrated a superior resolution of carotenoids including cisisomers within a short length of time. For example, de Faria,de Rosso, and Mercadante (2009) developed an HPLC methodfor separation of 19 carotenoids in jackfruit by using a shorterYMC C30 column (150 × 4.6 mm ID) containing smaller par-ticle size (3 μm). Though the separation time was reduced to

Fig. 1 – HPLC chromatograms of carotenoids extracted from S. barbata. See Table 4 for peak identification.

103j o u rna l o f f un c t i ona l f o od s 8C ( 2 0 1 4 ) 1 0 0 – 1 1 0

Table 1 – Retention time (tR), retention factor (k), separation factor (α), peak purity and quality control data of carotenoids extracted from S. barbata byHPLC-DAD-MS-APCI.

Peakno.

Compound tR/min Retentionfactor (k)

Separationfactor (α)

Peakpurity (%)

Intra-day variabilityc Inter-day variabilityc

Mean ± SD(μg/g)

RSD(%)d

Mean ± SD(μg/g)

RSD(%)

1 13-or 13′-cis-neoxanthin 7.2 1.10 1.11(1,2)a 99.9 3.44 ± 0.18 5.2 3.39 ± 0.16 4.72 di-cis-neoxanthin 7.6 1.22 1.24(2,3)a 99.9 1.30 ± 0.08 6.1 1.24 ± 0.06 4.83 all-trans-neoxanthin 8.6 1.51 1.19(3,4)a 99.9 2.67 ± 0.11 4.1 2.70 ± 0.09 3.34 all-trans-violaxanthin 9.6 1.81 1.21(4,5)a 89.8 88.1 ± 1.5 1.7 88.7 ± 1.3 1.45 9-or 9′-cis-neoxanthin 10.8 2.19 1.12(5,6)a 97.4 68.9 ± 1.1 1.5 68.8 ± 0.9 1.36 luteoxanthin 11.7 2.44 1.31(6,7)a 99.5 28.6 ± 0.8 2.7 28.5 ± 0.5 1.77 13-or 13′-cis-Lutein 14.3 3.19 1.12(7,8)a 99.9 25.0 ± 0.6 2.4 24.8 ± 0.6 2.48 13-or 13′-cis-Lutein 15.5 3.56 1.15(8,9)a 99.9 5.90 ± 0.29 4.9 5.83 ± 0.24 4.19 all-trans-lutein 17.4 4.10 1.18(9,10)a 94.6 715 ± 11 1.5 718 ± 5 0.6

10 all-trans-zeaxanthin 19.9 4.84 1.11(10, 11)a 99.9 1.38 ± 0.06 4.3 1.37 ± 0.06 4.311 β-apo-8′-carotenal (IS)b 21.6 5.36 1.20(11,12)a 99.912 β-carotene-5,6-epoxide 25.2 6.42 1.03(12,13)a 99.9 1.38 ± 0.05 3.6 1.37 ± 0.04 2.913 β-carotene-5,6-epoxide 25.9 6.61 1.06(13,14)a 99.9 3.58 ± 0.13 3.6 3.59 ± 0.13 3.614 β-carotene-5,8-epoxide 27.2 7.00 1.05(14,15)a 99.9 1.16 ± 0.05 4.3 1.16 ± 0.04 3.415 13-or 13′-cis-β-carotene 28.3 7.32 1.04(15,16)a 99.9 5.45 ± 0.22 4.0 5.53 ± 0.19 3.416 15-or 15′-cis-β-carotene 29.3 7.62 1.08(16,17)a 99.9 36.9 ± 0.8 2.1 37.4 ± 0.6 1.617 cis-β-carotene 31.3 8.20 1.13(17,18)a 99.9 4.06 ± 0.19 4.6 4.12 ± 0.16 3.818 all-trans-β-carotene 35.0 9.29 1.04(18,19)a 97.7 378 ± 9 2.3 381 ± 8 2.219 9-or 9′-cis-β-carotene 36.3 9.68 1.04(18,19 )a 99.9 9.83 ± 0.42 4.2 9.80 ± 0.40 4.0

a Numbers in parentheses represent values between two neighboring peaks.b IS = internal standard.c Mean of triplicate analysis ± standard deviation.d RSD% = (SD/mean) × 100%.

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be less than 40 min, the resolution remained insufficient. Like-wise, a total of 25 carotenoids including cis isomers were sepa-rated in Rhinacanthus nasutus (L.) Kurz within 54 min by a YMCC30 column (250 × 4.6 mm ID, 5 μm particle size) (Kao et al., 2011).However, the retention time was much longer. A C18 reversed-phase column (Vydac 218TP54, 250 × 4.6 mm ID) was used toanalyze maize seed carotenoids with a gradient mobile phaseand detection at 450 nm and flow rate at 1 mL/min, but onlyfive compounds including α-carotene, β-carotene, lutein, zea-xanthin and β-cryptoxanthin were separated and identified.The major drawback of this method is that no cis isomers ofcarotenoids were resolved and quantified, as well as no inter-nal standard was applied for quantitation (Kuhnen et al., 2009).In a recent study Pop et al. (2014) developed an UHPLC-PAD-ESI-MS technique to determine free and esterified carot-enoids in berries and leaves from six Romanian sea buckthornvarieties by employing a Waters Acquity UPLC BEH Shield RP18column (150 × 2.1 mm ID, 1.7 μm particle size) with a gradi-ent system and flow rate at 0.4 mL/min and detection at 450 nm.A total of 27 compounds were separated within 55 min;however, the resolution remains inadequate. In another studythe C30 column (150 × 2.0 mm, 3 μm particle size) was shownto perform superiorly over the C18 column (100 × 3.0 mm ID,2.6 μm particle size) by separating carotenoid isomers includ-ing numerous E- from all-Z-lycopene isomers in a plasmaextract within 40 min (Franke, Morrison, Custer, Li, & Lai, 2013).However, many peaks are overlapped and should decreasequantitation accuracy greatly. The simultaneous determina-tion of geometrical isomers of fucoxanthin, diatoxanthin and5,8-epoxydiadinoxanthin diasteroisomers in microalgae wasattained by developing an HPLC/DAD/MS technique and areversed-phase C30 column (250 × 3 mm ID, 5 μm particle size,YMC Inc) with a gradient elution system and flow rate at 0.5 mL/min and detection at 447 nm (Crupi et al., 2013). A total of 15carotenoids and chlorophylls were separated within 25 min;however, four compounds remained unidentified. In a laterstudy Simonovska, Vovk, Glavnik, and Cernelic (2013) com-pared the separation efficiency of spinach pigments by Kinetexcore-shell C18 column (100 × 4.6 mm ID, 2.6 μm particle size) withgradient elution and ProntoSIL C30 column (250 × 4.6 mm ID,5 μm particle size) with isocratic elution. A total of six carot-enoids were separated by the former within 14 min and fivecarotenoids by the latter within 20 min.The retention time wasreduced substantially when compared to some other pub-lished reports; however, the cis isomers of carotenoids re-mained unresolved.

3.2. Identification of neoxanthin and violaxanthin

As mentioned before, both neoxanthin and violaxanthin stan-dards were prepared from spinach by TLC. After collection ofneoxanthin and violaxanthin bands, both were subjected toabsorption spectra analysis without and with addition of 0.1NHCl by a spectrophotometer. A hypsochromic shift of about20 nm occurred for the former accompanied by a color changefrom yellow to green as neoxanthin contains only one 5,6-epoxy group, indicating conversion of neoxanthin to neochromeunder acidic treatment (Kao et al., 2011). Similarly, ahyposchromic shift of about 40 nm occurred for the latter ac-companied by a color change from yellow to blue as

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Table 3 – UV and MS spectral identification data for all-trans plus cis forms of lutein, zeaxanthin, β-carotene standards during iodine-catalyzed illumination at 25 °C for1, 1 and 5 h, respectively.

Peak no. Compound tR (min) ka PeakPurity (%)

λmaxb (on-line) λmax (reported) Q-ratioc

foundQ-ratioreported

[M+H]+ (m/z)found

[M+H]+ (m/z)reported

1 cis-lutein 10.5 2.09 97.4 328 408 436 452 332 410 434 458e 0.07 0.31e 569 569j

2 cis-lutein 11.9 2.50 99.7 340 412 436 460 338 422 446 470e 0.10 0.28e 569 569j

3 13- or 13′-cis-lutein 14.2 3.18 94.2 332 416 440 464 332 415 440 464e 0.47 0.4e 569 569k

4 13- or 13′-cis-lutein 15.5 3.56 98.9 332 416 440 468 332 416 440 464e 0.38 0.39e 569 569k

5 all-trans-lutein 17.9 4.26 88.7 332 420 444 472 332 420 444 472f 0.07 0f 569 569j

6 cis-lutein 19.9 4.85 99.6 340 424 452 480 344 422 446 476e 0.04 0.18e 569 569j

7 9- or 9′-cis-lutein 20.4 5.00 99.8 330 416 440 468 332 416 440 470e 0.07 0.13e 569 –8 9- or 9′-cis-lutein 22.0 5.47 99.8 340 416 444 472 332 421 446 470e 0.09 0.12e 569 –1′ 13- or 13′-cis-zeaxanthin 13.7 3.03 99.9 340 424 444 472 338 422 446 472g 0.12 0.37g 569 569.4g

2′ 13- or 13′-cis-zeaxanthin 14.3 3.21 99.9 340 424 444 472 338 422 446 472g 0.10 0.37g 569 569.4g

3′ 15- or 15′-cis-zeaxanthin 15.0 3.41 99.9 340 416 444 468 338 422 446 470g 0.15 0.45g 569 569.4g

4′ cis-zeaxanthin 15.9 3.68 99.9 340 404 428 452 – 0.26 – 569 –5′ cis-zeaxanthin 16.5 3.85 99.9 340 408 428 452 – 0.05 – 569 –6′ 15- or 15′-cis-zeaxanthin 17.1 4.03 99.9 340 416 444 472 338 422 446 470g 0.37 0.45g 569 569.4g

7′ all-trans-zeaxanthin 19.7 4.79 84.4 340 428 448 480 –d 426 450 478f 0.06 – 569 569.4g

8′ 9- or 9′ -cis-zeaxanthin 23.4 5.88 84.4 340 424 448 476 338 422 446 474g 0.10 0.12g 569 569.4g

1″ β-carotene-5,6-epoxide 25.3 6.44 99.9 340 420 452 480 – 420 445 471h 0.17 0h 553 553e,h

2″ β-carotene-5,6-epoxide 25.8 6.59 99.8 344 420 452 476 – 420 445 471h 0.17 0h 553 553e,h

3″ β-carotene-5,8-epoxide 27.1 6.97 99.9 340 402 428 456 – 404 428 453i 0.13 0i 553 553j

4″ 13- or 13′-cis-β-carotene 28.3 7.32 99.0 340 420 452 480 344 422 446 476e 0.33 0.46e 537 537j

5″ 15- or 15′-cis-β-carotene 29.3 7.62 99.0 340 420 448 472 337 420 449 470j 0.42 0.6e 537 537j

6″ 9- or 9′-cis-β-carotene 29.8 7.76 98.6 340 428 452 480 344 423 452 476e 0.09 0.14e 537 –7″ cis-β-carotene 31.5 8.26 99.8 340 420 448 472 344 410 446 470e 0.16 0.39e 537 –8″ 9- or 9′-cis-β-carotene 33.0 8.71 99.9 340 424 448 476 344 423 452 476e 0.11 0.14e 537 –9″ all-trans-β-carotene 35.0 9.29 89.6 340 428 456 480 350 430 458 482e 0.07 0.09e 537 537j

10″ 9- or 9′-cis-β-carotene 36.4 9.71 97.1 340 424 452 476 344 428 452 476e 0.12 0.16e 537 537j

a k: retention factor.b A gradient mobile phase of methanol–acetonitrile (86:14,v/v) and methylene chloride (from 100:0,v/v to 68:32,v/v) was used.c Q-ratio is defined as the height ratio of the cis peak to the main absorption peak.d ’–’ Data not available.e Based on a reference by Inbaraj et al. (2006).f Based on a reference by de Faria et al. (2009).g Based on a reference by Inbaraj et al. (2008).h Based on a reference by de Rosso and Mercadante (2007).i Based on a reference by Zanatta and Mercadante (2007).j Based on a reference by Zepka and Mercadante (2009).k Based on a reference by Mendes-pinto, Ferreira, Caris-veyrat, and de Pinho (2005).

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Table 4 – Identification data and contents (μg/g)a for carotenoids in S. barbatab.

Peakno.

Compound λmax (on-line)/nm λmax (reported)/nm Q-ratioc

foundQ-ratioreported

[M+H]+(m/z)found

[M+H]+(m/z)reported

Fragmentions (m/z)found

Fragment ions (m/z) reported Content

1 13-or 13′-cis-neoxanthin 328 412 432 460 328 416 438 464e 0.34 0.38e 601 – – – 3.44 ± 0.182 di-cis-neoxanthin 328 404 428 452 328 408 430 456e 0.26 0.23e 601 – – – 1.30 ± 0.083 all-trans-neoxanthin 312 420 444 468 – 418 440 468e 0.13 – 601 601i 565, 583, 601 583[M+H–18], 565[M+H–18–18], 547[M+H–18–18–

18], 509[M+H–92], 491[M+H–18–92], 221i,l,601[M+H]r

2.67 ± 0.11

4 all-trans-violaxanthin 328 416 440 468 – 416 440 468f 0.05 – 601 601k 509, 565, 583,601

583[M+H–18], 565[M+H–18–18], 509[M+H–92],491[M+H–18–92], 221i, 601[M+H]r,s

88.1 ± 1.5

5 9-or 9′-cis-neoxanthin 328 412 436 464 328 414 436 464e 0.05 0.07e 601 601l 491,547, 565,583

583[M+H–18], 565[M+H–18–18], 547[M+H–18–18–18], 509[M+H–92], 491[M+H–18–92], 393, 221l

68.9 ± 1.1

6 luteoxanthin 312 400 420 448 – 398 423 449g 0.06 0g 601 601m 491, 509, 583,601

583[M+H–18]i,q,t,509[M+H–92], 491[M+H–18–92],221q, 601[M+H]s

28.6 ± 0.8

7 13-or 13′-cis-Lutein 332 404 436 468 332 415 440 464h 0.41 0.44h 569 569n – – 25.0 ± 0.68 13-or 13′-cis-Lutein 332 416 440 468 332 416 440 464h 0.37 – 569 569n – – 5.90 ± 0.299 all-trans-lutein 336 420 444 472 326 416 440 464h 0.10 – 569 569n,o 459, 533, 551 551[M+H–18], 533[M+H–18–18], 477[M+H–92],

463[M+H–106] , 459[M+H–18–92]i715 ± 11

10 all-trans-zeaxanthin 340 424 452 476 – 425 450 476i 0.29 – 569 569o 551, 569 551[M+H–18]i,q, 533[M+H–18–18], 463[M+H–106]i,569u,

1.38 ± 0.06

11 β-apo-8′-carotenal (IS)d – – 464 – – – 464j – – – 417 417p – – –12 β-carotene-5,6-epoxide 340 424 452 480 – 420 445 471i 0.06 0i 553 553i 461, 535 535[M+H–18], 461[M+H–92], 205i 1.38 ± 0.0513 β-carotene-5,6-epoxide 344 420 452 476 – 420 445 471i 0.07 0i 553 553i – – 3.58 ± 0.1314 β-carotene-5,8-epoxide 340 402 428 456 – 404 428 453g 0.17 0g 553 553q 535 535[M+H–18], 461, 205i,q 1.16 ± 0.0515 13-or 13′-cis-β-carotene 340 424 452 476 344 422 446 476h 0.27 – 537 537q – – 5.45 ± 0.2216 15-or 15′-cis-β-carotene 340 424 448 472 340 430 452 480h 0.60 0.66h 537 537q 537 537v 36.9 ± 0.817 cis-β-carotene 340 424 444 468 344 410 446 470h 0.43 0.39h 537 – – – 4.06 ± 0.1918 all-trans-β-carotene 340 424 456 480 350 430 458 482h 0.28 – 537 537q – – 378 ± 919 9-or 9′-cis-β-carotene 340 424 448 476 344 428 452 476h 0.07 – 537 537q 537 537[M+H], 444u 9.83 ± 0.42

Total 1381 ± 26

a Mean of triplicate analysis ± standard deviation.b A gradient mobile phase of methanol–acetonitrile (86:14,v/v) and methylene chloride (from 100:0,v/v to 68:32,v/v) was used.c Q-ratio is defined as the height ratio of the cis peak to the main absorption peak.d IS = internal standard.e Based on a reference by Meléndez-Martínez et al. (2008).f Based on a reference by Meléndez-Martínez et al. (2007).g Based on a reference by Zanatta et al. (2007).h Based on a reference by Inbaraj et al. (2006).i Based on a reference by de Rosso and Mercadante (2007).j Based on a reference by Kao et al. (2011).k Based on a reference by Ornelas-Paz, Yahia, and Gardea-Bejar (2007).l Based on a reference by de Faria et al. (2009).m Based on a reference by Maoka, Fujiwara, Hashimoto, and Akimoto (2002).n Based on a reference by Mendes-pinto et al. (2005).o Based on a reference by Pop et al. (2014).p Based on a reference by Taylor, Brackenridge, Vivier, and Oberholster (2006).q Based on a reference by Zepka et al. (2009).r Based on a reference by Matsumoto, Ikoma, Kato, Kuniga, Nakajima, and Yoshida (2007).s Based on a reference by Dugo et al. (2008).t Based on a reference by Crupi, Milella, and Antonacci (2010).u Based on a reference by Inbaraj et al. (2008).v Based on a reference by Kurz, Carle, and Schieber (2008).

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violaxanthin contains two 5,6-epoxy groups, implying conver-sion of violaxanthin to auroxanthin under acidic treatment (Kaoet al., 2011). After HPLC analysis of both neoxanthin andviolaxanthin bands, the former was found to contain 13- or13′-cis-neoxanthin, all-trans-neoxanthin, di-cis-neoxanthin, all-trans-neochrome, and 9- or 9′-cis-neoxanthin, whereas the lattercontained all-trans-violaxanthin, 9- or 9′-cis-violaxanthin,luteoxanthin and cis-luteoxanthin. Table 2 shows the reten-tion time, retention factor, purity and Q-ratio of neoxanthinand violaxanthin bands prepared by TLC, which were rangedfrom 7.2 to 16.7 min, 1.10–3.91, 90.1–99.9% and 0.02–0.48, re-spectively. Similar carotenoid identity was also reported inspinach by TLC by Meléndez-Martínez, Vicario, and Heredia(2007) and Meléndez-Martínez, Britton, Vicario, and Heredia(2008). In addition, the identification data of all-trans-neochromeand luteoxanthin were also similar to that reported by de Rossoand Mercadante (2007) and Dugo et al. (2008).

3.3. Photoisomerization of carotenoid standards

As indicated earlier, carotenoid standards including lutein, zea-xanthin and β-carotene were photoisomerized for further iden-tification of cis isomers. Table 3 shows retention time, retentionfactor, purity, absorption spectra, Q-ratio and mass spectra ofphotoisomerized standards of lutein, zeaxanthin and β-carotene.Peaks 1–8 show the same mass spectra (m/z 551 [M+H-H2O]+),with all-trans-lutein (peak 5) dominated and the other peaksbeing cis isomers of lutein based on absorption spectra char-acteristics. Likewise, peaks 1′–8′ show the same mass spectra(m/z 569 [M+H]+) with peak 7′ being the major all-trans-zeaxanthin and the other peaks cis isomers of zeaxanthin. In-terestingly, the β-carotene profile was different from lutein andzeaxanthin after illumination as peaks 1′ and 2″ were identi-fied to be β-carotene-5,6-epoxide and peak 3″ to be β-carotene-5,8-epoxide based on mass spectra (m/z 553 [M+H]+) andabsorption spectra after treatment with 0.1N HCl, as a hypso-chromic shift of about 18 nm occurred for β-carotene-5,8-epoxide when compared to β-carotene-5,6-epoxide. For peaks4″–10″, all showed the same mass spectra (m/z 537 [M+H]+) withpeak 9″ being the dominant all-trans-β-carotene and the otherpeaks being cis isomers of β-carotene. After identification, allthese carotenoid isomers were then compared with unknownpeaks in Fig. 1 for further identification of cis isomers inS. barbata.

3.4. Identification data and contents of carotenoids inS. barbata

On the basis of the identification criteria as described above,S. barbata was found to contain all-trans-neoxanthin and itsthree cis isomers (13- or 13′-cis, di-cis and 9- or 9′-cis), all-trans-violaxanthin and its derivative luteoxanthin, all-trans-lutein andits two cis isomers (13- or 13′-cis), all-trans-zeaxanthin, twoβ-carotene-5,6-epoxides and one β-carotene-5,8-epoxide, andall-trans-β-carotene and its four cis isomers (9- or 9′-cis, 13- or13′-cis, 15- or 15′-cis), which amounted to 76.3, 117, 746, 1.38,6.12 and 434 μg/g, respectively. Apparently, all-trans-lutein andits cis isomers constituted the largest position in S. barbata, fol-lowed by all-trans-β-carotene and its cis isomers, all-trans-violaxanthin and luteoxanthin, all-trans-neoxanthin and its cisisomers, β-carotene-5,6-epoxide and β-carotene-5,8-epoxide, andall-trans-zeaxanthin (Table 4).

3.5. Quality control data

Table 5 shows the regression equations, correlation coeffi-cient (r2), LODs, LOQs and recoveries of carotenoid standardsincluding 9- or 9′-cis- neoxanthin, all-trans-violaxanthin, all-trans-lutein, all-trans-zeaxanthin and all-trans-β-carotene, withthe LODs being 0.05, 0.05, 0.025, 0.025 and 0.05 ppm, respec-tively, and the LOQs being 0.15, 0.15, 0.075, 0.075 and 0.15 ppm.The correlation coefficient for these carotenoid standards wereall higher than 0.99, whereas the recoveries were all higher than93%, demonstrating a high accuracy of this developed method.In several literature reports, the recoveries of all-trans formsof zeaxanthin, β-cryptoxanthin and β-carotene were 92, 92 and87%, respectively, for carotenoid analysis in Lycium barbarumfruit (Inbaraj et al., 2008). Similarly, in a study dealing with analy-sis of carotenoids in R. nasutus, the recoveries for all-trans formsof lutein, zeaxanthin and α-carotene were 87.0, 90.1 and 83.7%,respectively (Kao et al., 2011). All these outcomes suggested thatour method can be applied to determine the various carot-enoids in Chinese medicinal plants.

4. Conclusion

In conclusion, an HPLC-DAD-MS-APIC method was devel-oped to determine the various carotenoids in S. barbata by using

Table 5 – Calibration data, LODs, LOQs and recoveries of 9- or 9’-cis-neoxanthin and four all-trans carotenoid standards.

Carotenoids Calibration curve r2 Test range (ppm) LODa (ppm) LOQb (ppm) Recovery (%) (RSD,%)c

9- or 9′-cis-neoxanthin y = 5.4768x − 0.0366 0.9902 1–20 0.05 0.15 93.04 (3.17)all-trans-violaxanthin y = 5.4418x − 0.0801 0.9905 1–15 0.05 0.15 96.93 (4.81)all-trans-lutein y = 2.9461x − 0.0268 0.9988 2–8 0.025 0.075 100.91 (4.38)

y = 2.2236x + 3.4186 0.9978 40–100all-trans-zeaxanthin y = 6.5725x + 0.0404 0.9955 0.05–2 0.025 0.075 95.9 (3.38)all-trans-β-carotene y = 2.8177x + 0.0089 0.9954 0.05–2 0.05 0.15 95.91 (3.55)

y = 1.9359x + 0.5951 0.9971 2–50

a LOD: Limit of detection.b LOQ: Limit of quantification.c RSD% = (SD/mean) × 100%.

108 j o u rna l o f f un c t i ona l f o od s 8C ( 2 0 1 4 ) 1 0 0 – 1 1 0

a YMC C30 column and a gradient solvent system composedof (A) methanol/acetonitrile (86:14, v/v) and (B) methylene chlo-ride (100%) with flow rate at 1 mL/min, detection wavelengthat 450 nm and column temperature at 25 °C. Internal stan-dard β-apo-8′-carotenal was used for quantitation. A total of19 carotenoids including β-apo-8′-carotenal were separatedwithin 37 min, and all-trans-lutein and its cis isomers waspresent in largest amount. A high accuracy and precision wasattained with this method, which can be applied to deter-mine the various carotenoids in Chinese herb plants.

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