Article pubs.acs.org/JAFC
Composition of Lutein Ester Regioisomers in Marigold Flower, Dietary Supplement, and Herbal Tea El-Sayed M. Abdel-Aal* and Iwona Rabalski Guelph Food Research Centre, Agriculture and Agri-Food Canada, 93 Stone Road West, Guelph, Ontario, Canada N1G 5C9 S Supporting Information *
ABSTRACT: Characterization of lutein and its esters in a health product is necessary for its efficacy. In the current study lutein ester regioisomers were quantified and identified in several dietary supplements and herbal teas in comparison with marigold flower, the commercial source of lutein. The products were extracted with three solvents and separated on a C30 column. The separated esters were identified/confirmed with LC-MS in APCI+ve mode with the use of synthetic lutein esters. The total content of lutein esters substantially varied among marigold flowers (167−5752 μg/g), supplements (88,000−110,700 μg/g), and herbal teas (12.4−91.3 μg/g). Lutein supplement had a lutein profile similar to that of marigold flower, whereas herbal tea showed an extremely different profile. Lutein dipalmitate was the dominant compound in supplements and marigold flowers followed by lutein 3′-O-myristate-3-O-palmitate and lutein 3′-O-palmitate-3-O-myristate. Lutein was the major compound in marigold herbal tea with small amounts of lutein mono- and diesters. Differences in the concentration and composition of lutein compounds among marigold products could indicate distinct product quality and lutein bioavailability. KEYWORDS: lutein esters, carotenoids, separation and quantification, dietary supplement, herbal tea, marigold
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INTRODUCTION Lutein is well-known for its potential in promoting the health of eyes and skin1 and in reducing the risk of age-related macular degeneration (AMD),2 cataracts,3 cancer,4 and cardiovascular disease.5 Lutein and zeaxanthin constitute the pigments found in the yellow spot of the human retina,6 and they provide several protective functions, that is, protecting the macula from damage by blue light,7 improving visual acuity,3 and scavenging harmful reactive oxygen species.8 However, dietary consumption of lutein is relatively low,9 particularly in comparison with the levels at which health benefits have been observed.2,10 In general, carotenoids including lutein are essential for human health and must be provided in the diet, and thus their abundance in a human body is entirely dependent on dietary intake. These factors rationalize the ongoing interest in highlutein foods1,11 and lutein supplements.12 Because zeaxanthin is usually found with lutein in plant and animal foods and its role in human health has become evident, it is important to quantify it in foods and dietary supplements. This is especially critical because it has a molecular weight and structure similar to those of lutein with a subtle difference as it has two β-ionone rings, whereas lutein contains a β-ionone ring and an ε-ionone ring (Supplementary Figure 1). Lutein is found at relatively high amount in some commonly consumed grains and grain foods such corn and einkorn ancient wheat and bakery products.11,13,14 It is the main xanthophyll in green leafy vegetables such as kale, spinach, broccoli, peas, and lettuce.15 Chicken egg yolk is also a rich source of lutein and zeaxanthin with increased bioavailability due to the high fat content in eggs.16 Lutein is also present at extremely high concentration in marigold (Tagetes spp.) flowers,17,18 which is commercially utilized in making dietary supplements and carotenoid-enriched foods and beverages.19 Several food-grade and health products have been made from marigold flowers in Published XXXX by the American Chemical Society
either free or bound (esterified with long-chain fatty acids) form. These products are being used to boost lutein consumption through food fortification and/or dietary supplements. Both free and esterified lutein compounds are absorbed from foods and dietary supplements but the ester form requires prior de-esterification by intestinal enzymes.20 Thus, it is important to characterize these products on the basis of their content and composition of lutein esters. This information would be valuable in developing strategies for enhancing lutein intake and managing AMD and cataracts. At present, little has been reported about the composition of lutein esters in marigold-derived products. In an earlier study we developed a method to separate, identify, and characterize lutein esters based on the use of an integrated analytical approach of HPLC, LC-MS, and NMR.21 Unlike zeaxanthin, lutein is an asymmetric molecule that forms regioisomers when esterified with fatty acids. For example, in the presence of palmitic acid lutein forms two monoester regioisomers (lutein 3′-O-palmitate and lutein 3-O-palmitate) in addition to lutein diester or dipalmitate in this case (Supplementary Figure 1). The developed method was capable of separating and identifying mixed fatty acid lutein mono- and diesters using a C30 column with a mixture of synthetic or laboratory-prepared mono- and diester lutein mixtures. In the current study this method was used to fully characterize lutein ester compounds in marigold-based dietary supplements and herbal teas commonly consumed in comparison with marigold flowers. Due to differences in matrix structure between Received: September 10, 2015 Revised: October 14, 2015 Accepted: October 23, 2015
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DOI: 10.1021/acs.jafc.5b04430 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Journal of Agricultural and Food Chemistry
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marigold flowers, supplements, and herbal teas several extraction solvents were also evaluated.
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degasser, a G1315B photodiode array detector (PDA), and a ChemStation v.8.04 data acquisition system with capability of conducting an isoabsorbance plot and three-dimensional graphic analyses. Waters specialty column C30, a 10 cm × 4.6 mm, i.d., 3 μm, YMC Carotenoid column (Waters, Mississauga, ON, Canada), was employed for the separation of carotenoids at 35 °C. The column was eluted at 1.0 mL/min with a gradient program consisting of solvent A, a mixture of 81% methanol/15% methyl tert-butyl ether/4% nano pure water, and solvent B, a mixture of 90% methyl tert-butyl ether/10% methanol. The gradient was programmed as follows: 0−44 min, from 100 to 51% A; 44−46 min, from 51 to 0% A; 46−49 min, hold of B at 100%; 49−54 min, A increased from 0 to 100%; and 54−55 min, hold at 100% A. The separated carotenoids were detected and measured at 450 nm, and the identity of carotenoids was based on the congruence of retention times and UV/vis spectra used with comparison of authentic lutein standard and laboratory-synthesized lutein esters.21 The stock standard lutein was prepared in acetonitrile and methyl tertbutyl ether at the 90:10 ratio. An accurate amount of approximately 1 mg of lutein was weighed and solubilized in 10 mL of a mixed solvent. The range of injected working standard solutions was from 2.5 to 50 ng per injection and was used for samples with lower levels of carotenoids. For samples with higher content of carotenoids the range was from 0.49 to 1.47 μg per injection (the lutein stock solution was used for samples with higher lutein content by injecting 5, 10, and 15 μL). Lutein standard solutions exhibited a linear relationship within the above given range by plotting area response and injected amounts. The coefficient of determination (R2) ranged from 0.9961 to 0.9975. The HPLC analytical method was also assessed by calculating the limit of detection (LOD), limit of quantification (LOQ), and recovery of lutein compounds based on a spiked sample technique with one of the marigold products. Ten spiked and seven nonspiked samples were carried out. A sample (0.25g) was spiked with laboratory-prepared lutein palmitate containing lutein, lutein 3′-O-palmitate, lutein 3-Opalmitate, and lutein dipalmitate (0.2 mL) prepared from 3.32 mg of lutein plus 6.2 mg of palmitic acid in 400 μL of solvent.21 The LOD and LOQ were obtained statistically as 3S and 10S, respectively, where S is the residual standard deviation of lutein, lutein 3′-O-palmitate, lutein 3-O-palmitate, and lutein dipalmitate determination replicates.22 LC-MS Confirmation of Lutein Esters. Confirmation of the identity of each peak was carried out by LC-MS (Thermo Finnigan, San Jose, CA, USA) equipped with a SpectraSystem UV6000LP ultraviolet detector scanning from 190 to 800 nm and an LCQ Deca ion trap mass spectrometer operated in the APCI positive ion (+ve) mode scanning from m/z 50 to 2000. The LC-MS conditions were the same as for the LC-UV/vis analyses. The mass spectrometer was tuned for maximum response to lutein. The instrument operating conditions were as follows: shear gas and auxiliary flow rates were set at 58 and 3 (arbitrary units); voltages on the source, capillary, tube lens offset, multipole RF amplifier, multipole 1 offset, multipole 2 offset, intermultipole lens, entrance lens, and trap DC offset were set at 6.0 kV, 45.5, 3.0, 290.0 (p-p), −6.1, −9.5, −59.0, −56.0, and −10.0 V, respectively; capillary and vaporizer temperatures set at 150 and 450 °C, respectively. Statistical Analysis. The data were subjected to regression analysis to estimate the method linearity and coefficient of determination (R2) using Minitab software (version 12, Minitab inc., State College, PA, USA). The data were reported as means of three ± standard deviation (SD).
MATERIALS AND METHODS
Chemicals. all-trans-Lutein (95% purity), zeaxanthin (95% purity), and β-cryptoxanthin (95% purity) standards were purchased from ChromaDex (Santa Ana, CA, USA); lauric acid was from Nu-Check Prep, Inc. (Elysian, MN, USA); myristic acid, palmitic acid, and stearic acid were from The Hormel Institute (Austin, MN, USA); 1,3dicyclocarbodiimide and 4-(dimethylamino) pyridine were from Sigma-Aldrich (Oakville, ON, Canada); and acetonitrile, 1-butanol, dichloromethane, methanol, and methyl tert-butyl ether were from Fisher Scientific (Ottawa, ON, Canada). All solvents are of HPLC grade. Preparation of Lutein Esters. Lutein esters were synthesized with fatty acids in the presence of 1,3-dicylclocarbodiimide and 4(dimethyamino)pyridine.21 For each fatty acid, four compounds were identified: unreacted lutein, two regioisomers of lutein monoester, and lutein diester. A typical HPLC chromatogram depicting the separation of synthetic lutein esters is presented in Supplementary Figure 2A. Materials. The materials investigated in the current study were seven marigold cultivars from two species, four lutein dietary supplements, and four herbal teas. Marigold seedlings were purchased from local garden centers (Guelph, ON, Canada) and were grown locally. Fresh flower petals of seven cultivars, Antigua Orange (Tagetes erecta L.), Antigua Yellow (T. erecta L.), Durango Red (Tagetes patula L.), Hero Bee Marigold Souci (T. patula L.), Janie Primrose Marigold Souci (T. patula L.), Safari Scarlet Marigold Souci (T. patula L.), and Safari Yellow Marigold Souci (T. patula L.), were harvested at maturation and were frozen immediately at −20 °C. Frozen petals were pooled together, and a representative sample was collected and used for carotenoid extraction. Four lutein dietary supplements were purchased from local food and health stores (Guelph, ON, Canada). They were three soft gel capsules containing lutein esters and one capsulated lutein microbeads. Soft gel capsules and dry powder capsules were emptied, and the contents of each product were well mixed. A representative sample from each product was taken for carotenoid extraction. Four herbal teas made from marigold petals and/or blossoms were purchased from the local herbal store (Guelph, ON, Canada) or online at eclecticherb.com. Tea in bag samples was emptied and mixed to obtain uniform samples. Extraction of Lutein and Other Carotenoids. For marigold flowers, a frozen sample of 0.3−0.8 g was homogenized in 5 mL of each solvent, water-saturated n-butanol (WSB), methyl tert-butyl ether (MtBE), or 2-propanol, for 45 s using a Polytron PT 10/35 at 5000 rpm (Kinematica Inc., Luzern, Switzerland), then capped, wrapped in a black plastic sheet, kept overnight to immerse the sample in solvents, and then rehomogenized for 45 s. Soft gel capsules were opened with a sharp scalpel, and pastes (3−5 mg) were extracted with 1 mL of solvent using an IKA Vibrax VXR shaker (Janke & Kunkel Co., Staufen, Germany) at 1800 rpm for 15 min twice. Capsules containing microbead powders were opened, and the powder contents (150 mg) were extracted with 5 mL of solvent using a polytron homogenizer as above. Approximately 0.25 g of dry petal tea or bagged tea was homogenized as above for 1 min, and then capped, wrapped with a black plastic sheet, and kept overnight in dim light in a fume hood to allow the solvent to permeate through the dispersed sample; later the sample was rehomogenized for 1 min. All extracts were centrifuged at 10,000 g for 5 min and filtered through a 0.45 μm Nylon Acrodisc syringe filter (Paill Gelman Laboratory, Ann Arbor, MI) before HPLC analysis. All the extraction steps were carried out in amber glassware wrapped with black plastic sheet under dim light to avoid degradation and isomerization of lutein compounds and other carotenoids. Analysis of Lutein and Lutein Esters. Lutein compounds and other carotenoids in marigold flower, supplement, and herbal tea extracts were separated and quantified with an 1100 series chromatograph (Agilent, Mississauga, ON, Canada) equipped with a G1311A quaternary pump, a G1329A temperature-controlled injector, a G1316A temperature-controlled column thermostat, a G1322A
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RESULTS AND DISCUSSION Extraction and Method Validation. The adequate selection of solvent would improve the extraction efficiency of lutein compounds because they are present in various forms, for example, esterified versus nonesterified and monoester versus diester. These compounds attain different degrees of polarity and solubility. In the current study three solvents (WSB, MtBE, and 2-propanol) exhibiting different polarities and solubility properties were assessed in extracting lutein and
B
DOI: 10.1021/acs.jafc.5b04430 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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optimize extraction of lutein from marigold flower petals along with simultaneous alkali hydrolysis, various organic solvents, amounts of solvent, particle sizes of material, and temperatures were investigated in a single-step procedure.26 The optimal operating parameters were found to be hexane as organic solvent, temperature of 40 °C, particle size < 0.315 mm, solvent/material 5 L/kg, and alkali/material 3.75 L/kg of KOH in ethanol (5%, w/v). In addition, the extraction process was governed by the intraparticle diffusion step, which indicates the importance of the particle size of plant materials. Thus, in the current study marigold flower petals and herbal teas were soaked in solvents and homogenized to improve sample uniformity and lutein extractability. For supplements, additional treatments during or prior to extraction may be required depending on the type of supplements and the purpose of analysis. In a study on commercial dietary supplements, carotenoids were analyzed separately in the excipient of the capsules (vegetable oil) and in the gelatin shells to determine potential migration of analytes into the shells.27 The gelatin shells were digested with papain or Pronase N at ambient temperature for 1 h for complete release of carotenoids to improve extraction efficiency. A mixture of acetone and hexane (1:1, v/v) containing BHT and BHA as antioxidants was used in the extraction of carotenoids. No migration of lutein into the shells was observed for lutein. In the current study only the excipient of the capsules (vegetable oil) or the capsule contents were used in the extraction of lutein compounds. A method that was earlier developed in our laboratory was used in the analysis of lutein regioisomers in marigold flowers, supplements, and herbal teas. The method uses combined LC, MS, and NMR analytical techniques for thorough identification of lutein regioisomers. Table 2 shows the method recovery,
lutein esters from marigold flowers, supplements, and herbal teas (Table 1). The samples tested differ in their physical state, Table 1. Concentration of Total Luteina of Selected Marigold Flower, Supplement, and Herbal Tea Samples Determined by HPLC with Different Extracting Solvents (“As-Is” Basis) product
water-saturated n-butanol
marigold flower (μg/g) Antigua Orange 345 ± Antigua Yellow 105 ± Durango Red 452 ± Hero Bee 301 ± Janie Primrose 166 ± Safari Scarlet 417 ± Safari Yellow 355 ± marigold supplement (mg/g) A (Jarrow) B (NOW) C (Swanson) D marigold herbal tea (μg/g) A (Australian) 91.3 ± B (Egyptian) 71.9 ± C (Hungarian) 12.4 ± D (calendula) tr
methyl tert-butyl ether
2-propanol
b
a b
11.5 3.3 10.7 9.1 4.5 13.1 10.9
2377 167 3990 1270 292 5752 1228
± ± ± ± ± ± ±
51.3 6.5 67.8 41.7 9.6 91.5 34.9
35 12 114 50 24 180 57
± ± ± ± ± ± ±
0.9 0.7 3.5 1.3 1.1 5.3 1.5
88.0 ± 3.1 105.3 ± 4.4 110.7 ± 4.3 tr 2.9 2.6 0.5
68.3 ± 1.8 69.1 ± 2.3 8.4 ± 0.5 tr
87.8 ± 2.1 85.3 ± 4.1 8.1 ± 0.5 tr
Summation of lutein compound peaks measured as lutein equivalents. Average moisture content of fresh flowers is approximately 85%.
being soft gel based in oil supplement, powder supplement, and dry or fresh plant materials, so it was important to ensure uniformity of each sample prior to extraction of carotenoids. This was done through consistent mixing and homogenization. Other factors such as dim light, temperature, and use of amber glassware wrapped with a black plastic sheet were also considered to avoid photodegradation and isomerization. The three solvents examined showed substantial differences in the amount of total lutein as measured on the basis of the summation of HPLC lutein compound peaks. For marigold flowers and supplements methyl tert-butyl ether was more effective than the other two solvents in extracting lutein and its esters, whereas water-saturated butanol was found to be more efficient for herbal teas. The lutein profiles of marigold flowers and supplements were similar, the major compounds being lutein dieters, whereas the carotenoid profile of herbal tea was different from them with free or nonesterified lutein being the dominant compound (Supplementary Figure 2). This clarifies the effectiveness of MTBE solvent with marigold flowers and supplements, whereas WSB was the appropriate solvent for the herbal tea products investigated. The polarity of WSB is higher than that of MTBE, and thus they have different solubility properties. Due to the relatively higher polarity of nonesterified lutein compared to lutein diester, the WSB solvent was more efficient, with materials containing lutein as the prevailing compound. This finding is in agreement with our previous studies, where WSB was effective in extracting carotenoids from cereal grains in which lutein is the primary carotenoid.14,23 Extraction of lutein esters from marigold flowers is always done with organic solvents to produce the crude extract (marigold oleoresin), and the oleoresin can be saponified and/or purified to yield lutein products with a given purity.17,24,25 In a study to
Table 2. Limit of Detection (LOD), Limit of Quantification (LOQ), and Recovery of Lutein, Lutein 3′-O-Palmitate, Lutein 3-O-Palmitate, and Lutein Dipalmitate compound
LOD (ng/10 μL)a
LOQ (ng/10 μL)a
recovery (%)
lutein lutein 3′-O-palmitate lutein 3-O-palmitate lutein dipalmitate
13 11 22 31
43 36 73 105
102 99 97 95
a
Injection volume.
LOD, and LOQ for lutein, lutein 3′-O-palmitate, lutein 3-Opalmitate, and lutein dipalmitate. The accuracy of these lutein compounds was found to be high, ranging from 95 to 102%. The lutein compounds and isomers showed very low levels of LOD at 13−31 ng per injection volume (10 μL) and LOQ at 36−105 ng per injection volume. Free lutein and lutein 3′-Opalmitate had lower LOD and LOQ values as compared to lutein 3-O-palmitate and lutein dipalmitate, possibly due to their better detector responses and slightly higher recoveries. The method is capable of resolving lutein 3′-O-palmitate and lutein 3-O-palmitate despite tiny differences in their structure. The NMR results showed that the monoester acylated at the 3′hydroxyl group elutes first before its regioisomer.21 In other words, using HPLC and a C30 column, lutein 3′-O-palmitate eluted first before lutein 3-O-palmitate (Supplementary Figure 2). Composition of Lutein Esters. Several marigold flowers, dietary supplements, and herbal teas were analyzed for their C
DOI: 10.1021/acs.jafc.5b04430 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Table 3. Composition of Lutein Ester Regioisomers of Marigold Flowers (μg/g, “As-Is” Basis)a lutein compound free or nonesterified carotenoids lutein zeaxanthin total of free lutein monoesters lutein 3′-O-myristate lutein 3-O-myristate lutein 3′-O-palmitate lutein 3-O-palmitate lutein 3′-O-stearate lutein 3-O-stearate total of lutein monesters mixed diesters lutein 3′-O-laurate-3-O-myristate lutein 3′-O-myristate-3-O-laurate lutein 3′-O-laurate-3-O-palmitate lutein 3′-O-palmitate-3-O-laurate lutein 3′-O-myristate-3-O-palmitate lutein 3′-O-palmitate-3-O-myristate lutein 3′-O-myristate-3-O-stearate lutein 3′-O-stearate-3-O-myristate lutein 3′-O-palmitate-3-O-stearate lutein 3′-O-stearate-3-O-palmitate total of lutein mixed diesters homogeneous diesters lutein dimyristate lutein dipalmitate lutein distearate total of lutein homogeneous diesters ratio di/mono esters a
Antigua Orange 28.2 ± 0.9 1.1 ± 0.05 29.3
Antigua Yellow 13.8 ± 0.2 0.6 ± 0.03 14.4
7.7 1.9 10.7 3.6 1.3 4.1 29.3
± ± ± ± ± ±
0.3 0.07 0.5 0.3 0.04 0.3
1.5 1.0 1.5 1.5 1.1 1.9 8.5
± ± ± ± ± ±
0.06 0.06 0.05 0.07 0.04 0.07
47.1 64.3 53.6 59.4 403.1 345.9 77.3 53.7 92.9 31.1 1228.4
± ± ± ± ± ± ± ± ± ±
1.7 2.6 1.9 2.3 11.9 11.1 3.1 2.5 3.2 1.5
2.6 4.7 2.7 2.6 19.7 16.8 9.2 8.6 8.1 14.6 89.6
± ± ± ± ± ± ± ± ± ±
0.1 0.2 0.1 0.1 0.8 0.7 0.4 0.4 0.3 0.7
312.3 ± 10.2 736.9 ± 19.7 41.3 ± 1.2 1090.5 79.1
Durango Red
20.7 ± 0.8 24.6 ± 1.1 8.6 ± 0.3 53.9 16.9
31.3 ± 1.1 0.8 ± 0.04 32.1
Hero Bee
Janie Primrose
Safari Scarlet
Safari Yellow
17.9 ± 0.6 0.7 ± 0.04 18.6
27.4 ± 1.1 2.9 ± 0.1 30.3
27.5 ± 1.2 3.1 ± 0.1 30.4
18.1 ± 0.7 1.2 ± 0.05 19.3
29.3 10.5 32.4 11.0 2.3 9.8 95.3
± ± ± ± ± ±
0.9 0.6 1.2 0.5 0.2 0.4
15.4 6.8 17.4 9.2 1.7 7.0 57.5
± ± ± ± ± ±
0.6 0.4 0.7 0.3 0.06 0.3
5.1 2.9 4.6 2.3 0.7 1.9 17.5
± ± ± ± ± ±
0.3 0.1 0.2 0.1 0.04 0.1
49.0 19.0 46.9 18.9 7.5 28.3 169.6
± ± ± ± ± ±
2.3 0.8 1.9 0.7 0.2 1.1
15.7 7.1 14.0 6.6 2.2 7.6 53.2
± ± ± ± ± ±
0.5 0.3 0.5 0.4 0.1 0.3
89.9 31.0 129.6 99.8 768.2 603.1 52.9 136.1 128.9 122.8 2162.3
± ± ± ± ± ± ± ± ± ±
3.1 1.0 3.3 3.1 15.2 15.5 1.8 4.0 3.1 3.9
38.9 32.4 40.3 23.5 198.6 159.9 69.2 57.0 76.3 21.1 717.2
± ± ± ± ± ± ± ± ± ±
1.5 1.3 1.9 0.7 4.1 3.7 2.9 2.3 2.5 0.8
13.8 7.9 14.0 7.2 36.2 32.7 16.9 15.8 11.1 2.4 158.0
± ± ± ± ± ± ± ± ± ±
0.5 0.4 0.5 0.4 1.7 1.8 0.8 0.6 0.4 0.1
138.9 136.2 452.7 114.7 856.1 717.5 205.0 208.0 249.2 88.2 3166.5
± ± ± ± ± ± ± ± ± ±
4.1 3.7 12.8 3.5 21.3 19.9 5.5 5.3 7.1 3.6
43.2 47.1 50.7 27.9 143.7 210.7 57.1 49.0 51.8 17.1 698.3
± ± ± ± ± ± ± ± ± ±
1.7 1.6 1.6 1.3 4.2 5.5 2.3 1.9 1.8 0.8
557.9 ± 11.5 1101 ± 23.3 41.3 ± 1.9 1700.2 40.5
166.9 ± 3.6 268.4 ± 4.9 41.0 ± 1.7 476.3 20.8
40.4 ± 1.2 32.6 ± 1.7 13.0 ± 0.6 86.0 13.9
620.4 ± 15.1 1654 ± 32.3 110.9 ± 4.3 2385.3 32.7
177.7 ± 5.6 249.5 ± 5.3 29.8 ± 1.3 457.0 21.7
Average moisture content of fresh flowers is approximately 85%.
content and composition of lutein, lutein monesters, and lutein diesters. We were able to separate, quantify, and identify up to 20 lutein compounds and free zeaxanthin in the products tested (Tables 3−5; Supplementary Figure 2). These include nonesterified lutein and zeaxanthin, lutein monoesters, lutein mixed diesters, and lutein homogeneous diesters. This is the first report that provides detailed characterization for lutein esters in marigold flowers and products without pretreatment, for example, saponification or enzyme cleavage. Previous studies have reported coeluted or lower numbers of lutein esters.28−30 Table 3 shows the composition of lutein esters in marigold flowers. Seven marigold varieties of different colors were analyzed. In general, dark marigold flowers (red) exhibited higher levels of lutein esters with lutein palmitate esters being the major ones. Marigold yellow flowers had lower contents of lutein esters as compared to the red ones. The flower materials can be categorized into three groups based on their content of lutein compounds: a high-lutein group, >3000 μg/g (e.g., Safari Scarlet and Durango Red); an intermediate group, >1000 and
