Accepted Manuscript

Marigold carotenoids: Much more than lutein esters

Daniele Bobrowski Rodrigues, Adriana Zerlotti Mercadante,

Lilian Regina Barros Mariutti

PII: S0963-9969(18)30829-9
DOI: doi:10.1016/j.foodres.2018.10.043
Reference: FRIN 8013
To appear in: Food Research International
Received date: 7 August 2018
Revised date: 8 October 2018
Accepted date: 11 October 2018

Please cite this article as: Daniele Bobrowski Rodrigues, Adriana Zerlotti Mercadante,
Lilian Regina Barros Mariutti , Marigold carotenoids: Much more than lutein esters. Frin
(2018), doi:10.1016/j.foodres.2018.10.043

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MARIGOLD CAROTENOIDS: MUCH MORE THAN LUTEIN ESTERS

Daniele Bobrowski Rodrigues1, Adriana Zerlotti Mercadante1 and Lilian Regina Barros
Mariutti2* lilianma@unicamp.br

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Department of Food Science, Faculty of Food Engineering, University of Campinas,
Campinas-SP, 13083-862, Campinas, Brazil
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Department of Food and Nutrition, Faculty of Food Engineering, University of

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Campinas, Campinas-SP, 13083-862, Campinas, Brazil
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*
corresponding author.
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Abstract

Carotenoids constitute a large group of lipophilic pigments whose health-promoting benefits have
been widely recognized. Hydroxyl-containing carotenoids can be found in both free form or
esterified with fatty acids in several plant matrices, but the native carotenoid profile is overall
poorly explored due to the difficulty of analyzing carotenoid esters. One of the main natural
sources of carotenoids is the marigold flower, which has been extensively used by the industry

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for the production of food colorants or supplements, both often manufactured with no

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saponification process. Although lutein esters are well established as the major compounds
naturally found in marigold petals and their products, carotenoid esters other than the lutein ones

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have not been extensively examined. We carried out a comprehensive identification of
carotenoids and carotenoid esters from marigold petals by LC-DAD-(APCI+)MS/MS. Whereas

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18 carotenoids were identified in the saponified extract, 56 were identified when no
saponification procedure was carried out: 6 free carotenoids, 20 monoesters and 30 diesters. This
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is the first time that esters of zeaxanthin, violaxanthin, auroxanthin, zeinoxanthin and β-
cryptoxanthin are identified in marigold. The structural information obtained through
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characteristic fragmentation patterns and diagnostic fragments in MS and MS/MS spectra

(APCI+) sustained the differentiation between carotenoid esters with similar characteristics.
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Therefore, the separation of carotenoids by reversed-phase liquid chromatography using C30

columns in combination with DAD and APCI-MS/MS detection allowed high sensitivity and
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selectivity for carotenoid ester analysis.

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Keywords: carotenoid esters; LC-MS; lutein; atmospheric pressure chemical ionization; mass
spectrometry; xanthophylls
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1 Introduction

Carotenoids constitute a large family of fat-soluble isoprenoid pigments widely

recognized as bioactive, responsible for many of the yellow-to-red colors in flowers, fruits,
vegetables, microorganisms and also some animals and animal products such as salmon and egg
yolk. Their basic structure consists of a symmetrical and linear carbon skeleton with a series of
conjugated double bonds (cdb), which generates a resonance system of -electrons and

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corresponds to the molecule chromophore. Besides the color, this structural feature is responsible

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for the molecular shape and chemical reactivity of carotenoids (Britton, 1995).

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Marigold (Tagetes spp.) is a genus of flowers from the Asteraceae family established as
one of the main commercial sources of natural carotenoids for the food, animal feed, chemical
and pharmaceutical industries. Although some species (particularly Tagetes patula and Tagetes

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erecta) have already been extensively used in poultry feed to color eggs and for the production of
food colorants and ophthalmologic preparations, the crescent body of epidemiological evidences
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on health outcomes related to carotenoid consumption has further boosted the market of
marigold-based dietary supplements and extracts for food and beverage enrichment. In fact,
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supplements containing lutein were related to decreased risk of developing age-related macular
degeneration (AMD), a degenerative disease responsible for the loss of central vision and the
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major cause of irreversible blindness in elderly (Johnson, 2014; Akuffo, Nolan, Howard, Moran,
Stack, Klein, Klein, Meuer, Sabour-Pickett, Thurnham, & Beatty, 2015). Moreover, lutein was
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correlated with cognitive function in adults, being also accumulated in human brain and macula,
both in infants and elderly (Feeney, Finucane, Savva, Cronin, Beatty, Nolan, & Kenny, 2013;
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Tanprasertsuk, Li, Bernstein, Vishwanathan, Johnson, Poon, & Johnson, 2016; Saint, Renzi-
Hammond, Khan, Hillman, Frick, & Hammond, 2018).
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Although recognized for simplifying carotenoid analysis in foods, the saponification

process has been increasingly avoided as it may cause carotenoid degradation and isomerization,
besides adding costs to the manufacture of commercial preparations through, for instance, power
needs and safety procedures for handling corrosive chemicals and waste management (Abdala
Gallardo, Olvera, & Silva, 2017). Lutein esters are well known as the predominant carotenoids
naturally found in marigold petals, extracts, oleoresins and derivative products (Table S1). In
fact, several lutein monoesters and diesters of (all-E)- and (Z)-lutein acylated with lauric (12:0),
myristic (14:0), palmitic (16:0) and stearic (18:0) acid moieties have been described, being lutein
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dimyristate, dipalmitate, myristate-palmitate and palmitate-stearate the major compounds

(Breithaupt, Wirt, & Bamedi, 2002; Tsao, Yang, Young, Zhu, & Manolis, 2004; Abdel-Aal &
Rabaski, 2015).
The elevated number of different lutein ester structures found in Tagetes is related to the
structure of this xanthophyll, as shown in Figure 1. Lutein displays an asymmetric structure with
terminal - and -rings hydroxylated, respectively, at 3- and 3’-position (Table 1) that can lead to
the formation of monoesters and diesters. Either lutein monoacylation or diacylation by two

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different fatty acids (FA) yields distinct regioisomers (Breithaupt et al., 2002). To illustrate,

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considering the acylation of the (all-E)-form of this xanthophyll only with saturated FA

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containing chain length between 10 and 18 carbons, theoretically, 35 different (all-E)-lutein
esters can be possibly formed (Figure 1). In addition, lutein can be often found as 9, 9’, 13 and

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13’(Z)-isomers, and further acylated with different saturated or unsaturated FA in diverse
combinations, leading to a quite high number of different structures with similar characteristics.
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Mono- and di-hydroxylated xanthophylls other than lutein can also be found esterified with FA in
the most diverse combinations, further enhancing the variability of carotenoid structures.
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Although carotenoid esters are widely spread in nature, the high number of possible
structures found in plant matrices contributes to making the analysis of these compounds more
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difficult and laborious than after saponification (Mercadante, Rodrigues, Petry, & Mariutti,
2017). Moreover, since all the esters of a given carotenoid present exactly the same chromophore
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(Figure 1) and consequently the same UV-Visible (UV-Vis) spectrum, their identification must
include tandem mass spectrometry (MS/MS) studies (Rodrigues, Mariutti, & Mercadante, 2016,
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Mercadante et al, 2017, Mariutti and Mercadante, 2018). Several ionization methods have been
employed to study the native carotenoid profile of marigold flowers up to date (Table S1). The
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atmospheric pressure chemical ionization (APCI) is the most used ionization source for
carotenoid analysis, and the same is true for carotenoid esters, since abundant molecular ions
(M−•) or protonated ([M+H]+) and deprotonated ([M−H]−) molecules of carotenoids are likely
formed (Van Breemen, Dong, & Pajkovic, 2012; Mercadante et al., 2017). APCI in negative
ionization mode and in-source collision-induced dissociation (CID) were successfully applied to
study the fragmentation of lutein diesters in a commercial marigold extract, with the detection of
molecular ions [M−•] and in-source fragment ions consistent to the losses of fatty acid moieties
(Tian, Duncan, & Schwartz, 2003). Nonetheless, the use of APCI in positive ionization mode is
the most reported since distinctive fragmentation patterns and diagnostic fragments useful to
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unequivocally assign carotenoids have been described (Van Breemen et al., 2012; Petry &
Mercadante, 2016; Mercadante et al., 2017; Pérez-Galvez, Sánchez-García, Garrido-Fernández,
& Ríos, 2018).
Consistent progress has been made over the last years towards the development of LC-MS
methods for the identification of carotenoid esters in different matrices, and the main analytical
approaches for identification of such compounds by LC-DAD-MS/MS in food and biological
samples were recently reviewed (Mercadante et al., 2017; Mariutti & Mercadante, 2018). Even

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though, due to a combination of factors, such as similarity among the structures and chemical

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properties, the presence of a high number of peaks generally in low amounts and peak co-elution,

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the identification of carotenoid esters remains a challenge.
Although marigold flowers are a well-studied sample, the presence of carotenoid esters

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other than lutein has been poorly explored in the literature. Taking into account that many
supplements, food and feed contain non-saponified carotenoid extracts or oleoresin obtained from
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marigold flowers, the native carotenoid profile of this source merits further investigation.
Considering these facts, the objective of this work was to carry out a comprehensive
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identification of carotenoid esters from marigold petals by LC-DAD-(APCI+)MS/MS.

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2 Material and Methods

2.1 Chemicals
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Standard of (all-E)--carotene was purchased from Sigma-Aldrich (St. Louis, MO, USA).
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(All-E)-lutein, (all-E)-zeaxanthin, (all-E)--cryptoxanthin, and (all-E)--carotene standards were

donated by DSM Nutritional Products (Basel, Switzerland). Solvents and reagents of analytical
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grade used during carotenoid extraction and saponification were purchased from Synth
(Diadema, SP, Brazil). HPLC grade methyl tert-butyl ether (MTBE) and methanol (MeOH) were
obtained from Tedia (Fairfield, OH, USA) and J.T. Baker (Phillipsburg, NJ, USA), respectively;
water was purified by a Milli-Q system (Billerica, MA, USA). Samples and solvents were filtered
prior to chromatographic analysis using, respectively, through Millipore membranes of 0.22 and
0.45 µm.
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2.2 Samples

Marigold flowers (Tagetes patula cultivars Carmen, Cresta flame, Deep orange, Bonanza
bee and Yellow boy) were acquired at CEASA – Campinas (Centrais de Abastecimento de
Campinas S.A), São Paulo State, Brazil. Petals were manually separated and all the cultivars
were pooled together and lyophilized (Liobras model K105, São Paulo, Brazil). Freeze-dried
petals were ground into a powder, vacuum packed and stored at -80 ºC until analysis.

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2.3 Carotenoid extraction

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Freeze-dried petals were extracted with acetone by vortex agitation (3 x 5min with 12 mL,

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followed by 3 x 5 min with 4 mL) and maceration with Hyflosupercel20 mL, once), followed
by centrifugation (9,000 g for 10 min at 10 ºC) (Allegra 64R, Beckman Coulter, Indianapolis, IN,

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USA) and vacuum filtration through a filter paper (4-12 µm pore, cod 501.1240, Unifil,
Germany). The filtered extracts were combined, and partitioned to petroleum ether/ethyl ether
(1:2, v/v). Acetone was removed by washing the ether phase with distilled water. Residual water
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was removed with anhydrous Na2SO4. One aliquot of the ethereal carotenoid extract was
concentrated in a rotary evaporator (T  30 °C), dried under N2 flux and immediately stored at -
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36 ºC (non-saponified extract), while a second aliquot was saponified with 10% (w/v) KOH in
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MeOH, overnight (~16h) at room temperature. The alkali was removed by washing the extract
with distilled water and the saponified extract was then concentrated in a rotary evaporator,
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flushed with N2 and kept at - 36 °C until chromatographic analysis (De Rosso & Mercadante,
2007).
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2.4 HPLC-DAD-APCI(+)MS/MS

Carotenoid separation was achieved on a C30 YMC column (5 μm, 250 x 4.6 mm i.d.)
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(Waters, Wilmington, NC) in a Shimadzu (Kyoto, Japan) high pressure liquid chromatograph
(HPLC) equipped with a quaternary pump (LC-20AD), online degasser (DGU-20A5), Rheodyne
injection valve with 20 μL loop (Rohnert Park, CA, USA), connected in series to a diode array
detector (DAD) (SPDM20A) and a mass spectrometer with an ion trap analyser with atmospheric
pressure chemical ionization (APCI) source (AmaZon speed ETD, Bruker Daltonics, Bremen,
Germany). Chromatographic and MS conditions applied to analyze marigold non-saponified
extract were those described in details by Rodrigues, Mariutti, & Mercadante (2016), while the
saponified extract was analyzed according to De Rosso & Mercadante (2007) and Chisté &
Mercadante (2012). Briefly, for analysis of carotenoid esters, a linear gradient of mixtures of
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MeOH, MTBE and water as the solvents A (81:15:4, v/v/v) and B (16:80.4:3.6, v/v/v) was
applied as follows: from 99% to 44% solvent A in 39 min, decreasing to 0% solvent A in 6 min,
returning to the initial conditions in 5 min and maintaining this proportion for 5 min, totaling a 55
min run at flow rate of 1.0 mL/min. The column was kept at 35 ºC. To analyze the saponified
extract, a linear gradient of MeOH and MTBE was applied from 95:5 (v/v) to 70:30 in 30 min,
reaching 50:50 in 20 min, holding this proportion for 15 min and returning to the initial
conditions in 5 min. The flow rate was 0.9 mL/min and the column temperature was set at 29 °C.

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The MS parameters were set as follows: APCI positive mode, corona current of 4000 nA, source

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temperature of 450 °C, dry gas (N2) temperature of 350 °C at 5 L/min, nebulizer at 60 psi. UV-

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Vis spectra were acquired from 200 to 600 nm and processed at 450 nm, 347 nm (for
phytofluene) and 400 nm (for auroxanthin). MS spectra were acquired in the range of m/z 100 to

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1200 and MS/MS was obtained in automatic mode. Carotenoid identification was carried out
considering the combination of the following information: chromatographic elution order on C30
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column, co-chromatography with authentic standards, UV-vis spectra features (maximum
wavelength absorbance (max), spectral fine structure (% III/II) and cis peak intensity (% AB/AII)),
mass spectra characteristics (protonated molecule ([M+H]+) and MS/MS fragments) and
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comparison with literature data (Breithaupt et al., 2002; Britton, Liaaen-Jensen, & Pfander, 2004;
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De Rosso & Mercadante, 2007; Zepka & Mercadante, 2009; Chisté & Mercadante, 2012;
Bijttebier, D’Hondt, Hermans, Apersb, & Voorspoelsa, 2013; Petry & Mercadante, 2016;
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Rodrigues et al., 2016; Mercadante et al., 2017).

3 Results and Discussion

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In the non-saponified extract of marigold, 40 peaks were separated and 56 carotenoids

were identified or tentatively identified: six free carotenoids, 20 monoesters and 30 diesters
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(Table 2, Figure 2). Besides (all-E)-lutein (peak 2) and (all-E)--carotene (peak 11b), with
identification confirmed by authentic standards, the other free carotenoids were (all-E)-
violaxanthin (peak 1), (Z)-phytofluene (peak 4), (all-E)-phytofluene (peak 6b) and (Z)--carotene
(peak 8).
Lutein monoesters acylated with lauric, myristic, palmitic and stearic acids, lutein
heterodiesters formed by different combinations of two of these FA, and lutein homodiesters
acylated with myristic, palmitic and stearic acids were tentatively identified in different
geometric and regioisomeric forms, aligned with the profile already described in the literature
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(Table S1). Nevertheless, to the best of our knowledge, this is the first time that the homodiesters
lutein dibutyrate, dicaprylate, dicaprate, (Z)- and (all-E)-lutein dilaurate (peaks 16b, 18b, 20b,
21a and 23a, respectively), and the heterodiester (all-E)-lutein-3-O-laurate-3’-O-caprate (peak
24) were found in marigold flowers. As a detailed description of the identification of lutein esters
through the interpretation of information obtained from chromatographic characteristics and
fragmentation pattern in MS and MS/MS spectra has already been reported (Breithaupt et al.,
2002; Rodrigues et al., 2016; Mercadante et al., 2017), only the identification of those peaks not

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previously described in marigold are discussed below.

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In spite of the absence of a protonated molecule in the MS spectra, lutein homodiesters

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were characterized by fragment ions corresponding to the neutral loss of one [M+H-FA]+ and two
fatty acid moieties [M+H-2FA]+ of either butyric (88 u) (peak 16b), caprylic (144 u) (peak 18b),

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capric (172 u) (peak 20b) or lauric (200 u) (peak 21a) acid, the latter fragment equivalent to the
lutein carbon backbone at m/z 533. The heterodiester assigned as (all-E)-lutein 3-O-laurate-3’-O-
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caprate (peak 24) showed in-source fragment ions at m/z 733 and m/z 705, corresponding to the
loss of one moiety of capric acid [M+H-172]+ and lauric acid [M+H-200]+, respectively, and
MS/MS fragment at m/z 533, after the loss of both fatty acid moieties [M+H-172-200]+ (Figure
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3A). The intensity of the fragment ion at m/z 733 was higher than that at m/z 705, indicating that
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the loss of capric acid results in the formation of a more stable fragment ion than the loss of lauric
acid. In fact, the linkage at the 3-O-position is possibly more stable, favoring the loss of the
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substituent in the ε ring due to the position allylic to the double bond. This profile indicates that
capric acid was acylated to the -ring (3’-O-position) because the loss of the fatty acid attached in
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allylic position to the double bond in this ring is favored, generating a more stable fragment ion
than when the fatty acid is acylated to the β-ring (3-O-position). Furthermore, characteristic
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fragmentation patterns of ε-ring-containing structures, such as the loss of 56 u generated by retro-

Diels-Alder fragmentation of the α-ionone moiety, and of the polyene chain of carotenoids, such
as elimination of toluene (92 u), were observed (Van Breemen et al., 2002; Rivera, Christou, &
Canela-Garayoa, 2014; Rodrigues et al., 2016). On the other hand, due to the co-elution of at
least two carotenoids in peaks 16, 18, 20, 21 and 23, UV-vis spectra of lutein esters were not
characteristic and did not allow the assignment of lutein geometric isomers because the MS
fragmentation of (all-E) and (Z)- isomers are essentially the same. Nonetheless, combined with
the information given by the MS detector, the elution order also assisted in the assignment of
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lutein esters, since the retention time rationally increased as the fatty acid molecular weights
increased (Khachik & Beecher, 1988, Mercadante et al, 2017).
As a matter of fact, although a considerable number of lutein esters have been identified
in the present study, including some of them for the first time, lutein laurate and lutein laurate-
palmitate previously described in the literature (Breithaupt et al., 2002; Tsao et al., 2004; Young
Abdel-Aal, Rabalski, & Blackwell, 2007; Abdel-Aal & Rabaski, 2015) were not found in the
analyzed marigold extract. These dissimilarities could be attributed to differences in

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species/varieties of the analyzed flower samples, but the applied extraction method and distinct

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characteristics of the HPLC and MS systems are also factors that may have influenced

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differences with others investigations.
Beyond the lutein esters, other carotenoid esters were found in small amounts in non-

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saponified extract and were detected mainly by MS. Even though the presence of zeaxanthin and
violaxanthin esters in marigold has been reported once, no identification of such compounds was
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carried out so far in this sample (Lapshova, Deineka, Deineka, Blinova, & Tret’yakov, 2013).
Furthermore, monoesters of auroxanthin, -cryptoxanthin and zeinoxanthin, not previously found
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in marigold, are described herein.

Peaks 13, 16c, 33a and 36 were assigned, respectively, as (all-E)-zeaxanthin myristate,
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zeaxanthin palmitate, zeaxanthin myristate-palmitate and (all-E)-zeaxanthin dipalmitate; all of

them presented the same chromatographic and spectroscopy features than those previously
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reported for the same carotenoid esters found in peppers, orange and murici fruits (Weller &
Breithaupt, 2003; Petry & Mercadante, 2016; Rodrigues et al., 2016). Peak 39 was assigned as
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(all-E)-zeaxanthin palmitate-stearate considering the presence of the protonated molecule

[M+H]+ at m/z 1074 in the MS spectrum and MS/MS fragment ions at m/z 817 and m/z 789,
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corresponding to the respective losses of palmitic [M+H-256]+ and stearic [M+H-284]+ acid
moieties, in addition to the fragment ion generated by the sequential loss of both fatty acids
[M+H-284-256]+ at m/z 533 (Figure 3B). Note that these fragments generated from losses of
palmitic and stearic acid moieties are similar to those found when lutein is acylated with the same
fatty acids (peaks 37 and 38, Table 2), and that the carbon backbone at m/z 533 is common for
both zeaxanthin and lutein esters (Figure 3), as these di-hydroxylated xanthophylls share the
same molecular weight (568 u). Thus, the differentiation between zeaxanthin and lutein esters
was achieved through the comparison of their MS, MS/MS and UV-Vis spectra. Zeaxanthin
displays a symmetric structure with two terminal -rings, therefore the double bonds in both rings
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are not allylic, but conjugated (Table 1). As a result, the consistent presence of protonated
molecule distinguish the MS spectra of zeaxanthin esters from those of lutein, in which
protonated molecule is present at very low abundance or absent, and intense in-source fragments
are observed instead, due to the facility of losing the substituent at allylic position (Figure 3). The
absence of diagnostic fragment ions related to the elimination of α-ionone moiety observed in
,-derivatives (-carotene related structures), such as the fragment ion [M+H-FA-56]+ at m/z
495, was also considered to differentiate zeaxanthin esters from lutein ones (Van Breemen et al.,

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2012; Rodrigues et al., 2016; Ziegler, Wahl, Würschum, Longin, Carle, & Schweiggert, 2016).

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Moreover, peak 39 showed UV-vis spectrum with max at 451 nm, consistent with a chromophore

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with 11 cdb and two -rings, longer than that exhibit by lutein and lutein esters at 446 nm (10
cdb, Table 1).

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A representative number of violaxanthin esters was found in the non-saponified extract of
marigold, comprising three monoesters (peaks 3, 5 and 6a) and nine diesters (peaks 15a, 16a, 17a,
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18a, 19a, 22, 23b, 26 and 30). The characteristic fragmentation pattern observed in violaxanthin
esters described in details elsewhere (Petry & Mercadante, 2016), such as losses of one water
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molecule from the free hydroxyl group and fatty acid moieties accompanied and not accompanied
by further loss of one water molecule from the epoxide group, was also observed (Figure 4A).
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For example, MS spectra of peaks 22 and 23b, tentatively identified as violaxanthin myristate-
palmitate isomers, exhibited the protonated molecule [M+H]+ at m/z 1050, and MS/MS fragment
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ions at m/z 821, m/z 793 and m/z 565, as a result of the losses of one myristic acid (228 u), one
palmitic acid (256 u) and both fatty acid moieties, respectively. In addition, further losses of one
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water molecule from the epoxide group were observed at m/z 1032 [M+H-18]+, m/z 803 [M+H-
18-228]+, m/z 775 [M+H-18-256]+ and m/z 547 [M+H-18-228-256]+. Although the UV/vis
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spectra of some peaks were not characteristic of a pure compound or even not detected, the
assignment of violaxanthin esters was supported by the combination of the characteristic MS data
with their elution order.
Auroxanthin esters (all-E)-auroxanthin palmitate (peak 7) and auroxanthin stearate
isomers (peaks 10 and 12) presented the same UV-vis characteristics than those of free
auroxanthin, i.e. max at 378, 400, and 424 nm. As expected, the fragmentation pattern of the
auroxanthin esters was similar to that exhibited by esters of its structural analogous violaxanthin
(MW 600 u, Figure 4). Peak 7 was assigned as (all-E)-auroxanthin palmitate taking into account
the presence of the protonated molecule [M+H]+ at m/z 839 in the MS spectrum, and MS/MS
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fragments at m/z 565 [M+H-18-256]+ and m/z 547 [M+H-18-18-256]+, corresponding to the
losses of water molecules from epoxide and hydroxyl groups along with palmitic acid (Figure
4B). The same compound was tentatively identified in orange juices and a similar fragmentation
pattern was reported (Giuffrida, Dugo, Salvo, Saitta, & Dugo, 2010). Peaks 10 and 12 were
assigned as isomers of (all-E)-auroxanthin stearate since equivalent fragments with loss of stearic
(284 u) instead of palmitic acid moiety were detected. Despite the similar MS spectra,
auroxanthin and violaxanthin esters were differentiated based on their typical UV-vis spectra and

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chromatographic behavior, given the presence of furanoid groups at 5,8,5’,8’-position in

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auroxanthin and epoxy groups at 5,6,5’,6’-position in violaxanthin. Besides the hypsochromic

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shift of about 40 nm in the max observed in auroxanthin esters in contrast to the violaxanthin
ones, 5,6-epoxycarotenoids generally eluted earlier in the chromatographic run than their 5,8-

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counterparts (De Rosso & Mercadante, 2007; Mariutti, Rodrigues, & Mercadante, 2013; Petry &
Mercadante, 2016). In conformity, (all-E)-violaxanthin palmitate (peak 6a) showed shorter
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retention time than (all-E)-auroxanthin palmitate (peak 7) (Figure 2).
Esters of the monohydroxy xanthophylls, zeinoxanthin (peaks 17b and 20a) and -
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cryptoxanthin (peaks 19b, 23c, 28b and 32), were also found in marigold petals. Peak 17b was
tentatively identified as zeinoxanthin laurate considering the protonated molecule [M+H]+ at m/z
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735 and MS/MS fragment ion [M+H-200]+ at m/z 535, the latter corresponding to the carbon
backbone of zeinoxanthin after the loss of one lauric acid moiety. Peak 20a was assigned as (all-
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E)-zeinoxanthin myristate based on the presence of equivalent fragments, [M+H]+ at m/z 763 and
[M+H-228]+ at m/z 535 resulting from the neutral loss of a myristic acid moiety (Figure 5A).
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Similarly, -cryptoxanthin laurate and myristate (peaks 19b and 23c) showed fragment ions
[M+H]+ at, respectively, m/z 735 and m/z 763, and MS/MS fragment ions [M+H-FA]+ at m/z 535,
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corresponding to the losses of either lauric (200 u) or myristic acid (228 u) moieties (the latter is
shown in Figure 5B). Clearly, zeinoxanthin and -cryptoxanthin esters cannot be differentiated
only by MS (Figure 5) (Schlatterer & Breithaupt, 2005; Da Costa & Mercadante, 2018). The
xanthophylls -cryptoxanthin and zeinoxanthin are, respectively, ,- and ,- derivatives that
share the same molecular weight (Table 1). However, the site of acylation in zeinoxanthin
structure is located on -ring as it is in -cryptoxanthin, i.e., no substituents are found in the
position allylic to the double bond of the -ring and no in-source fragmentation is observed for
esters of both compounds. In spite of the similar MS spectra, zeinoxanthin and -cryptoxanthin
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esters exhibit distinct UV-vis features and elution order behavior, in analogy to lutein and
zeaxanthin. Even though the visualization of the UV-vis spectra was generally impaired due to
co-elution, the tentative peak assignment was possible considering the earlier elution of
zeinoxanthin esters on C30 column compared to -cryptoxanthin esters (Schlatterer & Breithaupt,
2005). Under the applied chromatographic conditions, zeinoxanthin esters (peak 17b and 20a)
eluted about 2 min before -cryptoxanthin esters acylated with the same fatty acid (peak 19b and
23c, Figure 2), and both -cryptoxanthin and zeinoxanthin esters acylated with lauric acid

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moieties eluted 3 min earlier than the same xanthophyll esterified with myristic acid. Retention
times of -cryptoxanthin myristate (peak 23c) and palmitate (peak 28b) were in agreement with

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those reported for the same esters analyzed under the same chromatographic conditions
(Rodrigues et al., 2016; Da Costa & Mercadante, 2018).

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Although the presence of -cryptoxanthin, a structural isomer (MW 552 u) of both
zeinoxanthin and -cryptoxanthin, has been reported in oleoresin of marigold flowers (Khachik,
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Steck, & Pfander, 1999, Table S1), the absence of -cryptoxanthin esters in the analyzed sample
was confirmed by MS analysis. In comparison with zeinoxanthin, -cryptoxanthin presents
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identical chromophore (10 cdb) and terminal - and -rings, but differs from the former in the
position of the hydroxyl group, attached to the -ring and thus, allylic to the double bond (Table
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1). As no in-source fragmentation was observed in the MS spectra of those carotenoid esters with
MW 552 u, the presence of -cryptoxanthin was excluded.
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A saponified extract of marigold petals was also prepared to support the occurrence of all
the free xanthophylls present in the esterified form in the non-saponified extract (Table 3, Figure
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6). Indeed, free (all-E)-zeinoxanthin (peak 8) and (all-E)--cryptoxanthin (peak 10a), but no -
cryptoxanthin, were positively identified, showing UV-vis, MS spectra and chromatographic
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behavior compatible with authentic standards and data available in the literature (De Rosso &
Mercadante, 2007; Zepka & Mercadante, 2009; Rodrigues, Mariutti, & Mercadante, 2013;
Mariutti et al., 2013). In total, 14 peaks were separated and 18 free carotenoids were identified or
tentatively identified in the saponified extract of marigold petals. Besides the aforementioned
zeinoxanthin and -cryptoxanthin, (all-E)- and (9Z)-violaxanthin (peaks 1 and 2b, respectively)
were found in considerable amounts after saponification (Figure 6). Remarkably, antheraxanthin
was detected in marigold extract after saponification, but no carotenoid ester compatible with
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MW 584 u and carbon backbone at m/z 549 was found during the analysis of the non-saponified
extract.
Given the complex composition of the native carotenoids found in flowers, fruits and
vegetables, compound co-elution in chromatographic separation and presence of several esters
only in small amounts are situations often reported (Schlatterer & Breithaupt, 2005; Petry &
Mercadante, 2016), as observed in the marigold chromatograms presented herein, turning the
visualization of well-defined and pure UV-vis and MS spectra and consequently the identification

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of some esters even more difficult. Analysis of a saponified extract in parallel may be useful to

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support the unequivocal assignment of the free xanthophylls present in the sample that may be

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found acylated with fatty acids.
In terms of fatty acids, only saturated fatty acids were found acylated to xanthophylls in

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marigold petals, aligned with the data available in the literature. The present study confirms the
presence of lauric, myristic, palmitic and stearic acids in carotenoid ester constitution, whereas
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the acylation with fatty acids with chain length lower than 12 carbons was described for the first
time in marigold.
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Conclusion

The applied analytical method allowed the assignment of three free carotenoids and 53
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carotenoid esters in the non-saponified extract and 17 free carotenoids in the saponified extract of
marigold petals. The current information on the carotenoid ester profile in marigold was
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confirmed and further expanded. Along with five lutein esters not previously described in
marigold, zeaxanthin, violaxanthin, auroxanthin, zeinoxanthin and -cryptoxanthin esters were
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separated and identified for the first time to the best of our knowledge. Therefore, separation of
carotenoids by reversed-phase liquid chromatography using a C30 column in combination with
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DAD and APCI-MS/MS detection allows high sensitivity and selectivity and is the technique of
choice for carotenoid ester identification in complex matrices. The analysis of marigold flowers
simultaneously with other matrices can be a very useful tool to help carotenoid esters
identification when no commercial standards are available.
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Acknowledgments

The authors acknowledge the financial support of FAPESP (grants #2013/23218-1 and
#2015/15238-8), CNPq (455748/2014-4) and FAEPEX-Unicamp (2084/17).

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Figure 1. Possible (all-E)-lutein esters formed with diverse fatty acids.

Figure 2. Chromatogram of the non-saponified extract of carotenoids from marigold (Tagetes

patula L.) petals, obtained by HPLC-DAD. Inset: chromatogram zooms ranging from 4.0 to 11.0
min and from 17.5 to 40.0 min. Peak characterization is given in Table 1.

Figure 3. MS and MS/MS spectra of (A) (all-E)-lutein 3-O-laurate-3’-O-caprate (peak 24) and

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(B) (all-E)-zeaxanthin palmitate-stearate (peak 39).

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Figure 4. MS and MS/MS spectra of (A) (all-E)-violaxanthin palmitate (peak 6a) and (B) (all-E)-

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auroxanthin palmitate (peak 7).

Figure 5. MS and MS/MS spectra of (A) (all-E)-zeinoxanthin myristate (peak 20a) and (B) (all-

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E)--cryptoxanthin myristate (peak 23c). AN
Figure 6. Chromatogram of the saponified extract of carotenoids from marigold (Tagetes patula
L.) petals, obtained by HPLC-DAD, processed at 450 nm. Peak characterization is given in Table
2. Inset: chromatograms processed at 286 nm and 347 nm.
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Table 1. Structural, UV-Vis and MS characteristics of free cyclic carotenoids found in

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Tagetes sp. and structural analogous

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a
Number of hydroxyl substituents; b molecular weight; c number of conjugated double bonds in the molecule, which
corresponds to the chromophore; d representative values for (all-E)-isomers in specific solvents according to Britton
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et al. (2004); e carotenoid chromophore (conjugated double bonds) is highlighted in grey. The table summarizes
structural, UV-Vis and MS characteristics of free cyclic carotenoids already described in Tagetes sp. and some of
their structural analogous. A relevant point to observe is that the chromophore of free carotenoids and their
respective esters are the same or very similar. Moreover, the MS spectra of esters show corresponding fragmentation
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patterns to those observed for their free xanthophylls (for instance, the presence of in-source fragmentation), while in
MS/MS losses of substituents attached to the rings, either hydroxyl groups or fatty acids, are seen rather than the
simply loss of hydroxyl groups.

Table 2. Mass spectrometry and UV-vis characteristics of carotenoids from non-saponified

extract of marigold petals
Peaka Carotenoid Rt λmax [M+H]+ MS/MS fragment ions (m/z)
(min)b (nm)c (m/z)
1 (all-E)-violaxanthin 5.7- 414, 438, 601.5 583.5 [M+H-18]+, 565.5 [M+H-18-18]+, 547.5
5.8 468 [M+H-18-18-18]+, 509.5 [M+H-92]+, 491.4 [M+H-
18-92]+, 221.1
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Peaka Carotenoid Rt λmax [M+H]+ MS/MS fragment ions (m/z)

(min)b (nm)c (m/z)
2 (all-E)-lutein 9.5- 421, 445, 569.5 551.5 [M+H-18]+ → 533.5 [M+H-18-18]+, 495.4
9.8 472 [M+H-18-56]+, 477.5 [M+H-92]+, 459.1 [M+H-18-
92]+, 429.3
3 (all-E)-violaxanthin 18.8- 418, 439, 811.8 793.8 [M+H-18]+, 775.8 [M+H-18-18]+, 719.5
myristate 19.3 468 [M+H-92]+, 701.7 [M+H-18-92]+, 583.6 [M+H-
228]+, 565.5 [M+H-18-228]+, 547.5 [M+H-18-18-
228]+, 221.0
4 (Z)-phytofluene 20.5- 331, 348, 543.6 461.5 [M+H-82]+, 449.6 [M+H-94]+, 405.4 [M-
21.1 367 137]+, 337.3 [M-205]+

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5 (9Z)-violaxanthin 21.8- 413, 436, 811.8 793.8 [M+H-18]+, 775.8 [M+H-18-18]+, 701.7
myristate 22.3 464 [M+H-18-92]+, 583.6 [M+H-228]+, 565.5 [M+H-18-

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228]+, 547.6 [M+H-18-18-228]+
6a (all-E)-violaxanthin 22.9- 415, 439, 839.8 821.8 [M+H-18]+, 803.6 [M+H-18-18]+, 747.8
palmitate 23.5 471 [M+H-92]+, 729.7 [M+H-18-92]+, 583.6 [M+H-

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256]+, 565.6 [M+H-18-256]+, 547.6 [M+H-18-18-
256]+
6b (all-E)-phytofluene 22.9- 332, 348, 543.6 461.5 [M+H-82]+, 405.4 [M-137]+, 337.3 [M-205]+
23.5 367

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7 (all-E)-auroxanthin 24.5- 378, 399, 839.8 821.8 [M+H-18]+, 803.6 [M+H-18-18]+, 747.8
palmitate 25.1 424 [M+H-92]+, 729.8 [M+H-18-92]+, 583.6 [M+H-
256]+, 565.5 [M+H-18-256]+,
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8 (13Z)- or (15Z)-β- 26.4- 421, 445, 537.6 457.4 [M+H-80]+, 444.4 [M-92]+, 430.6, 413.4,
carotene 27.2 473 400.4, 177.1
9 (all-E)-lutein 3'-O- 27.7- 420, 446, nd 551.5 [M+H-228]+ → 533.5 [M-+H-228-18]+, 495.4
myristate 28.3 473 [M+H-228-56]+, 459.4 [M+H-228-92]+, 429.4, 411.4
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10 auroxanthin stearate 28.9- 380, 401, 867.9 849.8 [M+H-18]+, 775.8 [M+H-92]+, 757.8 [M+H-
isomer 1 29.2 425 18-92]+, 583.6 [M+H-284]+, 565.5 [M+H-18-284]+,
547.5 [M+H-18-18-284]+
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11a (all-E)-lutein 3-O- 29.2- 423, 449, nd 761.8 [M+H-18]+ → 533.5 [M+H-18-228]+, 477.4
myristate 29.8 473 [M+H-18-228-56]+, 411.4
11b (all-E)-β-carotene 537.6 457.5 [M+H-80]+, 444.5 [M-92]+, 413.4, 400.4,
177.0
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12 auroxanthin stearate 30.3- 379, 401, 867.8 849.9 [M+H-18]+, 775.8 [M+H-92]+, 757.8 [M+H-
isomer 2 31.0 426 18-92]+, 583.6 [M+H-284]+, 565.5 [M+H-18-284]+,
547.5 [M+H-18-18-284]+
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13 (all-E)-zeaxanthin 30.7- 426, 448, 779.8 761.8 [M+H-18]+, 687.8 [M+H-92]+, 551.5 [M+H-
myristate 31.4 472 228]+, 533.5 [M+H-18-228]+, 459.4 [M+H-228-92]+
14 (all-E)-lutein 3'-O- 31.5- 420, 445, nd 551.5 [M+H-256]+ → 533.5 [M+H-256-18]+, 495.4
palmitate 32.1 473 [M+H-256-56]+, 429.4
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15a (all-E)-violaxanthin 33.4- 420, 441, 994.0 976.0 [M+H-18]+, 901.9 [M+H-92]+, 884.0 [M+H-
laurate-myristate 34.1 470 18-92]+, 793.8 [M+H-200]+, 775.9 [M+H-18-200]+,
isomer 1 765.8 [M+H-228]+, 747.8 [M+H-18-228]+, 565.6
[M+H-200-228]+, 547.6 [M+H-18-200-228]+
15b (all-E)-lutein 3-O- nd 789.8 [M+H-18]+ → 733.8 [M+H-18-56]+, 697.7
palmitate [M+H-18-92]+, 533.5 [M+H-18-256]+, 477.4 [M+H-
18-256-56]+, 411.4
16a violaxanthin 34.6- 415, 437, 994.0 976.0 [M+H-18]+, 902.9 [M+H-92]+, 883.8 [M+H-
laurate-myristate 35.4 464 18-92]+, 793.8 [M+H-200]+, 775.8 [M+H-18-200]+,
isomer 2 765.8 [M+H-228]+, 747.7 [M+H-18-228]+, 565.5
[M+H-200-228]+, 547.5 [M+H-18-200-228]+
16b lutein dibutyrate nd 621.6 [M+H-88]+ → 533.5 [M+H-88-88]+
16c zeaxanthin 807.8 789.8 [M+H-18]+, 715.8 [M+H-92]+, 551.5 [M+H-
palmitate 256]+, 533.5 [M+H-18-256]+, 459.4 [M+H-256-92]+
17a violaxanthin 35.1- nd 1022.0 1004.0 [M+H-18]+ → 912.0 [M+H-18-92]+, 775.9
dimyristate isomer 1 35.9 [M+H-18-228]+, 547.6 [M+H-18-228-228]+
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Peaka Carotenoid Rt λmax [M+H]+ MS/MS fragment ions (m/z)

(min)b (nm)c (m/z)
17b zeinoxanthin laurate 735.7 643.6 [M+H-92]+, 535.6 [M+H-200]+, 479.4 [M+H-
200-56]+, 443.4 [M+H-200-92]+
18a (all-E)-violaxanthin 36.2- 418, 441, 1022.0 1004.0 [M+H-18]+, 930.0 [M+H-92]+, 912.0 [M+H-
dimyristate isomer 2 36.8 470 18-92]+, 793.9 [M+H-228]+, 775.8 [M+H-18-228]+,
565.6 [M+H-228-228]+, 547.5 [M+H-18-228-228]+
18b lutein dicaprylate nd 677.8 [M+H-144]+ → 533.5 [M+H-144-144]+
19a violaxanthin 37.3- 414, 438, 1022.0 1004.1 [M+H-18]+, 931.0 [M+H-92]+, 912.0 [M+H-
dimyristate isomer 3 38.1 465 18-92]+, 793.8 [M+H-228]+, 775.9 [M+H-18-228]+,
565.6 [M+H-228-228]+, 547.6 [M+H-18-228-228]+
β-cryptoxanthin 643.7 [M+H-92]+, 535.5 [M+H-200]+, 443.4 [M+H-

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19b 735.8
laurate 92-200]+

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20a (all-E)-zeinoxanthin 38.4- 426, 447, 763.8 671.7 [M+H-92]+, 535.5 [M+H-228]+, 443.5 [M+H-
myristate 39.2 473 228-92]+
20b lutein dicaprate nd 705.7 [M+H-172]+ → 533.5 [M+H-172-172]+

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21a (Z)-lutein dilaurate 39.2- 426, 447, nd 733.8 [M+H-200]+ → 533.5 [M+H-200-200]+
39.5 473
21b (all-E)-lutein 3’-O- nd 551.7 [M+H-284]+ → 533.6 [M+H-18-284]+
stearate

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21c (all-E)-lutein 3-O- nd 817.8 [M+H-18]+ → 533.5 [M+H-18-284]+, 477.4
stearate [M+H-18-284-56]+, 411.4
22 (all-E)-violaxanthin 39.7- 418, 441, 1050.0 1032.1 [M+H-18]+, 958.0 [M+H-92]+, 940.0 [M+H-
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myristate-palmitate 40.0 471 18-92]+, 821.9 [M+H-228]+, 803.8 [M+H-18-228]+,
793.8 [M+H-256]+, 775.8 [M+H-18-256]+, 565.5
[M+H-228-256]+, 547.5 [M+H-18-228-256]+
23a (all-E)-lutein 40.3- 425, 450, nd 733.7 [M+H-200]+ → 533.5 [M+H-200-200]+
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dilaurate 41.2 474

23b violaxanthin 1050.0 1032.1 [M+H-18]+, 959.0 [M+H-92]+, 941.0 [M+H-
myristate-palmitate 18-92]+, 821.9 [M+H-228]+, 803.9 [M+H-18-228]+,
ED

793.8 [M+H-256]+, 775.9 [M+H-18-256]+, 565.6

[M+H-228-256]+, 547.6 [M+H-18-228-256]+
23c (all-E)-β- 763.9 671.7 [M+H-92]+, 535.5 [M+H-228]+, 443.5 [M+H-
cryptoxanthin 228-92]+
PT

myristate
24 (all-E)-lutein 3-O- 40.5- 421, 446, nd 733.8 [M+H-172]+ → 677.6 [M+H-172-56]+, 641.7
laurate-3’-O-caprate 41.4 473 [M+H-172-92]+, 533.5 [M+H-172-200]+, 477.4
[M+H-172-200-56]+, 441.4 [M+H-172-200-92]+,
CE

411.4
705.7 [M+H-200]+ → 613.6 [M+H-200-92]+, 533.5
[M+H-200-172]+, 477.4 [M+H-200-172-56]+, 411.4
AC

25 (Z)-lutein 41.1- nd nd 761.8 [M+H-228]+ → 533.5 [M+H-228-228]+, 477.4

dimyristate 41.9 [M+H-228-228-56]+, 411.3
26 violaxanthin 42.0- nd 1078.1 1061.1 [M+H-18]+, 986.0 [M+H-92]+, 967.9 [M+H-
dipalmitate 42.8 92-18]+, 821.9 [M+H-256]+, 803.8 [M+H-18-256]+,
565.6 [M+H-256-256]+, 547.6 [M+H-18-256-256]+
27 (all-E)-lutein 3-O- 42.9- 423, 445, nd 761.8 [M+H-200]+ → 533.5 [M+H-200-228]+, 477.4
laurate-3’-O- 43.5 473 [M+H-200-228-56]+, 411.4
myristate 733.8 [M+H-228]+ → 533.5 [M+H-228-200]+, 477.5
[M+H-228-200-56]+, 411.4
28a (all-E)-lutein 3-O- 43.6- 423, 446, nd 761.8 [M+H-200]+ → 533.5 [M+H-200-228]+, 477.4
myristate-3’-O- 44.1 473 [M+H-200-228-56]+, 411.4
laurate 733.8 [M+H-228]+ → 533.5 [M+H-228-200]+, 477.5
[M+H-228-200-56]+, 411.4
28b β-cryptoxanthin 791.9 698.8 [M+H-92]+, 535.5 [M+H-256]+, 443.4 [M+H-
palmitate 256-92]+
 ACCEPTED MANUSCRIPT

Peaka Carotenoid Rt λmax [M+H]+ MS/MS fragment ions (m/z)

(min)b (nm)c (m/z)
29 (all-E)-lutein 44.3- 422, 446, nd 761.8 [M+H-228]+ → 533.5 [M+H-228-228]+, 477.4
dimyristate 44.7 474 [M+H-228-228-56]+, 411.3
30 violaxanthin 44.7- nd 1106.0 1088.1 [M+H-18]+
palmitate-stearate 45.0 821.8 [M+H-284]+ → 803.8[M+H-284-18]+, 729.8
[M+H-284-92]+, 565.5 [M+H-284-256]+, 547.5
[M+H-284-256-18]+
31a (all-E)-lutein 3-O- 45.3- 420, 446, nd 761.8 [M+H-256]+, 789.8 [M+H-228]+
myristate-3’-O- 45.7 474
palmitate
761.8 [M+H-256]+ → 669.7 [M+H-256-92]+, 533.5

T
31b (all-E)-lutein 3-O-
palmitate-3’-O- [M+H-256-228]+, 477.4 [M+H-256-228-56]+, 441.4

IP
myristate [M+H-256-228-92]+, 411.4
789.8 [M+H-228]+ → 733.7 [M+H-228-56]+, 697.7
[M+H-228-92]+, 533.5 [M+H-228-256]+, 477.4

CR
[M+H-228-256-56]+, 441.4 [M+H-228-256-92]+,
411.4
32 β-cryptoxanthin 45.7- nd 819.8 727.7 [M+H-92]+, 535.5 [M+H-284]+, 443.4 [M+H-
stearate 46.0 284-92]+

US
33a zeaxanthin 46.1- 423, 446, 1018.1 926.0 [M+H-92]+, 789.8 [M+H-228]+, 761.8 [M+H-
myristate-palmitate 46.4 474 256]+, 697.8 [M+H-228-92]+, 669.8 [M+H-256-92]+,
533.6 [M+H-228-256]+, 441.5 [M+H-228-256-92]+
AN
33b lutein dipalmitate nd 789.8 [M+H-256]+, 533.6 [M+H-256-256]+
34 (all-E)-lutein 3-O- 46.3- 420, 446, nd 817.8 [M+H-228]+, 761.8 [M+H-284]+, 533.6
myristate-3’-O- 46.5 474 [M+H-228-284]+
stearate
M

35 (all-E)-lutein 3-O- 46.4- 421, 446, nd 817.8 [M+H-228]+ → 533.5 [M+H-228-284]+, 477.5
stearate-3’-O- 46.7 473 [M+H-228-284-56]+, 441.4 [M+H-228-284-92]+,
myristate 411.4
ED

761.8 [M+H-284]+
36 (all-E)-zeaxanthin 46.6- 423, 450, 1046.1 954.0 [M+H-92]+, 789.9 [M+H-256]+, 697.8 [M+H-
dipalmitate 46.9 477 256-92]+, 533.5 [M+H-256-256]+
37 (all-E)-lutein 3-O- 46.8- 421, 446, nd 817.8 [M+H-256]+ → 533.5 [M+H-256-284]+, 477.5
PT

palmitate-3’-O- 47.1 474 [M+H-256-284-56]+, 441.5 [M+H-256-284-92]+,

stearate 411.4
789.8 [M+H-284]+ → 533.5 [M+H-284-256]+, 411.3
817.9 [M+H-256]+, 789.8 [M+H-284]+, 533.6
CE

38 (all-E)-lutein 3-O- 46.9- 421, 446, nd

stearate-3’-O- 47.2 474 [M+H-284-256]+
palmitate
39 (all-E)-zeaxanthin 47.3- 423, 451, 1074.1 1057.3 [M+H-18]+, 982.0 [M+H-92]+, 817.9 [M+H-
AC

palmitate-stearate 47.6 478 256]+, 789.8 [M+H-284]+, 697.7 [M+H-284-92]+,

533.5 [M+H-284-256]+, 441.5 [M+H-284-256-92]+
40 (all-E)-lutein 47.5- 422, 446, nd 817.9 [M+H-284]+ → 533.5 [M+H-284-284]+, 477.4
distearate 47.7 474 [M+H-284-284-56]+, 441.4 [M+H-284-284-92]+,
411.4
a Numbered according to the chromatogram shown in Figure 1. b Retention time on C30 column. c Gradient of methanol, methyl
tert-butyl ether and water. Ion fragments in bold indicate in-source fragmentation. Underlined ion fragments indicate the peak
with the highest intensity in MS spectra between the 2 peaks representing the loss of one fatty acid molecule. nc: not calculated;
nd: not detected. * Loss of fatty acids: 88u = butyric acid (4:0), 144 u = caprylic acid (8:0), 172 u = capric acid (10:0), 200 u =
lauric acid (12:0), 228 u = myristic acid (14:0), 256 u = palmitic acid (16:0), 284 u = stearic acid (18:0).
 ACCEPTED MANUSCRIPT

Table 3. Mass spectrometry and UV-Vis characteristics of carotenoids from saponified

extract of Marigold petals
Peaka Carotenoid Rt λmax (nm)c % %AB/AIIe [M+H]+ MS/MS fragment
(min)b III/IId (m/z) ions (m/z)
1 (all-E)- 7.8 415, 438, 95 0 600.6 583.5 [M+H-18]+,

T
violaxanthin 468 565.5 [M+H-18-18]+,

IP
547.5 [M+H-18-18-
18]+ , 491.5 [M+H-18-
92]+, 221.2

CR
2a (all-E)- 10.9 327, 415, 56 6 584.9 566.5 [M+H-18]+,
antheraxanthin 440, 466 548.4 [M+H-18-18]+
2b 601.9 583.6 [M+H-18]+,
(9Z)- 565.7 [M+H-18-18]+,

US
violaxanthin 509.1 [M+H-92]+,
491.6 [M+H-18-92]+,
393.2
3a (all-E)- 11.5 330, 416, 36 25 601.5 583.4 [M+H-18]+,
AN
auroxanthin 438, 465 565.4 [M+H-18-18]+,
3b 568.6 221.4
(13’Z)-lutein 551 [M+H-18]+, 533.5
[M+H-18-18]+,
M

495.5[M+H-18-56]+,
459.2[M+H-18-92]+,
429.2
ED

4 (all-E)-lutein 13.1 421, 444, 67 0 568.7 551.6 [M+H-18]+,

472 533.6 [M+H-18-18]+,
477.3 [M+H-92]+,
PT

430.3
5 (all-E)- 15.5 421, 450, 38 0 569.6 551.5 [M+H-18]+,
zeaxanthin 477 533.6 [M+H-18-18]+
6 (9Z)-lutein 15.8 329, 416, 67 nc 568.8 551.6 [M+H-18]+,
CE

441, 467 533.4 [M+H-18-18]+,

495.0 [M+H-18-56]+,
477.3 [M+H-92]+,
429.5
AC

7 phytoene 18.2 285 0 0 545.7 489.7, 435.5, 395.4,

339.4 [M+H-205]+
8 (all-E)- 19.8 422, 445, 58 0 553.5 535.7 [M+H-18]+,
zeinoxanthin 473 496.8, 443.4,
9 (Z)-phytofluene 20.4 331, 348, 72 nc 543.6 461.5, 405.7 [M+H-
367 137]+, 337.5 [M+H-
205]+
10a (all-E)-β- 23.4 427, 451, 38 0 553.6 535.6 [M+H-18]+,
cryptoxanthin 479 460.5

10b (all-E)- 331, 348, 95 0 543.7 461.5, 405.6 [M+H-

phytofluene 368 137]+, 337.4 [M+H-
205]+
11 (all-E)-β- 33.0 430, 451, 29 0 537.6 481.6
carotene 479
 ACCEPTED MANUSCRIPT

Peaka Carotenoid Rt λmax (nm)c % %AB/AIIe [M+H]+ MS/MS fragment

(min)b III/IId (m/z) ions (m/z)
12 -carotene 1’,2’- 33.9 431, 455, 31 0 553.5 535.6 [M+H-18]+,
epoxide 480 497.4 [M+H-56]+,
461.6 [M+H-92]+,
429.4 [M+H-123]+
13 -carotene 1’,2’- 40.7 438, 461, 64 0 553.6 535.5 [M+H-
epoxide 1 492 18]+,461.7 [M+H-92]+
14a -carotene 1’,2’- 41.6 438, 461, 64 0 553.6 535.5 [M+H-
epoxide 2 492 18]+,447.7 [M+H-
14b -carotene 536.7 106]+

T
nd
a b c
Numbered according to the chromatogram shown in Figure 5. Retention time on C30 column. Gradient of methanol and

IP
d
MTBE. Spectral fine structure: Ratio of the height of the longest wavelength absorption peak (III) and that of the middle
absorption peak (II). e Ratio of the cis peak (AB) and the middle absorption peak (II). Ion fragments in bold indicate in-source

CR
fragmentation. nc: not calculated. nd: not detected.

Lutein esters possible to be generated by acylation of the (all-E)-lutein with some even-chain saturated fatty acids:

US
capric (10:0), lauric (12:0), myristic (14:0), palmitic (16:0) and stearic acid (18:0). Assuming no enzyme specificity,
the number of different lutein esters possible to be found is (n+1) 2-1, where n = the number of FAs. The (all-E)-
lutein molecule presents two possible sites of acylation, in the 3-O-position of the -ring and 3’-O-position in the -
ring. Lutein chromophore (10 conjugated double bonds), highlighted in grey, is responsible for the pattern of
AN
absorption of light in the electromagnetic spectrum; esterification does not change the carotenoid chromophore, thus,
all the lutein esters present the same UV-vis spectrum of the free-lutein. The UV-vis spectrum of free (all-E)-lutein
in mixture of MeOH:MTBE:H2O is represented (mobile phase for carotenoid ester separation, see chromatographic
conditions in the text).
M

Highlights
ED

 Comprehensive identification of free and acylated carotenoid from marigold by LC-

MS/MS.
PT

 Zea-, viola-, auro-, zeino- and β-cryptoxanthin esters identified for the first time.

CE

18 free carotenoids were identified in the saponified extract from marigold.

 56 carotenoids were identified in the non-saponified extract.
AC
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6