molecules

Article
Simultaneous LC/MS Analysis of Carotenoids and
Fat-Soluble Vitamins in Costa Rican Avocados (Persea
americana Mill.)
Carolina Cortés-Herrera 1, *, Andrea Chacón 1 , Graciela Artavia 1 and
Fabio Granados-Chinchilla 2
1 Centro Nacional de Ciencia y Tecnología de Alimentos, Universidad de Costa Rica, Ciudad Universitaria
Rodrigo Facio, 11501-2060 San José, Costa Rica; [email protected] (A.C.);
[email protected] (G.A.)
2 Centro de Investigación en Nutrición Animal (CINA), Universidad de Costa Rica, Ciudad Universitaria
Rodrigo Facio, 11501-2060 San José, Costa Rica; [email protected]
* Correspondence: [email protected]; Tel.: +506-2511-7226

Academic Editor: Severina Pacifico




Received: 26 August 2019; Accepted: 5 October 2019; Published: 10 December 2019

Abstract: Avocado (a fruit that represents a billion-dollar industry) has become a relevant crop
in global trade. The benefits of eating avocados have also been thoroughly described as they
contain important nutrients needed to ensure biological functions. For example, avocados contain
considerable amounts of vitamins and other phytonutrients, such as carotenoids (e.g., β-carotene),
which are fat-soluble. Hence, there is a need to assess accurately these types of compounds. Herein we
describe a method that chromatographically separates commercial standard solutions containing both
fat-soluble vitamins (vitamin A acetate and palmitate, Vitamin D2 and D3 , vitamin K1 , α-, δ-, and
γ-vitamin E isomers) and carotenoids (β-cryptoxanthin, zeaxanthin, lutein, β-carotene, and lycopene)
effectively (i.e., analytical recoveries ranging from 80.43% to 117.02%, for vitamins, and from 43.80%
to 108.63%). We optimized saponification conditions and settled at 80 ◦ C using 1 mmol KOH L−1
ethanol during 1 h. We used a non-aqueous gradient that included methanol and methyl tert-butyl
ether (starting at an 80:20 ratio) and a C30 chromatographic column to achieve analyte separation
(in less than 40 min) and applied this method to avocado, a fruit that characteristically contains
both types of compounds. We obtained a method with good linearity at the mid to low range of
the mg L−1 (determination coefficients 0.9006–0.9964). To determine both types of compounds in
avocado, we developed and validated for the simultaneous analysis of carotenoids and fat-soluble
vitamins based on liquid chromatography and single quadrupole mass detection (LC/MS). From actual
avocado samples, we found relevant concentrations for cholecalciferol (ranging from 103.5 to 119.5),
δ-tocopherol (ranging from 6.16 to 42.48), and lutein (ranging from 6.41 to 15.13 mg/100 g dry
weight basis). Simmonds cultivar demonstrated the higher values for all analytes (ranging from 0.03
(zeaxanthin) to 119.5 (cholecalciferol) mg/100 g dry weight basis).

Keywords: avocado; LC/MS; fat-soluble vitamins; carotenoids

1. Introduction
Avocado represents a billion-dollar industry, the projection of the apparent per capita consumption
of avocado sets the top six world importers of avocado to be US, Netherlands, France, United Kingdom,
Spain, and Canada with 3.64, 1.62, 2.10, 2.21, 2.30, and 2.55 kg in a given year, respectively [1].
Avocado exports have made some countries like Mexico, Dominican Republic, Peru, Chile, Colombia,
and Costa Rica increase their cultivated area [1].

Molecules 2019, 24, 4517; doi:10.3390/molecules24244517 www.mdpi.com/journal/molecules

Molecules 2019, 24, 4517 2 of 17

The topmost exporter countries have directed their offer to destinations such as the US, Europe,
and Asia, especially China [2], where import growth is in the order of 250%, from 154 tons in 2012, to
25,000 tons in 2016 [3,4]. For example, Costa Rican avocado harvested area was estimated at 1 888 ha
in 2014 and increased to 3092 ha in 2017, which represents 845 tons of Costa Rican avocado (or 448900
USD) [5].
As a complex matrix, performing chemical analysis on the avocado flesh presents an additional
difficulty. The ripe avocado fruit has a firm, oily, and yellow to light green colored mesocarp that
contains both fat-soluble vitamins and carotenoids associated with other lipids [6]. Usually, both types
of analysis have to be performed separately using different chromatographic conditions altogether,
which represents an additional expense, both economic and in terms of labor. Analyzing both types of
fat-soluble compounds is relevant, especially in fruits such as avocado in which there is evidence that
carotenoid absorption might be improved by the addition of avocado and avocado oil [7–9].
Interestingly, the Association of Official Analytical Chemists (AOAC) Official Methods of Analysis
(OMASM ) does not have any method established for neither fat-soluble vitamins nor carotenoids in
fruits. Several papers have already annotated the relevance of including avocado in the diet as it
has been related to health benefits [10–19]. Previously, another report analyzed carotenoid content in
avocado using spectrophotometry [20]. A later report has used a similar approach to measure total
carotenoid content [21]. On another hand, a research group has analyzed avocado pigments in oil [22]
and tissue [22,23] using HPLC coupled with a photodiode array detector using a triphasic organic
solvent gradient. Carotenoids in avocado seed have also been described [24,25].
The application of chromatography and mass spectrometry to carotenoid analysis in fruits is not
new [23,26,27]. However, few papers have been dedicated solely to the study of both fat-soluble and
carotenoid content in avocado fruits. For example, research assayed both types of compounds (i.e.,
carotenoids and tocopherol) using two independent chromatographic techniques [28].
Among the papers dedicated to assessing specifically carotenoids or vitamins in avocado, the most
relevant include advantages such as that most researchers use acetone (a versatile and low boiling point
solvent) during primary extraction [22,23,28,29] and ethyl ether or hexane after saponification [22,23,29].
For saponification at room temperature, the use of 2,6-di-tert-butyl-4-methylphenol, and nitrogen
flushing seems to be a norm [28,29], thus protecting the target analytes. Diversity of carotenoids studies
is ample (including epoxides [28], isomers of carotene [20], chlorophylls [22], phytoene [23]). Mobile
phases are usually simple and environmentally friendly [22,23,29], which include methanol, water,
methyl tert-butyl ether, and ethyl acetate. Yano and coworkers were able to apply their method to 75 and
15 different fresh and processed fruits, respectively [23]. Solid-phase extraction has been used to reduce
interferences [22]. Mass spectrometry has been used to recognize unidentified compounds [20,28].
Disadvantages of these methods include the presence of water in the mobile phase (which increases
mobile phase polarity), several approaches exhibit some issues with chromatographic resolution for
some of the signals [28,29] and in some cases saponification time [29], base concentration [29],
and chromatographic run [23] are excessive or the identification of compounds is based on light
absorption [20,22,29]. Finally, vitamin analysis of the fruit is scarce at best [11,28].
Lastly, very little information has been gathered regarding varieties of Costa Rican avocados,
proximate analysis, mineral content, and some vitamins have been explored [30]. Some papers have
also focused on standing out differences between avocado varieties [21,28], including varieties of
Guatemalan race (e.g., Hass [11] and Nabal [20]), as avocadoes originated from New Zealand [22],
California [28], and Mexico [29] and those commercially available from Israel [20] and Japan [23].
Herein, we report a liquid chromatography and single quadrupole mass detection (LC/MS) based
method using a C30 column and methanol and methyl tert-butyl ether to assay and quantitatively
separate both fat-soluble vitamins (vitamin A acetate and palmitate, Vitamin D2 and D3 , vitamin K1 ,
α-, δ-, and γ-vitamin E isomers) and carotenoids (β-cryptoxanthin, zeaxanthin, lutein, β-carotene, and
lycopene) simultaneously in avocado fruit.
Molecules 2019, 24, 4517 3 of 17

2. Results and Discussion

2.1. Stationary Phase Selection and Green Chemistry

Where other alkyl modified stationary phases failed (Table 1), the C30 allowed an excellent
separation of fat-soluble compounds even between structurally related molecules using a
MeOH/tert-butyl methyl ether (MTBE)-based mobile phase (Table 2). The solvent selection not
only ensured good compound solubility and chromatographic separation, but it also helped improve
column life span as no water was involved and the column could be safely stored under 80% MeOH.

Table 1. Performance of other stationary phases and conditions tested to try to separate calciferol and
tocopherol isomers.

Stationary Phase C8 C18 C30

90:7:3
Solvent System 95:5 MeOH:H2 O 95:5 MeOH:H2 O
MeOH:CH3 CN:2-propanol
Flow, mL min−1 0.75 1.00 0.50
Temperature, ◦C 50 50 35
Compound tR , min Rs tR , min Rs tR , min Rs
Retinyl acetate 3.50 4.75 5.42
Ergocalciferol 4.74 0 8.41 8.96 0.78
Cholecalciferol 4.74 0 8.81 1.12 9.27 0.78
δ-tocopherol 4.67 1.47 8.05 1.20 7.42 2.96
γ-tocopherol 5.17 1.47 9.52 2.07 8.28 1.85
α-tocopherol 7.05 15.14 12.19
Phylloquinone 7.84 19.87 13.17
Retinyl palmitate 13.09 48.33 35.22

Table 2. Optimized Mass Spectrometry (MS) parameters for the assayed compounds, in order of m/z.

Detector Set
Compound tR , min Selected SIM Ion, m/z Fragmentor, V Dwell Time, ms
Time, min
269.3 [C20 H29 ]•+ /325.2
Retinyl acetate 2.99 100
[C20 H29 OH + K]+
From 0 to 5 95
Ergocalciferol 3.59 398.3 [M + H]+ 220
Cholecalciferol 4.02 385.3 [M + H]+ 160
δ-tocopherol 5.26 402.5 [M+] 220
From 5 to 8 γ-tocopherol 5.78 416.4 [M+] 140 71
α-tocopherol 6.61 430.4 [M+] 80
Phylloquinone 8.07 451.4 [M + H]+ 140
Astaxanthin 9.07 597.4 [M + H]+ 160
From 8 to 13 56
Lutein 9.97 569.4 [M + H]+ 140
Zeaxanthin 11.49 568.4 [M+] 140
Retinyl 269.3 [C20 H29 ]•+ /563.4
11.19 100
palmitate [M + K]+
After 13 β-cryptoxanthin 16.58 552.6 [M+] 120 95
β-carotene 22.51 536.4 [M+] 120
Lycopene 37.39 536.1 [M+] 160

Some methods have selected chlorinated solvents as an effective way to extract [28] or separate
carotenoids [31]. However, we chose MTBE as a greener alternative to chlorinated solvents [32].
Additionally, our chromatographic separation was mostly based on MeOH. The high degree of
shape recognition of the C30 was validated early [33] and was, once again, here, demonstrated. It is
recommended for analysis of retinoid as well as carotenoid molecules [34]. However, herein, we
exploited the versatility of the C30 column further. Furthermore, as avocado lacks the presence of
Molecules 2019, 24, 4517 4 of 17

lycopene [20,28], this compound when introduced into the separation (especially in its deuterated form),
can be used as an internal standard (IS). Though lycopene is considered a relative liable carotenoid,
it has been used successfully as an IS [35]. Other more stable molecules have been used as well and
could be considered (e.g., canthaxanthin [35], sudan I [36]; 80 -apo-80 -β-carotenal [37], echinenone [38]).

2.2. Singular Ion Monitoring Parameter Selection

As expected, using reverse phase chromatography, fat-soluble vitamins eluted during the first
8 min (except for retinyl palmitate, an esterified compound with an extra C15 alkyl chain) (Table 2).
Both retinoids assessed were prone to retain monovalent cations such as K+ ([M + K]+ ). Retinyl acetate
and retinyl palmitate share a similar fragmentation pattern, which includes ion 269 m/z as a base
peak (Table 2). The retinoid alkyl chain did not seem to affect ionization voltage, indicating that the
C20 H29 OH base was governing their behavior. Ion 296 m/z found for retinoids corresponded to the
protonation and elimination of water and acetate during positive ion electrospray [34].
All tocopherol isomers, as well as zeaxanthin, β-cryptoxanthin, β-carotene, and lycopene (the
heavier analogs), exhibited deprotonated species as molecular ions ([M+]). Electrospray ionization
(ESI+ ) ions are usually preformed in solution by acid/base reactions (i.e., [M + nH]n+ ), some ions
are probably formed by a field desorption mechanism at the surface. Hence, the production of
abundant cations, with little fragmentation [34]. Meanwhile, phylloquinone, the acidic carotenoid
astaxanthin [34], and lutein all ionized through protonated species ([M + H]+ ) (Table 2). Interestingly,
the highly related compounds α-, γ-, and β-tocopherols exhibited very different ionization energies
(i.e., 80, 140, and 220 V, respectively) (Table 2).

2.3. Method Performance Data

For the vitamin group, the higher sensitivity was exhibited by α-, γ-, and retinyl palmitate
(Table 2). On the other hand, carotenoids with a lower limit of detection were astaxanthin, lutein, and
zeaxanthin (Table 3). Electrospray analysis of carotenoids, since some years ago, has demonstrated
high sensitivity (i.e., in the pmol range) [39]. These compounds, as expressed in the matrix of
interest, could be detected as low as 1 µg/100 g dry matter (Table 3). The resolution obtained between
the compounds analyzed ranged from 2.18–71.90; the lowest value recommended is 2 (Table 2,
Figure 1A) [40]. Several compounds were very close to the theoretical value of 1 (i.e., a perfect peak
with a Gaussian distribution and completely symmetrical) though ergocalciferol (1.707), and lutein
(1.348) exhibited some tailing. Fronting (leading peak) can also be observed for retinyl acetate (0.709)
(Table 3). Column efficiency was very high for compounds eluted above 8 min, such as phylloquinone,
zeaxanthin, retinyl palmitate, β-cryptoxanthin, and β-carotene (Table 3). A good baseline definition
was observed for all compounds analyzed (i.e., αs ranging from 1.07 to 1.76) (Table 3).

Table 3. Method performance parameters obtained during validation.

Sensitivity
LoD, µg/100 g LoQ, LoD, µg/100 g LoQ, µg/100 g
Compound LoD, µg L−1 LoQ, µg L−1
fat µg/100 g fat dry matter dry matter
Retinyl
3.00 × 102 9.20 × 102 1.00 × 102 3.07 × 102 1.50 × 101 4.60 × 101
acetate
Ergocalciferol 1.00 × 102 2.90 × 102 3.30 × 101 9.70 × 101 0.50 × 101 1.50 × 101
Cholecalciferol 2.70 × 102 8.20 × 102 9.00 × 101 2.73 × 102 1.40 × 101 4.10 × 101
δ-tocopherol 1.70 × 102 5.10 × 102 5.70 × 101 1.70 × 102 0.90 × 101 2.60 × 101
γ-tocopherol 1.30 × 101 3.80 × 101 0.40 × 101 1.30 × 101 0.10 × 101 0.20 × 101
α-tocopherol 0.70 × 101 2.40 × 101 0.20 × 101 0.80 × 101 0.10 × 101 0.10 × 101
Phylloquinone 4.30 × 102 1.29 × 103 1.43 × 102 4.30 × 102 2.20 × 101 6.50 × 101
Astaxanthin 2.20 × 101 1.25 × 102 0.70 × 101 4.20 × 101 0.10 × 101 0.60 × 101
Lutein 1.00 × 101 2.90 × 101 0.30 × 101 1.00 × 101 0.10 × 101 0.10 × 101
Zeaxanthin 0.90 × 101 2.80 × 101 0.30 × 101 0.90 × 101 0.10 × 101 0.10 × 101
Molecules 2019, 24, 4517 5 of 17

Table 3. Cont.

Retinyl
2.40 × 101 7.40 × 102 0.80 × 101 2.47 × 102 0.10 × 101 3.70 × 101
palmitate
β-cryptoxanthin 3.30 × 102 9.90 × 102 1.10 × 102 3.30 × 102 1.70 × 101 5.00 × 101
β-carotene 8.80 × 102 2.67 × 103 2.93 × 102 8.90 × 102 4.40 × 101 1.34 × 102
Lycopene 1.56 × 102 4.71 × 102 5.20 × 101 1.57 × 102 0.80 × 101 2.40 × 101
Molecules 2019, 24, x FOR PEER REVIEW Sensitivity 5 of 16

−12 −1
LoD, µg/100 g LoQ, LoD, µg/100 g LoQ, µg/100 g
Compound
β-cryptoxanthin LoD, µg×L10
3.30 LoQ, × 10L2
9.90 µg 1.10 ×fat
102 3.30 × 102g fat 1.70
µg/100 dry× matter
101 5.00
dry×matter
101
β-carotene 8.80 × 102 2.67 × 103 2.93 × 102 8.90 × 102 4.40 × 101 1.34 × 102
Chromatographic 1Parameters
Lycopene 1.56 × 102 4.71 × 102 5.20 × 10 1.57 × 102 0.80 × 101 2.40 × 101
−1 Area, Height, Parameters
Chromatographic Peak N,
Compound tR , min [[], mg L SymmetryRs k α
×105 ×104 Peak Width ×104
[], mg Area, Height Symmetr
Compound
Retinyl tR, min Widt Rs k α N, ×104
3.00 L−1
5.00 ×10 5
8.98 , ×10 4
12.96 y
acetate h 0.12 0.71 3.67 0.35 1.61 1.08
Retinyl acetate
Ergocalciferol 3.483.00 5.00
0.50 8.98
5.34 12.96
5.12 0.12 0.15 0.71
1.71 3.67
3.54 0.35
0.57 1.61
1.39 1.08 0.92
Cholecalciferol
Ergocalciferol 3.973.48 1.00
0.50 1.76
5.34 1.78 0.15
5.12 0.13 0.91
1.71 7.96
3.54 0.79
0.57 1.71
1.39 0.92 1.45
δ-tocopherol
Cholecalciferol 5.213.97 1.00
1.00 0.67
1.76 0.63 0.13
1.78 0.18 0.95
0.91 2.85
7.96 1.35
0.79 1.16
1.71 1.45 1.34
γ-tocopherol
δ-tocopherol 5.715.21 1.00
1.00 9.07
0.67 7.65 0.18
0.63 0.17 0.77
0.95 3.52
2.85 1.57
1.35 1.22
1.16 1.34 1.87
α-tocopherol
γ-tocopherol 6.495.71 1.00
1.00 21.99
9.07 13.27 0.17
7.65 0.28 0.99
0.77 7.20
3.52 1.92
1.57 1.33
1.22 1.87 0.88
Phylloquinone
α-tocopherol 7.896.49 1.00
1.00 0.29
21.99 0.32 0.28
13.27 0.11 0.80
0.99 4.75
7.20 2.56
1.92 1.18
1.33 0.88 7.77
Astaxanthin
Phylloquinone 8.947.89 0.05
1.00 0.78
0.29 0.40 0.11
0.32 0.33 0.88
0.80 2.18
4.75 3.03
2.56 1.11
1.18 7.77 1.19
Lutein
Astaxanthin 9.688.94 0.05
0.05 4.19
0.78 1.95 0.33
0.40 0.36 1.35
0.88 4.59
2.18 3.37
3.03 1.19
1.11 1.19 1.17
Zeaxanthin
Lutein 11.099.68 1.00
0.05 0.66
4.19 0.44 0.36
1.95 0.25 1.06
1.35 2.87
4.59 3.40
3.37 1.07
1.19 1.17 3.09
Retinyl
Zeaxanthin 11.09 1.00 0.66 0.44
11.75 0.20 5.68 4.52 0.25 0.21 1.06
1.08 2.87
26.91 3.40
4.30 1.07
1.49 3.09 5.03
palmitate
Retinyl
β-cryptoxanthin 11.75
16.40 0.20
0.20 5.68
0.03 4.52
0.04 0.21
0.14 1.08
0.80 26.91
27.10 4.30
6.40 1.491.40 5.0323.14
palmitate
β-carotene 22.14 1.00 0.38 0.22 0.29 0.76 71.90 8.98 1.76 9.54
β-cryptoxanthin 16.40 0.20 0.03 0.04 0.14 0.80 27.10 6.40 1.40 23.14
Lycopene 37.39 1.00 0.28 0.34 0.14 1.04 - - - 118.31
β-carotene 22.14 1.00 0.38 0.22 0.29 0.76 71.90 8.98 1.76 9.54
Lycopene 37.39 1.00 0.28 0.34 0.14 1.04 - - - 118.31

Figure1.1.Selected
Figure Selectedionionmonitoring
monitoring(SIM)
(SIM)chromatogram
chromatogramfor for(A).
(A).(A)
(A)AAstandard
standardmixture
mixtureofof1414analytes
analytesat
at 1 μg−1 mL −1 for liposoluble vitamins and 0.05 −1 μg mL −1 for carotenoids. 1. Retinyl acetate. 2.
1 µg mL for liposoluble vitamins and 0.05 µg mL for carotenoids. 1. Retinyl acetate. 2. Ergocalciferol.
3.Ergocalciferol. 3. Cholecalciferol.
Cholecalciferol. 4. δ-Tocopherol.4.5.δ-Tocopherol.
γ-Tocopherol.5. 6.
γ-Tocopherol.
α-Tocopherol. 6. α-Tocopherol. 7. Phylloquinone.
7. Phylloquinone. 8. Astaxanthin.
9.8.Lutein.
Astaxanthin. 9. Lutein. 10.
10. Zeaxanthin. 11. Zeaxanthin. 11. Retinyl
Retinyl palmitate. palmitate. 12. β-Cryptoxanthin.
12. β-Cryptoxanthin. 13. β-Carotene.13.14.
β-Carotene.
Lycopene.
14.The
(B) Lycopene.
saponified(B) avocado
The saponified avocado sample
sample analyzed using theanalyzed
proposed using the proposed method
chromatographic chromatographic
(blue line),
method
and (bluespiked
a sample line), and
witha sample
5 µmol spiked
for eachwith 5 μmol
vitamin and for1 each
µmolvitamin
for eachand 1 μmol for
carotenoid each
(red carotenoid
line).
(red line).
The method was successfully applied to avocado fruits (Figure 2B). The lack of available certified
The materials
reference method was forsuccessfully applied
matrices such to avocado
as fruits obliges fruits
the use(Figure 2B).solutions
of spike The lack of
to available certified
demonstrate both
reference
matrix materials
effects (if any)for matrices
and suchefficiency
extraction as fruits obliges the use
of vitamins andofcarotenoids
spike solutions to demonstrate
in avocado both
specifically.
matrix effects (if any) and extraction efficiency of vitamins and carotenoids in avocado specifically.
 method (blue line), and a sample spiked with 5 μmol for each vitamin and 1 μmol for each carotenoid
(red line).

The method was successfully applied to avocado fruits (Figure 2B). The lack of available certified
reference
Molecules materials
2019, for matrices such as fruits obliges the use of spike solutions to demonstrate6 both
24, 4517 of 17
matrix effects (if any) and extraction efficiency of vitamins and carotenoids in avocado specifically.

Figure 2. Average calibration curves and error bars depicting variability obtained for two of the target
analytes (A) δ-tocopherol and (B) zeaxanthin. Mean and standard deviations (used as error) calculated
from n = 3 independently constructed calibration curves injected on different days.

2.4. Performance during Saponification

Saponification-wise, using the same starting mass and sample, we proceeded to optimize the
conditions needed (i.e., base concentration and time) to improve analyte recovery. The differential
analysis showed that, overall, samples treated at 80 ◦ C for one hour and using 1 mol KOH L−1
exhibited the best results in the case of the analytes of interest, for avocado (Table 4). Variables tested
showed a profound and significant effect over analyte recovery (p < 0.05 for all cases) (Table 4).
Reaction parameters must be optimized to ensure proper hydrolysis within the complex, considerably
oily (ranging from 35.3 to 39.1 g fat/100 g dry weight basis), food matrix that is found in the ripe
avocado fruit [41]. Carotenoids are increasingly sensitive to heat. Hence, hot saponification was carried
out using an organic solvent with a relatively low boiling point [6] (i.e., 78.37 ◦ C at Standard Pressure
and Temperature [STP] for ethanol) and pyrogallol was used as a radical sink to protect the compounds
of interest. However, the improvement in recovery was achieved by increasing the temperature to
80 ◦ C. A procedure that might be justified as (i) the thermal effect must be sufficient to break the cell
walls from the avocado fruit and provide a rapid molecular diffusion to promote reaction; (ii) higher
temperatures increase solubility of lipophilic compounds and enhance kinetics of saponification (i.e.,
favors localized “hot spots” which deliver sufficient energy for the molecules to react) [42]. We chose
hot saponification to diminish reaction time; a similar approach has been reported elsewhere [43].

Table 4. Optimization of saponification conditions.

Temperature, ◦ C 60 80 95
Base Concentration, mol KOH L−1 1 2 1 2 1 2
Compound tR , min mg/100 g a
Ergocalciferol 3.59 18.89 (−0.14) 10.98 (−0.50) 22.06 8.38 (−0.62) 1.74 (−0.92) ND (−1.00)
δ-tocopherol 5.26 9.65 (−0.89) 88.22 (−0.02) 90.06 67.32 (−0.25) 0.24 (−1.00) 88.17 (−0.02)
γ-tocopherol 5.78 0.45 (−0.93) 0.50 (−0.92) 6.14 2.48 (−0.60) 0.11 (−0.98) 9.62 (0.57)
180.96 102.35
α-tocopherol 6.61 267.77 90.77 (−0.66) 74.00 (−0.72) 70.63 (−0.74)
(−0.32) (−0.62)
Astaxanthin 9.07 18.69 (−0.26) 16.82 (−0.33) 25.14 17.12 (−0.32) 7.64 (−0.70) 14.16 (−0.44)
Lutein 9.97 0.08 (0.00) 0.07 (−0.13) 0.08 0.12 (0.50) 0.02 (−0.75) 0.12 (0.50)
Zeaxanthin 11.49 26.60 (−0.13) 37.70 (0.23) 30.73 47.44 (0.54) 7.85 (−0.74) 20.38 (−0.34)
β-cryptoxanthin 16.58 24.66 (−0.22) 12.13 (−0.61) 31.50 27.02 (−0.14) 5.54 (−0.82) 16.93 (−0.46)
β-carotene 22.51 18.89 (−0.14) 10.98 (−0.50) 22.06 8.38 (−0.62) 1.74 (−0.92) ND (−1.00)
p values 0.044 0.040 - 0.037 0.011 0.028
a Brackets indicate bias, expressed as a fraction, with respect to the saponification treatment of 1 h at 80 ◦ C using a
1 mol L−1 base concentration. ND: not detected.
Molecules 2019, 24, 4517 7 of 17

2.5. Quantification, Linearity, and Calibration Curve Construction

Five-point calibration curves were constructed to quantitate the analytes. Slopes (mx ) ranging
from 4.05 × 103 (cholecalciferol) to 5.51 × 104 (ergocalciferol) and 5.45 × 104 (β-cryptoxanthin) to
6.49 × 106 (zeaxanthin) for vitamins and carotenoids, respectively, were obtained. Determination
coefficients spoke toward excellent linearity ranging from 0.9906 to 0.9964 (Figure 2A,B). For vitamins,
standard calibration curves were constructed as follows: from 1.00 to 10.00 mg L−1 for retinyl acetate,
ergocalciferol, γ-, and α-tocopherol and retinyl palmitate; from 2.50 to 30.00 mg L−1 for cholecalciferol,
δ-tocopherol, and phylloquinone. Finally, considering lower concentrations found in the target matrix
and carotenoid sensitivity, standard calibration curves were constructed as follows: 0.05 to 0.80 mg
L−1 for astaxanthin, β-cryptoxanthin, and β-carotene and from 0.1 to 2 mg L−1 for zeaxanthin and
lutein (Figure 2A,B). Concentrated stock solutions were prepared by dissolving the solid standard in
2-propanol for vitamins and chloroform for carotenoids; dilutions performed after that were matched
with the mobile phase (i.e., the gradient at the start of the chromatographic run).

2.6. Analyte Recovery and Method Accuracy

Overall, vitamin recovery, in spiked avocadoes, exhibited better performance than for carotenoids
(except for β-carotene with a recovery of 108.63%). Structurally, the lower recoveries (ranging from
43.80% to 63.68%) were found for those carotenoids that were oxygenated (Table 5). Noteworthy, when
zeaxanthin and β-cryptoxanthin were extracted using a mixture of ethyl ether, and hexane (50:50)
obtained/experimental mass increased between 12.35% and 15.56%. Furthermore, when nitrogen
flushing was performed, in conjunction, an additional increment between 13.35% to 19.66%, was
observed (data not shown). Carotenoids, among several mechanisms of transformation [44,45], are
susceptible to thermal degradation [46,47], β-carotene may suffer from symmetrical oxidative cleavage
(which generates, the apocarotenoid, retinol) [48], and β-cryptoxanthin can undergo light-induced
oxidation and isomerization [49].

Table 5. Spiked avocado samples and recovery for representatives for fat-soluble vitamins
and carotenoids.

Concentration, µmol
Compound tR , min Recovery, % a
Theoretical/Added Experimental/Obtained
Ergocalciferol 3.59 5.00 × 101 4.00 × 101 81.21 (70–110)
α-tocopherol 6.61 4.60 × 101 3.70 × 101 80.43 (70–110)
Phylloquinone 8.07 3.50 × 101 4.20 × 101 117.02 (70–110)
Astaxanthin 9.07 1.70 × 101 1.00 × 101 62.27 (60–120)
Lutein 9.97 0.35 × 101 0.23 × 101 63.68 (60–120)
Zeaxanthin 11.49 0.23 × 101 0.10 × 101 43.80 (60–120)
β-cryptoxanthin 16.58 1.09 × 102 6.50 × 101 59.51 (70–110)
β-carotene 22.51 1.23 × 101 1.33 × 101 108.63 (60–120)
a Brackets represent recovery values recommended by AOAC [50].

2.7. Mass Spectra Analysis

Retinyl acetate and β-cryptoxanthin showed, under our conditions, a higher degree of
fragmentation compared to tocopherol and an oxygenated carotenoid. Additional to the signals
analyzed above for retinoids, ion 369.3 represented the retinyl acetate molecular ion plus a potassium
ion, [M + K]+ (Figure 3A). On another hand, tocopherols usually are characterized to present few
fragmentation processes [51] (Figure 3B). Major ions are formed by cleavage through the non-aromatic
portion of the chromanol ring, both with and without hydrogen transfer and by a loss of the
isoprenoid side chain [51]. In contrast, we found none of the usual fragments reported elsewhere
for the fragmentation of γ-tocopherol (e.g., C9 H11 O2 + m/z 151 amu; [51]) probably because the total
Molecules 2019, 24, 4517 8 of 17

ion chromatogram was obtained using lower energy than required. Also, there may have been
some instrument limitations as a single quadrupole was used throughout the experiments, and the
fragmentation ions were the result of molecule degradation in the ion source not in the collision
chamber used during tandem mass spectrometry. Hence, the lack of tandem mass detection justified the
absence, in some cases, of multiple fragmentation patterns [52]. However, the molecular ion [M+] for
γ-tocopherol was unmistakable and could be used to differentiate nuances among tocopherols (e.g., the
difference between γ-tocopherol and α-tocopherol is a methyl group in the aromatic ring (∆15 amu)).
As an example of calciferol identification, cholecalciferol total ion chromatogram (TIC)-mass spectra
showed relevant fragments at 365.1 (loss of H2 O), 337.1 (subsequent loss of both –CH3 from the
isopropyl moiety), 301.2, and 227.0 (loss of –CH3 and =CH2 or C17 H23 3• ) m/z (data not shown).
Molecules 2019, 24, x FOR PEER REVIEW 8 of 16

Figure
Figure 3.
3. Experimental
Experimentalmass
massspectra
spectraobtained
obtainedfor
for(A)
(A)retinyl
retinylacetate,
acetate,(B)
(B)γ-tocopherol,
γ-tocopherol,(C)
(C)Lutein,
Lutein,(D)
(D)
β-cryptoxanthin.
β-cryptoxanthin. Total
Total ion
ion chromatograms
chromatograms at
at 100
100 mg −1each.
mg LL−1 each.

On another
On another hand,
hand, Atmospheric-pressure
Atmospheric-pressure chemical chemical ionization
ionization (APCI),(APCI), Fast
Fast atom
atom bombardment
bombardment
(FAB), electron
electron ionization
ionization(EI),(EI),andandESIESIallall
havehave mass-based
mass-based techniques
techniques used to assess
used carotenoids
to assess [53].
carotenoids
WhileWhile
[53]. APCIAPCI
is a popular approach
is a popular for thefor
approach ionization of lipophilic
the ionization compounds,
of lipophilic we selected
compounds, ESI+ . InESI
we selected this
+.

scenario, some modifications were introduced into the mobile phase

In this scenario, some modifications were introduced into the mobile phase to enhance ionization. Into enhance ionization. In this
specific
this case,case,
specific formic acidacid
formic was wasselected as itas
selected hasit been described
has been described to decrease signal
to decrease suppression
signal suppression[54].
For the
[54]. Forcase of lutein
the case the the
of lutein signal 551551
signal m/zm/z (i.e., [M[M
(i.e., +H +H − −18]
18]which
whichcorresponds
correspondsto to the
the loss
loss of H22O)O)
and 463
and 463 m/z ([M + H − 106], loss of two water molecules and xylene
+ H − 106], loss of two water molecules and xylene from the polyene chain) werefrom the polyene chain) were
observable ([53,55];
observable ([53,55]; Figure
Figure 3C).
3C). Additionally,
Additionally, as as zeaxanthin
zeaxanthin and and lutein
lutein differed
differed from each other other byby aa
position of aadouble bond, in a ε-ring, they rendered similar spectra [53]. Further
double bond, in a ε-ring, they rendered similar spectra [53]. Further fragmentation for fragmentation for both
compounds
both responded
compounds respondedto thetoloss
the of
losssaid
of rings.
said rings.Finally, for β-cryptoxanthin,
Finally, for β-cryptoxanthin, signal 552.5552.5
signal ([M+]) and
([M+])
and m/z ([M
553.2553.2 + H]++H]
m/z ([M ) were evident,
+) were fragments
evident, fragments 425.1425.1 H3240H]+40]and
[C32[C + and 399
399[M[M++HH−− 153]
+ m/z responded
153]+ m/z responded
to the
to the partial
partial breakup
breakup of both β-rings and the
β-rings and the complete
complete lossloss of the oxygenated β-ring plus aa methyl
β-ring plus methyl
group (Figure 3D).
group

2.8. Chromatographic
2.8. Chromatographic Separation
Separation of
of Tocopherol
Tocopherol Isomers
Isomers
Under our
Under our chromatographic
chromatographic conditions,
conditions,tocopherol
tocopherolisomers
isomerswere
wereeasily
easilysegregated
segregated(Figure
(Figure4A).
4A).
Using the
Using the tocopherol
tocopherol mixture
mixture containing
containing α-,
α-, β-,
β-, δ-, and γ-tocopherols,
δ-, and we were
γ-tocopherols, we were able
able to
to assess
assess further
further
that no additional [M+] signal was necessary for the detection of β-tocopherol, as β- and γ-tocopherol
that no additional [M+] signal was necessary for the detection of β-tocopherol, as β- and γ-tocopherol
share the same molecular mass (Figure 4B,C). The signal for β-tocopherol was observed at a tR of
5.387 min (Figure 4A). Hence, the four tocopherol isomers eluted as follows: δ-, β-, γ-, and α-
tocopherol (Table 1, Figure 4A).
 Molecules 2019, 24, 4517 9 of 17

share the same molecular mass (Figure 4B,C). The signal for β-tocopherol was observed at a tR of
5.387 min (Figure 4A). Hence, the four tocopherol isomers eluted as follows: δ-, β-, γ-, and α-tocopherol
(Table
Molecules 1, 24,
2019, Figure 4A).
x FOR PEER REVIEW 9 of 16

Figure 4. (A)
Figure 4. Chromatogram
(A) Chromatogram of aofmixture of tocopherols
a mixture of tocopherols (W530066
(W530066 70,70,
13,13,
523, 105
523, 105mg g−1
mgg−1 forfor
α-α-(peak
+
+ H]ionion
4), β-(peak
(peak4),2), (peak
β- γ- 2), γ-
(peak 3),(peak 3), and δ-tocopherol
and δ-tocopherol (peak (peak 1), respectively).
1), respectively). Selected
Selected [M[M+ H] + forfor
(B) δ-
(B) δ-tocopherol (402.1 m/z), (C) β/γ-tocopherol (416.4 m/z), and (D) α-tocopherol (430.4
tocopherol (402.1 m/z), (C) β/γ-tocopherol (416.4 m/z), and (D) α-tocopherol (430.4 m/z). Selected ion m/z). Selected
ion monitoring −1 .
monitoring (SIM) at(SIM)
700,at 700,5230,
130, 130, 5230,
1050 1050
μg mLµg−1mL
.
2.9. Method Application in Real Samples
2.9. Method Application in Real Samples
Simmonds variety characterizes itself for having an oblong oval to pear-shape large-sized fruit of
Simmonds
light varietysmooth
green-colored characterizes
skin, anditself
a seedfor having an
of medium size,oblong
usuallyoval
tight.toMeanwhile,
pear-shapeGuatemala
large-sizedhasfruit
of light green-colored
a medium smooth
to large size skin, and
and nearly rounda seed
shape,ofsmooth,
medium size,granular,
thick, usually andtight. Meanwhile,
green skin with Guatemala
a small
seed. Finally, Hass fruit possesses an ovoid to pear shape, is of medium
has a medium to large size and nearly round shape, smooth, thick, granular, and green skin with a size, with tough, leathery,
smallpebbled, thin, dark
seed. Finally, purple
Hass to black
fruit (whenan
possesses ripe) skin, to
ovoid with a small
pear seedis[30,56].
shape, of medium size, with tough,
We found in avocado both non-provitamin A (lutein and
leathery, pebbled, thin, dark purple to black (when ripe) skin, with a small zeaxanthin) and provitamin A (β-carotene
seed [30,56].
and β-cryptoxanthin) carotenoids [57] (Table 6). All carotenoids encountered have been previously
We found in avocado both non-provitamin A (lutein and zeaxanthin) and provitamin A (β-
identified for the fruit (see, for example, [11,20,21,23,28,29]). Our data indicated that, overall, all
carotene and β-cryptoxanthin) carotenoids [57] (Table 6). All carotenoids encountered have been
three varieties of avocado exhibited concentrations, of carotenoids and vitamins, in line (except for
previously identified for the fruit (see, for example, [11,20,21,23,28,29]). Our data indicated that,
provitamin A carotenoids which levels were considerably higher for Costa Rican avocadoes) with
overall, allreported
those three varieties
elsewhere of [28].
avocado exhibited
Considering theconcentrations,
above, Costa Rican of carotenoids
avocado would andsupply
vitamins, in line
to diet
(except forequivalents
retinol provitamin [58]A carotenoids
ranging which
from 1085.21 levels
to 425.01 µg were
on a dryconsiderably
weight basis. higher for Costa
Though three differentRican
avocadoes) with those reported elsewhere [28]. Considering the above, Costa
varieties of avocado have been examined, it should be clear that any number of factors can affect theRican avocado would
supply to diet retinol
concentrations equivalents
of such molecules,[58] ranging
including from 1085.21
edaphoclimatic to 425.01genotypic
conditions, μg on adifferences,
dry weight andbasis.
Thoughnutritional status of the
three different plants [59].
varieties Hass and
of avocado Guatemala
have varietals showed
been examined, it shoulda somewhat
be clear similar
that anyprofile
number
for both fat-soluble vitamins as carotenoids. Meanwhile, the Simmonds variety
of factors can affect the concentrations of such molecules, including edaphoclimatic conditions, showed higher levels
for almost
genotypic all analytes
differences, and tested (except for
nutritional lutein)
status (Table
of the 6). Interestingly,
plants [59]. Hass andthe δ/α ratio of isomers
Guatemala is close
varietals showed
to 1 for all varieties (i.e., 0.75–1.22).
a somewhat similar profile for both fat-soluble vitamins as carotenoids. Meanwhile, the Simmonds
variety showed higher levels for almost all analytes tested (except for lutein) (Table 6). Interestingly,
the δ/α ratio of isomers is close to 1 for all varieties (i.e., 0.75–1.22).

Table 6. Fat-soluble vitamins and carotenoids obtained from Costa Rican avocadoes.

Simmonds Hass Guatemala

Compound
Concentration, mg/100 g dry Weight Basis
Ergocalciferol 1.84 ± 0.18 0.31 ± 0.16 0.53 ± 0.33
Cholecalciferol 119.50 ± 1.20 103.50 ± 15.50 108.50 ± 33.40
δ-tocopherol 42.48 ±7.96 8.31 ±1.63 6.16 ± 2.88
γ-tocopherol 4.81 ± 0.48 2.42 ± 0.37 0.92 ± 0.16
Molecules 2019, 24, 4517 10 of 17

Table 6. Fat-soluble vitamins and carotenoids obtained from Costa Rican avocadoes.

Simmonds Hass Guatemala

Compound
Concentration, mg/100 g dry Weight Basis
Ergocalciferol 1.84 ± 0.18 0.31 ± 0.16 0.53 ± 0.33
Cholecalciferol 119.50 ± 1.20 103.50 ± 15.50 108.50 ± 33.40
δ-tocopherol 42.48 ±7.96 8.31 ±1.63 6.16 ± 2.88
γ-tocopherol 4.81 ± 0.48 2.42 ± 0.37 0.92 ± 0.16
α-tocopherol 34.80 ± 14.50 8.16 ± 0.97 8.20 ± 3.67
Astaxanthin 2.23 ± 0.14 0.64 ± 0.23 0.98 ± 0.55
Lutein 6.41 ± 2.03 15.13 ± 8.66 10.79 ± 2.77
Zeaxanthin 0.03 ± 0.01 0.02 ± 0.01 0.02 ± 0.01
β-cryptoxanthin 6.10 ± 1.39 3.37 ± 0.20 1.66 ± 0.85
β-carotene 3.43 ± 2.09 2.28 ± 1.56 1.71 ± 0.21
Molecules 2019, 24, x FOR PEER REVIEW 10 of 16

2.10.
2.10.Method
MethodApplication
Applicationin
inOther
OtherSamples
Samples
As
Asstated
statedbefore,
before,no
nostandard
standardreference
referencematerial
materialisisavailable
availablefor
foravocado.
avocado.However,
However,asasaaquality
quality
control material, we subjected our method to infant formula to further assess method
control material, we subjected our method to infant formula to further assess method accuracy. We accuracy.
We obtained
obtained values
values according
according toto theprovider,
the provider,and
andno
noappreciable
appreciablematrix
matrix effects
effects were
were observed
observed for
forthis
this
food either (Figure 5A). Also, our method showed promise to extrapolate to other matrices, especially
food either (Figure 5A). Also, our method showed promise to extrapolate to other matrices, especially
fruits.
fruits. Unambiguous
Unambiguous signals
signals of
of several
several vitamins
vitamins of
of interest
interest can
can be
beseen
seenwhen
whenaachloroform
chloroformextract
extract
obtained from green tomatoes was injected (Figure
obtained from green tomatoes was injected (Figure 5B). 5B).

Figure5.5. (A)
Figure (A) Infant
Infant formula
formula with
with certified
certified values
valuesforforfat-soluble
fat-solublevitamins.
vitamins. The
The box
box shows
shows the
the
amplificationofofthe
amplification theregion
regionfrom
from1 1toto1010min
minofofthe
thesaid
said chromatogram.FAPAS
chromatogram. FAPAS ®®®® reference material
reference material
TYG009RM.Vitamin
TYG009RM. VitaminA, A,vitamin
vitaminDD 3 ,3,and
andvitamin
vitaminEEatat508
508±±12,12,7.16
7.16±± 0.33,
0.33, and
and10,900
10,900±± 400
400 µg/100
μg/100 g.
g.
(B) Non-saponified sample of green tomatoes extracted after mechanical shearing
(B) Non-saponified sample of green tomatoes extracted after mechanical shearing and chloroform and chloroform
extractionand
extraction andanalyzed
analyzedusing
usingthe
theproposed
proposedmethod.
method.

3.3.Materials
Materialsand
andMethods
Methods

3.1. Reagents
3.1. Reagents
tert-Butyl methyl ether (99%, catalog 34875, MTBE, chromatographic grade), methanol (≥99.9%,
tert-Butyl methyl ether (99%, catalog 34875, MTBE, chromatographic grade), methanol (≥99.9%,
catalog 646377, MeOH, chromatographic grade) and potassium hydroxide (ACS reagent, catalog
catalog 646377, MeOH, chromatographic grade) and potassium hydroxide (ACS reagent, catalog
1050210250) were acquired from Merck Millipore (Burlington, MA, USA). Retinyl acetate (catalog
1050210250) were acquired from Merck Millipore (Burlington, MA, USA). Retinyl acetate (catalog
46958), retinyl palmitate (catalog 46959-U), cholecalciferol (catalog C9774), ergocalciferol (catalog
46958), retinyl palmitate (catalog 46959-U), cholecalciferol (catalog C9774), ergocalciferol (catalog
47768), 3-phytylmenadione catalog (95271), α-tocopherol (catalog 47783), δ-tocopherol (catalog 47784),
47768), 3-phytylmenadione catalog (95271), α-tocopherol (catalog 47783), δ-tocopherol (catalog
γ-tocopherol (catalog T1782), tocopherols (mixed, W530066), lutein (catalog 071068), β-carotene (catalog
47784), γ-tocopherol (catalog T1782), tocopherols (mixed, W530066), lutein (catalog 071068), β-
C4582), β-cryptoxanthin (≥97%, catalog C6368), zeaxanthin (catalog 14681), all-trans-astaxanthin
carotene (catalog C4582), β-cryptoxanthin (≥97%, catalog C6368), zeaxanthin (catalog 14681), all-
(catalog 41659), and lycopene (catalog 75051) from Sigma-Aldrich (unless stated otherwise, all
trans-astaxanthin (catalog 41659), and lycopene (catalog 75051) from Sigma-Aldrich (unless stated
standards
otherwise,were of analytical
all standards weregrade, St. Louis,grade,
of analytical MO, USA). Chloroform
St. Louis, (ACS
MO, USA). reagent, catalog
Chloroform (ACS 366919),
reagent,
ethanol (200 proof, ACS reagent, ≥99.5%, 459844), 2-propanol (HPLC Plus, 650447), pyrogallol
catalog 366919), ethanol (200 proof, ACS reagent, ≥99.5%, 459844), 2-propanol (HPLC Plus, 650447), (ACS
pyrogallol (ACS reagent, ≥99%, catalog 16040), and sodium sulfate (anhydrous, granular, free-
flowing, Redi-Dri™, ACS reagent, ≥99%, catalog 798592) were also acquired from Sigma-Aldrich.

3.2. Sample Treatment and Preparation

Three different varieties of avocado collected from Costa Rica farms, two varietals from low and
Molecules 2019, 24, 4517 11 of 17

reagent, ≥99%, catalog 16040), and sodium sulfate (anhydrous, granular, free-flowing, Redi-Dri™, ACS
reagent, ≥99%, catalog 798592) were also acquired from Sigma-Aldrich.

3.2. Sample Treatment and Preparation

Three different varieties of avocado collected from Costa Rica farms, two varietals from low and
one highland, i.e., Simmonds (from 0 to 1000 m amsl), Guatemala (from 600 to 1500 m amsl), and Hass
(from 1000 to 2000 m amsl), respectively [30,56]. Three batches of each variety were analyzed, each
with eight days of maturation. A previously homogenized subsample (2 g) of freeze-dried material
was weighed, and chloroform (20 mL, ACS reagent, Sigma-Aldrich 366919) was added. After that,
the mixture was stirred continuously (30 min) using an Ultraturrax® (T25, at 7500 rpm, IKA Works
Staufen, Germany). The remnant suspension was centrifuged, and the supernatant liquid recollected.
The extraction procedure was repeated twice. The solvent was evaporated using a rotary evaporator
(Multivapor™ P-6, Büchi, Flawil, Switzerland) until the lipid fraction was attained, which was then
processed immediately for saponification.

3.3. Optimization of Saponification Conditions

An additional experiment was performed to enhance the recovery of the analytes of interest
during the saponification reaction. The experimental design consisted of maintaining constant reaction
time (1 h), the concentration of radical protection agent (0.1 g/100 mL), and the extraction solvent
(hexane). Base concentration was contrasted (1 vs. 2 mol KOH L ethanol−1 [43] for each temperature),
and the temperature was progressively increased (60, 80, and 95 ◦ C). The conditions that rendered the
most recoveries were selected to process the samples.

3.4. Sample Saponification

A small portion of the fat fraction (0.3 g) was weighed and quantitatively transferred to a conical
centrifuge tube (50 mL, CLS430829, polypropylene, Corning® , New York, USA). Afterward, KOH in
ethanol (1 mmol mL−1 ) and containing pyrogallol (0.1 g/100 g) was added (10 mL). The mixture was
let to saponify (during 1 h at 80 ◦ C) in a heat bath (1229U55, Boekel Scientific, Feasterville, PA, USA).
The resulting mixture was let to cool to room temperature and transferred to a Squibb separatory funnel
(PYREX® , 250 mL, Corning® 6402). The above ethanol/aqueous layer was subjected to a liquid-liquid
extraction using hexane. The extraction procedure was repeated twice. The totality of the organic
solvent layer was then collected and filtered through sodium sulfate (used as a desiccant). The resulting
solution was evaporated to dryness under a nitrogen flow (Ultra-High Pure Nitrogen was purchased
from Praxair Technology Inc., Danbury, Connecticut, USA) and then reconstituted with MTBE (1 mL)
and 2-propanol (1 mL) used at the start of the chromatographic separation and transferred to an HPLC
vial (Agilent technologies, Santa Clara, CA, USA).

3.5. Stationary Phase and Selection of Chromatographic Conditions

The major obstacle in vitamin separation is the segregation of isomers. With this in mind, as a
starter setup, we used a mobile phase based on MeOH and water using an eight carbon-based alkyl
stationary phase (0.75 mL min−1 , Eclipse Plus C8 , 4.6 mm ID × 150 mm, 3 µm, Agilent Technologies).
As the resolution was insufficient, a C18 column was selected, and only flow was modified (1 mL min−1 ,
Eclipse Plus C18 , 4.6 mm ID × 150 mm, 3 µm, Agilent Technologies). Then we substituted the column
for a C30 , removed water, and used a less polar solvent in acetonitrile and 2-propanol, reducing yet
again the solvent flow (0.5 mL min−1 ) (Table 1). Finally, retaining a similar proportion of MeOH, we
substituted acetonitrile and isopropanol for MTBE. The C30 column was kept as it already had excellent
capabilities reported for highly lipophilic compounds (e.g., carotenoids) [39].
Molecules 2019, 24, 4517 12 of 17

3.6. Chromatographic Conditions

All assays performed using an Agilent Technologies LC/MS system equipped with 1260 infinity
quaternary pump (61311C), column compartment (G1316A), automatic liquid sampler modules (ALS,
G7129A) and a 6120-single quadrupole mass spectrometer with electrospray ionization ion source
(Agilent Technologies, Santa Clara, CA, USA). Gradient elution was used to separate all the compounds.
The solvent gradient was optimized using MeOH (solvent A) and MTBE (solvent B), both acidified
with formic acid (0.1 mL/100 mL). Solvent proportions were set as follows: at 0 min 80% A, at 5 min 80%
A, at 7 min 73% A, at 15 min 62.5% A, at 20 min 62.5% A, at 30 min 45% A, at 35 min 10% A, at 40 min
10% A, at 45 min 80% A and 50 min 80% A. Flow rate was kept constant at 0.6 mL min−1 . Injection
volume was held at 10 µL. The column compartment was held at a temperature of 10.0 ± 0.8 ◦ C.
Considering the need for the separation of structurally similar compounds, a 30-carbon alkyl chain
based chromatographic column was used to achieve the analytical separation (YMC Carotenoid,
4.6 mm ID × 150 mm, S-3 µm, YMC Co., Ltd., Kyoto, Japan).

3.7. MS Detection System Conditions

The fragmentor was initially cycled to assess the voltage (from 20 to 300 V) that rendered the
highest sensitivity for the compounds; omitting column interaction (Figure 6A). Afterward, total ion
Molecules 2019, 24, x FOR PEER REVIEW 12 of 16
chromatographs (TIC) allowed us to obtain the MS spectra for each of the compounds (scan mode
using
using aa mass
massrange
rangeand anddetector
detectorgain
gain setset
toto
50–750
50–750 m/z, andand
m/z, 10.00, respectively)
10.00, respectively)(Figure 6B,C).
(Figure EachEach
6B,C). TIC
was used to identify the molecular ion signal. Drying gas, nebulizer pressure,
TIC was used to identify the molecular ion signal. Drying gas, nebulizer pressure, drying gas drying gas temperature,
and capillary and
temperature, voltage was set,
capillary respectively,
voltage was set, to 12.0 L min−1
respectively, to, 12.0
50 psi, 350−1◦, C,
L min 50 4000 V for
psi, 350 °C,positive
4000 V ion
for
mode electrospray ionization (ESI + ). Selected ion monitoring was used to corroborate each compound
positive ion mode electrospray ionization (ESI ). Selected ion monitoring was used to corroborate
+

identity, remove interferences

each compound identity, removeand interferences
improve sensitivity (SIM mode
and improve with peak
sensitivity (SIMwidth
modeand cycle
with peaktime set
width
to 0.05 min, andset 0.30 −1
and cycle time to s0.05
cycle
min,, and
respectively) (Table
0.30 s cycle 2, Figure 6D).
−1, respectively) (Table 2, Figure 6D).

Figure 6.6.Example
Exampleofof thethe voltage
voltage cycling
cycling for phylloquinone
for phylloquinone to obtain
to obtain the mostthe most sensitivity,
sensitivity, data
data obtained
obtained
at 100 µg at mL 100 −1 .μg mLA−1.parameter
(A) (A) A parameter was selected
was selected wherewhere the signal
the signal delivered
delivered the most
the most area area
under under
the
the curve.
curve. (B) (B)
Mass Mass spectra
spectra obtained
obtained fromfrom a total
a total ionion chromatogramfor
chromatogram forphylloquinone
phylloquinoneatat100 100µgμgmL mL−1−1..

Fragmentation as follows follows (molar

(molar mass mass 450.7
450.7ggmol mol −1−1):): 473.3
473.3 ([M K]++), 451.4
([M ++ K] 451.4 ([M H]++), 381.3
([M ++ H] 381.3
H3535OO2]2•]),• ),353.4
26H
([C26 353.4([C HH
([C2424 30O
302O ]2•225
]2•2), ([C([C
), 225 15H15 O13
13H 2]•Oand
• partial
2 ] and alkyl
partial isoprenoid
alkyl isoprenoidchain [C16[C
chain H3316]H
•
•), and
33 ] ),
and 186 (quinone • ) m/z
186 (quinone ring, ring,
[C12H[C 9O122] H
• O2 ][60].
) 9m/z (C)[60]. (C) Chromatogram
Chromatogram for phylloquinone
for phylloquinone was using
was obtained obtained the
using the[M selected [Mm/z+ H] + 451 m/z and (D) selected ion monitoring (SIM) for the target analyte−1at
selected + H]+ 451 and (D) selected ion monitoring (SIM) for the target analyte at 20 μg mL .
20 µg mL−1 .
Sensitivity is greatly improved using a SIM targeted scan. For example, the same standard 34.4
mg phylloquinone L-1 in TIC throws 37870 vs. 457785 area under the curve in SIM. Furthermore,
within curve sensitivity reaches only 24.1 mg L−1 for TIC while the signal for 4.31 mg L−1, in SIM, is
still appreciable (i.e., 80471 area under the curve, 0.54 mg L−1 within curve sensitivity) (Figure 7A,B).
Absolute sensitivity to vitamin K increases almost 50 fold (24.1/0.54). For carotenoids, the change is
more dramatic as 100 mg L−1 standard has to be prepared in TIC for a detectable signal while 0.136
 the curve. (B) Mass spectra obtained from a total ion chromatogram for phylloquinone at 100 μg mL−1.
Fragmentation as follows (molar mass 450.7 g mol−1): 473.3 ([M + K]+), 451.4 ([M + H]+), 381.3
([C26H35O2]•), 353.4 ([C24H30O2]2•), 225 ([C15H13O2]• and partial alkyl isoprenoid chain [C16H33]•), and
186 (quinone ring, [C12H9O2]•) m/z [60]. (C) Chromatogram for phylloquinone was obtained using the
selected
Molecules [M24,
2019, + H] + 451 m/z and (D) selected ion monitoring (SIM) for the target analyte at 20 μg13
4517 mL 17.
of −1

Sensitivity is greatly improved using a SIM targeted scan. For example, the same standard 34.4
Sensitivity is greatly improved using a SIM targeted scan. For example, the same standard 34.4 mg
mg phylloquinone
phylloquinone L-1 in TIC throws 37870 vs. 457785 area under the curve in SIM. Furthermore,
L-1 in TIC throws 37870 vs. 457785 area under the curve in SIM. Furthermore, within
withincurvecurve sensitivity
sensitivity reaches reaches onlymg
only 24.1 24.1
L−1mg
forLTICfor
−1 TICthe
while while thefor
signal signal for L4.31
4.31 mg mg
−1 , in L−1,isinstill
SIM, SIM, is
still appreciable
appreciable(i.e., (i.e.,80471
80471 area under the curve, 0.54 mg −1 L −1 within curve sensitivity) (Figure 7A,B).
area under the curve, 0.54 mg L within curve sensitivity) (Figure 7A,B).
Absolute
Absolute sensitivity
sensitivitytotovitamin
vitaminKKincreases almost50
increases almost 50fold
fold(24.1/0.54).
(24.1/0.54). ForFor carotenoids,
carotenoids, the change
the change is is
more dramatic
more dramatic asas100
100mg mg L −1
L standard
−1 hastotobebe
standard has prepared
prepared in TIC
in TIC for a for a detectable
detectable signal
signal while while
0.136 mg 0.136
L−1 is still noticeable (Figure 2B), which represents ca. 750-fold in increased sensitivity.
mg −1 is noticeable (Figure 2B), which represents ca. 750-fold in increased sensitivity.

Figure
Figure 7. Calibration
7. Calibration curveand
curve andsensitivity
sensitivity comparison
comparisonusing (A).
using (A).TIC andand
TIC SIMSIM
modes for vitamin
modes K1
for vitamin K1
both signals tested at 137.8, 68.9, 34.4, 17.2, 8.61, and 4.31 mg L −1 and using a fragmenter of 140 V. No
both signals tested at 137.8, 68.9, 34.4, 17.2, 8.61, and 4.31 mg L and using a fragmenter of 140 V. No
−1

signal is noticeable at 17.2 mg L−1−1 for TIC (blue line in panel (A)). Meanwhile, a calibration curve is
signal is noticeable at 17.2 mg L for TIC (blue line in panel (A)). Meanwhile, a calibration curve is
easily constructed in SIM mode with the lowest point in 4.31 mg L−1 (purple line panel (B)).
easily constructed in SIM mode with the lowest point in 4.31 mg L−1 (purple line panel (B)).
Retention times and mass spectra were collected by the centroid of the chromatographic peak.
Quantitation was carried out by comparing the peak areas found in the samples with those of standard
solutions. The identification and quantification of targeted compounds analyzed by LC-ESI+ -MS were
performed using OpenLab Chemstation C.01.07 (Agilent Technologies) for the processing of MS data
sets. Confirmation of target analytes was based on the retention time (± 0.2 min as accepted time
deviation), measurement of the molecular ion in a specific timeframe (Table 2).

3.8. Statistical Analysis

Calibration curves parameters (i.e., slopes and intercepts), coefficients of determination, limits
of detection, and standard errors were computed as a linear fit model using SAS JMP 13 (Marlow,
Buckinghamshire, England). An ANOVA with a post-hoc Dunnet test was used to assess differences
among treatments during the optimization of the conditions during saponification. Concentrations
obtained using the conditions 1 h and 1 mmol KOH mL ethanol−1 at 80 ◦ C, were used as the control
parameters; the test considered if the data was below the control, with α = 0.05 significance level.
The statistical analysis was performed using IBM SPSS®® Statistics 23 (Armonk, NY, USA).

4. Conclusions
The proposed method was regarded as a greener option by replacing chlorinated solvents and
allowed two nutritionally relevant families of bioactive compounds (i.e., carotenoids and fat-soluble
vitamins) to be analyzed together (which is not usually the case) and offered an adequate resolution in
the case of tocopherol and calciferol isomers to improve their differential quantification. We obtained
an accurate, sensitive, robust, and highly specific multi-analyte method that was successfully applied
to avocadoes, a fruit of high economic value, of dietary interest, and a staple of Latin-American
cuisine. The use of separation based entirely on organic solvents and the C30 column retention
capability rendered a versatile method that can facilitate the incorporation of other pigments that have
been reported present in avocado fruit (e.g., neoxanthin, trollichrome, chrysanthemaxanthin) [14,21]
to further extend its chemical characterization. Saponification was paramount in the recovery of
fat-soluble compounds. Therefore, optimized conditions should be assessed for each matrix to be
tested. The method may be extended to evaluate fat-soluble vitamins and carotenoids in other matrices.
Molecules 2019, 24, 4517 14 of 17

Mass spectrometry was a crucial tool in enabling the discrimination of structurally related compounds
(e.g., all three tocopherol isomers could be easily accounted for in avocado).

Author Contributions: Conceptualization, F.G.-C., G.A., and C.C.-H.; methodology, F.G.-C. and C.C.-H.; software,
F.G.-C. and C.C.-H.; validation, F.G.-C., G.A., A.C., and C.C.-H.; formal analysis, F.G.-C., G.A., A.C., and
C.C.-H.; investigation, F.G-C.; resources, F.G.-C., G.A., and C.C.-H.; data curation, C.C.-H., F.G-C., and G.A;
writing—original draft preparation, F.G.-C.; writing—review and editing, F.G.-C., G.A., A.C., C.C.-H.; visualization,
F.G.-C.; supervision, F.G.-C. and C.C-H; project administration, F.G.-C. and C.C-H.; funding acquisition, C.C-H.
Funding: This research received no external funding except for the APC, which was funded by the Vice Provost
Office for Research of the Universidad de Costa Rica.
Acknowledgments: Laura Arroyo is acknowledged for acquiring the avocado samples and preliminary integration
of a few avocado samples and María Sabrina Sánchez for their suggestions, revising the manuscript and for
language editing.
Conflicts of Interest: The authors declare no conflict of interest.

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Sample Availability: Samples of the fat-soluble vitamins and carotenoids are available from the authors.

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