Separations 07 00036 v2

separations
Review
Recent Advances in the Analysis of Vitamin D and Its
Metabolites in Food Matrices
Bárbara Socas-Rodríguez 1, * , Margareta Sandahl 1 , Cecilia Holm 2 and Charlotta Turner 1
1 Department of Chemistry, Centre for Analysis and Synthesis, Lund University, P.O. Box 124,
221-00 Lund, Sweden; Margareta.Sandahl@chem.lu.se (M.S.); Charlotta.Turner@chem.lu.se (C.T.)
2 Department of Experimental Medical Science, Faculty of Medicine, Lund University, P.O. Box 124,
221-00 Lund, Sweden; cecilia.holm@med.lu.se
* Correspondence: barbara.socas_rodriguez@chem.lu.se

Received: 22 May 2020; Accepted: 22 June 2020; Published: 24 June 2020
Abstract: Vitamin D and its analogues are fat-soluble vitamins that carry out important functions
in human and animal organisms. Many studies have pointed out the relationship between the
deficiency of these substances and the development of both skeletal- and extra-skeletal diseases.
Although vitamin D is fundamentally derived from the bio-transformation of its precursor,
7-dehydrocholesterol, through the action of UV-B radiation in the skin, dietary intake also plays
an important role in the regulation of its status in an organism. For this reason, the application
of reliable methodologies that enable monitoring the content of vitamin D and its analogues in
food and supplements constitutes an aspect of special relevance to establish adequate habits,
which avoid the deficiency of these substances in organisms and, consequently, the appearance of
related diseases. The use of chromatographic techniques in combination with conventional and novel
sample pre-treatments has become a suitable strategy to achieve this aim. This review compiles
the most relevant methodologies reported in the last ten years for vitamin D analogues analysis in
food matrices. Particular attention has been paid to provide a general overview of the most suitable
approaches in terms of reliability, sensitivity and simplicity, used in the field of food analysis.
Keywords: vitamin D; vitamin D metabolites; food analysis; sample preparation; chromatographic

techniques; saponification; conventional extraction; miniaturized techniques
1. Introduction
Vitamin D3 (cholecalciferol) (D3 ) and vitamin D2 (ergocalciferol) (D2 ) are fat-soluble vitamins
with a steroidal structure. They play an important role in calcium and phosphate absorption, as well
as bone metabolism, and have many other pleiotropic functions in humans and other species [1]. D3
is fundamentally derived from the transformation of its precursor, 7-dehydrocholesterol (7-DHC),
present in the skin, through the action of ultraviolet radiation B (UV-B, 290–300 nm). It is converted
into previtamin D3 (Pre-D3 ) which finally is thermally isomerized to D3 . However, synthesis in the
skin is not the only source of D3 . In fact, around 10–20% of this substance is obtained through dietary
intake by absorption in the intestine, although it also depends on the area and the season around
the world [2]. In contrast, D2 is principally obtained from the consumption of edible plants, fungi
or yeast, which also transform previtamin D2 (Pre-D2 ) into D2 by UV radiation action [1]. Both D3
and D2 are inactive substances that are transported through the blood stream to the liver, where the
most stable metabolites, 25-hydroxyvitamin D (25-OHD) forms, are generated. Those are further
hydroxylated into the most active metabolite, 1,25-dihydroxyvitamin D (1,25-OHD), in kidney and
other organs such as breast, colon, brain, pancreas and prostate. Additionally, other less active forms,
as, for example, 24,25-dihydroxyvitamin D (24,25-(OH)2 D) or 1-hydroxyvitamin D (1-OHD) and their
Separations 2020, 7, 36; doi:10.3390/separations7020036 www.mdpi.com/journal/separations

Separations 2020, 7, 36 2 of 27
epimeric forms, are also generated [3]. Apart from hydroxylated metabolites, it is believed that ester
forms are produced in different tissues of the organism, although the routes and tissues in which these
metabolites are obtained and the roles that they play in the organism are not very well-known yet.
Despite the fact that D2 is not produced by mammals, the active forms of D2 and D3 are considered
equivalent, although this is an issue of controversy [4].
Currently, there is a global concern about the status of vitamin D and its metabolites in the
population since it has been reported that insufficiency and deficiency in these vitamins in humans is
linked not only to the development of bone diseases, such as rickets and osteoporosis, but also to many
extra-skeletal diseases, including diabetes, hypertension, cardiovascular diseases, multiple sclerosis,
psoriasis, Crohn’s disease, neuropsychiatric illnesses or even breast and colon cancer [3,5–7]. This is
why increasing the consumption of foods which contain these vitamins, with a daily intake of 5–20 µg
in adults, is essential to tackle this problem, especially for the population that lives at high latitudes,
where the subcutaneous synthesis of vitamin D is absent during winter months. However, it should be
emphasized that the recommendations for daily intake vary considerably between different population
groups and advisory guidelines [3]. Besides, intake of excessive doses can cause other health problems,
such as hypercalcemia, hypercalciuria or hyperphosphatemia [8].
Fatty fish, fish oil, meat or egg yolk are generally considered to be food items with a high content
of vitamin D, although it can also be found in fruit, edible plants and seeds [8–10]. Nevertheless,
the content of these substances in the diet is limited and their stability in food matrices is also questioned
under certain conditions such as light, heat or oxidant conditions [1]. For this reason, there are
international recommendations for food fortification including D3 , D2 and 25-OHD analogues [11].
In this sense, diverse studies have been carried out in order to enrich widely consumed foodstuffs by
the addition of these fat-soluble vitamins or by “bio-addition” enhancing the bio-production process in
the products [2,11]. In the same direction, the US Food and Drug Administration (FDA) revised the
food labelling guidelines in 2016 to make vitamin D content required information in conventional food
and supplement packages with the aim of promoting vitamin D awareness among consumers [12].
Additionally, the Codex Alimentarius recommends that food for infants and young children is fortified
with D2 and D3 [13], and many countries, including United Kingdom, Canada, Australia, United States,
Finland, Denmark and Ireland, have mandatory or voluntary food fortification regulations [14]. This is
why a strict evaluation of the presence of vitamin D and its metabolites in food items is an issue of
special relevance for the scientific community and also for general society, in order to get an accurate
knowledge of the population intake.
In this regard, the determination of vitamin D and its metabolites in food matrices has
been traditionally carried out using bio-assay methods. However, these kinds of techniques are
time-consuming and, additionally, cross-reactions occur as a result of the similar structures of the
different analogues, hampering the analysis of the different individual bioactive compounds [10]. This is
why the application of chromatographic techniques has become one of the most adequate alternatives
since the 1980s because such applications allow the selective separation and analysis of each compound
individually. Indeed, the official methods for determining D3 and D2 in dried skimmed milk and in
general commodities, proposed by the International Dairy Federation (IDF) [15] and the European
Commission (EC) [16], respectively, are based on the use of liquid chromatography (LC) combined with
UV detection. In addition, the Association of Official Analytical Chemists (AOAC) has also proposed
other methods for the determination of these two analytes in nutritional formulas using LC hyphenated
with tandem mass spectrometry (MS/MS) [17,18], after the proposal of diverse methodologies based
on high-performance liquid chromatography (HPLC)-UV analysis [19–21]. Apart from that, the Codex
Alimentarius proposed in 2018 the endorsement of the AOAC Official Method 2016.05 as type II—the
most suitable method—for the analysis and sampling of vitamin D in infant formulas displacing the
EN 12821, based on HPLC-UV analysis, as type III, only necessary for verification in case of controversy.
However, it is worth mentioning that most of the applications developed so far in this field have
been focused on the determination of D2 and D3 forms [8,18,22–39] and only a few applications have
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Molecular Molecular
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NameName Structure
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NameName Structure
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Formula
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Molecular
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of vitamin
MolecularanaloguesName
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Structure
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Molecular
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Molecular
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Molecular
Weight
Weight
(g/mol)
Formula
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Molecular Molecular
Name
NameName Structure
Structure Formula
Molecular Weight Weight
(g/mol) Name
Name Structure
Structure Formula
Molecular Weight
(g/mol)
Weight
Structure Formula (g/mol)
Formula
Molecular
Weight
Molecular
Name Structure Formula
Formula
Molecular
(g/mol)
Weight
Molecular
Name Structure Formula (g/mol) (g/mol) Name Structure Formula (g/mol)
(g/mol)
D3 D3 C27Formula
HC4427OH44O 384.65 (g/mol)
Weight (g/mol) 1OH-D
384.65 1OH-D
2 2 Formula (g/mol)
CC2828HH4444OO22 Weight
412.65
(g/mol)
D3 D3 C27HC4427OH44O 384.65 384.65 1OH-D
1OH-D 2 2 CC2828HH4444OO22 412.65
412.65
D3 C27H44O 384.65 1OH-D2 C28H44O2 412.65
D3 D3 C27HC4427OH44O 384.65 384.65 1OH-D 2
1OH-D 2
CC2828HH4444OO22 412.65
412.65
D3 DD3 3 C27CHC 44
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D2 D2 C28HC4428OH44O 396.66
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24,25-(OH) 2D23D3 CC2727HH4444OO33 416.64
416.64
D2 C28H44O 396.66 24,25-(OH)2D3 C27H44O3 416.64
D2 D2 C28HC4428OH44O 396.66
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24,25-(OH)2D3 CC2727HH4444O O33 416.64
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428.70
25-OHD
25-OHD 25-OHD
2 22 C28C C44H
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412.65
412.65 1,25-(OH)
1,25-(OH) 2D
1,25-(OH) 2D
2D 2 2 CC C28H
28 28H H44
44 3O33
O44O 428.70
428.70
25-OHD
25-OHD 2
C28HCC 28H44O2 412.65 1,25-(OH) 2D2
CCC282828HH44O33 428.70
2
25-OHD 2 28O
44 H244O2 412.65
412.65 1,25-(OH)
1,25-(OH)2D22D2 H4444OO3 428.70
428.70
25-OHD2 C28H44O2 412.65 1,25-(OH)2D2 C28H44O3 428.70
25-OHD3 C27H44O2 400.64 1,25-(OH)2D3 C27H44O3 416.64

25-OHD
25-OHD
25-OHD
3 33 C27C
H27
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442O2 400.64
400.64
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1,25-(OH)2D
1,25-(OH) 23D
2D 3 3 CCC27H
27 H
27 O44O
H44
44 3O33 416.64
416.64
25-OHD
25-OHD 3
3
C27HC4427OH2 44O2 400.64
400.64 1,25-(OH)
1,25-(OH) 2D3
2D3
CC2727HH4444O
O33 416.64
416.64
25-OHD3 C27H44O2 400.64 1,25-(OH)2D3 C27H44O3 416.64
25-OHD
25-OHD 3 3 C27HC4427OH244O2 400.64
400.64 1,25-(OH)
1,25-(OH) 2D23D3 C H
C27H44O33
27 44 O 416.64
416.64
25-OHD3 C27H44O2 400.64 1,25-(OH)2D3 C27H44O3 416.64
1-OHD
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400.64
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400.64
1-OHD3 C27H44O2 400.64
1-OHD
1-OHD
3 3 C27HC4427OH2 44O2 400.64
400.64
1-OHD3 C27H44O2 400.64
1-OHD3 C27H44O2 400.64
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among manybefore
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low low
of concentration
these
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polysaccharides,
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of levels
of at
using which
atatwhich
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proteins
chromatographic
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sample
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or analogues
lipids,
DDanalogues
preparation
preparation among
techniques.
procedures
procedures are
are present
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However,
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before
before
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the components,
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determination of concentration
these substances levels
using which vitamin
chromatographic analogues
techniques. are present
However,
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The
in suchchemical
complexity
samples,
components, instability,
as of
makefood
well dealing
matrices
necessary
as the low with
the certain
containing
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of such
polysaccharides,
effective
levels at as light,
sample
which oxygen
proteins
preparation
vitamin D or pH,
oranalogues
lipids, and
among
proceduresaretheir easy
many
before
present
in
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their the samples,
separation
chemical
separation
in such
their and make
samples,
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instability,determination
determination
make
instability, dealing
necessary the
of application
of
with
these
the
dealing with these
certain
substances
application
certain of
substanceseffective
factors
of using
using such
effective
factors such sample
as preparation
chromatographic
light,
chromatographic
sample oxygen procedures
techniques.
or
techniques.
preparation
asconditions
light, oxygen pH,
procedures
or pH,it and before
However,
their easy easy
and their
However,
before
other isomerization
components,
the separation as
and high
well temperature
as the
determination low make
concentration
of these this step
levels
substances and at the
using which vitamin
chromatographic atD which
analogues
techniques. is are
developed,
present
However,
in
the such
their samples,
their
separation
chemical
isomerization
the chemical
and
separation
isomerization make
at high
and necessary
instability,
determination
instability, dealing
temperature
determination
atinhigh the
dealing
of application
with
of
temperature make with
these certain
certain
make
these of
substances
factors
this
substances
this effective
factors
using
step such
such
and
using sample
as as
light,
the preparation
light,
chromatographic oxygen
oxygen
conditions
chromatographic oror
at procedures
pH,
techniques.
pH,
which and
and
techniques.
step and the conditions at which it is developed, isbefore
their
However,
their
it easy
developed,
However,
in suchdecisive
their aspects
chemical
samples, make the
instability,determination
necessary dealing
the with process
certain
application [3].
factors
of such
effective as
samplelight, oxygen
preparation or pH, and
procedures their easy
before
the
their isomerization
separation
chemical
their
isomerization
decisive and
chemical
at
aspects
decisive aspects at
instability,high
determination
instability,
high
inatin
the temperature
dealing
temperature of
dealing
determination
the these
withwith
make
determinationprocess make this
substances
certain
certain
this step
factors
factors
step
[3].
process [3]. and and
using
such
such
the the asconditions
chromatographic
as light,
light,
conditions oxygenat
oxygen
at which
or
which pH, it
techniques.
or pH, it and
and
isis developed,
However,
their easy
easy
developed,
the isomerization
separation
decisive and
aspects high
determination
in the temperatureof
determination these make this
substances
process step
[3]. and
using the conditions
chromatographic at which it
techniques. is developed,
However,
their chemical
isomerization
isomerization
decisive In aspects
generalinstability,
at at
high
interms,
the high dealing
temperature
temperature
determination
conventional with
make certain
make
process this factors
this stepand
step
[3].
solid-liquid such
and the asconditions
the light, oxygen
conditions
andatat or pH,ititand
which
which their easy (LLE)
is developed,
is developed,
decisive
their decisive
chemical aspects in the
instability, determination
dealing with certain [3]. extraction
processfactors such
(SLE)
asconditions
light, oxygen
liquid-liquid
or pH,it and
extraction
their easy
isomerization at high
aspects
widelyin temperature
the
applied determination
decisive aspects in the determination process [3].
have been theatmost techniques make
for this
process
thethe
extraction step
[3]. 3
of this and
vitaminthe at which is developed,
isomerization
decisive aspects inhigh temperature
the determination make this
process 3and
[3].step3 conditions at which it and its metabolites
is developed,
from food
decisive matrices.
aspects in theHowever, solid-phase
determination processextraction
[3]. 3 3 (SPE) and preparative chromatography combined
with SLE and LLE have also been applied as a common
3 3 sample pre-treatment in this field. Additionally,
a few applications of miniaturized techniques have
3 been performed with the same aim, although to a
3 presence of antioxidant agents was carried out at the
lesser extent. In most cases, saponification in the
beginning of the process in order to favor the separation of the analytes from the matrix and remove
interferences [3], although other hydrolysis procedures or deproteinization have also been applied to
accomplish this aim.
The application of saponification is focused on the chemical degradation of complex lipids present
in this type of sample that can preclude the determination of vitamins. This procedure is carried out
by a hydration reaction, in which ester bonds are broken by the action of an alkali agent, obtaining
hydrosoluble free fatty acids and glycerol that can be easily removed in the aqueous phase [43,44].
With this aim, a certain concentration of KOH in ethanol (EtOH) was used in most cases as a strong
alkali agent to carry out the reaction, and a high temperature (around 60–80 ◦ C) was applied in
order to reduce the process time [7,24–26,30,33,34,41] and avoid overnight reactions, which were
necessary when room temperature was applied [9,10,42]. However, some authors have reported short
reaction times (30–60 min) under room temperature with good results for the evaluation of milk-based
formulas, fruit juice and vegetable beverages, after a thorough optimization of time and temperature
factors [28,29]. In this sense, the work carried out by Kwak et al. [29] should be highlighted. In this
case, saponification using 150 mg of KOH, 150 mg of NH3 and 2.4 g of NaCl was performed during
5 min at room temperature, after dilution and subsequently extraction using 10 mL of isopropanol (IPA)
for the extraction of D3 from milk-based formulas. The extract was then evaporated and injected in a
HPLC-MS/MS system after reconstitution. The methodology was compared with the AOAC official
method [17], in which high-temperature (75 ◦ C) saponification during 30 min was necessary for the
determination of D2 and D3 in infant formula and adult nutritionals.
Additionally, due to the instability of these vitamins during the procedure [3], antioxidant
agents are usually added to the reaction media to assure their correct determination. In this sense,
ascorbic acid [9,24,34,35], pyrogallol [10,26], sodium ascorbate [7,36,42] and butylated hydroxytoluene
(BHT) [23,28] have been the most commonly antioxidant agents applied with this aim.
2.1. Conventional Extraction Techniques

As previously indicated, despite the fact that conventional SLE and LLE are complex and time
consuming, they have been the most widely applied extraction techniques for the evaluation of
food matrices (see Table 2). Nevertheless, other alternative procedures have also been sporadically
applied. In this sense, the use of the dilute-and-shoot strategy should be remarked. As an example,
Byrdwell et al. [27] applied this pre-treatment in combination with HPLC-mass spectrometry (MS)
and HPLC-UV determination for the evaluation of D3 in dietary supplements. The procedure
avoids the application of complex steps, limiting the method to sample weighing, addition of an
internal standard (IS) and dilution with the adequate solvent [43]. In this case, oil contained in the
supplement gel caps was diluted to 100 mL using methanol (MeOH) and dichloromethane (DCM)
in the composition MeOH/DCM (6/4, v/v), and was directly injected in the chromatographic system.
This study demonstrated the suitability of the methodology for the evaluation of not only D3, but also
triacylglycerols in supplement gel caps and oil samples in combination with both LC-MS and LC-UV
systems, showing that UV detection provided lower sample-to-sample relative standard deviation
than atmospheric pressure chemical ionization (APCI)-MS detection.
Table 2. Recent applications of conventional extraction techniques for the analysis of vitamin D-related compounds in food matrices.
Matrix Recovery
Analytes Sample Pre-Treatment Determination Technique LOD Comments Reference
(Amount) %
-APCI (+) was used as
ionization source.
-NH4 HCOO in MeOH was used as
make-up solvent.
-Different columns were evaluated.
-Derivatization was carried out
D2 , D3 , 25-OHD2 , Deproteinization (40 ◦ C, 1 h, using PTAD.
3-epi-25-OHD2 , Commercial breast milk EtOH (1 mL)); LLE 2 X -Deuterated compounds were used
UHPSFC-(QqQ)-MS/MS 70–107 0.02 µg/L (a) [4]
25-OHD3 , 3- (1 mL) (hexane/EtOAc (9/1, v/v) as ISs.
epi-25-OHD3 (2.5 mL)) -Comparison between saponification
and deproteinization.
-SRM was used for method validation.
-SFC conditions: fluoro-phenyl
column, mobile phase
(MeOH/NH4 HCOO/H2 O/CO2 ),
45 ◦ C.
-Vitamin A, E and amino acids and
pesticides were also evaluated.
Rice SLE (hexane (4 mL), 5 min
D3 HPLC-UV - - -LC conditions: C18 column, mobile [22]
(1 g) under N2 )
phase (ACN/H2 O), 30 ◦ C.
-Occurrence: 13.8–28.6 mg/kg.
ionization source.
-NH4 HCOO in MeOH was used as
make-up solvent.
-SRM was used for method validation.
Hydrolysis (α-amylase (50 -Vitamin K, A and D were
Infant formula, cereals,
mg): 45 ◦ C, 30 min, papain also evaluated.
adult nutritionals,
D 2 , D3 solution (5 mL)); LLE UHPSFC-(QqQ)-MS/MS 90–110 40 pg (a) -Derivatization was carried out [23]
mixed meals
(acidified MeOH (20 mL), using PTAD.
(2 g)
BHT in isooctane (10 mL)) -Isotopically labelled compounds
were used as surrogate ISs.
-SFC conditions: 1-AA column,
mobile phase
(MeOH/NH4 HCOO/H2 O/CO2 ),
45 ◦ C.
Table 2. Cont.
Matrix Recovery
(Amount) %
Milk and infant formulas:
VA-LLE
-ESI (+) was used as ionization source.
(MeOH/H2 O/isooctane
-Deuterated D3 was used as
(6.25/1.25/2.50, v/v/v), 2 min).
surrogate IS.
Fortified bread, bovine Bread: homogeneization
-Comparison of D2 and D3
D 2 , D3 milk, infant formula (EDTA (2 mL), ascorbic acid HPLC-(QqQ)-MS/MS - - [24]
supplement efficiency.
(4 g) (100 mg), vortex),
-LC conditions: C18 column, mobile
saponification (KOH in
phase (MeOH/H2 O/NH4 HCOO),
water, 60 ◦ C, 30 min), LLE
40 ◦ C.
(MeOH/isooctane, (2/1, v/v),
(30 mL))
Folch method
-Mineral and other fat-soluble
(CHCl3 /MeOH, (2/1, v/v)
vitamins were also evaluated.
Finfish, shellfish (450 mL)); saponification
D3 HPLC-UV - - -LC conditions: C18 column, mobile [25]
(30 g) (KOH in MeOH, 30 min,
phase (MeOH/ACN), -.
refluxing): LLE (petroleum
-Occurrence: 7.72–23.28 µg/kg.
ether, (50 mL))
-Derivatization was carried out
Milk powder, infant Saponification (KOH, using PTAD.
formulas, nutritional pyrogallol, EtOH, 1 h, -AOAC Official Method 2016.05.
D 2 , D3 UHPLC-(QqQ)-MS/MS 96–101 1.2–1.6 µg/kg [26]
formulas 70 ◦ C); LLE (isooctane (10 -Deuterated standards were used
(1.8–21.0 g or 10 mL) mL)); washing (H2 O) as ISs.
phase (MeOH/H2 O/FA), 40 ◦ C.
-Different columns were tested.
D3 , D2 , 25-OHD2 , -Derivatization was carried out
Deproteinization (ACN (8
25-OHD2 , using PTAD.
Human, cow, mare, goat mL), vortex 2 min, room
24,25-(OH)2 D2 , -Comparison between saponification
and sheep milk temperature (15 min)); HPLC-(QqQ)-MS/MS 88–99 0.27–0.47 pM [39]
24,25-(OH)2 D3 , and deproteinization.
(4 mL) LLE 2 X (hexane/DCM (1/4,
1,25-(OH)2 D2, -Deuterated standards were used
v/v) (12 mL))
1,25-(OH)2 D3 as ISs
phase (MeOH/H2 O/FA), -.
Table 2. Cont.
Matrix Recovery
(Amount) %
ionization source.
-SIM and full scan modes
were compared.
HPLC-(TSQ)-MS, -APCI and ESI were compared.
Dietary supplements Dilute-and-shoot strategic
D3 HPLC-(TSQ)-MS/MS, - - -D2 was used as IS. [27]
(-) (-)
HPLC-UV -Triacylglycerols were
also evaluated.
-LC conditions: hydrophilic
endcapping-C18 column, mobile
phase (MeOH/ACN/DCM), 10 ◦ C.
Method 1:
Saponification/hydrolysis
(KOH, sodium ascorbate,
NaOH, 1 h, 80 ◦ C, refluxing);
LLE (diethyl ether (50 mL);
EtOH/pentane (1/5, v/v) (60
D2 , D4 , pre-D2 , ionization source.
mL), pentane (50 mL and
pre-D4 , -Analysis was carried out after
20 mL)).
tachysterol2 , UV-B treatment.
Method 2:
tachysterol4 , Oyster mushroom HPLC-UV, -D3 was used as surrogate IS.
Saponification/hydrolysis 97 0.02–0.06 mg/kg [7]
lumisterol2 , (5 g) HPLC-(QqQ)-MS/MS -Different extraction methods
(KOH, sodium ascorbate,
lumisterol4 , were compared.
NaOH, 20 h, 18 ◦ C,
ergosterol, 22,23- -LC conditions: hydrophilic
refluxing); LLE (diethyl
dihydroergosterol endcapping-C18 column, mobile
ether (50 mL); EtOH/pentane
phase (MeOH/H2 O/FA), 40 ◦ C.
(1/5, v/v) (60 mL), pentane
(50 mL and 20 mL)).
Method 3: US-LLE X 3
(pentane (100, 100 and 50
mL), 10 min, 13 ◦ C)
-ESI (+) was used as
ionization source.
Fortified cereals,
-Deuterated D3 was used as IS.
cereals-based food, milk, Saponification (KOH, H2 O,
-SRMs were used for validation.
Pre-D3 , D3 fruit juice, yogurt, sun EtOH, 20 min, 80 ◦ C); LLE 2D-HPLC-UV-(QqQ)-MS/MS 96–105 0.5–0.8 µg/kg [41]
-Pre-D3 , D3 were analyzed together.
flower oil, egg yolk (ciclohexane, (41 mL))
-LC conditions: C8 and C18
(5–30 g)
columns, mobile phase
(MeOH/ACN/H2 O/FA), 35 ◦ C.
Table 2. Cont.
Matrix Recovery
(Amount) %
-Vitamin E was also evaluated.
Milk, infant formula,
Saponification (KOH, EtOH, -C8 and C18 columns
fruit juice,
D 2 , D3 BHT, 1 h, room temperature), HPLC-DAD - 0.82–1.57 ng (b) were compared. [28]
vegetable beverage
LLE (2× hexane (50 mL)) -LC conditions: C18 column, mobile
(20–25 g)
phase (MeOH/ACN), -.
-ESI (+) was used as
Dilution (H2 O, 10
ionization source.
mL);VA-LLE (IPA (10 mL)
-SRMs were used for validation.
Milk-based formula with (NH4 )2 SO4 );
D3 HPLC-(QqQ)-MS/MS 93–110 0.84 µg/kg -Comparison with official method. [29]
(0.5 g) saponification (KOH, NaCl,
NH3 , 30 min, room
phase (MeOH/H2 O/NH4 HCOO),
temperature)
40 ◦ C.
(a)limit of quantification (LOQ); (b) instrumental limit of detection (LOD); 1-AA:1-aminoanthracene; 2D-HPLC: two dimensional-high performed liquid chromatography; 3-epi-25-OHD2 :
3-epi-25-hydroxyvitamin D2 ; 3-epi-25-OHD3 : 3-epi-25-hydroxyvitamin D3 ; 25-OHD2 : 25-hydroxyvitamin D2 ; 25-OHD3 : 25-hydroxyvitamin D3 ; ACN: acetonitrile; AOAC: Association of
Official Analytical Chemists; APCI: atmospheric pressure chemical ionization; BHT: butylated hydroxytoluene; C8 : octasylane; C18 : octadecylsylane; D2 : vitamin D2 ; D3 : vitamin D3 ; D4 :
vitamin D4 ; DAD: diode array detector; DCM: dichloromethane; EDTA: ethylenediaminetetraacetic acid; ESI: electrospray ionization; EtOAc: ethyl acetate; EtOH: ethanol; FA: formic acid;
HPLC: high-performance liquid chromatography; IPA: isopropanol; IS: internal standard; LC: liquid chromatography; LLE: liquid–liquid extraction; LOD: limit of detection; MeOH:
methanol; MS: mass spectrometry; MS/MS: tandem mass spectrometry; Pre-D2 : previtamin-D2 ; Pre-D3 : previtamin-D3 ; Pre-D4 : previtamin-D4 ; PTAD: 4-phenyl-1,2,4-triazoline-3,5-dione;
QqQ: triple quadrupole; SIM: selected ion monitoring; SLE: solid–liquid extraction; SRM: standard reference material; TFA: trifluoroacetic acid; TSQ: tandem sector quadrupole; UHPLC:
ultra-high performance liquid chromatography; UHPSFC: ultra-high performance supercritical fluid chromatography; US-LLE: ultrasound assisted liquid–liquid extraction; UV: ultraviolet;
VA-LLE: vortex-assisted liquid-liquid extraction.
However, for other samples with higher complexity, the application of multiple step methodologies
has been found to be unavoidable so far. SLE and LLE have been applied to a wide variety of food
matrices including milk and milk-based formulas, rice, cereals, bread, different kinds of fish, mushrooms,
fruit juice, Separations
vegetables beverages,
2020, 7, oil or eggs, among others [4,7,22–26,28,29,40,41]. In those
x FOR PEER REVIEW 10 ofcases,
32 a great
range of solvents such as hexane, ethyl acetate (EtOAc), MeOH, isooctane, chloroform, petroleum
However, for other samples with higher complexity, the application of multiple step
ether, diethyl ether, EtOH,
methodologies has beenpentane,
found tocyclohexane,
be unavoidableisooctane or IPA,
so far. SLE and as well
LLE have beenas mixtures
applied of them have
to a wide
been used,variety
applyingof food volumes in the range
matrices including milk and1–100 mL andformulas,
milk-based carrying rice,out several
cereals, bread,repetitions
different in most
kinds of
cases, in order tofish,
get mushrooms,
a quantitative fruit juice, vegetables
extraction beverages,
of the targetoilanalytes
or eggs, [4,7,28].
among others [4,7,22– all cases
In almost
26,28,29,40,41]. In those cases, a great range of solvents such as hexane, ethyl acetate (EtOAc),
reported so far, the application of not only saponification [7,24–26,28,29,41], but also other hydrolysis
MeOH, isooctane, chloroform, petroleum ether, diethyl ether, EtOH, pentane, cyclohexane,
treatmentsisooctane
for the or evaluation
IPA, as wellof ascereals
mixturesand nutritional
of them have beenformulas [23]volumes
used, applying or deproteinization
in the range 1–100 for different
milk samples mL and[4,40] has out
carrying been carried
several out, prior
repetitions in most tocases,
extraction,
in order to get
favor the cleaning
a quantitative of the
extraction samples and
of the
to removetarget analytes [4,7,28].
interferences. WithInrespect
almost alltocases reported so far,
the efficiency the application
of these of not only despite
three strategies, saponification
saponification
[7,24–26,28,29,41], but also other hydrolysis treatments for the evaluation of cereals and nutritional
being the one most extensively applied, some studies have demonstrated the higher effectiveness of
formulas [23] or deproteinization for different milk samples [4,40] has been carried out, prior to
deproteinization
extraction,in releasing
to favor thevitamins
cleaning offrom
the milk
samples matrices
and to [4,40].
remove In this sense,With
interferences. it is respect
worth to mentioning
the the
work carried out byofGomes
efficiency these threeet al. [40], who
strategies, developed
despite a careful
saponification beingcomparative study of both
the one most extensively strategies for
applied,
the effectivesome studies have
extraction demonstrated
of eight vitamin theDhigher effectiveness
analogues of deproteinization
(i.e., D in releasing vitamins
3 , D2 , 25-OHD2 , 25-OHD2 , 24,25-(OH)2 D2 ,
from milk matrices [4,40]. In this sense, it is worth mentioning the work carried out by Gomes et al.
24,25-(OH)[40],
2 D3who
, 1,25-(OH)
developed 2 Da2 careful
and 1,25-(OH)
comparative 2 D3study
) from human, cow, mare, goat and sheep milk samples
of both strategies for the effective extraction of
prior to their determination by HPLC-MS/MS.
eight vitamin D analogues (i.e., D3, D2, 25-OHD2, 25-OHD With this aim, 4 mL 2of
2, 24,25-(OH) D2, sample
24,25-(OH) was
2D3, deproteinized
1,25-
using 8 mL of 2acetonitrile
(OH) D2 and 1,25-(OH) (ACN), vortexed
2D3) from human,for cow,2 min,
mare, incubated
goat and sheep for milk
15 min at room
samples prior temperature
to their and
subsequently determination
centrifuged. by HPLC-MS/MS. With this aim,
For saponification, 4 mL 4 mL
of 8ofMsample was deproteinized
ethanolic KOH solution usingand8 mL 2.8ofmL of 1 M
acetonitrile (ACN), vortexed for 2 min, incubated for 15 min at room temperature and subsequently
ascorbic acid were added and, after vortexing for 2 min, the sample was hatched at room temperature
centrifuged. For saponification, 4 mL of 8 M ethanolic KOH solution and 2.8 mL of 1 M ascorbic
for 12 h, before
acid were centrifugation. In both for
added and, after vortexing cases,
2 min,a the
subsequent
sample wasextraction
hatched at room of the obtained
temperature for supernatant
12
was carried h, out
before using 12 mL ofInhexane/DCM
centrifugation. (1/4, v/v)extraction
both cases, a subsequent twice, priorof thetoobtained
determination.
supernatantAs was can be seen
carried out using 12 mL of hexane/DCM (1/4, v/v) twice, prior to determination.
in Figure 1, larger efficiency in terms of concentration was obtained for all target analytes using the As can be seen in
Figure 1, larger efficiency in terms of concentration was obtained for all target analytes using the
deproteinization treatment, which demonstrated the higher suitability of this procedure, at least for
deproteinization treatment, which demonstrated the higher suitability of this procedure, at least for
the analysis of milk-based
the analysis of milk-based matrices.
matrices.
Deproteinization
Saponification
Figure 1. Comparison of deproteinization and saponification effectiveness for the release of vitamin
Figure 1. Comparison of deproteinization and saponification effectiveness for the release of vitamin D
D analogues from milk samples. Reproduced from [40], with permission from Elsevier, 2020.
analogues from milk samples. Reproduced from [40], with permission from Elsevier, 2020.
Although in general terms, saponification, hydrolysis or deproteinization in combination with
Although in extraction
only one general terms, saponification,
technique hydrolysis
have been found sufficient or correct
for the deproteinization in combination
release of the analytes from with
the complextechnique
only one extraction food samples, in some
have beencases,
foundthe sufficient
performanceforof the
additional
correcttreatments
release or of extraction
the analytes from
procedures has been necessary [24,25]. As an example, Mohanty et al. [25] developed a
the complex food samples, in some cases, the performance of additional treatments or extraction
methodology for the extraction of D3 from finfish and shellfish based on a modified Folch method
procedures has 450
using been
mLnecessary [24,25].
of the mixture CHClAs an example, Mohanty et al. [25] developed a methodology
3/MeOH (2/1, v/v) followed by alkaline saponification under
for the extraction of D3 from finfish and shellfish 10 based on a modified Folch method using 450 mL of
the mixture CHCl3 /MeOH (2/1, v/v) followed by alkaline saponification under reflux for 30 min and
LLE of fish oil using 50 mL of petroleum ether prior to determination by HPLC-UV. The thorough
extraction developed and clean extract obtained from the application of the described method allowed
the authors to determine D3 and other fat-soluble vitamins in a great variety of fishes with a high fat
content and, therefore, the evaluation of the contribution of these food commodities to the vitamin D
daily intake in the population with an occurrence in the range 7.72–23.28 µg/kg in seventeen especies
evaluated, including marine and freshwater fishes and moluscs.
However, despite the necessity of carrying out strong digestive treatments to get an efficient
release of vitamin D and their analogues from food samples, there exists a remarkable trend to avoid
them in order to simplify the procedures and keep the whole information about the matrix vitamin
forms distribution in this kind of analysis [23]. In this regard, the fact is that in only a few cases,
LLE and SLE have been directly applied for the evaluation of these analytes in food matrices [22,24].
Especially remarkable is the work carried out by David et al. [22], in which the authors applied a
previously developed methodology [45], for the determination of vitamin D, as well as other fat-soluble
vitamins in rice samples from different parts of Nigeria and Thailand by direct extraction of the
matrix using 4 mL of hexane under N2 steam for 5 min. After centrifugation, 1 mL of supernatant
was evaporated, reconstituted in butanol and subsequently injected in the HPLC-UV system for the
separation and determination of the target compounds. Although the Thailand species presented
lower occurrence (13.8 mg/kg) than the Nigerian products (23.2–28.6 mg/kg), all species turned out to
be poor sources of vitamin D, confirming the previous studies carried out in this area, in which the
content of this vitamin has been associated with food commodities with a higher amount of fat such as
fish, eggs and whole milk, among others [8–10].
Despite the fact that conventional extractions are the most commonly applied for the
analysis of these analytes in food samples, the high amount of solvents used, as well as the
complexity of the procedure, constitute clear disadvantages of their application. Apart from that,
additional pre-treatments to carry out an effective extraction and clean-up of the sample, including
hydrolysis or deproteinization, are still fundamental steps to achieve the successful analysis of
compounds. For this reason, the search of new protocols that allow the miniaturization of such
methods is an issue of special interest for the scientific community.
2.2. Solid-Phase Extraction

As shown in Table 3, SPE has been applied in this area for the extraction and/or sample
clean-up of cereals and flour products [30], edible plants, seaweeds, fruit and fruit seeds [9,31],
dietary supplements [31], porcine fat, meat and liver [42], cod liver supplements [8], beef, egg,
chicken and fish [10]. The development of a previous digestion step, generally saponification,
was the procedure most commonly carried out [9,10,30,42], although in some cases, the complexity
of the evaluated samples also requires the performance of a solvent-based extraction between both
stages [10,42], whereas in only one report, SPE has been applied directly after dilution of the sample using
hexane [8]. In this last case, Bartoluccia et al. carried out a reliable methodology for the confirmation of
the content of D3 in cod liver oil-based supplements after the detection of D3 intoxication in several
consumers of this kind of pill. Authors pointed out the necessity of using a normal phase SPE with
an apolar solvent eluent as the sample pre-treatment due to the lipidic nature of the sample since
other alternatives, such as the application of C18 sorbent in combination with polar eluent, have not
demonstrated to yield an adequate release of fat-soluble vitamins from this type of sample. With this
aim, 0.2% of the content of each capsule was diluted up to 1 mL with hexane and the sample was loaded
onto a NH2 -propyl-SPE cartridge previously conditioned with the same solvent. After washing with
hexane, the target compound was eluted with 1 mL of EtOAc. Finally, the extract was dried and the
residue reconstituted with IPA prior to the separation and determination by HPLC-triple quadrupole
(QqQ)-MS/MS. After validation of the methodology, the analysis of the samples showed a concentration
of D3 three orders of magnitude higher than the one indicated in the product (1.5 µg/capsule) for
some of the batches evaluated. These results emphasize the necessity of the development of selective
and reliable methodologies that allow the correct evaluation of all kinds of commercialized dietary
supplements which guarantees food safety and protects consumer health.
Table 3. Recent applications of solid-phase extraction for the analysis of vitamin D-related compounds in food matrices.
Matrix Sample Elution Solvents Determination

Analytes Sorbent Recovery % LOD Comments Reference
(Amount) Pre-Treatment (Volume) Technique
-Online SPE was carried out.
Saponification (KOH, -Vitamin K was
EtOH, hexane, 60 ◦ C, also evaluated.
Cereal and 30 min); vortex, MeOH using a -SRM was used for
C18 colum
D2, D3 flour-based products centrifugation, gradient at different UHPLC-PDA >70 0.05–0.12 mg/L method validation. [30]
(-)
(1.5 g) upper layer drying, flow rates -Occurrence: 1.0–2.9 mg/kg.
reconstitution and -LC conditions: C18 column,
filtration mobile phase (MeOH/H2 O),
30 ◦ C.
- ESI (+) was used as
ionization source.
-Deuterated compounds
Saponification (KOH,
Plants, seaweeds, Diatomaceous were used as ISs.
D2 , D3 , 25-OHD2 , ascorbic acid in H2 O, Petroleum ether
fruit seed earth column HPLC-(QqQ)-MS/MS 94–101 0.5 µg/kg -Derivatization was carried [9]
25-OHD3 EtOH, overnight, (2 × 30 mL)
(-) (-) out using PTAD.
room temperature)
-LC conditions: C18 column,
mobile phase (MeOH/H2 O),
-.
Saponification (KOH, -ESI (+) was used as
sodium ascorbate, ionization source.
EtOH; overnight, -Derivatization was carried
Porcine fat and liver room temperature); Silica column 6% IPA in heptane; out using PTAD.
D3 , 25-OHD3 HPLC-(QqQ)-MS/MS 72–124 <0.1 µg/kg (a) [42]
(0.2–1 g) LLE (20% EtOAc in (500 mg) 10% IPA in heptane -LC conditions: C18 column,
heptane); drying; mobile phase
reconstitution (1% (MeOH/H2 O/FA/methyl
IPA in heptane) amine), 50 ◦ C.
-9 more vitamins were
SLE (MeOH); also analyzed.
Fruit, fruit juices, 60% MeOH in water
centrifugation; C18 column -Occurrence: 2.5–74 µg/kg.
D3 dietary supplements (3 mL); MeOH (3 HPLC-DAD 86–95 5.28 mg/L(b) [31]
filtration; hydrolysis (500 mg) -LC conditions: C18 column,
(2 g) mL); DCM (3 mL)
(carrez solutions) mobile phase
(MeOH/H2 O/TFA), 30 ◦ C.
ionization source.
Cod liver oil-based -Deuterated D3 was used
NH2 -propyl
dietary supplements Dilution in hexane EtOAc as IS.
D3 column HPLC-(QqQ)-MS/MS - - [8]
(0.2% of (up to 1 mL) (1 mL) -Occurrence: 1.31 mg/pill.
(200 mg)
each capsule) -LC conditions: C18 column,
mobile phase
(MeOH/H2 O), -.
Table 3. Cont.
Matrix Sample Elution Solvents Determination

Analytes Sorbent Recovery % LOD Comments Reference
(Amount) Pre-Treatment (Volume) Technique
ionization source.
-D2 and 25-OHD2 were used
as ISs.
Saponification (KOH,
-CRM and ground pork
pyrogallol, EtOH;
control sample were used for
overnight, room
method validation.
temperature); LLE X
Pork, beef, egg, -Poultry was also analyzed.
4 (petroleum
chicken, turkey, D3: HPLC-DAD, -Further purification was
ether/diethyl ether, Silica colum 0.4–3.0% IPA
D3 , 25-OHD3 dolphinfish, salmon, 25-OHD3 : 73–88 0.4 µg/kg carried out after SPE using [10]
2/8, v/v); washing (5% (1000 mg) in DCM
tilapia HPLC-(QqQ)-MS/MS normal phase
KOH); drying (N2 );
(20 g) semi-preparative HPLC.
reconstitution (0.1%
-Occurrence: D3
IPA in ciclohexane
(1.1–9.2 µg/kg);
(0.5 mL)/DCM
25OHD3 (0.9–3.6 µg/kg).
(0.6 mL))
-LC conditions: C18 column,
mobile phase D3 :
(MeOH/H2 O),-; 25-OHD3 :
(MeOH/H2 O/acetic acid), -.
(a)LOQ, (b) instrumental LOD; 25-OHD2 : 25-hydroxyvitamin D2 ; 25-OHD3 : 25-hydroxyvitamin D3 ; ACN: acetonitrile; APCI: atmospheric pressure chemical ionization; C18 : octadecylsylane;
CRM: certified reference material; D2 : vitamin D2 ; D3 : vitamin D3 ; DAD: diode array detection; DCM: dichloromethane; ESI: electrospray ionization; EtOAc: ethyl acetate; EtOH: ethanol;
FA: formic acid; HPLC: high-performance liquid chromatography; IPA: isopropanol; IS: internal standard; LC: liquid chromatography; LLE: liquid–liquid extraction; LOD: limit of detection;
MeOH: methanol; MS: mass spectrometry; MS/MS: tandem mass spectrometry; PDA: photodiode array detector; PTAD: 4-phenyl-1,2,4-triazoline-3,5-dione; QqQ: triple quadrupole; SLE:
solid–liquid extraction; SPE: solid-phase extraction; SRM: standard reference material; TFA: trifluoroacetic acid; UHPLC: ultra-high performance liquid chromatography.
Regarding the type of sorbent applied in the different methods developed so far, it should by
highlighted that, as indicated in the previous example, the use of silica as normal phase sorbent [10,42]
has been commonly applied for the evaluation of samples with a high fat content such as porcine fat
and liver [42], as well as meat, fish and eggs [10]. Indeed, the AOAC proposed in 1996 the AOAC 995.05
method [20] for the determination of cholecalciferol in infant formulas and enteral products using a
silica-SPE after saponification and extraction using hexane, which pointed out the improvement with
respect to previously developed methods in which LLE or SLE had been applied [19]. On the contrary,
non-polar cartridges based on C18 have been applied for the extraction of vitamin D analogues in
cereal and flour products [30] or fruit-based commodities [31], and diatomeous earth for the evaluation
of plants, seaweeds and fruit seeds [9]. The application of this sorbent in combination with 60 mL of
petroleum ether as the elution solvent was used for the determination of D2 , D3 , 25-OHD2 and 25-OHD3
in several types of seaweeds and different parts of Australian plants like seeds, leaves or stems after
saponification of the samples overnight at room temperature. The whole methodology was validated
and applied for the determination of the target compounds with recovery values in the range 94–101%
and a limit of detection (LOD) of 0.5 µg/kg. However, the authors remarked that the determination of
25-OHD3 in roasted and milled seeds of wattleseeds could not be successfully performed due to the
high amount of matrix interferences obtained in the final extract, thus demonstrating the inefficiency
of the selected sorbent to carry out the release of 25-OHD3 from this commodity.
As indicated at the beginning of this section, in some cases, the characteristics of the evaluated
samples have made necessary the combination of SPE with other extraction and clean-up procedures to
get an efficient extraction. Especially remarkable is the work carried out by Bilodeau et al. [10], in which
the evaluation of D3 and its metabolite 25-OHD3 was carried out in pork, beef, egg, chicken, turkey,
dolphinfish, salmon and tilapia, using a HPLC-diode array detector (DAD) for the quantification
of D3 and HPLC-(QqQ)-MS/MS for 25-OHD3 . Sample pre-treatment consisted of an initial alkaline
saponification of the sample at room temperature using pyrogallol as the antioxidant agent, followed by
LLE four times with 150 and 75 mL of petroleum ether/diethyl ether (2/8, v/v), washing with 5% KOH
(one time) and water (twice), subsequent SPE, using 1 g of silica and 0.4–3% of IPA/DCM as the
elution solvent, and semi-preparative HPLC using a normal phase column. Recovery values in the
range 73–88% and an LOD of the method of 0.4 µg/kg demonstrated the suitability of the developed
methodology. The reduction of matrix interferences and noise was considerable after de-application of
the semi-preparative HPLC clean-up, especially for the determination of D3 by UV, as can be seen in
Figure 2, in which the HPLC-UV chromatograms of the separation between D3 and the IS, used for its
determination (D2 ), in a solvent standard and in a real sample submitted to the developed methodology
are shown.
As it has been demonstrated throughout the discussed literature, the application of SPE involves
an improvement in the extraction and clean-up efficiency for the analysis of vitamin D analogues.
In fact, the AOAC included in 1996 this procedure as alternative to SLE and LLE, commonly used with
this aim. However, despite the mentioned advantages, it is true that the application of hydrolysis
treatments and even previous solvent-based extractions are still necessary in many of the developed
approaches, as well as the use of a high amount of organic solvents as the eluents. It is remarkable
the low variety of sorbents tested in this area, which is an aspect that could considerably enhance the
relevance of this technique, extending their application to a wider variety of matrices and improving
the extraction and clean-up efficiency achieved.
2.3. Preparative Chromatographic Techniques

Preparative liquid chromatography has also been proposed as a suitable technique to get an
adequate cleaning of the sample and to avoid the presence of interferences that could preclude the
determination of vitamin D and its analogues in food matrices (see Table 4). In fact, the AOAC Official
Method 2002.05 [21] describes the use of this technique for the evaluation of D3 and D2 in fatty food,
adding a UV detector at the outlet position that allows establishing the most accurate collection window
and removing the2020,
Separations highest
7, x FORamount of matrix interferences.
PEER REVIEW 17 of 32
Figure 2. HPLC-UV chromatograms of a solvent standard (A) and in a real sample extracted using
Figure 2. HPLC-UV chromatograms of a solvent standard (A) and in a real sample extracted using the
the developed methodology (B). Reproduced from [10], with permission from Elsevier, 2020.
developed methodology (B). Reproduced from [10], with permission from Elsevier, 2020.
As it has been demonstrated throughout the discussed literature, the application of SPE
Following
involvestheanguidelines
improvement of in
thetheofficial method,
extraction all applications
and clean-up ofthe
efficiency for theanalysis
technique carried
of vitamin D out so far
have beenanalogues.
performed using
In fact, thesilica
AOAC asincluded
the stationary
in 1996 phase for theasevaluation
this procedure alternative ofto cacao-based
SLE and LLE, food [32],
commonly
yogurt, dietary used with this
supplements andaim. However, despite
margarine the mentioned
[33], fish and seafood advantages,
[34], and it isorange
true thatjuice
the [35] and
application of hydrolysis treatments and even previous solvent-based extractions are still necessary
applying an initial step of saponification followed by LLE, before LC separation. As an exception,
in many of the developed approaches, as well as the use of a high amount of organic solvents as the
Byrdwell eluents.
et al. [35] carried out
It is remarkable the the determination
low variety of sorbents of D3ininthis
tested fortified orange
area, which juicethat
is an aspect applying
could an LLE
directly to the sample using ethyl ether and petroleum ether with ascorbic acid as the antioxidant agent
considerably enhance the relevance of this technique, extending their application to a wider variety
of matrices and improving the extraction and clean-up efficiency achieved.
and subsequent clean-up by preparative LC with a (25.0 cm × 4.6 mm, 5 mm) silica column using a
mixture of2.3.
IPA/methyl tert-butyl ether
Preparative Chromatographic (MTBE)/cyclohexane/heptane (0.05/0.2/4.875/4.875, v/v/v/v) as
Techniques
the elution solvent. In this case, the particular characteristics of the sample, which presents a very low
Preparative liquid chromatography has also been proposed as a suitable technique to get an
fat contentadequate
in comparison with the rest of the analyzed
cleaning of the sample and to avoid the presence matrices shown inthat
of interferences Tablecould4, allowed the omission
preclude the
of a saponification step.of The
determination authors
vitamin D andremarked
its analoguesthatinthe
foodapplication
matrices (seeofTable
LLE4). andIn preparative
fact, the AOAC LC together
Official
with the use of DMethod 2002.05 [21] describes the use of this technique for the evaluation of D3 and D2 in
2 as IS and the analysis by HPLC-UV and HPLC-MS constitutes the best combination
fatty food, adding a UV detector at the outlet position that allows establishing the most accurate
of elements taken from the AOAC Official Methods 992.26 and 200.05 for the determination of D3 in
collection window and removing the highest amount of matrix interferences.
the selected matrix.
On the contrary, both Kühna et al. [32], who carried out the evaluation of cacao-based products,
Nestola and Thellmann [33], who analyzed yogurt, dietary supplements and margarine, as well
as Byrdwell et al. [34], who determined the content of vitamin D analogues in fish and seafood,
described the necessity of applying a saponification step followed by LLE using hexane or a mixture
of petroleum ether/diethyl ether to accomplish an adequate release of the analytes from the samples
analyzed in each case. Especially remarkable is the evaluation of the influence of the saponification step
carried out by Byrdwell et al., in which a comparison of of the amount of alkaline solution (30 and 60 mL
of 50% KOH solution) effect on the reproducibility17 of the method was done, even though subsequently
cleaning with the semi-preparative LC was applied. Results of the National Institute of Standards and
Technology (NIST) standard reference material (SRM) of infant/adult nutritional formula and pulled
salmon samples demonstrated that a stronger saponification increases the reproducibility between
samples due to the increase in the matrix digestion [34].
Table 4. Recent applications of preparative chromatography for the analysis of vitamin D-related compounds in food matrices.
Matrix Sample Determination

Analytes Column Mobile Phase Recovery % LOD Comments Reference
(Amount) Pre-Treatment Technique
-ESI (+) used as
ion source.
-Occurrence:
1.5–54.8 µg/kg.
Cacao, cacao-based Saponification -Deuterated D3 and D2
D 2 , D3 food (KOH, -); Silica column - HPLC-(QTrap)-MS/MS - - were used as ISs. [32]
(-) LLE (hexane) -Derivatization was
carried out using PTAD.
-LC conditions: C18
(ACN/H2 O/NH4 HCOO/FA), -.
-EI used as ion source.
-Comparison with
HPLC-MS and
HPLC-UV
determination were
Homogeneization carried out using
(hexane; different sample
water/EtOH (1/1, pre-treatments.
Yogurt, dietary
v/v)), saponification -Online-HPLC-GC was
supplement,
D 2 , D3 (KOH, 60 ◦ C, 30 Silica column 0.1% IPA in DCM GC-(ToF)-MS - 50–150 pg performed reducing [33]
margarine
min); washing of procedure complexity.
(-)
hexane phase -Deuterated D2 and D3
(water/EtOH (1/1, were used as ISs.
v/v)) -Determination was
carried out using pyro-
isomers due to the
transformation reaction
occuured at
high temperature.
Table 4. Cont.

Analytes Column Mobile Phase Recovery % LOD Comments Reference
-APCI (+) used as
ion source
-D2 used as IS.
Saponification -Study of the
(KOH, EtOH, improvement of the
ascorbic acid, 75 ◦ C, saponification process.
Finfish, shellfish MeOH/ACN (2/8, HPLC-DAD;
D3 30 min, refluxing); Silica column - <2.0 µg/kg (a) -SRM was used for [34]
(5–10 g) v/v) HPLC-(TSQ)-MS
LLE (petroleum method validation.
ether/diethyl ether: -Occurrence:
AOAC 992.26) (0.5–1.8 µg/kg)
-LC conditions: C18
(MeOH/ACN), -.
ionization source.
IPA/MTBE/ UV: -Occurrence:
Fortified orange LLE (Ethyl ether,
ciclohexane/heptane HPLC-UV-(TSQ)-MS, 0.298 µg/kg 10.7–16.63 µg/kg.
D3 juice petroleum ether, Silica column - [35]
(0.05/0.2/4.875/4.875, HPLC-(IT)-MS MS: -D2 was used as IS.
(30 mL) ascorbic acid)
v/v/v/v) 1.175 µg/kg -LC conditions: C18
(MeOH/ACN), 40 ◦ C.
(a) LOQ; ACN: acetonitrile; AOAC: Association of Official Analytical Chemists; APCI: atmospheric pressure chemical ionization; C18 : octadecylsylane; D2 : vitamin D2 ; D3 : vitamin D3 ;
DAD: diode array detection; DCM: dichloromethane; EI: electron impact; ESI: electrospray ionization; EtOH: ethanol; FA: formic acid; HPLC: high-performance liquid chromatography;
IPA: isopropanol; IS: internal standard; IT: ion trap; LC: liquid chromatography; LLE: liquid–liquid extraction; LOD: limit of detection; MeOH: methanol; MTBE: methyl tert-butyl-ether;
MS: mass spectrometry; MS/MS: tandem mass spectrometry; PTAD: 4-phenyl-1,2,4-triazoline-3,5-dione; QTrap: quadrupole linear-ion trap; SRM: standard reference material; ToF: time-of
flight; TSQ: tandem sector quadrupole; UV: ultraviolet.
This procedure constitutes an adequate complement step to both solvent and sorbent-based
extractions in order to improve the clean-up step, particularly in fat content samples for which normal
stationary phases have shown good results. In fact, it could be considered as an alternative to the
application of SPE since the use of off-line set-ups usually simplifies the procedures and reduces the
consumption of solvents.
2.4. Miniaturized Techniques

The current trends in analytical chemistry are focused on the development of environmentally
sustainable methodologies that enable a decrease in the consumption of harmful chemicals and diminish
the impact of chemical activity on the environment. In this sense, miniaturized techniques that allow
a reduction of cost, time and toxic and hazardous organic solvents consumption, as well as which
require lower amounts of samples, are being increasingly used [46]. However, despite the undeniable
advantages obtained by the application of miniaturized techniques and their wide application in
other areas, their use for the determination of vitamin D and vitamin D analogues is strikingly low.
As it is shown in Table 5, there are only a few reports in the area for the determination of these
substances in food samples using magnetic-micro-dispersive solid-phase extraction (m-µ-dSPE) [37,39]
and dispersive liquid–liquid microextraction (DLLME) [36,38] for the evaluation of milk, infant
formulas, green vegetables, milk/fruit-based beverages and cereals. It is worth mentioning that
only in one case, saponification was necessary prior to the application of the extraction process [36],
whereas deproteinization was applied in the case of cereals and fruit/milk-based beverages [36,39].
A fast and simple methodology was developed by Viñas et al. [38] based on the application of
DLLME for the evaluation of infant formulas (50–250 mg) and green vegetables (i.e., spinach and cos,
iceberg and lamb’s lettuce) (0.2–2.0 g). With this aim, an initial SLE using 3 mL of ACN as the extraction
solvent was developed. Then, the extract obtained was used as the dispersant and mixed with 150 µL
of carbon tetrachloride (extracting solvent). The mixture was rapidly injected in 6 mL of water and
manually shaken for some seconds to favor the cloudy spread formation enabling the simultaneous
preconcentration of the target analytes (D2, D3 ) and clean-up of the sample. After centrifugation, the
small carbon tetrachloride drop (around 50 µL) containing the target compounds was taken from the
bottom of the tube, dried and reconstituted in the same volume of ACN prior to injection in a HPLC-ion
trap (IT)-MS. The good recovery values obtained in the range 88–103% and the LODs between 3.1
and 4.0 µg/L demonstrated the efficiency of the technique for the evaluation of the complex samples
selected. In addition, the method demonstrated the simplification of the procedure in comparison with
methods described until now and a reduction in the extraction time and organic solvents consumption.
Table 5. Recent applications of miniaturized extraction techniques for the analysis of vitamin D-related compounds in food matrices.

Analytes Technique Recovery % LOD Comments Reference
Saponification (H2 O, KOH,
DLLME
Wheat flour, EtOH, sodium ascorbate; -) -LC conditions: C18
(dispersant: EtOH
D3 powder bread (ultrasounds, 5 min, 25 ◦ C); HPLC-UV-Vis 87–94 0.7 µg/kg column, mobile phase [36]
(650 µL); extractant:
(2.0 g) deproteinization (MeOH), 25 ◦ C.
octanol (80 µL))
(carrez solutions)
m-µ-dSPE -Occurrence: D2 : 6.5–10.2
(sorbent: µg/L; D3 : 2.7–8.8 µg/L.
Milk Dilution (phosphate buffer,
D2, D3 Fe3 O4 @PPy (30 mg); HPLC-UV 72–90 0.02–0.05 µg/L -LC conditions: C18 [37]
(1 mL) 9 mL)
elution solvent: column, mobile phase
ACN (1 mL)) (MeOH/ACN), -.
ionization source.
-Vitamin K1 , K2 and K3
were also evaluated.
-CRM were used for
Infant formula DLLME
method validation.
(50–250 g), green (dispersant: ACN
D2, D3 SLE (ACN, 3 mL) HPLC-(IT)-MS 88–103 3.1–4.0 µg/L -Different LC columns [38]
vegetables (3 mL); extractant:
were evaluated.
(0.2–2 g) CCl4 (150 µL))
-DAD was also applied
as detector.
-LC conditions: C18
(ACN/H2 O/IPA), 25 ◦ C.
m-µ-dSPE -Other fat-soluble vitamins
Fruit juice, milk (sorbent: were also evaluated.
Deproteinization (ACN,
D2 beverage C18 @Fe3 O4 (30 mg); CLC-UV -LC conditions: C18 [39]
0.9 mL)
(0.1 mL) elution solvent: column, mobile phase
ACN (-)) (ACN), -.
ACN: acetonitrile; APCI: atmospheric pressure chemical ionization; C18 : octadecylsylane; CLC: capillary liquid chromatography; CRM: certified reference material; D2 : vitamin D2 ; D3 :
vitamin D3 ; DAD: diode array detection; DLLME: dispersive liquid–liquid microextraction; EtOH: ethanol; HPLC: high-performance liquid chromatography; IPA: isopropanol; IT: ion trap;
LC: liquid chromatography; LOD: limit of detection; MeOH: methanol; m-µ-dSPE: magnetic-micro-dispersive solid-phase extraction; MS: mass spectrometry; PPy: polypyrrole; SLE:
solid–liquid extraction; UV: ultraviolet.
The use of nanomaterials as sorbents in sample pre-treatments of food matrices is a strategy

widely applied due to the particular characteristics of such materials that allows the miniaturization of
the extraction and clean-up procedures, minimizing the sample and solvent amount requirements,
simplifying the methodologies and providing very good efficiencies with a minimal environmental
impact. Particular interest has reached the magnetic nanomaterials. Those not only provide a high
surface area, increasing the active point, in which the sorbent can interact with the substances of
interest and present high tuneable capacity, favoring the versatility and specificity of the extractions as
occurs with the rest of nanomaterials, but their use also simplifies even more the procedures as a result
of their magnetic properties, which allows the easy separation of the sorbent and the sample using an
external magnet [47]. These advantages were precisely the ones provided by the sorbents developed by
Jiao et al. [37] and Hu et al. [39] for the determination of vitamin D using m-µ-dSPE. Particularly simple
was the methodology described by Jiao et al. [37] using polypyrrole (PPy)-coated Fe3 O4 nanoparticles
(PPy@Fe3 O4 ) to carry out the extraction and preconcentration of D3 and D2 from milk samples prior to
their determination by HPLC-UV. With this aim, 1 mL of sample was initially diluted with 9 mL of
buffer phosphate solution. Then, the m-µ-dSPE was directly performed without any additional step by
using 30 mg of PPy@Fe3 O4 . After stirring for 10 min, the sorbent was magnetically separated from
the sample solution with the assistance of a magnet and then washed with 2 mL of water. Finally,
the analytes were desorbed with 1 mL of ACN by ultrasonication for 5 min. The sorbent was again
separated using an external magnet and the eluate dried, reconstituted and submitted to HPLC-UV
analysis. The good recovery values obtained in the range 72–90% and the low LODs (0.02–0.05 µg/L)
demonstrated the effectiveness of the π–π interactions between the sorbent commodities and the target
analytes, as well as the good performance of the developed methodology with a pre-treatment time of
only 15 min and avoiding the digestion steps.
The advantages of these techniques in terms of simplicity and chemicals consumption are
undeniable and the great variety of sorbent and solvent materials that could be applied increases
exponentially the range of application of these methods. However, their application to solid samples
still involves the use of an initial pre-treatment using a high volume of solvents which could be
considered as a disadvantage of their application. In this sense, only a few approaches have been
applied so far. The application of other miniaturized procedures such as matrix solid phase dispersion
could be an interesting alternative to solve this problem.
3. Recent Applications of Chromatographic Techniques

In the last thirty years, the use of chromatographic techniques for the separation and determination
of vitamin D and its analogues has become a common trend in this area due to the advantages that
these techniques offer with respect to bio-assay methods, particularly the possibility of carrying out
the analysis of individual analytes, providing an accurate evaluation of the levels of each analogue in
the organism [10].
Regarding the most recent applications, as shown in Tables 1–4, the combination of LC with
UV or MS detection have been the most common strategies [3], whereas gas chromatography (GC)
applications have been hardly reported due to the easy isomerization of vitamin D analogues at
high temperature [3], which would preclude the individual determination of this group of analytes.
The application of other chromatographic techniques in the last ten years has been reduced to the
use of supercritical fluid chromatography (SFC) hyphenated with MS. However, it has been applied
in few cases owing to the current stage of development of this technique in terms of commercial
instrumentation, etc. [48].
3.1. Liquid Chromatography Applications

Regarding the use of LC, different modalities have been applied, including HPLC [7–10,
22,24,25,27–29,31,32,34–38,40,42], two dimensional-HPLC (2D-HPLC) [41], ultra-high performance
liquid chromatography (UHPLC) [26,30] and capillary liquid chromatography (CLC) [38], although,
among them, HPLC has been the most extended as a consequence of its higher availability in
most laboratories. Aqueous–organic mixtures with ACN and MeOH, fundamentally, as well
as IPA [7–10,22,24,26,29–32,34,38,40–42] or pure organic solvents and mixtures of them have also
been used [25,27,28,35–37,39] as mobile phases without [8–10,22,25,27,28,30,34–39] or with additives
including formic acid (FA) [7,26,40–42], ammonium formate [24,29] or both of them [32], acetic acid [10],
methyl amine [42] and trifluoro acetic acid (TFA) [31], especially for LC-MS hyphenations. With respect
to the columns usually selected for this type of application, despite the fact that the use of normal phases
has been previously reported for the analysis of these compounds, the latest publications describe,
fundamentally, the separation using reversed-phase columns with very good results. However, it is
worth mentioning that in many cases, a clean-up step using polar sorbents in SPE or normal phases in
preparative chromatography has been necessary in order to remove lipid interferences, especially for
high-fat content sample analysis [1].
The combination of LC with UV detection or DAD was also very common in the previous decades.
However, the high matrix effect that precludes the correct determination of these vitamins in food
samples, as well as the limitations in terms of sensitivity and selectivity have brought about the
necessity to move on to the use of more suitable systems such as LC-MS or LC-MS/MS which have
become the gold tools for the simultaneous determination of vitamin D analogues [3]. In this sense,
UV has been used for the determination of only one or two analytes simultaneously (D2 and D3 ) in most
of cases [22,25,28,30,31,36,37,39,41] using, fundamentally, 265 nm as the most suitable wavelength,
and their combination with MS has been necessary in some cases to reach the levels at which some
analytes are present in the samples analyzed [10]. As a remarkable exception, Wittig et al. [7] carried
out the determination of ten vitamin D analogues (i.e., D2 , D4 , previtamin-D2 (Pre-D2 ), previtamin-D4
(Pre-D4 ), tachysterol2 , tachysterol4 , lumisterol2 , lumisterol4 , ergosterol, 22,23-dihydroergostero), as well
as D3 used as the surrogate, in oyster mushrooms after UV-B treatment by HPLC-UV. As can be seen
in Figure 3, clean chromatograms of the evaluated samples treated with hot alkaline saponification
followed by LLE and subsequent separation by HPLC were obtained with an acceptable separation
of all analytes. The good recovery (98%) and the good sensitivity of the developed methodology
(LODs: 0.02–0.06 mg/kg) enabled its successful application to the monitoring of these compounds in
mushroom samples submitted to UV-B exposition in order to enhance their content in vitamin D-active
metabolites with therapeutic functions. However, although analytes quantification was performed
using UV detection, it should be highlighted that MS determination was also carried out to confirm
their identification.
Figure 3. HPLC-UV chromatogram of an oyster mushroom sample submitted to saponification and

subsequent LLE after UV-B treatment. Analytes identification: (1) pre-D2 ; (2) tachysterol2 ; (3) D2 ;
(4) D3 (IS); (5) lumisterol2 ; (6) pre-D4 ; (7) tachysterol4 ; (8) vitamin D4 ; (9) lumisterol4 ; (10) ergosterol
(provitamin D2 ); (11) 22,23-dihydroergosterol (provitamin D4 ). Adapted from [7], with permission
from Elsevier, 2020.
The development of methodologies based on the coupling of LC with MS systems has been carried
out in combination with all kinds of sample pre-treatments previously described as a consequence
of the advantages that this strategy provides in terms of sensitivity and selectivity. Both MS
detection [27,34,35,38] and MS/MS [7–10,24,26,27,29,41,42] have been applied using QqQ in most
cases [7–10,24,26,29,32,40–42]. Nevertheless, other analyzers such as tandem sector quadrupole

(TSQ) [34,35], quadrupole linear-ion trap (QTrap) [32] and IT [35,38] have also been used recently.
Regarding the ionization sources, both electrospray ionization (ESI) [9,24,26,29,32,40–42] and
APCI [7,8,10,27,34,35,38] in positive mode have been applied, using a great variety of additives as
mentioned at the beginning of this section. However, FA has been preferred in the majority of cases for
both modes.
Other usual practice in the evaluation of this type of compound by MS is the application of a
derivatization step in order to increase the sensitivity, especially for those analogues which are present
at a lower concentration and when the amount of sample available also is small. In recent years,
4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) has been the chosen reagent in all cases in the area of food
analysis since it presents a high effectiveness, increasing sensitivity between 7 and 100 times depending
on the compound [40].
Finally, it should also be highlighted the use of deuterated [8,9,24,26,32,40,41] and
non-deuterated [7,10,27,34] surrogates and ISs for the validation of a great number of the evaluated
matrices using not only MS, but also UV or DAD detectors in order to guarantee the reliability of the
obtained results.
3.2. Gas Chromatography

GC has been hardly applied as a separation technique for the analysis of vitamins in food matrices
due to the low stability of these compounds at high termperature [33]. This was pointed out by Nestola
and Thellmann in their work reported in 2015, in which the analysis of D2 and D3 was carried out using
an online hyphenation between a prepartive HPLC and GC-time of flight (ToF)-MS, after saponification,
in yogurt, dietary supplements and margarine [33]. Authors highlighted the simplicity of the online
procedure and compared it to HPLC-MS/MS, indicating that similar results were obtained with less
manual clean-up performance. However, the isomerization of the analytes as a consequence of the
high temperature applied in the GC separations could not be avoided, generating double peaks which
hindered the correct identification and quantification of vitamin D. This constraint made necessary to
carry out the determination of the target analytes using their isomerized pyro-structures in combination
with deuterated ISs by a “cross-contribution” procedure. Despite the advantages that the online
procedure provided, the limitations of using GC for the analysis of thes kinds of substances are evident,
which explains the higher use of other techniques such as LC.
3.3. Supercritical Fluid Chromatography

SFC is a separation technique which integrates the characteristics of LC and GC. Developed
in the 1960s, it did not gain popularity until the 1980s due to infrastructure limitations. Since 2009,
new nstruments that allow the hyphenation of the technique with different detectors, as well as other
improvements such as a reduction of dead volume, higher control of pressure or the use of stationary
phases with smaller particle size have been developed by different companies, which has increased the
number of applications and publications using this technique. SFC is based on the separation of the
analytes using an eluent based on liquid and/or supercritical carbon dioxide mixed with a co-solvent or
solvent mixture. Carbon dioxide is used as the eluent almost always owing to its particular advantages
in terms of low toxicity, availability and accessible critical point, among others. The separation of
analytes not only depends on the identity of the stationary phase ligand, the nature of the eluent
constituents and how these are mixed in the gradient elution, but also on the density of the mobile
phase which has an important influence on analytes solubility. The customized modification of such
parameters allows the comprehensive separation of a wide variety of analytes with different polarities
and also the selective separation of compounds with similar structures, as occur with enantiomers.
The large range of polarities that cover this technique involving all compounds with Log P between
−2.11 and −10.12 makes fat-soluble vitamins (Log P 6–9) ideal candidates for their separation using
SFC [3,48–51].
Despite the recent development of equipment that guarantee the robustness and efficiency of this
technique [3], in the area of food analysis and, particularly, for the evaluation of vitamin D analogues,
the application of SFC is limited to a few studies developed in recent years for the evaluation of milk [4]
and infant and adults nutritionals, as well as cereal samples [23]. In both cases, the hyphenation of
ultra-high-performance supercritical fluid chromatography (UHPSFC) with MS has been applied using
a particle size lower than 2 µm in order to improve the efficiency and rapidness of the technique,
as well as decrease the consumption of solvents and reduce void volumes [51,52]. QqQ as analyzer
and APCI ionization source in positive mode have been chosen on both publications. Besides, as a
consequence of the particular characteristics of the mobile phase used in this technique, the coupling
with MS involves the application of a make-up solvent added to favor the transfer of the compounds to
the MS and guarantee the determination of the analytes. This solvent presents an important influence
on the sensitivity of the methodology. In these cases, ammonium formate in MeOH was selected to
achieve an adequate ionization of the analytes including D2 and D3 [4,23], as well as hydroxylated
metabolites [4]. As described for LC separations, derivatization using PTAD was also selected for the
determination of these analogues with low levels of concentration in the samples of interest [4,23].
Column selection is considered a big challenge in SFC since the specific interactions established
between the analytes and stationary phases are important to achieve the successful separation of
vitamin D isomers. In this sense, fluoro-phenyl columns with charged surface hybrid (CSH) particles [4]
and 1-aminoanthracene (1-AA) columns [23] have been the ones selected so far. An interesting study
about column selection was the one carried out by Oberson et al. [4]. In order to achieve the separation
of PTAD-derivatized D2 , D3 , 25-OHD2 , 3-epi-25-OHD2 , 25-OHD3 and 3- epi-25-OHD3 , the authors
checked nine different columns with a great variety of stationary phases including fluoro-phenyl-CSH,
ethylene bridged hybrid (BEH)-C18 , BEH-2-ethylpyridine (2-EP), 1-AA-C18 , high-strength silica
(HSS)-C18 , Diol-C18 , 2,6-cholesteryl-C18 , C30 and amylose tris(3,5-dimethylphenylcarbamate) (AD-3)
columns. Most of them provided a poor separation of the analytes and low sensitivity, except in
the cases of the fluoro-phenyl-CSH and AD-3 columns. However, the fluoro-phenyl-CSH column,
using MeOH and water as the cosolvents and ammonium formate as the additive at 45 ◦ C, was finally
selected for the validation of the whole methodology. As can be seen in Figure 4, the baseline separation
of isomers was obtained with better peak shape than the results obtained using the AD-3 phase,
although double peaks were observed for the 3-epi-25-OHD forms.
Finally, it should also be highlighted that mixtures of water and MeOH using ammonium formate
as the additive were selected in the articles recently published in the area of vitamin D analysis in food
matrices. The higher sensitivity provided by MeOH as compared with ACN has been demonstrated
previously by some studies developed for the determination of these compounds by SFC-MS [4].
Although the application of this technique is still scarce, the great comprenhensivity of SFC and
the excellent sensitivities obtained when it is hyphenated with MS systems makes this approach one of
the most promising techniques for the analysis of vitamin D and its metabolites in food matrices.
Separations 2020, 7, x FOR PEER REVIEW 28 of 32
Figure
Figure 4.4. UHPSFC-MS/MS
UHPSFC-MS/MS chromatograms
chromatograms of the
of separation of PTAD-derivatized
the separation 25-OHD
of PTAD-derivatized 2, 3-epi-
25-OHD 2,
25OHD 2, 25-OHD3 and 3-epi-25OHD3 using fluoro-phenyl column-CSH (1.7 μm, 3 × 100 mm).
3-epi-25OHD2 , 25-OHD3 and 3-epi-25OHD3 using fluoro-phenyl column-CSH (1.7 µm, 3 × 100 mm).
Gradient
Gradient of of MeOH/water
MeOH/water(98/2,
(98/2,v/v)
v/v) containing
containing 10 10
mM mM ammonium
ammonium formate
formate as organic
as organic modifier
modifier on CO on.
2
CO 2. Column temperature: ◦ 45 °C. Atmospheric back pressure regulator: 128 bar.
Column temperature: 45 C. Atmospheric back pressure regulator: 128 bar. Make-up solvent was Make-up solvent
was
10 Mm 10 ammonium
Mm ammoniumformate formate
in MeOH, in MeOH,
flow rateflow rate 0.4 Reproduced
0.4 mL/min. mL/min. Reproduced frompermission
from [4], with [4], with
permission from Springer,
from Springer, 2020. 2020.
4. Conclusions and Future

Finally, it should alsoRemarks
be highlighted that mixtures of water and MeOH using ammonium
formate as the additive were selected
The important role that vitamin in the
D and its articles recently
metabolites play published in the
in several vital area of for
functions vitamin
human D
analysis in food matrices. The higher sensitivity provided by MeOH as compared with
beings and the influence that their status in the organism has on the development of a great number of ACN has
been demonstrated
diseases previously
have made vitamin by some
D deficiency studies
a global healthdeveloped
concern. Thefor
lowthe determination
subcutaneous of these
bio-production
compounds by SFC-MS [4].
of vitamin D, especially for populations at high latitudes during winter months, makes the diet the
mainAlthough
source ofthe application
these compounds. of this
Fortechnique
this reason,is still scarce, the great
the determination comprenhensivity
of vitamin of SFC
D and vitamin D
and the excellent sensitivities obtained when it is hyphenated with MS systems
analogues contents in commonly consumed food items, as well as the identification of food products makes this
approach one of therich
that are especially most inpromising techniques
those substances, foruttermost
is of the analysis of vitamin Additionally,
importance. D and its metabolites in
each time,
food matrices.
the most common use of supplements or fortified foods that guarantee the adequate levels of these
compounds in the population also urges a reliable assessment of these commercialized products for
4. Conclusions and Future Remarks
ensuring food safety.
The important
All the role that vitamin
abovementioned makesDevident
and its metabolites
the necessity play
of in several vital
developing functions
reliable for human
methodologies
beings and the
that enable an influence
accurate that their status of
determination in the organism
content ofhas on the D
vitamin development
in foodstuffs.of a great number
In this sense,
of diseases have made
chromatographic systems vitamin
have D deficiency
become a global
the most health
suitable concern. The
techniques low out
to carry subcutaneous bio-
the individual
production
evaluation of ofvitamin
vitaminDD, especially
and for populations
its analogues at high
in food matrices latitudes other
displacing during winter initially
methods months,applied
makes
the
suchdiet the main source
as bio-assays. of thesethe
Particularly, compounds.
hyphenation For
of this reason,
different the determination
modalities of LC withof vitamin D and
conventional UV
vitamin
detectorsDand,
analogues contentswith
most recently, in commonly
MS systems consumed
have been food
theitems,
most as well as strategies
recurrent the identification of
due to the
food products
advantages thatthat
thisare especially
technique rich inwith
presents those substances,
respect to GC inis of uttermost
which importance.
analytes’ Additionally,
isomerization occurs at
each time, the most
high temperature. common the
In addition, use commercialization
of supplements oroffortified
robust SFCfoods that guarantee
systems the adequate
and the advantages that
levels of these
this technique compounds
provides in termsin the population also
of comprehensiveness, urges and
selectivity a reliable assessment
peak capacity of these
has allowed the
commercialized products for ensuring food safety.
All the abovementioned makes evident the necessity of developing reliable methodologies that
enable an accurate determination of the content of vitamin D in foodstuffs. In this sense,
28
performance of suitable methodologies based on the use of supercritical fluids, which have become a
promising alternative for the evaluation of vitamins.
Nevertheless, the great complexity of food matrices makes sample analysis difficult without
pre-treatment. In this sense, the application of digestive treatments, such as saponification or
deproteinization, is frequent in most applications. Those are usually combined with conventional SLE
or LLE. However, other widely known techniques, such as SPE or preparative LC, have also been applied
in recent years with considerable improvements in the efficiency of clean-up performance. However,
the current trends in analytical chemistry based on the developed methodologies supported by the
principles of green chemistry have come up with the application of miniaturized techniques also in this
field, reducing the costs and time, as well as the sample and solvent amount requirements of conventional
methodologies. So far, the techniques applied, DLLME and µ-dSPE, have provided promising results,
although their use for the analysis of solid samples still requires previous pre-treatments that involve
the use of high volumes of organic solvents. In this sense, it could be interesting the use of other
approaches more suitable for this kind of sample such as matrix solid-phase extraction that would
allow the direct application in the matrix and the accomplishment of green chemistry principles.
Ultimately, it is evident that there is much to be done in the field. There are limitations that hamper
the individual and simultaneous determination of the different kinds of analogues including D3 , D2 ,
precursor forms, hydroxylated metabolites and ester analogues, for which there is scarce information
up to date. Besides, the effective release of the analytes from the complex food samples without matrix
interferences is still a big challenge for the scientific community. However, the current developments
focus on the combination of simple, fast and effective sample pre-treatment with the state-of-the-art
chromatographic equipment and the most sensitive and versatile detection systems constituting the
most promising alternatives to achieve the goals posed in this area.
Author Contributions: All Authors contributed equally to the present work. All authors have read and agreed to
the published version of the manuscript.
Funding: This research was funded by the Carl Trygger Foundation (project: CTS 18:395).
Acknowledgments: B.S.-R. would like to thank Carl Trygger Foundation for funding her postdoctoral stipend.
This work was supported by the Carl Trygger Foundation (project: CTS 18:395).
Conflicts of Interest: The authors declare no conflict of interest.
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© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).

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separations

Keywords: vitamin D; vitamin D metabolites; food analysis; sample preparation; chromatographic

Separations 2020, 7, 36; doi:10.3390/separations7020036 www.mdpi.com/journal/separations

25-OHD2 C28H44O2 412.65 1,25-(OH)2D2 C28H44O3 428.70

25-OHD3 C27H44O2 400.64 1,25-(OH)2D3 C27H44O3 416.64

2.1. Conventional Extraction Techniques

2.2. Solid-Phase Extraction

Matrix Sample Elution Solvents Determination

Matrix Sample Elution Solvents Determination

2.3. Preparative Chromatographic Techniques

Matrix Sample Determination

Matrix Sample Determination

2.4. Miniaturized Techniques

Matrix Sample Determination

The use of nanomaterials as sorbents in sample pre-treatments of food matrices is a strategy

3. Recent Applications of Chromatographic Techniques

3.1. Liquid Chromatography Applications

Figure 3. HPLC-UV chromatogram of an oyster mushroom sample submitted to saponification and

cases [7–10,24,26,29,32,40–42]. Nevertheless, other analyzers such as tandem sector quadrupole

3.2. Gas Chromatography

3.3. Supercritical Fluid Chromatography

4. Conclusions and Future

50. Tarafder, A. Metamorphosos of supercritical fluid chromatography to SFC: An overview. TrAC-Trend.

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