Trends in Analytical Chemistry 87 (2017) 82e97

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Trends in Analytical Chemistry

journal homepage: www.elsevier.com/locate/trac

Advanced analytical techniques for fat-soluble vitamin analysis

Chiara Fanali a, Giovanni D'Orazio b, Salvatore Fanali b, Alessandra Gentili c, *
a
Centro Integrato di Ricerca, Campus Bio-Medico University, Roma, Italy
b
Istituto di Metodologie Chimiche, Consiglio Nazionale delle Ricerche (C.N.R.), Monterotondo, Roma, Italy
c
Department of Chemistry, University of Rome “La Sapienza”, Roma, Italy

a r t i c l e i n f o a b s t r a c t

Article history: This review presents advancements in sample preparation and liquid chromatographic methods made
Available online 20 December 2016 during the last 10 years for the fat-soluble vitamin (FSV) analysis in foods. Since activity and bioavail-
ability of organic micronutrients (MOs) depend on the form in which they are present in a food, special
Keywords: emphasis has been given to the most recent techniques, instruments and approaches which have
Fat-soluble vitamins allowed the extraction and separation of various vitamin homologues as well as the comprehensive MO
HPLC
profiling in foods. Due to the numerous ancillary advantages, miniaturized techniques which have been
UHPLC
applied in this analysis field are also discussed in this review. A number of selected applications are
Supercritical fluid chromatography
Convergence chromatography
proposed to enable readers to access the most recent developments and trends aimed at gaining more
Miniaturized chromatographic techniques in-depth knowledge of the vitamin composition of foods.
Core-shell technology © 2016 Elsevier B.V. All rights reserved.
Sample preparation
Miniaturized sample preparation
techniques
Vitamin profiling

1. Introduction metabolites, all of them with a different bioavailability, biological

potency and/or physiological role [4].
Since the publication of “The Seven Countries Study” by Ancel Vitamin A is a generic term including two classes of compounds:
Keys on the beneficial effects of the Mediterranean diet, there has
been an international consensus on protective properties expli- i) retinoids (retinol, retinal, retinoic acid, retinyl esters), found in
cated by specific foods [1]. However, to this day, the adoption of foods of animal origin;
healthy dietary choices has still been limited by the fragmentary ii) provitamin A carotenoids (all carotenoids with a 11-carbon
knowledge on the composition of many foods. This is why the latest polyene chain and, at least, one unsubstituted b-ionone ring),
research in nutrition science has been aimed at the identification of present in fruits and vegetables [5].
bioactive components (antioxidants, minerals, vitamins, peptides,
etc.) in the so-called “smart foods”, i.e. foods whose regular intake Consequently, the number of vitamin A vitamers occurring in a
can reduce the risk of chronic disease and increase life expectancy food can be considerably high and variegated. Lack of authentic
[2,3]. standards and subtle chemical heterogeneity are the main diffi-
FSVs are a large family of essential micronutrients with a variety culties in identifying, separating and quantifying such compounds,
of functions crucial for the human organism. Compared to macro- especially retinyl esters. For these reasons, the literature on this
nutrients, their natural distribution has been known more roughly issue is quite limited [6e8], while it is rich of methods concerning
due to the considerable analytical complexity to which multiple the total vitamin A determination through the hydrolysis of all
factors contribute. First of all, number and variety of forms: the bound forms [9]. The practical advantages which ensue from the
FSVs are classified within the groups A, D, E and K, each of which dosage of total retinol are related to the simplification of the
embraces a large number of homologues (vitamers) and chromatographic separation, to the increased sensitivity (the entire
signal is concentrated on only one homologue) and, finally, to the
cutback in costs of analysis (only one authentic standard is neces-
sary). On the other hand, the downside of such an approach is that
* Corresponding author. Department of Chemistry, University of Rome “La Sapi-
enza”, P.le A. Moro 5, 00185 Roma, Italy. the information pertaining to the natural distribution of retinyl
E-mail address: alessandra.gentili@uniroma1.it (A. Gentili). esters is lost.

http://dx.doi.org/10.1016/j.trac.2016.12.001
0165-9936/© 2016 Elsevier B.V. All rights reserved.
 C. Fanali et al. / Trends in Analytical Chemistry 87 (2017) 82e97 83

Abbreviations LOD Limits of detection

LC Liquid chromatography
1a,25-(OH)2D2 1a,25-dihydroxy vitamin D2 LLE Liquid-liquid extraction
1a,25-(OH)2D3 1a,25-dihydroxy vitamin D3 MS Mass spectrometry
1a-OHD3 1a-hydroxy vitamin D3 MSPD Matrix solid-phase dispersion
24,25-(OH)2-D2 24,25-dihydroxyvitamin D2 MK-4 Menaquinone-4
24,25-(OH)2-D3 24,25-dihydroxyvitamin D3 MK-n Menaquinones
25-OHD2 25-hydroxy vitamin D2 MEEKC Microemulsion electrokinetic chromatography
25-OHD3 25-hydroxy vitamin D3 MP Mobile phase
3-epi-25OHD3 3-epi-25-hydroxy vitamin D3 MRM Multireaction monitoring
NPTAD 4-(4-nitrophenyl)-1,2,4-triazoline-3,5-dione nano-LC Nanoliquid chromatography
MBOTAD 4-[2-(6,7-dimethoxy-4-methyl-3-oxo-3,4-4-[4-(6- NARP Non-aqueous reversed phase
methoxy-2-benzoxazolyl)phenyl]-1,2,4-triazoline-3,5- NP Normal phase
dione NMR Nuclear magnetic resonance
FMTAD 4-ferrocenylmethyl-1,2,4-triazoline-3,5-dione PEDAS Pentaerythritol diacrylate monostearate as monomer
ASE Accelerated solvent extraction PFP Pentafluorophenyl
ATF Active Flow Technology ODM Poly(octadecyl methacrylate)
APCI Atmospheric pressure chemical ionization PLE Pressurized liquid extraction
BHT Butylated hydroxytoluene RP Reversed phase
CEC Capillary electrochromatography SIM Selected ion monitoring
CO2 Carbon dioxide SLE Solid-liquid extraction
CC Convergence chromatography SPE Solid phase extraction
DMEQ-TAD Dihydroquinoxalyl)ethyl]-1,2,4-triazoline-3,5- ST Stationary phase
dione SFC Supercritical fluid chromatography
DAD Diode array detection SFE Supercritical fluid extraction
DLLME Dispersive liquid-liquid microextraction Ts Tocopherols
EL Electrochemical t3s Tocotrienols
CEC Electrochromatography C30 Triacontyl
EOF Electroosmotic flow 2D-LC Two-dimensional liquid chromatography
ESI Electrospray ionization UHPLC Ultra-high performance liquid chromatography
ES Electrospray UPCC or UPC2 Ultra-performance convergence chromatography
ELSD Evaporative light scattering UPLC Ultra-performance liquid chromatography
FSV Fat-soluble vitamin UV/Vis Ultraviolet/Visible
FL Fluorescence D2 Vitamin D2
GC Gas-chromatography D3 Vitamin D3
HPLC High performance liquid chromatography K Vitamin K
I.D. Internal diameter a-TAc a-tocopherol acetate
IMMS Ion mobility mass spectrometry

Vitamin D is represented by two main forms: natural spread of vitamin D and on which the scientific commu-
nity has successfully being worked in the last few years
i) vitamin D3 or cholecalciferol (D3); [10,12e14].
ii) vitamin D2 or ergocalciferol (D2). Vitamin E collects eight tocochromanols, all of them of plant
origin:
All of them are derived from the UV irradiation of provitamin D
sterols in the animal skin and plants, respectively. In the human/ i) four tocopherols (Ts), having a saturated isoprenoid side chain;
animal body, both homologues metabolise to 25-hydroxy vitamin ii) four tocotrienols (T3s), with a side chain analogous to Ts but
D (25-OHD3 and 25-OHD2) and to the biologically active 1a,25 containing three trans double bonds.
dihydroxy vitamin D (1a,25-(OH)2D3 and 1a,25-(OH)2D2); other
metabolites of varying degrees of activity are 3-epi-25-hydroxy Ts and T3s are designated as a-, b-, g- and d-according to the
vitamin D (3-epi-25OHD3), 1a-hydroxy vitamin D3 (1a-OHD3) number and position of the methyl substituents in the chromanol
and 24,25-dihydroxy vitamin D (24,25-(OH)2-D2 and 24,25-(OH)2- ring. Difficulties hindering the speciation of the vitamin E homo-
D3) [10]. In total, to date, over 50 naturally-occurring metabolites logues have mainly concerned the chromatographic separation of
of vitamin D are known and some of them may interfere in the positional isomers b- and g which have often been determined
analytical measurements with other metabolites of interest [11]. globally [15e17].
Besides a high chromatographic efficiency, indispensable to Vitamin K is represented by two families of compounds with a
separate isomeric metabolites, another essential requirement in common naphthoquinone nucleus and a differing side chain:
profiling vitamin D-active compounds is the sensitivity of the
detection system due their very low natural concentrations. These i) vitamin K1 or phylloquinone (K1), having a phytyl chain, from
are the two major drawbacks that have delayed insights into the plant foods;
84 C. Fanali et al. / Trends in Analytical Chemistry 87 (2017) 82e97

ii) vitamin K2, including the group of menaquinones (MK-n, with n v) Interactions with other food macro-components, such as
number of unsaturated isoprene unit varying from 4 to 13), from polysaccharides, proteins, and lipids.
bacterial sources [18,19].
In foods of animal origin, FSVs and carotenoids occur in the lipid
The major analytical hurdles which, so far, have hampered the fraction [4], which is mainly composed of triglycerides and partly of
MK-n analysis in foods of animal origin (they are absent in plant sterols and phospholipids. All of these substances have similar
foods) are: the very low concentrations, the co-extraction of lipids solubility which complicates the vitamin isolation and constitute a
interfering with the menaquinone-4 (MK-4) determination, and source of interference during the LC analysis [4,21]. In food of plant
the limited availability and/or high cost of their authentic standards origin, carotenoids and FSVs are tightly associated with the thyla-
[20]. koid membranes of the chloroplasts [19]. Saponification is an
Chiefly owing to the large number of vitamin forms naturally- effective means for removing unwanted lipids and chlorophylls, but
occurring in foods, liquid chromatography (LC) in all its variants it should only be used when indispensable, i.e. for the analysis of
(high performance liquid chromatography (HPLC), ultra-high per- foods of animal origin. Enzymatic digestion with lipase is a more
formance liquid chromatography (UHPLC), nano-liquid chroma- expensive option, which has especially been applied for extracting
tography (nano-LC), two-dimensional liquid chromatography (2D- vitamin K from milk and dairy products [22,23]. In the last years,
LC)) is the more suitable technique for the FSV analysis. Pivotal is these classical procedures have been modified (Section 3.1) and/or
also its versatility related to the variety of chromatographic modes combined with other recent techniques (Sections 3.1 and 3.2). In
(normal phase (NP), reversed phase (RP), non-aqueous reversed the case of vegetables and fruits, which have low-lipid contents,
phase (NARP)), stationary phases (SPs) and column packings extraction techniques alternative or synergic to hydrolysis have
(2e5 mm porous particles, sub-2-mm particles, coreeshell particles, been experimented to preserve analytes in their natural forms so as
and monoliths). In few words, LC, coupled with a vast assortment of to obtain a higher level of information (Section 3.1).
detectors (ultraviolet/Visible (UV/Vis), fluorescence (FL), electro-
chemical (EL)), mass spectrometry (MS)), offers an array of tech- 2.1. Conventional-scale sample preparation techniques
nical solutions for dealing with the vitamin characterization of
complex matrices such as foods. In particular, LCeMS provides both Hot saponification [4,21] has still been the most effective tool for
the selectivity and the sensibility suited to identify the very low the extraction of FS micronutrients from foods. It is also applied to
concentrations of some vitamin forms/metabolites and to perform simplify the analysis of vitamin A and carotenoids, since the alka-
profiling studies. For these peculiarities, the number of LC-MS- line conditions induce hydrolysis of retinol and carotenols esteri-
based methods has been increased meaningfully in the last decade. fied with fatty acids. Usually, saponification is carried out with a
This review presents the latest advancements in vitamin anal- mixture of ethanol and 50% (w/v) aqueous KOH solution in the
ysis for what concerning extraction and separation techniques and presence of an antioxidant (BHT more frequently) for 30 min at
outlines how these novel methodologies may be applied to solve about 80
C. Then, FSVs and carotenoids are extracted with hexane
the more relevant problems affecting vitamin determination. An in- or other water-immiscible organic solvents (petroleum ether or
depth overview will also be provided on the most relevant appli- petroleum ether: diethyl ether 1:1, v/v). Hot saponification can be
cations of the last years and on the emerging trends aimed at the used for individual and simultaneous extraction of carotenoids and
definition of micronutrient food fingerprints/profiling by using vitamins A, D, and E, but it is not advisable for the K homologues,
multidimensional approaches (multidimensional LC and/or multi- which are quickly decomposed by alkalis at high temperature;
dimensional detection). moreover, vitamin E, retinol and xanthophylls can be partly
degraded, whereas carotenes and vitamin D can be subjected to
thermal isomerization [4,21]. In the very last years, the trend in
2. Recent developed extraction techniques vitamin analysis has been that to obtain distribution profiles in
foods of interest. In this respect, saponification possesses all the
Extraction is the key step in vitamin analysis because of a series characteristics for the simultaneous extraction of all FS micro-
of difficulties that can be summed up in the following points: nutrients, vitamin K included, provided that it is carried out under
mild conditions of temperature and alkaline concentration. Over-
i) Chemical instability towards light, oxygen, heat, alkalis and night cold saponification has successfully been applied to this end
acids. Table 1 lists the main factors to keep under control to when digestion was conducted for longer time (15 h instead of
avoid significant losses during analysis. 30 min), but at room temperature, in the dark and by using an
ii) Intra-group and inter-groups chemical heterogeneity. amount of KOH equal or less than the stoichiometric one, roughly
iii) Different and/or low concentration levels in food samples. calculated on the basis on fat composition of food [20,24].
iv) Matrix complexity. Under these conditions, twelve target micronutrients (retinol, a-

Table 1
Chemical stability of fat-soluble micronutrients.

Fat-soluble micronutrient Parameters affecting the chemical Main precautions during the analysis
group stability of vitamins

Carotenoids Sensitive to light, oxygen, heat and acids Low actinic amber glassware and subdued light.
Xanthophylls Sensitive to light, oxygen, heat, acids and alkalis Centrifuge tubes wrapped with aluminium foils.
Vitamin A Sensitive to light, oxygen and acids Addition of a proper antioxidant to the solvents employed for the preparation
Vitamin D Sensitive to light, oxygen and acids. of standard solutions and for the extraction (this precaution is not necessary
It isomerizes reversibly when heated. for vitamin K).
Stable to alkalis
Vitamin E Sensitive to light and oxygen.
Stable to alkalis if protected by light and oxygen.
Vitamin K Sensitive to light (UV), acids and alkalis.
Stable to heat and oxygen.
 C. Fanali et al. / Trends in Analytical Chemistry 87 (2017) 82e97 85

tocopherol, d-tocopherol, g-tocopherol, ergocalciferol, cholecalcif- Pressurized liquid extraction (PLE) is an automated technique
erol, phylloquinone, menaquinone-4, lutein, zeaxanthin, b-cryp- which makes use of solvents at elevated temperature and pressure
toxanthin and b-carotene) were recovered from cow's milk with to increase isolation yields, to reduce extractant volume and to
relative yields ranging between 54% (MK-4) and 100%, while other shorten extraction time; the additional advantage for vitamins and
untargeted carotenoids could be simultaneously isolated and carotenoids is that the extraction is carried in a stainless steel cell,
tentatively identified by HPLC-DAD-MS/MS; for the first time, the i.e. in an oxygen and light-free environment. Solid samples are
authors characterized the cow's milk carotenoid profile compre- simply crushed [33e35] or prepared as explained for MSPD
hensively and highlighted the only occurrence of lutein and zeax- [36e40] and then packed in the cell. In the last recent years, PLE has
anthin traces in buffalo's, goat's, ewe's and donkey's milk [20]. In been used as sample preparation technique for vitamin extraction
order to use chemical hydrolysis in place of enzymatic hydrolysis from foods in a larger extent than SF extraction (SFE) [41]. It has
for vitamin K speciation, K2CO3 was compared to KOH [24]. especially been used for the vitamin E extraction by using methanol
Although the weaker base was supposed to cause a less extensive or a methanol:isopropanol (1:1, v/v) solution at 50
C and 1600 psi
degradation, actually, it resulted to be less effective in hydrolysing as an extractant and hydromatrix celite as a dispersing medium/
glycerides of milk (an interfering peak was detected almost at the drying agent in order to prevent the aggregation of sample parti-
same retention time of K1) and freeing the vitamin. cles; the extracts were diluted to 50 mL with methanol, filtered and,
For its peculiarities, matrix solid-phase dispersion (MSPD) is the then, directly injected into the LCeMS system without any other
sample-preparation technique most suitable for the profiling of FS treatment [36e40]. For the extraction of vitamin K1 from fruits and
micronutrients in foods. The use of mild extraction conditions vegetables, C18 was used as an sorbent/dispersing medium and
(room temperature and atmospheric pressure) allows preserving heptane:ethyl acetate (4:1) at 75
C and 1500 psi as a solvent sys-
endogenous forms (e.g. esters of retinol and carotenols) and tem; in this case, the extract was cleaned-up on a silica SPE car-
avoiding creation of artefacts (i.e. cis/trans isomerization). The tridge to remove lipophilic pigments, concentrated at a final
operating instructions of MSPD, introduced about 20 years ago by volume of 500 mL and analysed via LC-APCI-MS [39]. Besides con-
Barker, are extremely simple [25,26]: a sample (e.g. a minced tissue ventional organic solvents (ethanol, hexane), hot water and su-
or a liquid) is blended with a solid support (C18, diatomaceous perheated water were tested to perform extraction of carotenoids
earth, or other materials) by using a pestle till to obtain a dry, fine from microalgae and to exploit the peculiarities of the green sol-
and evenly coloured solid mixture; the latter is handled as a vent for excellence; although water in subcritical extraction has a
chromatographic phase and used to pack a cartridge/column suit- dielectric constant (3 ) similar to that of methanol, ethanol at 115
C
able for performing sequential elution with solvents. Besides the and 1500 psi was the better solvent system [34,35].
potential in omic approaches, practical advantages of MSPD are: Partial shortcomings of PLE are related to the high cost of the
speed; low cost instrumentation and operations; reduced volumes instrumentation and to the variability of the extracted volumes,
of organic solvents; ability to perform one-step extraction/clean- which are different even if samples are always prepared in the same
up. In recent years, this technique has proved to be very appropriate way; this implies a step of the volume measurement of the extracts
for the micronutrient characterization of plant foods [27e29]. Since when they have to be injected directly.
fruits and vegetables are rich of water and own a low-lipid content, In recent years, a large varieties of microextraction techniques
preferred dispersing media have been neutral alumina combined have been developed to replace the conventional extraction
with sea sand [29] or diatomaceous earth mixed with a highly methods, providing the significant advantage of using volumes of
hygroscopic salt (sodium sulphate) and stratified over a layer of C18 solvents in the microliter order. So far, many methods for vitamin
sorbent (the latter is useful to retain proteins and avoid foaming in extraction have been based on liquid-liquid extraction (LLE), whose
the extract) [27,28]. Recently, overnight cold saponification and the dispersive liquid-liquid microextraction (DLLME) is the minia-
MSPD have been carried out in parallel to perform a large-scale turized evolution [42,43]. This effective preconcentration tech-
screening of carotenoids in tomato fruits by HPLC-DAD-MS/MS nique makes use of a ternary solvent system, i.e. a sample aqueous
[28]. The comparison of the LC-DAD-MS chromatograms, ac- phase, a disperser solvent and an extraction solvent. The rapid in-
quired after applying the two different extraction protocols, was jection of the mixture of the organic solvents (e.g. acetonitrile or
used as an easy and quick way to exclude the occurrence of ester- methanol as dispersing solvent and a chlorinated solvent as
ified carotenols in tomato fruits. The greatest enrichment factor extractant) into the aqueous sample generates a cloudy solution
achieved by means of saponification allowed detecting and iden- composed by minute dispersed droplets of extraction solvent. The
tifying also minor micronutrients [28]. very high contact area between the two phases favours the quick
extraction of analytes; moreover, since the solvent droplets are
2.2. Eco-friendly sample preparation techniques heavier than water, they settle on the bottom of the falcon and are
easily recovered after centrifugation. Fig. 1 illustrates the main
Conventional solvent extraction techniques employ large vol- steps of a classical DLLME experiment.
umes of organic solvents, are poorly selective, can be labour- and DLLME, used alone [44e46] or preceded by alkaline digestion
time-demanding, can provide low recoveries but, overall, can pro- [47,48], has been used for the extraction of FSVs from a large va-
long the extract exposure to heat, light, and air, so significant losses rieties of foods, attaining a high enrichment factor. The pre-
of vitamins can be recorded if the precautions indicated in Table 1 liminary saponification step was applied either to convert all
are not respected. In the last decades, sample preparation has been forms of vitamin A to retinol (for example, retinyl acetate, retinyl
the focus of a constant and unending research which has been palmitate in enriched fruit juices) [47] or to isolate tocols from
following six major trends: simplification, speediness, miniaturi- cereal samples [48]. A modified version of DLLME was developed
zation/wastage reduction, automation, costs and safety. In this for the extraction of vitamin A and vitamin E from oils [46]. In this
context, compressed fluids-based techniques and miniaturized case, the extraction was realized in RP by using 1,4-dioxane, as
extraction techniques have been developed to meet these demands disperser solvent, and ethanol:water (80:20, v/v), as extraction
which the classical techniques not always can satisfy. All of them, solvent, while sonication favoured the dispersion formation; after
assembling many of the above-mentioned qualities, are considered centrifugation, 5 mL of the settled aqueous phase was directly
green techniques and have been finding increasing application in injected for the RP HPLC analysis. DLLME was also combined with
both food analysis and natural products analysis [30e32]. PLE: depending on the food concentration of Ts and T3s, 1/250 or
86 C. Fanali et al. / Trends in Analytical Chemistry 87 (2017) 82e97

and T3s in particular, whose identification is also confirmed by the

elution order: a-T < a-T3 < b-T < g-T < b-T3 < g-T3 < d-T < d-T3
[37,49,52].

3.1.2. Reversed phase HPLC

Compared to NP, RP-HPLC offers a series of advantages encom-
passing the use of MPs suitable for more sensitive MS detection,
greater robustness of the chromatographic columns, reproduc-
ibility of retention times, fast equilibration, and improved peak
shape. For these reasons, the majority of the FSV HPLC separations
have been carried out on C18 columns [36,37,46,54e57,59,60].
Moreover, C18 SPs with a high carbon loading can differentiate D2
from D3 [65], making possible the use of one homologue as an in-
ternal standard for the other when possible [66]. On the other hand,
standard C18 microparticulate phases cannot separate the b- and g-
tocols under routine conditions, i.e. by using aqueous acetonitrile or
methanol, since their partitioning is very similar: d-T3 < (b þ g)-
Fig. 1. Main steps in a typical DLLME experiment. Reproduced from Ref. [42] with the T3 < a-T3 < d-T < (b þ g)-T < a-T. Nevertheless, recently, their
permission of Springer.
partial resolution was achieved on a conventional C18 column under
RP-HPLC conditions [54]. Elution was done with water: 2-propanol
mixtures at low temperature (7
C). The major pitfalls of the method
1/25 of the PLE extract was further purified by means of the
are the use of a HPLC column chiller (an expensive equipment not
miniaturized technique [38].
always available in a lab) and the long run times (more than 1 h)
DLLME is also advantageous for its specific compatibility with
due to the low flow rates (0.3 mL/min) imposed by the high column
modern chromatographic instruments able to analyse volumes of
pressure.
extracts in the pico-litre to the micro-litre range.
Other RP SPs, alternative to C18, which enable the tocol sepa-
ration on HPLC instruments are triacontyl (C30) [67] and penta-
3. Modern analytical chromatographic techniques fluorophenyl (PFP) phases [68]. The HPLC C30 column has since long
been the “golden standard” in the chromatographic separation of
3.1. Conventional-scale liquid chromatography carotenoids [69], whereas it has been a bit less used for FSVs
[58,70e72]. The major advantage of this sorbent is its superior
NP [47,49e52], RP [36,37,44e46,53e60] and NARP [8,20,24,27] shape selectivity which permits the partial or complete resolution
are the most common HPLC modes have been used for the sepa- of geometric isomers [73] and positional isomers. The resolution
ration of FSVs [9,13,21,61,62]. Kind of food matrix, vitamin forms grade depends on the MP composition but, overall, on the column
and sample treatment are the crucial factors in the identification of temperature since the C30 selectivity improves significantly at
the most convenient chromatographic mode to be used. subambient temperature due to a chain straightening phenomenon
[74,75]. Moreover, if compared with a C18 column, a C30 column is
3.1.1. Normal phase HPLC less efficient and exhibits broader chromatographic peaks. This
NP is expedient for the selectivity towards geometric and po- entails higher limits of detection (LODs) and a less aptitude for
sitional isomers which are usually well-resolved on silica SPs due to detecting and quantifying minor micronutrients in a food. In recent
the different steric fitting of these molecules with the adsorption years, the use of fluorinated SPs under RP LC conditions has become
sites. Actually, NP has successfully been applied to the separation of significant because of the alternative mechanisms of retention
cis and trans isomers of retinol [47,63] as well as of all eight vitamin which provide selectivity not readily achievable on traditional alkyl
E congeners, allowing the individual quantification of the b- and g phases. In particular, the PFP bonded phase, besides favouring
positional isomers [49,51]. Another advantage of NP columns retention of polar and ionic compounds (due to the strong dipole-
consists in tolerating relatively high loads of lipid material which dipole, H-bonding and pep interactions), exhibits a relative rigidity
are not strongly adsorbed and which can be easily removed from which provides superior shape selectivity of analytes similar for
the column by the non-polar MP. When saponification is not solubility but differing in size and spatial attributes. So, the recent
essential for the analyte isolation, this quality can be exploited for commercial availability of PFP SPs based on a fully porous particles
the direct injection of extracts obtained from fatty foods (marga- has allowed separating b- and g-tocols with an efficiency greater
rine, milk, oil) by means of sample extraction/dilution with hexane than that of the conventional silica columns [76,77].
[49,64,65]. In these cases, the FL detection is preferred to the ab- NARP chromatography [78] is a kind of RP-LC mode which has
sorption detection since it is less affected by interfering lipids co- been conceived for the separation of low polar compounds [79] and
extracted from matrix [49]. However, one substantial limitation of that has successfully been employed for the separation of FSVs and
NP is the not complete suitability for the MS detection. In fact, the carotenoids [8,20,24,27,28]. This chromatographic mode makes use
most used MPs for the FSV analysis employ a binary solvent system of both low polar eluents to improve the analyte solubility and a
based on hexane together with polar organic modifiers, including highly retentive SP (e.g. C18 with a high carbon loading (20%) or C30)
ethers or very low percentages of alcohols, which favour neither to balance the increased analyte affinity for the NARP MP. The more
electrospray (ES) nor atmospheric pressure chemical ionization polar solvent is usually methanol or acetonitrile, while the modifier
(APCI). Despite this and the incapability of NP in separating vitamin is a stronger solvent (e.g. dichloromethane) or a mixture of solvents
D2 and vitamin D3 each other, Strobel et al. applied this chro- (e.g. 2-propanol:hexane, 50:50, v/v). The selectivity increases as the
matographic mode and exploited the APCI-ion trap detection for difference in polarity between MP and SP becomes smaller. How-
distinguishing and determining vitamin D2, vitamin D3, 25-OHD2 ever, in general, a NARP MP is hardly compatible with the APCI, the
and 25-OHD3 in meat [52]. However, NP-HPLC coupled with golden standard technique for the FSV ionization. In fact, in addi-
UVeVis, FL and EL detection has still been used for analysis of Ts tion to the nebulizer gas and make-up gas, also the MP vapour acts
 C. Fanali et al. / Trends in Analytical Chemistry 87 (2017) 82e97 87

as a reactant gas, so its composition (and flow rate) assumes a key analysing also real samples, the authors verified the superior
role in supporting the APCI. In their works, Gentili et al. selectivity of the HPLC C30 column for cisetrans isomers and an
[8,20,24,27,28] studied the NARP-MP composition in order to meet overall better performance in the separation of carotenoids (see
both chromatographic and mass-spectrometric demands for the Fig. 2). On the other hand, the peak capacity of the HPLC column
profiling of FS micronutrients in several foods. At this end, meth- was about a third lower in comparison to those of the UHPLC col-
anol was selected as polar solvent, while the mixture 2- umns. Moreover, under RP conditions, analysis on the HPLC C30
propanol:hexane (50:50, v/v) as modifier: the hexane was needed column took about four times longer compared to the UHPLC
to reduce the retention time of carotenes and to avoid the peak methods (100 min versus 23 min, respectively); nevertheless,
broadening due to longitudinal diffusion; methanol and 2-propanol shorter runs on HPLC C30 columns have been obtained using NARP
were effective in supporting the APCI due to their involvement in MPs composed for the APCI-MS screening of carotenoids in food
acid-base reactions in the gas phase. This MP composition guar- samples [28]. Another shortcoming of UHPLC columns is their
anteed the solubility of analytes as well as the complete dissolution limited ruggedness which demands an exhaustive sample prepa-
of the extracts obtained from particularly fatty foods. ration to avoid matters arising from intolerably high backpressure
and/or column clogging.
3.1.3. Reversed phase UHPLC It's important to highlight that many of the UHPLC separations
It is since a little more than one decade that the UHPLC tech- of FSVs carried out so far [81e83] have involved the use of columns
nology has been available and has allowed the employment of based on Fused-Core or core-Shell technology. Nevertheless, the
columns packed with sub-2 mm particles [80]. Compared to the peculiarities of this revisited technology, whose origin dates back to
standard HPLC columns usually packed with 2e5 mm particles, the the late 1960s [87], make it particularly advantageous for the
use of UHPLC columns gives rise to small peak widths responsible possibility of using also conventional HPLC systems already present
for: in a laboratory, without the necessity of investing in a new
expensive instrumentation [88,89]. In fact, core-shell particles are
i) an enhanced resolution, potentially useful to separate composed by an inner solid core (silica, aluminium, gold, carbon,
vitamin homologues more efficiently; polymer or copolymer) covered by a superficial porous shell,
ii) lower LODs (higher signal-to-noise ratios), useful to detect available in different total diameters: 1.3 mm and 1.7 mm (packings
the small endogenous concentrations of minor forms; only for UHPLC columns); 2.6 mm (packing suitable both for UHPLC
iii) shorter run times. and HPLC columns); 5 mm (packing specific for HPLC columns).
Such columns are characterized by smaller van Deemter co-
Despite these advantages, the number of UHPLC applications for efficients (A term is less than 40% in comparison with fully porous
the analysis of FS micronutrients in foods is still limited [81e86]. particles, B decreases of 20% and also the C value is reduced) and
The two main reasons are related to the high cost instrumentation provide speed and efficiency comparable to sub-2 mm particles, but
and to the current commercial unavailability of UHPLC columns at approximately half the backpressure for the same column length.
containing SPs (e.g. C30) fulfilling the selectivity requirements for This lower pressure is the reason for which the 5 mm and 2.6 mm
the FS micronutrient separations. In this respect, Bijttebier et al. core-shell columns can be run on HPLC instruments, especially if
[69] compared a set of six UHPLC ACQUITY columns (BEH C18, HSS the column temperature is kept between 30
and 40
C and/or the
T3, HSS C18 SB, CSH C18, BEH Shield RP C18, BEH Phenyl; flow rate is  0.5 mL/min. Both C18 [82,83,90,91] and PFP [38,58]
2.1  100 mm; 1.7 or 1.8 mm; from Waters, Milford, MA) with an SPs, all of them with 2.6 mm core-shell particles, have successfully
HPLC YMC C30 (2.0  250 mm, 3 mm; from YMC, Dinslaken, Ger- been applied in the area of vitamin analysis. The overall selectivity
many) to study the separation of 15 model carotenoids. By is similar to that of fully porous particles, but the remarkable

Fig. 2. Extracted ion chromatograms of a carotenoid standard mixture on an HPLC C30 column (a) and on two C18 UHPLC columns (b and c). Carotenoids; 1: violaxanthin, 2:
astaxanthin, 3: antheraxanthin, 4: zeaxanthin, 5: lutein, 6: tunaxanthin, 7: canthaxanthin, 8: lycopene, 9: a-carotene, 10: b-carotene, 11: phytofluene, 12: phytoene, 13: astaxanthin
dipalmitate. Stationary phases: (a) HPLC YMC C30 (2.0  250 mm, 3 mm); (b) ACQUITY UPLC HSS T3 (2.1x; (c) 100 mm, 1.8 mm); ACQUITY UPLC HSS C18 SB (2.1x; C: 100 mm, 1.8 mm).
MS detection: Orbitrap equipped with an APCI source operated in both positive and negative mode. Reproduced from Ref. [69] with the permission of Elsevier.
88 C. Fanali et al. / Trends in Analytical Chemistry 87 (2017) 82e97

increase in efficiency is due to the Fused-Core particle support. 3.2.1. Nano-LC/CLC employing monolithic or packed capillary
Lately, Knecht et al. compared core-shell-based-PFP and C30 col- columns
umns on a HPLC equipped with a FL detector for the separation of Monolithic capillary columns, also called continuous bed, have
tocochromanols extracted from raw and cooked vegetables by been used in nano-LC and CLC for the separation of several classes
means of direct acetone extraction [58]. On the PFP column, the of compounds also including vitamins. These columns are inter-
elution order was d-b-g-a, whereas on the C30 column it was d-g-b- esting because they have a lower backpressure and, therefore, can
a by using methanol:water (85:15, v/v for PFP and 91:9, v/v for C30 operate at higher flow rate. In addition, the features of the SP can be
as phase A) and methyl tert-butyl ether:methanol:water (80:18:2; tailored modifying the chemistry. Monolithic columns can be silica-
v/v/v as phase B). Better resolution and a shorter run time were or polymeric- (methacrylates or others) based.
obtained using the PFP column, moreover the separation of the g- The usefulness of silica monolithic columns for the separation of
and b-isomer on the C30 column was possible only when it was Ts has been reported by Tanaka's group in 2007. The silica was
cooled and kept at 18
C. coated with poly(octadecyl methacrylate), ODM column. With this
column b- and g-T were almost baseline separated eluting with a
mixture of methanol:water (95:5, v/v) [98]. Later, the analysis of
3.2. Miniaturization in the FSV analysis phytonadione (man-made vitamin K1), a-T, retinyl palmitate and b-
carotene was carried out with nano-LC on a silica C18 monolithic
Miniaturization has been introduced in separation science column. An in-column pre-concentration method was used to treat
with the aim to i) achieve fast separations, ii) improve chro- the corn samples in order to increase the sensitivity [99]. A
matographic efficiency, iii) reduce costs and iv) reduce space monolithic silica C18 column with an I.D. of 0.2 mm was also used
occupied by the conventional instrumentation. Nano-scale tech- for the separation of D2, a-T and phytonadione, by eluting with
niques such as nano-LC or capillary electrochromatography (CEC) methanol at a relatively high flow rate of 50e100 mL/min. The
have been studied since some decades ago and successfully validated method (LOD 0.9 ng/mL; relative standard deviations of
applied to the analysis of several compounds including drugs, retention time in the range 1.2e1.4%; recovery between 55 and
enantiomers, proteins, peptides, vitamins etc. [92e95]. These two 84%) was then applied for the determination of the vitamins after
nano-scale techniques make use of thin capillaries (internal extraction with magnetic C18 particles [100]. Notwithstanding the
diameter (I.D.) <100 mm) containing the SP. The MP is delivered at excellent work done by Cerretani et al. employing nano-LC for the
low flow rate (nL/min). In nano-LC the flow is controlled by a separation of Ts and T3s in the same run, the b- and g-tocols sep-
dedicated pump and has a parabolic profile, while in CEC the aration failed both employing a lauryl methacrylate ester-based
movement of the MP is generated by an electroosmotic flow (EOF) monolithic column [101] and a monolithic silica column 100 mm
as the result of an application of a high electric field. The profile of I.D [102].
the flow has a flat shape and, therefore, higher efficiency can be Although excellent results are obtained with monolithic mate-
expected in CEC. The EOF is influenced by some parameters, e.g. rial, columns packed with silica or titanium based particles are used
the double layer on the charged surface (capillary wall or particles not only in HPLC but also in other miniaturized LC techniques.
or polymeric material-monolith) and the MP composition (pH, Actually, CLC has been applied successfully for the analysis of FSVs
buffer type and concentration, organic additive etc.). One impor- in biological [103,104] and cosmetic samples [105], while applica-
tant feature of the two techniques is the low flow rate tions related to food samples are missing. For example, Fanali et al.
(10e800 nL/min). Just for the reduced flow, CEC and nano-LC can [104] separated d-T, g-T, a-T, a-tocopherol acetate (a-TAc) and BHT
offer some advantages over conventional techniques, e.g. higher in less than 5 min by nano-LC using capillaries packed with silica
mass sensitivity, lower running costs, better compatibility with C18 particles (3 or 5 mm) and applied the optimized method for the
MS etc. Higher mass sensitivity is achieved by reducing the col- analysis of biological and pharmaceutical samples. The efficiency of
umn diameter with a consequent reduction of the chromato- CLC in separating retinoids was studied by Molander et al. [106].
graphic dilution [96]. The volumes of MPs usually used are very All-trans-retinol, all-trans-retinoic acid and 13-cis-retinoic acid
small compared to the ones involved in HPLC and, therefore, less were separated utilizing an on-column focussing large sample
solvent is needed and less waste produced. Finally, the lower volume injection. The CLC was performed at a relatively high
volumes of MP allow a perfect coupling with MS using appro- temperature (50
C). This approach was helpful to reduce the
priate interfaces such as nano-ES. backpressure and operate at higher flow rates reducing the analysis
In principle the same capillary columns can be used in both time to 25 min.
techniques, however it is worth noting that in CEC charged or In the above reported applications of nano-LC/CLC to vitamins'
chargeable groups must be present on the MP surface to generate analysis, UV detector was used. Next example shows the potenti-
the EOF. In addition the electrode compartments are usually pres- ality in coupling the described miniaturized techniques with
surized (8e12-bar) in order to avoid bubble formation that can be spectroscopic ones such as nuclear magnetic resonance (NMR). This
generated after the application of the high electric field. tool can be particularly helpful in the identification of isomeric
Detection is usually done on line with UV or MS detectors. micronutrients. This has been demonstrated by Albert's group
Because of the small volumes of solvents involved with these which analysed Ts and a-TAc on a silica capillary (250 mm I.D.) with
miniaturized techniques, the ordinary instrumentation used in immobilized poly(ethylene-co-acrylic) acid. The relatively high
HPLC cannot be employed and, therefore, dedicated apparatus have capillary I.D. was necessary to connect the CLC apparatus to a NMR
to be considered. However, laboratory-assembled instruments can instrument. The authors did not separate b-T and g-T into the col-
also be prepared. In this case, attention must be paid in minimizing umn, but they could differentiate them by NMR [107].
dead volumes, selecting detector cell with low volumes, appro-
priate valve injection (10e60 nL) and, last but not least, tube con- 3.2.2. Vitamins separation using electromigration techniques
nections with small I.D. A very promising solution to this problems Silica based SPs containing either polymeric or monomeric C30
is the use of chips (also commercial available) containing packed or were used for the CEC of Ts. The authors studied the MP compo-
monolithic SPs [97]. sition and achieved quite good selectivity for all studied analytes
Due to their features, nano-LC and CEC have been successfully also including b- and g-isomers. The use of methanol with the
applied to the separation and analysis of vitamins. addition of small concentration of water allowed the base line
 C. Fanali et al. / Trends in Analytical Chemistry 87 (2017) 82e97 89

resolution. The optimized method was applied to the analysis of a results and applied to the analysis of the vitamins in vegetable oil
vitamin E supplement [108]. samples [111].
An amphiphilic monolithic capillary column with pentaery- Interesting results, dealing with the separation of the same
thritol diacrylate monostearate as monomer (PEDAS) was used for analytes, have been reported by Carabias-Martínez et al. using CEC.
the separation of Ts. The SP, containing both hydrophilic hydroxyl Capillary columns of 100 mm I.D. were packed with three different
and hydrophobic C17 groups, could be easily used in CEC for the silica based SPs: C18, C30 and polar embedded phase (ULTIMA C18).
separation of the four Ts and a-TAc [109]. Under optimized conditions, baseline resolution of the four Ts was
Fig. 3 reports as an example of the potentiality of CEC to the obtained using C30 and ULTIMA C18. It was also observed that the
separation of Ts by using CEC. two SPs were interacting in a different way with b-T and g-T; using
CEC was capable to separate Ts and T3s, also including the pairs the same experimental conditions (100% methanol and 5 mM TRIS)
b-T/g-T and b-T3/g-T3, utilizing a SP based on PFP silica in aqueous an inversion of retention time was achieved [112].
methanol. A different retention order was observed with a tri- d-T, a-T and a-TAc were separated by CEC using a lauryl meth-
acontylsilica SP in non-aqueous methanol. The method was applied acrylate monolith with embedded silver nanoparticles. The pres-
to the analysis of rice bran oils and soybean oil [110]. ence of the nanoparticles in the material influenced the
Columns packed with silica modified with C8 or C18 allow the morphological and chromatographic properties of the polymer, e.g.
separation of only three forms of vitamin E (b- and g-tocols are good electric conductivity [113].
overlapped). Our group demonstrated the usefulness of CEC for the Chang et al. [114] reported a nice separation and analysis a-,g-,
separation of d-T, g-T, a-T and a-TAc in less than 2.5 min. The short d-Ts and a-TAc using microemulsion electrokinetic chromatog-
analysis time was obtained utilizing the short end packed capillary raphy (MEEKC). A quite complicated MP, containing 4% (w/w) so-
(7 cm) and silica C18 particles of 3 mm. The optimum MP was dium dodecyl sulphate, 6.6% (w/w) 1-butanol, 0.8% (w/w) n-octane,
composed by methanol:acetonitrile (50:50, v/v) containing 0.01% 20% (w/w) 2-propanol, 68.6% (w/w) phosphate (25 mM, pH 2.5),
ammonium acetate. The method was validated obtaining good was selected considering the hydrophobicity of the studied vita-
mins. However, the analytes were not resolved due to the quite
similar chemical structure. Therefore, some cyclodextrins (CDs)
were studied and added to the MP in order to modify the vitamin
electrophoretic mobility promoting inclusion complexation with
the cavity of the CD. The most effective CD resulted to be hepta-
kis(2,6-di-O-methyl)-b-cyclodextrin. This additive, at a concentra-
tion of 25 mM, allowed the complete separation of all studied
compounds. After validation, the optimized method was applied to
the analysis of a-, b-, g- and d-Ts. Unfortunately b-T and g-T were
not separated, therefore the concentration of the second form was
overestimated. A similar approach was adopted for separating vi-
tamins A, D, E and K. After optimization, the MEEKC method was
applied to the analysis of the four vitamins spiked in animal feed
[115].

3.3. Supercritical fluid chromatography

Supercritical fluid chromatography (SFC) is a separative tech-

nique which, integrating characteristics of gas-chromatography
(GC) and LC, exhibits an excellent selectivity for separating non-
polar and moderate-polar compounds such as FS MOs. Over the
years, due to its complementary nature, SFC has also been desig-
nated as high-pressure GC, dense GC or convergence chromatog-
raphy (CC) [116]. Lately, two big companies have invested in this
technology, combining its advantages with those of HPLC/UPLC to
obtain a hybrid LC/SFC system (Agilent in 2009 and 2012) and a
holistic SFC system (Waters and Agilent in 2012). In the latter case,
the technique has been named ultra-performance convergence
chromatography (UPCC or UPC2) [117] and, during the last four
years, have successfully been tested in several application areas.
In CC, the primary MP is carbon dioxide (CO2) in either a su-
percritical state (gas and liquid phases are indistinguishable) or
subcritical state (liquefied state). Despite the low critical point of
CO2 (74 bar and 31
C), most separations defined as “SFC” have
actually been carried out in subcritical conditions, partially or
totally. In fact, working in gradient elution with a temperature
between 40
and 60
C, the MP can pass from a supercritical to a
subcritical state upon increasing the modifier percentage (usually
from 2 to 40% of methanol) [118]. In CC, the role of co-solvent is that
Fig. 3. Separation of BHT, four Ts and a-TAc on the PEDAS-EDMA monolithic column. of enhancing polarity, selectivity, solvating power and eluting
Capillary column, 23.5 cm effective length, 32 cm total length  100 mm I.D.; applied
voltage, 30 kV; column temperature, 35
C; electrokinetic injection at 110 kV for 30 s;
strength of pure CO2.
detection wavelength, 200 nm. Reproduced from Ref. [109] with the permission from The analyte retention mainly depends on the MP composition,
Wiley. but it is also influenced by the MP density, i.e. by pressure and
90 C. Fanali et al. / Trends in Analytical Chemistry 87 (2017) 82e97

temperature. CC can replace NP LC and NARP LC to obtain fast and

green separations by varying composition and density of the binary
MP (unlike LC, an increase of temperature implies an increase of
retention time since the MP density is reduced). Moreover, CC is
compatible with elution gradients enabling utilization of detection
techniques such UV, diode array detection (DAD) and MS. Its hy-
phenation with MS is currently gaining in popularity and, in this
respect, the post-column addition of a make-up solvent to the MP
enhances the ionization efficiency significantly. The APCI detection
is also influenced by the MP flow-rate/pressure and the split-ratio
since it is a mass sensitive ionization technique. Fig. 4 shows a
typical equipment to carry out a UPC2-MS analysis; in the specific
case, the CC conditions were optimized for the separation of nine
vitamin D metabolites within 8 min [119].
CC exhibits an enhanced selectivity for the separation of com-
pounds with structural similarity (enantiomers, positional isomers
and structural analogues and conjugates). Since all compounds
with log P values ranging between 2 and 9 are suitable for CC,
carotenoids (logP > 8) and FS vitamins (logP from 6 to 9) are the
Fig. 5. UPC2-MS chromatogram of a working standard solution. Containing the four Ts
ideal candidates [120,121]. The majority of papers, published so far, and the four T3s. Reproduced from Ref. [123] with the permission of Elsevier.
treat the analysis of vitamin E in particular [119,122e127]. An im-
mediate advantage of UPC2 is the reduction of time analysis: from
30 s to 5.5 min for the complete separation of Ts, compared to the a potent means of identification. Nevertheless, in this work, the
typical analysis times of about 20 min by NPLC and of about 10 min authors highlighted the problematic nature of the MS detection of
by NARP chromatography (Fig. 5). retinyl esters which, in the APCI as well as in other ionization
sources, suffer an extensive fragmentation/degradation responsible
4. Selected applications for an information loss on the molecular mass. For any retinyl ester,
the most abundant detected ion was [MH-fatty acid-H2O]þ, i.e.
This section is devoted to illustrate and discuss some applica- protonated dehydroretinol, while more diagnostic ions, such as
tions related to FSV analysis in foods, reported in literature between [M]þ and [MþH]þ, were observed with poor intensity. For these
2006 and 2016, which have been selected either for the innovative reasons, the only way to monitor the endogenous concentrations of
approach or for the use of advanced techniques of extraction, retinyl esters in the real samples was the employment of the
separation and/or detection. Additional information (i.e. extraction multireaction monitoring (MRM) transitions shared with retinol,
technique, LC technique, experimental conditions, SPs, MPs, and i.e. m/z 269/119 and m/z 269/199. Owing to this partial selectivity of
detector) are available in Table 2 and Table 3. the APCI detection, the full chromatographic separation was the
It is noteworthy discussing the reasons for the few papers unique solution to identify and quantify the target retinoids (reti-
dealing with the retinoid profiling in milk and the recent de- noic acid, retinal, retinol, and fourteen retinyl esters); this goal was
velopments achieved in this ambit [6e8]. The main difficulty in reached by assembling a tandem C18/C30 column system and using
realizing this objective is related to the complex profile of fatty a NARP MP. In all kind of milk, retinyl palmitate was found to be the
acids, and hence, to the high number of resulting retinyl esters most abundant vitamin A vitamer, but retinyl oleate was the
which occur in foods of animal origin. The main shortcoming of the prevalent form in the caprine milk; moreover, retinoid composition
LC-UV methods is the interference of co-extracted lipids with the of buffalo and ewe's milk was characterized for the first time.
retinoid identification at 325 nm, combined with a chromato- Based on what has been explained in the Introduction, it is clear
graphic efficiency not enough to separate the wide varieties of that the analysis of vitamin D in food and biological samples is a
forms [6,7]. Lately, the HPLC-DAD-APCI-MS/MS hyphenation was daunting task, even when MS is used as chromatographic detection
used to make the retinoid identification more reliable and to system. In fact, the ionization efficiencies of the D homologues are
elucidate the composition of cow, buffalo, ewe, and goat's milk [8]. low both in ESI and APCI, so chemical derivatization has been used
In general, MS is a highly selective detection system and constitutes to increase their MS response and to shift their m/z ratios to higher

Fig. 4. The figure schematizes the major components in a SFC system. Experimental settings at typical initial chromatographic conditions are also illustrated. Reproduced from
Ref. [119] with the permission from Elsevier.
Table 2
Selected LC methods for analysis of fat-soluble vitamins in foods.

Extraction technique Analyte Matrix Extraction Recovery LC method Ref.

PLE Retinyl acetate,d-T, (bþg)-T and a-T Infant formulas One cycle of extraction with a static 92e106% HPLC with electrochemical detection [37]
time of 5 min, methanol as extraction (ELD) in amperometric mode
solvent, T ¼ 50
C
PLE a-, b-, g-, and d-Ts Cereals One cycle of extraction with a static 91e109% HPLCeESIeMS [36]
time of 5 min, methanol as extraction
solvent, T ¼ 50
C, 110 bar
PLE and DLLME a-, b-, g-, and d-Ts and eT3s Fruits and vegetables (spinach, corn, PLE: one cycle of extraction with a static 90e108% HPLCeFL and APCIeMS [38]
cranberry, pomegranate and mango time of 5 min, methanol:isopropanol
juice) (1:1, v/v) as extraction solvent,
T ¼ 50
C, 1600 psi. DLLME: organic
fraction resulting from the PLE
extraction was recovered and used as
dispersant solvent, carbon tetrachloride
(extraction solvent)
Solvent extraction All-trans-lutein, all-trans-zeaxanthin, Milk from different animal species Overnight cold saponification solvent 55e100% HPLCeDAD and APCIeMS/MS [20]
all-trans-b- cryptoxanthin, all-trans-b- extraction (n-hexane)

C. Fanali et al. / Trends in Analytical Chemistry 87 (2017) 82e97

carotene, all-trans-retinol, a-T, g-T, d-T,
D2, D3, K1, and MK-4
Overnight cold saponification All-trans-b-carotene, all-trans-lutein, Tomato fruit Overnight cold saponification: ethanol e HPLC-DAD and APCI-MS/MS [28]
and MSPD all-trans-zeaxanthin, all-trans-b- containing BHT and aqueous KOH.
cryptoxanthin, and all-trans-lycopene MSPD: diatomaceous earth, as a
dispersing medium and methanol, 2-
propanol, and hexane as elution
solvents
MSPD a-, b-, g-, and d-Ts and eT3s Whole grain barley Neutral alumina as the dispersion agent 74e91% HPLCeFL [29]
and methanol as elution solvent
Overnight cold saponification cis-retinol and trans-retinol Fruit-juices and milk-containing Saponification: ascorbic acid, methanol e HPLCeFL [47]
and DLLME fruit juices and KOH; pH at 6.5e7 with HCl;
filtration for subsequent DLLME.
DLLME: 2 mL of methanolic digest
(dispersive solvent), 100 mL of
tetrachloroethane (extractant solvent),
water solution; centrifugation and
recovery of the settled phase
(tetrachloroethane).
Solid liquid extraction (SLE) D2, D3, K1, MK-4, menadione(K3) Spinach, lettuce, infant cereals SLE: with acetonitrile (3 mL; 0.2e2 g of 88e105% HPLCeDAD [45]
and DLLME crushed samples). Centrifugation. HPLCeMS
DLLME: The supernatant (dispersive
solvent) þ 150 mL of carbon
tetrachloride (extraction solvent) was
rapidly injected into water.
Centrifugation. Recovery and
evaporation of the settled phase.
Reconstitution with acetonitrile.
Ultrasound-assisted a-Tocopherol and retinol acetate Oil samples Dilution of an oil sample with e HPLCeUV [46]
and RPeDLLME cyclohexane. A mixture of 1,4-dioxane
(disperser solvent) and ethanol/water
(80:20 v/v) (extraction solvent) was
rapidly injected into the test tube
containing the oil sample.
Ultrasonication for 10 min at 25
C.
Centrifugation until to the complete
phase separation. Recovery and analysis
of the settled aqueous phase.

91
Table 3

92
Selected LC methods for analysis of fat-soluble vitamins in foods.

Analyte Matrix Extraction Technique Separation Detection LOD and LOQ Ref.

D2, D3, 25-OHD2 and 25- Meat Saponification and solid HPLC NP APCIeMS e [52]
OHD3 phase extraction (SPE)
a-, b-, g-, and d-Ts and Olive oil Dilution with n-hexane HPLC NP - FL and DAD connected LOD: 0.0001e0.0072 mg/ [49]
-T3s in series mL (fluorescence/DAD);
- UV 0.13e0.94 mg/mL (UV);
- Evaporative light 0.35e5.40 mg/mL (ELSD)
scattering (ELSD)
a-, b-, g-, and d-Ts and Rice Solideliquid extraction HPLC NP FL e [50]
-T3s and g-oryzanol (SLE)
a-, b-, g-, and d-Ts and 6 vegetables (raw and Extraction with solvent HPLC NP FL LOD: 21.0e48.0 ng/mL for [51]
-T3s cooked), 3 herbs/spices, (hexane:ethyl acetate, Ts and 56.0e67.0 ng/mL
raw and cooked eggs, 85:15, v/v) for T3s; LOQ: 105.0
vegetable oils (canola, e240.0 ng/mL for Ts and
olive and soybean), 280.0e335.0 ng/mL for
flaxseed and sorghum T3s
(flour and seeds) and soy

C. Fanali et al. / Trends in Analytical Chemistry 87 (2017) 82e97

(flour)
D2, D3, K1, K2, K3 Infant foods and green DLLME HPLC RP DAD and APCIeMS LOD: 0.6 (vit. D2) and 0.4 [47]
vegetables (vit. D3) ng/mL; LOQ: 2.0
(vit. D2) and 1.3 (vit. D3)
ng/mL
a-, b-, g-, and d-Ts and Cereals (durum wheat, SPE HPLC RP FL LOD: 0.47e0.75 and 0.27 [54]
-T3s bread wheat, rice, barley, e0.76 mg/mL for standard
oat, rye, and corn) and spiked cereal
samples; LOQ: 1.42e2.26
and 0.82e2.32 mg/mL for
standard and spiked
cereal samples
K Fresh and dried herbs, Solvent extraction and HPLC RP FL e [57]
spices and seeds, SPE
seasoning blends, and
other flavour enhancers
K, MK-4 and MK-7 Human milk Overnight cold HPLC NARP APCIeMS LOD: 0.6e0.8 ng/mL; [24]
saponification and LOQ: 1.0e1.4 ng/mL
solvent extraction
(hexane)
15 carotenoids Carrot Accelerated solvent UHPLC six columns (BEH C18, HSS APCIeMS e [69]
extraction (ASE) T3, HSS C18 SB, CSH C18,
BEH Shield RP C18, BEH
Phenyl)
a-, b-, g-, and d-Ts and Raw and cooked Different homogenization HPLC RP (core-shell-based-PFP FL LOQ: 0.01e0.02 mg/100 g [58]
-T3s vegetables (carrot, and/or stabilization and C30 ST) fresh weight for d-T and
broccoli, red pepper, procedures followed by d -T3; 0.01e0.04 mg/
green pepper, spinach, direct solvent extraction 100 g fresh weight for b-
green beans, kohlrabi, (acetone) or T, g-T, g-T, and b-T3; 0.05
tomato, celery) saponification and e0.2 mg/100 g fresh
solvent extraction (n- weight for a-T and a-T3
hexane)
D3 Fish and shellfish Saponification and HPLC RP UV and selected ion LOD: 1.22 mg/100 g; LOQ: [55]
solvent extraction (using monitoring MS (SIMeMS) 5.30 mg/100 g
the ethyl ether/
petroleum ether)
g-oryzanol compounds Crude rice bran oil Solvent extraction (ACN/ HPLC RP (C18 and C30 silica SPs) DAD and APCI-MS e [67]
methanol/propan-2-ol
(50:45:5, v/v/v))
a-, b-, g-, and d-Ts and Plasma and liver Liver: saponification and HPLC RP (PFP) FL LOD: 27e156 pg; lower [68]
-T3s solvent extraction. LOQ: 92e519 pg; upper
Plasma: solvent LOQ: 31e173.
extraction
Nine fat soluble vitamins Infant formula and Dietary supplements: UFLC RP (C30 silica SP) DAD and APCI-MS/MS LOD: 0.001e0.063 mg/mL; [72]
and their derivatives dietary supplements solvent extraction. Infant LOQ: 0.003e0.210 mg/mL.
(retinol, retinyl formula: saponification
acetate, retinyl and solvent extraction.
palmitate, D3,a-,b-,g-,
and d-Ts, a-TAc) and
three carotenoids (b-
carotene, lutein and
zeaxanthin)
a-, b-, g-, and d-Ts and Margarine Melting, dilution and HPLC RP (C30 silica SP) FL LOD: 0.018e0.061 mg/L [76]
-T3s centrifugation
Retinoic acid, retinal, Raw milk (cow, buffalo, Solvent extraction HPLC NARP (tandem C18/C30 DAD and APCIeMS/MS LOD: 6.4e260.4 mg/L; [8]
retinol, and fourteen ewe, and goat) column system) LOQ: 21.3e868.0 mg/L.
retinyl esters
All-trans-lutein, all-trans- Milk from different Cold saponification and HPLC Carotenoid: NARP (C30 DAD and APCIeMS/MS LOD: 0.90e15.6 mg/L; [20]
zeaxanthin, all-trans- animal species solvent extraction silica SP); fat soluble LOQ: 2.70e46.80 mg/L

C. Fanali et al. / Trends in Analytical Chemistry 87 (2017) 82e97

b- cryptoxanthin, all- (hexane) vitamins: RP (tandem C18
trans-b-carotene, all- column system)
trans-retinol, a-T, g-T,
d-T, D2, D3, K1, and MK-
4
Four carotenoids (lutein, Maize flour, green and Matrix solid-phase HPLC RP (C30 silica SP) DAD and APCIeMS/MS LOD: 0.0004e0.075 mg/g: [27]
zeaxanthin, b- golden kiwi dispersion (MSPD) (C18 LOQ: 0.001e0.226 mg/g
cryptoxanthin, and b- sorbent as dispersing
carotene) and six medium in the case of
compounds with fat- maize flour and
soluble activity (a-T, d- diatomaceous earth for
T, g-T, D2, K1 and MK- kiwi)
4)
D3 Fortified infant formula, Solvent extraction UPLC RP (C18 core shell ESIeMS LOD: 0.067 mg/100 g; [83]
milk and milk powder (isooctane and particles SP) LOQ: 0.117 mg/100 g
acetonitrile)
Five carotenoids, four Broccoli Solvent extraction UPLC RP (two C18 sub two DAD LOD: 05e8.0 ng/5 mL; [82]
chlorophylls and one (ethanol) micron particle core-shell LOQ: 2e16 ng/5 mL
tocopherol SP)
All-trans-lutein, Tomato Solvent extraction UPLC RP (C18 sub two micron DAD LOD: 07e2.3 ng; LOQ: 2.5 [85]
lycopene,b-carotene (ethanol and hexane) particle core-shell SP) e6.2 ng.
and their 22 cis-
isomers
Carotenoids, A and E Forages, bovine plasma, Forage: saponification UPLC RP (tandem C18 column DAD LOD: 1.0e3.0 ng; LOQ: 7.0 [86]
and milk and solvent extraction; system) e13.3 ng
Plasma: solvent
extraction (hexane);
Milk: solvent extraction.
K and MK-n Feces, serum and baby Solvent extraction and HPLC RP (C18 ST) APCI-MS LOD: 1e30 pmol/g; LOQ: [90]
food SPE 5e90 pmol/g
Retinol and a-T Breast milk Deproteinization, HPLC Five various porous shell DAD and FL LOD: 0.004e0.078 mmol/ [91]
saponification and liquid and monolithic columns L; LOQ: 0.012
eliquid extraction e0.182 mmol/L

93
94 C. Fanali et al. / Trends in Analytical Chemistry 87 (2017) 82e97

mass range where chemical noise is lower [12,81,128,129]. set at 12.4 MPa, the phase state of the MP was supercritical at the
Cookson-type reagents (4-substituted 1,2,4-triazoline-3,5-dione) start, then it was presumably changed to subcritical and, finally,
have proved to be effective, since they react with the s-cis-diene liquid by increasing the modifier composition. This application has
structure of D vitamers rapidly and quantitatively to give Diel- demonstrated a considerable potential in vitamin analysis as well
seAlder adducts containing proton-affinitive sites with a sensitivity as in approaches involving the simultaneous analysis of a large
gain up to 100e1000-fold. 4-Phenyl-1,2,4-triazoline-3,5-dione range of compounds significantly differing for their logP by means
(PTAD) has been the dienophile most used so far, also because it of a single chromatographic technique.
is commercially available unlike other its variants (Fig. 6). Never-
theless, Kamao et al. [129] used 4-[2-(6,7-dimethoxy-4-methyl-3- 5. Current trends and future outlooks
oxo-3,4-dihydroquinoxalyl)ethyl]-1,2,4-triazoline-3,5-dione
(DMEQTAD) for derivatising vitamins D2, D3 and their 25-OH me- Over the last 10 years, there have been considerable advances in
tabolites and for dosing their low endogenous concentration in the vitamin analysis field, especially stimulated by the parallel
human breast milk by LC-APCI-MS/MS. In this way, vitamin D remarkable progresses made in sample preparation and separation
compounds could be detected with LODs of 1e2 pg, while the other techniques. Consequently, more accurate values of concentration
underivatised FSVs (retinol, b-carotene, a-tocopherol, phylloqui- have been obtained both for endogenous forms and for vitamins
none and menaquinone-4) exhibited LODs ranging between 50 and added to fortified foods and supplements. Beyond this conventional
250 pg. Derivatization was also used by Abernethy [83] to detect analysis approach, one of the most recent trends in this research
vitamin D3 in fortified milk and milk-based products without area is the profiling of micronutrients that naturally occur in a food.
having recourse to an extensive sample preparation. This method Very complex profiles of vitamins and carotenoids can be found in
makes use of a simple derivatization with PTAD to avoid the nature and often their characterization can be further hampered by
evaporation step and to improve selectivity without necessity of an interfering isomeric/isobaric compounds co-extracted from the
effective chromatography to separate D3 from isobaric nonsaponi- matrix. Till today, as we have illustrated, only few papers dealing
fiable interferences. with this topic have been published; however, since this emerging
Among the different chromatographic techniques, CC chroma- trend is becoming apparent, more sophisticated analytical
tography is particularly attractive for offering fast, clean and well- methods, based on highly-resolved separation and/or enhanced
resolved separation of non- and low polarity compounds. Very detection systems, are needed to perform unambiguous screening
recently, a single method based on this technique was developed to on large-scale. High resolution MS guarantees adequate selectivity
separate 17 vitamins (retinyl acetate, retinyl palmitate, b-carotene, and identification power, but many structural and/or geometric
D2, a-T, K1, MK-4, thiamine, riboflavin, nicotinic acid, nicotinamide, isomers exist among both carotenoids and vitamins. In this ambit, a
pantothenic acid, pyridoxine, biotin, cyanocobalamin, and ascorbic very promising technological solution seems to be the ion mobility
acid) covering a wide polarity range (logP from 2.11 for thiamine MS (IMMS). IMMS is an analytical technique which separates ions
up to 10.12 for retinyl palmitate) [127]. MS equipped with an ESI on the basis of their size/charge ratios as well as their interactions
source was used as detection system. During the 4-min run, the with a buffer gas; therefore, it represents an alternative strategy for
column was kept at 40
C and the MP was varied from almost 100% resolving isobaric and isomeric compounds as well as interferences
CO2 up to 100% methanol. With the active back pressure regulator from analytes of interest. Another possibility is related to the

Fig. 6. Representative Cookson-type reagents: (a) derivatization of 25(OH)D3 with PTAD and major product ion (m/z 298) in MS/MS, (b) derivatization of 1a-OHD3with 4-
ferrocenylmethyl-1,2,4-triazoline-3,5-dione (FMTAD) and major product ion (m/z 199) in MS/MS and (c) chemical structures of 4-[4-(6-methoxy-2-benzoxazolyl)phenyl]-1,2,4-
triazoline-3,5-dione (MBOTAD), DMEQTAD and 4-(4-nitrophenyl)-1,2,4-triazoline-3,5-dione (NPTAD). Reproduced from Ref. [12] with the permission of Elsevier.
 C. Fanali et al. / Trends in Analytical Chemistry 87 (2017) 82e97 95

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