Food Chemistry 373 (2022) 131436

Contents lists available at ScienceDirect

Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Lipid-soluble vitamins from dairy products: Extraction, purification, and

analytical techniques
Emad Karrar a, Isam A. Mohamed Ahmed b, Muhammad Faisal Manzoor c, Wei Wei a,
Frederick Sarpong d, Xingguo Wang a, *
a
National Engineering Research Center for Functional Food, Collaborative Innovation Center of Food Safety and Quality Control in Jiangsu Province, School of Food
Science and Technology, Jiangnan University, Wuxi 214122, Jiangsu, People’s Republic of China
b
Department of Food Science and Nutrition, College of Food and Agricultural Sciences, King Saud University, Riyadh 11451, Saudi Arabia
c
School of Food and Biological Engineering, Jiangsu University, 301 Xuefu Road, Zhenjiang 212013, Jiangsu, People’s Republic of China
d
Value Addition Division, Oil Palm Research Institute, Council for Scientific and Industrial Research, Kade, Ghana

A R T I C L E I N F O A B S T R A C T

Keywords: Milk and dairy products are considered as essential sources of lipid-soluble vitamins (LSVs) for human nutrition.
Lipid-soluble vitamins Due to the lower concentrations, complexity, and instability of LSVs during extraction, their quantification re­
Milk mains challenging. This review focus on advances in the extraction and quantification of LSVs from different
Sample preparation
dairy products. Saponification, and liquid-liquid (LLE), solid-phase (SPE), and supercritical fluid (SFE) extraction
Chromatographic analysis
methods, as well as dispersive liquid-liquid microextraction, are the most common techniques. Liquid
chromatography-mass spectrophotometry (LC-MS) has unique advantages for LSVs determination and quanti­
fication due to its high sensitivity and specificity.

Lipids are a large group of natural compounds, including fatty acids,

1. Introduction
Milk is a primary food source due to its nutritional properties that
humans require for growth and development (Zhu & Guo, 2021; Zhu
et al., 2021). According to the Food and Agriculture Organization (FAO),
global milk production increased from 391.2 million metric tons in 1969
to 883.3 million metric tons in 2019 (FAOSTAT, 2021). Global milk
production in 2019 could be divided into five categories including cow
(81.1%), buffalo (15.1%), goat (2.3%), sheep (1.2%), and camels (0.4%)
(FAOSTAT, 2021). Milk production from these animals has increased
significantly in recent years (Fig. 1), and its consumption has increased
in both developed and developing countries due to increases in popu­
lation growth, income, and uses and forms of dairy products (OECD-
FAO, 2021). Milk composition attributes, including proteins, bioactive
peptides, carbohydrates, lipids, vitamins, and minerals such as calcium,
potassium, phosphorous, and zinc, make it an important component of
healthy diets (Dominguez-Salas, Omore, Omosa, & Ouma, 2019). Lipids
are among the most important constituents of milk for nutritional and
economic reasons. They are considered as a good source of energy and
provide unique sensory and physical attributes to dairy products
(Gutiérrez-Peña, Fernández-Cabanás, Mena, & Delgado-Pertíñez, 2018).
monoglycerides, diglycerides, sterols, carotenoids, lipid-soluble vita­
mins (vitamin A, vitamin D, vitamin E and vitamin K), phospholipids,
and others. The amounts of lipid-soluble vitamins (LSVs) in food can
vary depending on the origin, preparation, and storage of the product,
making it difficult to predict the exact concentrations of these micro­
nutrients in the processed product. Moreover, natural concentrations do
not always match the values shown on the food nutrition label due to the
inherent instability of LSVs (Tan & McClements, 2021; Yaman et al.,
2021). LSVs are classified into four categories: vitamin A, vitamin D,
vitamin E, and vitamin K. They have important roles in several functions
of the human body, such as vision (vitamin A), calcium absorption
(vitamin D), antioxidative protection in cell membranes (vitamin E), and
blood coagulation (vitamin K) (Bernal, Mendiola, Ibáñez, & Cifuentes,
2011; Chen et al., 2011; Fanali, D’Orazio, Fanali, & Gentili, 2017; Zhang
et al., 2018). Research on milk LSVs has attracted widespread interest,
and consequently, the number of publications on milk LSVs has
increased significantly in recent decades. Therefore, critical reviews on
the separation and quantitation of LSVs in milk and dairy products are of
high importance.
Extraction is the critical step in the determination of LSVs in milk
samples due to the sensitivity of vitamin A, vitamin D, vitamin E, and

* Corresponding author at: National Engineering Research Center for Functional Food, Collaborative Innovation Center of Food Safety and Quality Control in
Jiangsu Province, School of Food Science and Technology, Jiangnan University, Wuxi 214122, Jiangsu, People’s Republic of China.
E-mail address: xingguow@jiangnan.edu.cn (X. Wang).

https://doi.org/10.1016/j.foodchem.2021.131436
Received 18 May 2021; Received in revised form 14 October 2021; Accepted 17 October 2021
Available online 20 October 2021
0308-8146/© 2021 Elsevier Ltd. All rights reserved.
E. Karrar et al. Food Chemistry 373 (2022) 131436

Abbreviations CO2 Carbon dioxide

PLE Pressurized liquid extraction
LSVs Lipid-soluble vitamins DLLME Dispersive Liquid-liquid microextraction
LC/MS Liquid chromatography-Mass Spectrophotometry LC/UV Liquid chromatography- ultraviolet
FAO Food and agriculture organization FL Fluorescence
VA Vitamin A MPs Mobile phase
VD Vitamin D APCI Atmospheric pressure chemical ionization
VE Vitamin E ES Electrospray
VK Vitamin K NP Normal phase
HPLC-UV High-performance liquid chromatography- Ultraviolet CPBA Competitive protein binding assay
HPLC/MS High-performance liquid chromatography/ Mass RP-HPLC Reverse-phase- high performance liquid chromatography
Spectrophotometry DAD Diode array detection
HPLC High-performance liquid chromatography LOD Limits of detection
FAs Fatty acids PAD Photodiode array detector
HPLC/DAD/MS/MS High-performance liquid- chromatography- LC/DAD Liquid chromatography/ diode array detection
diode array detection-tandem mass spectrometry ESI Electrospray ionization
LLE Liquid-liquid extraction LC-APCI-MS/MS Liquid chromatography-atmospheric pressure
VD3 Cholecalciferol chemical ionization- tandem mass spectrometry
VD2 Ergocalciferol QqQ triple quadrupole
UV Ultraviolet MS-MS Tandem mass spectrometry
CPBA Competitive protein binding assay LOQ Quantification limit
RIA Radio immunoassay MRM Multireaction monitoring
EIA Enzyme immunoassay ESI/MS/MS Electrospray ionization-tandem mass spectrometry
PUFA Polyunsaturated fatty acid CEC Capillary electrochromatography
VLDLs Very low-density lipoproteins EOF Electroosmotic flow
VKDPs Vitamin K -dependent proteins I.D. Internal diameter
LC-F Liquid chromatography- fluorescence spectroscopy GC Gas-chromatography
LC Liquid chromatography CC Converge chromatography
MK-4 Menaquinone-4 SFC Supercritical fluid chromatography
MK-n Menaquinone LC/SFC Liquid chromatography/ supercritical fluid
TAG Triglycerides chromatography
BHT Butylated hydroxytoluene UPLC Ultra-high-performance liquid chromatography
SPE Solid-phase extraction MPa Megapascal pressure unit
MS Mass spectrometry RSD Relative standard deviation
RP- HPLC Reverse-phase- high-performance liquid chromatography DTAG Di- triglycerides
DE Direct extraction MTAG Mono-triglycerides
SFE Supercritical fluid extraction FFA Free fatty acid

electrochemical MS, and DAD-MS/MS detectors have been used for the
1000 determination of LSVs in foods (Fanali et al., 2017; Gentili et al., 2013).
Cow Buffalo Goat Sheep Camel These methods require unique preconditioning treatments of the sam­
900
ples, such as extraction and separation of LSVs from complex matrices
800 (Karaźniewicz-Łada & Główka, 2016). The duration of sample prepa­
700 ration and extraction approaches is critical as most vitamins quickly
Million tones

decompose in solution under light and oxygen conditions (Kar­

600
aźniewicz-Łada & Główka, 2016). Heat, cold, alkaline saponification,
500 lipase hydrolysis, solvent, and supercritical extraction treatments were
400 used to hydrolyze the lipid resides and extract vitamins from different
300
types of food matrix (Blake, 2007; Fanali et al., 2017).
A literature survey on LSVs in milk and its determination methods
200
from 2001 to May 2021 was conducted on the Web of Science database,
100 and the results are depicted in Fig. 2. The number of publications on
0 LSVs in milk has increased (a total of 541 articles) in recent years due to
1969 1979 1989 1999 2009 2019 the recognition of the importance of these micronutrients for human
Years health (Fig. 2A). In addition, the number of publications related to the
determination and analysis methods of LSVs in milk has also been
Fig 1. Global milk production in the last fifty years (FAOSTAT, 2021).
increased (a total of 206 articles) in the past twenty years (Fig. 2B).
Among these publications, a minimal number of review articles were
vitamin K. The chemical nature of LSVs has a high sensitivity to UV light, found regarding the extraction and determination of LSVs (Arachchige
oxygen, heat, acids, and alkalis (Lazzarino et al., 2017; Oberson, Cam­ et al., 2020; Fanali et al., 2017; Karaźniewicz-Łada & Główka, 2016;
pos-Giménez, Rivière, & Martin, 2018). To date, different liquid chro­ Kasalová, Aufartová, Krčmová, Solichová, & Solich, 2015; Luque-Garcı́a
matography (LC) methods such as normal phase- HPLC, reverse phase- & Luque de Castro, 2001; Turner, King, & Mathiasson, 2001; Zhang
HPLC, UHPLC systems coupled with UV/VIS, fluorescence, et al., 2018) and vitamin E (Cervinkova, Krcmova, Solichova, Melichar,

2
E. Karrar et al. Food Chemistry 373 (2022) 131436

Fig 2. Number of publications from the literature survey of publications in the period 2001–2021 (May, 14) on Web of Science with (A) lipid soluble vitamins (LSVs)
in milk or dairy products and (B) analysis or determination of LSVs in milk or dairy products.

& Solich, 2016) in biological materials and vitamin D in milk (Kasalová
et al., 2015; Perales, Alegría, Barberá, & Farré, 2005). However, we
were unable to locate any review about the extraction and determina­
tion of LSVs in milk from different species. This review thus examines
the most recent advancements in extraction and separation methods for
milk lipid-soluble vitamins. It helps demonstrate how these cutting-edge
methodologies can help solve some of the most pressing issues in
vitamin analysis (Fig. 3).
2. Extraction techniques
It has been a challenge to develop a general standard method for
extraction and quantification of all LSVs from milk samples due to the
variation in the chemical structures, and stability of LSVs, moreover
variation in structure and composition of milk matrices (Arachchige
et al., 2020). During the investigation, interactions with poly­
saccharides, lipids, proteins, and other macro-components of food
should be considered to avoid significant losses of LSVs (Fanali et al.,
2017). The key factors to keep under control are listed in Table 1. The
lipid fraction of milk containing LSVs is primarily the triglycerides
(TAG), with minor phospholipids, sterols, and other subaltern compo­
nents. These compounds share similar solubility with LSVs, thereby
affecting the extractability and analysis of these micronutrients (Fanali
et al., 2017). Several extraction techniques have been used to extract
LSVs from dairy products.

3
E. Karrar et al. Food Chemistry 373 (2022) 131436

Fig 3. The major extraction methods of LSVs from milk (A) direct extraction (DE), (B) liquid–liquid extraction (LLE), (C) solid-phase extraction (SPE), and (D)
supercritical fluid extraction (SFE) methods.

Table 1
Chemical stability of lipid-soluble vitamins (Fanali et al., 2017).
Lipid-soluble vitamins Parameters that affect vitamins chemical Principal precautions during the determination
stability

Sensitive to acids, oxygen, and light To the solvents used to prepare standard, a proper antioxidant
compound should be added (this precaution is not necessary
for vitamin K).

Sensitive to acids, oxygen, and light.

When hot, it reversibly isomerizes. Stable
to alkalis

Responsive to oxygen and light. Stable

with alkalis if covered by oxygen and
light.

Sensitive to acids, alkalis, and light (UV).

Stable to oxygen and heat.

2.1. Liquid-liquid extraction (LLE) extract the target compounds from samples with minimum contami­
nants (Poole, 2020a). It is one of the basic techniques for extracting/
LLE is one of the oldest extraction methods. The target compound is purifying LSVs from milk samples for a long time (Luque-Garcı́a & Luque
transferred from the liquid phase sample to another liquid phase to de Castro, 2001). LLE processes are being extensively used to extract

4
E. Karrar et al.
LSVs from different milk samples (Table 2). Before LLE, hot or cold
saponification is applied as the most effective pre-extraction method for
LSVs extraction from milk (Blake, 2007; Levêques et al., 2019). Hot
saponification is usually conducted with a 50% (w/v) aqueous solution
of KOH and a mixture of ethanol for 30 min at about 80 ◦ C in Butylated
hydroxytoluene (BHT) antioxidant. Cold saponification involves treat­
ing the sample with aqueous or ethanolic KOH at 25 ◦ C under slow
constant stirring overnight. To avoid the oxidation of vitamin D pyro­
gallol, BHT as an antioxidant is used (Gentili et al., 2013) (Table 2).
Extraction of vitamin K from milk by pretreatment with lipase enzyme is
also used; however, it is more expensive than saponification and solvent
extraction methods (Indyk & Woollard, 2000). In addition, precipitation
of protein with a solvent such as methanol, ethanol, acetone, acetoni­
trile, and isopropanol has been reported as an effective method for
improving the extractability and quantification of retinol and
α-tocopherol in milk (Kašparová et al., 2012; Kučerová, Krčmová, Sol­
ichová, Plíšek, & Solich, 2013; Sunarić, Lalić, & Spasić, 2017). Alkaline
hydrolysis of the fat from 5 mL of human milk to measure the tocoph­
erols and their different forms (α-tocopherol, γ-tocopherol, δ-tocoph­
erol) before HPLC analysis has been reported (Wei et al., 2018).
Observed that earlier methods for the analysis of vitamins in human
milk lipid using a high volume of sample, from 5 to 20 mL, while Lev­
êques et al. (2019) used low volume of the human milk (950 µL; 200 µL
for vitamin A and 750 µL for vitamin E and vitamin K) and the recovery
rates were within 90–105%. We found this is the first report that allows
the characterization and quantification of vitamin A, vitamin E, and
vitamin K in <1 mL human milk. Chemical hydrolysis using K2CO3 and
KOH was applied before analysis of vitamin K in milk, and the results
indicated that K2CO3 hydrolysis resulted in less vast degradation, low
hydrolysis of milk glyceride, and low release of vitamin (Gentili et al.,
2016).
After hydrolysis processes, the sample can be subjected to solvent
extraction with different solvents. Hexane extraction and two portions
washing with butylated hydroxytoluene and methanol-water were used
for the simultaneous determination of LSVs such as vitamin A, vitamin
D, vitamin E, vitamin K, retinyl acetate, tocopherol acetate, ergosterol,
retinyl palmitate, and 7-dehydrocholesterol in milk (Blanco, Fernández,
& Gutiérrez, 2000; Gomis, Fernández, & Gutiérrez Alvarez, 2000). In
these processes, robust recoveries (89–107%) of LSVs were achieved due
to the low sample size, less solvent intake, and the extraction of LSVs and
their precursors in one procedure (Gomis et al., 2000). The recovery of
methanol-dichloromethane, ethanol, and ethyl-acetate, was less than
72% (Table 2). A study by Kamao et al. (2007) described that the LSVs
recovery was 91–105% after performing two extractions with (hexane-
ethyl acetate/ pyrogallol-ethanol).
In most studies, internal standards were added to milk samples
before the extraction with benzene, alkali-saponification, and analytic
procedures to monitor the recovery of LSVs (Abernethy, 2012; Jakobsen
& Saxholt, 2009; Kamao et al., 2007). The samples preparation was not
appropriate for routine research due to the time requirements of various
extractions procedures for vitamins and dihydroxy metabolites. Barba,
Esteve, and Frígola (2011) compared diethyl ether/ hexane as extrac­
tions solvent for determining vitamin E and vitamin D. In their study, a
double extraction of hexane was used to achieve the most generous
yield, and since diethyl ether is unstable and more volatile than hexane,
it was recommended to use hexane instead. The optimum condition was
found to be adding BHT and saponification with potassium hydroxide in
ethanol (Barba et al., 2011). This technique can assess vitamin D and
vitamin E levels in various liquids, making it suitable for regular testing
of both vitamins in various samples using the same extraction technique.
Abernethy (2012) described a quick process (Diels-Alder derivati­
zation) for analyzing vitamin D3 in fortified infant formula, milk, and
milk powder. In just one step, the vitamins were extracted and converted
to isooctane. And the deuterium-labeled vitamin D3 was used to
compensate for extractions loss. It is one of the best (LLE) experiments
since it only needs 4 mL of sample and 35 mL of solvent. Only a precise
5
Food Chemistry 373 (2022) 131436
volume of the internal norm and the sample residue’s slow reaction to
the derivatizing reagent is needed for accurate analysis. Another more
suitable extractions technique with high recovery (%) and less sample
and solvents consumption were developed by (Kaushik, Sachdeva,
Arora, & Wadhwa, 2014). These extractions methods, however, have
just been recommended for vitamin D. The most appropriate methods
focused on liquid-liquid extraction showed near (100%) recovery with
lower than a 15% coefficient of variation. The extractions methods
described above can be used to analyze vitamins in dairy products.
Despite this, significant issues such as high solvent consumption during
multi-stage extraction and the lengthy preparation of the sample times
persist in such application.
2.2. Dispersive liquid-liquid microextraction (DLLME)
DLLME methods have recently been developed to replace traditional
extractions techniques, with the advantage that they use solvent con­
centrations in the microliter range (Fanali et al., 2017; Sadrykia,
Shayanfar, Valizadeh, & Nemati, 2019). A ternary solvent method in­
cludes a sample’s aqueous phase, disperser solvents, and extraction
solvents; this successful preconcentration technique is used. By rapidly
injecting a mixture of the organic solvent (e.g., methanol or acetonitrile
as a dispersing solvent and a chlorinated solvent as an extractant) into an
aqueous sample, a cloudy solution containing minutely distributed
droplets of the extraction solvent is formed. Solvents droplets are much
heavier than H2O, so they collect on the bottom of Falcon tubes. After
centrifugation, the solvent is quickly extracted, and the large contact
area between the two phases facilitates analyte extraction (Fanali et al.,
2017; Sadrykia et al., 2019; Viñas, Bravo-Bravo, López-García, &
Hernández-Córdoba, 2013; Viñas, Campillo, López-García, & Hernán­
dez-Córdoba, 2014). A DLLME unique compatibility with modern
chromatographic methods can analyze the volumes of pico-liter to
micro-liter extracts (Fanali et al., 2017; Sadrykia et al., 2019). The
extensive contact surface of tiny droplets and analytes speeds up the
mass transferring processes of analytes from the aquatic to the organic
phase in a DLLME process, which increases extraction efficiency and
removes the time-consumption issue (Berton, Monasterio, & Wuilloud,
2012; Reema, Itishree, Shantaram, & Jagdish, 2013). Compared to other
extraction methods, DLLME has higher enrichment factors and lower
solvent consumption due to microdroplet production (Sadrykia et al.,
2019). This might be because a dispersive solvent was used, which
lowers the partition coefficients of analytes into extraction solvents. In
the study of sample preparation techniques, parameter optimization is
crucial. Even if a method’s optimization is extensive, it still has a
drawback. Some studies have used the fractional factorial design to
investigate the effects of extraction conditions on fast and efficient
extraction or clean-up of target compounds from the sample matrix to
find the conditions that allow for fast and efficient extraction or clean-up
of target compounds from the sample matrix (Chen et al., 2011). DLLME
has been used widely within the last decade to extract analytes from
milk (Alshana, Ertaş, & Göğer, 2015; Altunay & Gürkan, 2016; Faraji &
Adeli, 2017; Sadrykia et al., 2019; Viñas et al., 2013).
Sadrykia et al. (2019) used 1 mL of milk sample with 2 mL aceto­
nitrile and 200 µL chloroform as dispersive and extraction solvents,
respectively, for the isolation and clean-up of vitamin E from infant
formula samples without the need for saponification and the recovery
rates were within 89.12–109.98% (Table2). The advantage of DLLME
compared with the saponification process and LLE is decreasing organic
solvent consumption and proposing a simple and fast method for anal­
ysis of vitamin E in infant formula. While Viñas et al. (2013) used 2 mL
of the methanolic extract containing 100 µL tetrachloroethane with
saponification, the recovery rates were within 89–103%. However,
DLLME is meager quantities of solvent, meaning that the procedure can
be described as environmentally friendly. In recent years, micro­
extraction methods have been developed to replace classic extraction
procedures, with the main advantage of using microliter-scale solvent
E. Karrar et al. Food Chemistry 373 (2022) 131436

Table 2
Extraction techniques used for the determination of LSVs.
Extraction Analyte Extraction solvent Matrix Recovery Method References
techniques

Overnight cold all-trans-retinol, α-tocopherol, 18 mL of ethanol Milk from 55–100% HPLC-APCI-DAD-MS/ Gentili et al. (2013)
saponification γ-tocopherol, δ-tocopherol, different animal MS
ergocalciferol, cholecalciferol, species (donkey,
phylloquinone, and cow, buffalo, goat,
menaquinone-4 bovine, sheep, and
ewe milk)
Alkaline hydrolysis Tocopherols (α-tocopherol, Ethanol (5 mL)/ ethyl ether Human milk – HPLC Wei et al. (2018)
γ-tocopherol, δ-tocopherol) (12.5 mL)/petroleum ether
(12.5 mL)
Alkaline α-, γ-, δ-tocopherols Ethanol + Methanol + H2O Cow milk – HPLC Czauderna and
saponification Kowalczyk (2007)
Alkaline hydrolysis Retinol α- and γ-tocopherol n-hexane: dichloromethane Ewe milk – UPLC Revilla et al. (2017)
(5:1)/isopropanol (4/1)
Saponification vitamins E (α-tocopherol, 15 mL of KOH in ethanol Milk and infant 85.8–108.7% HPLC Barba et al. (2011)
γ-tocopherol, δ-tocopherol) (50%, w/v) formula
and D (cholecalciferol and
ergocalciferol)
Saponification Vitamins A, D, E, and K 30 mL of hexane/ethyl Breast milk 90.9–105% LC–MS/MS Kamao et al. (2007)
acetate (9:1, v/v)
LLE vitamin D3 (cholecalciferol) 10 mL of Isopropyl alcohol Milk-based infant 93.11–110.65% LC–MS/MS Abernethy (2012)
(IPA) formulas
LLE Retinol α-, γ-tocopherol, 1000 μL ethanol Human milk >95% LC-UV/FLD LC-MS/ Levêques et al.
phylloquinone, and MS (2019)
menaquinone-4
LLE Vitamin D2 2 mL hexane Bovine + buffalo 99.6% HPLC Kaushik et al.
milk (2014)
LLE Retinol, α- and γ-tocopherol 1000 μL ethanol human milk – NP-HPLC/UV Redeuil et al. (2021)
LLE Vitamin A, E 5 mL of ethanol Cow and goat milk – HPLC Guneser and
Karagul Yuceer
(2012)
LLE Vitamin K (K1, MK-4, and MK- 3 mL of ethanol Breast milk 66–104% HPLC–MS/MS Gentili et al. (2016)
7)
LLE vitamins A, D, D, E and K, 15 mL hexane Bovine milk 89–107% HPLC/UV Gomis et al. (2000)
retinyl acetate, retinyl Microcolumn LC
palmitate, tocopherol acetate,
ergosterol and 7-
dehydrocholesterol
LLE/SPE/ Alkaline Vitamins A, D2, D3, E and K1, 15 mL hexane Bovine milk 85–105% HPLC Blanco et al. (2000)
hydrolysis retinyl acetate, retinyl
palmitate, tocopherol acetate,
ergosterol and 7-
dehydrocholesterol
Saponification/LLE Vitamin D Hexane: dichloromethane Human, cow, 88.2–105% LC-MS/MS Gomes, Shaw,
(4:1, v/v) mare, goat and Whitfield, and
sheep milk Hewavitharana
(2015)
Deproteinization, Retinol and α-tocopherol Ethanol and n-hexane Breast milk 73.4–116.3% HPLC Plíšek et al. (2013)
saponification and
LLE
DLLME Vitamin E(α-tocopherol) 2 mL acetonitrile Infant formula 89.12–109.98% HPLC-UV Sadrykia et al.
(2019)
DLLME Vitamins D2, D3, K1, K2 and K Dispersive solvent, to which Infant formula 89–103% LC–DAD/ LC–APCI- Viñas et al. (2013)
150 mL of carbon MS
tetrachloride (extractant
solvent)
Solvent extraction Retinoic acid, retinal, retinol, 12 mL of hexane Raw milk (cow, 70–108% HPLC-DAD-MS/MS Rocchi et al. (2016)
and fourteen retinyl esters buffalo, ewe, and
goat)
Solvent extraction Vitamin A, E n-hexane/ethyl acetate (9:1) Milk – UPLC/ HPLC Chauveau-Duriot,
Doreau, Nozière,
and Graulet (2010)
Solvent extraction α-tocopherol, and γ-tocopherol Hexane Donkey milk – GC Martini et al. (2021)
SPE Vitamins A, D3 and E 100 mL of n-hexane Fortified infant 96–105% LC–APCI-MS Heudi et al. (2004)
formula
SPE/Silica cholcalciferol (D3) and 6 mL hexane/ethylacetate Bovine milk 60–90% LC–MS/MS Trenerry et al.
ergocalciferol (D2) (80:20) (2011)
SPE/ Silica Vitamin D3, 25-hydroxyvita­ Diethyl ether: petroleum Bovine milk 93–101% HPLC Jakobsen and
min D3 (25OHD3), vitamin D2, ether (1:1) Saxholt (2009)
and 25-hydroxyvitamin D2
(25OHD2)
PFSPE Retinyl esters, cholecalciferol, 100 µL of ethanol Bovine milk >90% HPLC Chen et al. (2011)
tocopherol acetate, and
phylloquinone
(continued on next page)

6
E. Karrar et al.
Table 2 (continued )
Extraction Analyte Extraction solvent
techniques
SFE Vitamins A and E 1.5 g of hydro flow SFE/ 1.0
mL of degassed methanol
PLE Retinyl acetate, α-, γ-, and One extraction cycle with a
δ-tocopherol static time of 5 min,
methanol as solvent
extraction, T = 50 ◦ C, at a
pressure of 1600 psi.
amounts. Liquid-liquid extraction (LLE) was previously used for many
vitamin extraction techniques, with dispersive liquid-liquid micro­
extraction (DLLME) being the most recent advancement (Fanali et al.,
2017).
2.3. Direct extraction (DE)
DE or solvent extraction is a process for the extraction of targeted
compounds. The milk is known as an immiscible or nearly immiscible
matrix, a solvent that exhibits a high preferential affinity toward tar­
geted compounds. Due to the affinity of solvent to targeted compounds,
they liberate quickly from the matrix to the solvent and consequently
either directly obtained by evaporation of the solvent or separated by
different chromatographic means. As shown in Table 2, DE has been
used over the years for extraction of LSVs from various milk types (cow,
buffalo, ewe, donkey, and goat milk) by using organic solvents for
deproteinization and extraction without a saponification pretreatment
(Martini, Altomonte, Licitra, Bartaloni, & Salari, 2021; Rocchi et al.,
2016). Ethanol was used for deproteinization, and hexane was also
applied as an organic solvent (Rocchi et al., 2016). Ultrasonication has
played a vital role in disrupting the lipid globule membrane that en­
capsulates the LSVs in conjunction with ethanol (Blanco et al., 2000;
Gomis et al., 2000). Direct extraction of vitamin D recoveries has yielded
close results to saponification (89–107%). The recoveries were assessed
at three distinct concentration levels by integrating criteria (Blanco
et al., 2000; Gomis et al., 2000).
2.4. Solid-phase extraction (SPE)
In the SPE method, the targeted compounds are transferred from a
gas, liquid, or supercritical fluid matrix to a solid sorbent, which retains
the targeted compounds by their interaction with the sorbent. After that,
the targeted compounds are recovered by solvent displacement or
thermal desorption (Poole, 2020b). As shown in Table 2, SPE has been
used extensively to extract LSV from bovine and human milk and infant
formula (Heudi, Trisconi, & Blake, 2004; Jakobsen & Saxholt, 2009;
Trenerry, Plozza, Caridi, & Murphy, 2011). SPE extraction of LSVs was
achieved using Sep-Pak silica and C18 as sorbents for milk samples
(Kasalová et al., 2015). C18 cartridges are used because these contents
have high potentials for extracting vitamins. The recoveries of LSVs
were in the 74–116% range (Blanco et al., 2000). Some authors have
used SPE to purify milk samples in more than one step. Liquid-liquid
extraction was used to achieve a purified extract after a (silica)-SPE
clean-up phase appropriate for (MS) detection (Trenerry et al., 2011).
The sensitivity was high with this complex multistep purification, but
the recovery was only 61–86%. Methanol and water were used in both
processes for the washing, elution, and mobile phase (Jakobsen & Sax­
holt, 2009). Trenerry et al. (2011) used a 500 mg Silica SPE cartridge to
remove potential interfering compounds from the sample, pre-
concentrate, and extract vitamin D. The cartridge was washed with 3
mL hexane, 3 mL hexane: ethyl acetate (90:10 v/v), and the vitamin D
eluted with 6 mL hexane: ethyl acetate (80:20 v/v). The solvents were
removed with a stream of nitrogen, and the residue was reconstituted
with 0.25 mL internal standard and filtered through a 0.45 µm. The
recoveries of vitamin D were in the range of 60–90%. Blanco et al.
7
Food Chemistry 373 (2022) 131436
Matrix Recovery Method References
Milk and milk 76–101% HPLC Berg et al. (2000)
powder
Milk powder 92–106% HPLC with Delgado-Zamarreño
electrochemical et al. (2006)
detection (ELD) in
amperometric mode
(2000) utilized Mega Bond Elut C18 cartridges to remove potential
interfering compounds from the sample and preconcentrate and extract
fat-soluble vitamins. The cartridge was previously made with 25 mL
methanol and 10 mL Milli-Q water. The sample was passed through the
Mega Bond Elut C18 cartridge at a controlled flow rate, ensuring that all
vitamins were entirely preserved. The cartridge was then cleaned with 5
mL methanol-water (1 + 9, v/v), and the fat-soluble vitamins eluted
with 6 mL methanol. The eluate was filtered with a 0.45 µm filter. This
was then evaporated to dryness under nitrogen. The residue was
reconstituted in 20 or 40 µL ethanol with 0.025 percent (m/v) BHT for
fresh and fortified milk samples. In a 5 µL aliquot, these solutions were
injected into the HPLC equipment. The recovery of vitamins was in the
range of 84–108%. SPE has many advantages compared with conven­
tional extraction methods, e.g., complete phase separation, excellent
recovery, and reduced organic solvent consumption.
2.5. Supercritical fluid extraction (SFE)
Pressure/temperature creates a supercritical fluid with improved
solvating properties (Sharif et al., 2014). The efficacy of SFE depends on
various factors such as temperature, pressure, sample matrix composi­
tion, and interaction of target compound with the matrix (Sharif et al.,
2014). CO2 is commonly used as an extraction solvent during the SFE
process. Since supercritical CO2 has a high diffusivity and low viscosity,
it can quickly diffuse through semi-solid materials. The properties of
supercritical fluids allow for faster extraction than traditional LC (Berg,
Turner, Dahlberg, & Mathiasson, 2000; Zhang et al., 2018).
Consequently, the process has been used to extract LSVs from milk
samples (Table 2). Berg et al. (2000) recorded vitamin E and vitamin A
determination in milk using methanol with modified CO2 as a super­
critical solvent, Hydro-matrix as a water adsorbent. SFE is a solid
alternative to LLE for evaluating LSVs in foods, as shown by the high
recoveries (76–101%) obtained from milk and milk powder. In addition,
Turner and Mathiasson (2000) developed and optimized a process to
extract vitamin A and E from milk powder. With slight modifications to
the SFE protocol, the technique could be applied to other kinds of food
formulas and other LSVs.
Moreover, the automated SFE process could be run when paired with
the saponification procedures compared with the traditional method­
ology. Since just a small amount of solvents has to be evaporated, sample
throughputs can be significantly increased. The process is less expensive,
allows faster extraction, and is environmentally friendly than solvents
extraction (Berg et al., 2000).
2.6. Pressurized liquid extraction (PLE)
The pressurized liquid extraction (PLE) is an automated method that
uses high pressure (1600 psi) and high-temperature (50 ◦ C) solvents to
decrease the extractant’s volume, maximize insulation yields and reduce
the extraction time. This process also has an advantage for LSVs, in
which, extraction takes place in a stainless-steel cell, i.e., in an envi­
ronment free of light and oxygen (Czauderna & Kowalczyk, 2007; Fanali
et al., 2017). The ability to automate, low solvent levels, and shorter
extraction times are advantages of PLE over other extraction methods.
PLE can be done in static, dynamic, or combination modes. The sample
E. Karrar et al.
and solvent are held at constant pressure and temperature for a user-
specified time in the stationary process. The solvent flows continu­
ously through the sample in the dynamic mode (Delgado-Zamarreño,
Bustamante-Rangel, García-Jiménez, Sánchez-Pérez, & Carabias-Martí­
nez, 2006). Recently, PLE has been used more than SFE for extractions of
vitamins (Jaime et al., 2015), and its application in the extraction of
LSVs from milk samples have been reported (Table 2). For vitamin E
extraction using methanol- isopropanol and methanol (1:1, v/v) solution
at 50 ◦ C and 1600 psi were used as an extractant, with the hydro matrix
as a drying agent or dispersing medium for the prevention of sample
particle aggregation. The extract was diluted to 50 mL with methanol,
filtered, and then injected directly into an LC/MS without any other
treatments (Bustamante-Rangel, Delgado-Zamarreño, Sánchez-Pérez, &
Carabias-Martínez, 2007; Delgado-Zamarreño, Bustamante-Rangel,
García-Jiménez, Sánchez-Pérez, & Carabias-Martínez, 2006; Viñas,
Bravo-Bravo, López-García, Pastor-Belda, & Hernández-Córdoba, 2014).
The disadvantage of PLE is related to the high cost of the equipment and
to the variability of the extracted volumes, which are different even if
samples are always prepared in the same way; this implies a step of the
volume measurement of the extracts when they have to be injected
directly(Fanali et al., 2017).
3. Chromatographic analysis techniques
Vitamin concentrations in milk are low (in μg/L to ng/L range),
necessitating selective isolation and detection techniques. The published
techniques are summarized in Table 3. The most currently used LSVs
analysis techniques are LC/MS, LC/UV, and immunological methods.
Liquid chromatography is probably the most popular technique for
vitamin separation. Liquid chromatography has been paired with
various detection methods, including UV/VIS, MS, and MS/MS, because
they are sensitive, exact, and supply high specificity. Chromatographic
approaches are the most commonly used analytical methods for classi­
fying LSVs in milk. Vitamin derivatives can be quantified independently
and concurrently using these techniques. Standard and reverse-phase
chromatography is used to evaluate milk vitamins, with normal phase
HPLC being used for vitamin separation.
3.1. Separation techniques
3.1.1. Normal phase HPLC (NP-HPLC)
Normal phase (NP) and reverse phase (RP) chromatography have
been used to determine vitamin amounts. The choice depends on the
extraction/purification procedures used and on the vitamers to be
measured. Isocratic elution was carried out in almost all studies. Hexane
or a mixture of hexane with a small percentage of a more polar solvent,
such as isopropyl alcohol or methyl dichloride, is the most utilized
mobile phase for normal phase HPLC (Fanali et al., 2017). In reality, the
normal phase has been used successfully to separate retinol (Fanali et al.,
2017; Redeuil et al., 2021). All vitamin E congeners from the cis and
trans isomers allow single quantification of positional isomers β- and γ
(Pinheiro-Sant’Ana et al., 2011). Another feature of the NP column is
tolerating comparatively high loads of fat material that are not strongly
adsorbed and can be easily removed from the column by the non-polar
mobile phase. When saponification is not essential for the analyte
isolation, this quality could be exploited for the direct injection of
extract obtained from fatty milk during the step of vitamin extraction
(Barba et al., 2011). Purification, commitment, and subsequent quan­
tification of vitamins in milk, on the other hand, has proven to be
difficult and takes more than two steps. Furthermore, those proposed
processes were solvent intensive, time-sensitive, and inefficient methods
(Kasalová et al., 2015).
3.1.2. Reversed-phase HPLC (RP-HPLC)
The most commonly used chromatographic method for the analysis
of LSVs in milk is RP-HPLC (Pulido et al., 2019). A non-polar stationary
8
Food Chemistry 373 (2022) 131436
phase with a polar mobile phase is used in this process. An octadecyl
(C18) column has been used mainly for this. Particle sizes in the
analytical columns typically range from 2.6 to 10 µm. A pre-column or
guard column has been used in several cases to protect the analytical
column (Trenerry et al., 2011). Narrow-bore columns can be used for
reduced solvent consumption compared to standard columns (Blanco
et al., 2000; Gomis et al., 2000) and allow the injection of smaller
amounts of samples. Barba et al. (2011) conducted a study comparing
three columns for quantifying vitamin E (α-, γ-, and δ-tocopherol) and
vitamin D. Three columns were assessed: C18 (5 µm, 150 mm × 4.6
mm), C18 (5 µm, 250 mm × 4.6 mm) and C8 (5 µm, 250 mm × 4.6 mm).
On the C8 column, vitamin E peaks (α-, γ-, and δ-tocopherol) and
vitamin D (D2 and D3) overlapped because C8 did not reach adequate
peak resolution (Barba et al., 2011). The highest peak resolution and
fastest study were achieved with a C18 (5 µm, 150 mm × 4.6 mm).
Revilla, Escuredo, González-Martín, and Palacios (2017) assessed the
suitability of UHPLC for the simultaneous determination of retinol and
α-, and γ-tocopherol using an Acquity UPLC HSS T3 (1.8 µm, 150 mm ×
2.1 mm) column. With the decrease in particles in the stationary phases,
the analysis time was much shorter. UPLC is faster, more sensitive, and
more environmentally friendly. More information about the chromato­
graphic condition and columns can be found in Table 3. The mobile
phase for LSVs separation is usually a mixture of water with acetonitrile,
ethanol, or methanol, but non-aqueous mobile phases have also been
used, such as acetonitrile/methanol (Barba et al., 2011) or 100%
methanol (Chen et al., 2011). The isolate was primarily accomplished by
mobile phase gradient elution. Methanol-water (A) and methanol-
tetrahydrofuran (B) were successfully used as a mobile phase gradient
elution to assess LSVs (Blanco et al., 2000; Gomis et al., 2000; Pulido
et al., 2019), and adding tetrahydrofuran to methanol resulted in
reduced retention time. Kaushik et al. (2014) optimized the mobile
method to estimate vitamins. They used three mobile phases: acetoni­
trile/methanol/chloroform (88:8:4) and acetonitrile/methanol/ethyl
acetate (88:8:4). The highest resolution was achieved using an 88:8:4
mixture of acetonitrile/methanol/chloroform at a 1 mL/min for flow
rate. This technique gives low limits for detection and quantification and
high reproducibility of vitamin D2.
3.1.3. Supercritical fluid chromatography (SFC)
SFC is a separation method that integrates gas-chromatography (GC)
characteristics and LC, and it exhibits an excellent selectivity for sepa­
rating vitamins (Fanali et al., 2017). Two major companies have
recently made significant investments in this design to create a hybrid
LC/SFC system and a holistic SFC system by integrating its advantages
with HPLC/UPLC. The method is known as UPCC or UPC2 (ultra-per­
formance convergence chromatography) (Nováková et al., 2014), and it
has been successfully examined in various applications. Compared with
LC, SFC is faster, has improved resolution, provides sharper peaks, and
has better efficiency (Oberson, Bénet, Redeuil, & Campos-Giménez,
2020). Only one study used SFC-MS/MS methods to analyze vitamin D
in milk (Oberson et al., 2020). For separation, a Waters CSHTM Fluoro
Phenyl column using a gradient of methanol-ammonium formate on
carbon dioxide with a LOD of 2 ng/100 mL was used. The recovery was
in the range of 85–115%. This method gives a good resolution, provides
sharper peaks, and is faster. SFC has many advantages over HPLC to a
determination of LSVs; these include (i) lower organic solvent con­
sumption; (ii) higher separation efficiency; (iii) separation of a broader
spectrum of analytes of various sizes and polarities in a single chro­
matographic run; (iv) separation of β- and γ-tocopherol on reversed-
phase columns; and (v) separation of cis/trans isomers in a single
chromatographic run. Future SFC applications on the milk analysis,
which allows for simultaneous determination of LSVs, triglycerides
(TAG), di- triglycerides (DTAG), mono-triglycerides (MTAG), free fatty
acid (FFA), cholesterol, and other steroids and fats esters, could be
recommended.
In addition to the approaches outlined above, effective techniques
 E. Karrar et al.
Table 3
An overview of the determination methodology of LSVs.
Matrix Analyte Technique Extraction Column Detector Mobile phase/flow rate Analysis time Injection References
(min) and volume
temperature

Human milk Retinol, α- and NP-HPLC/ LLE NP column UV – – – Redeuil et al.

γ-tocopherol UV (2021)
Human milk α-tocopherol, HPLC Alkaline Silica column (250 SPD-M10AV diode The mobile phase was n-hexane/isopropanol 10 min 20 µL Wei et al.
γ-tocopherol, hydrolysis mm × 4.6 mm × 5 µm) array detector (295 (98:2, v/v) with a flow rate of 0.5 mL* min− 1. (25 ◦ C) (2018)
δ-tocopherol nm)
Infant formula Vitamin E HPLC DLLME ODS column (150 mm UV (296 nm) The mobile phase was 91:8:1acetonitrile: 15 min 20 μL Sadrykia
× 4.6 mm) methanol: water. The flow rate was 1.5 mL/min (30 ◦ C) et al. (2019)
fortified milk Retinyl esters, HPLC PFSPE C18 column (5 μm; SPD-M20A PAD (327 The mobile phase, 100% methanol, was used at the 20 min 20 µL Chen et al.
power cholecalciferol, 250 mm × 4.6 mm) nm for retinyl esters, a flow rate of 1.0 mL/min. (2011)
tocopherol acetate, 264 nm for
phylloquinone cholecalciferol, 284
nm for tocopherol
acetate, and 247 nm
for phylloquinone
Milk and infant Vitamins E HPLC Saponification Three columns were UV A mixture of 90% acetonitrile and 10% methanol. (16–28 ◦ C) – Barba et al.
formula (α-tocopherol, assayed: C18 (5 µm, Flow rate (1.00 and 1.5 mL.min− 1 265 nm). (2011)
γ-tocopherol, 150 × 4.6 mm), C18
δ-tocopherol) and D (5 µm, 250 × 4.6 mm)
(cholecalciferol and and C8 (5 µm, 250 ×
ergocalciferol 4.6 mm)
Bovine milk all trans-retinol, HPLC/MS Solvent C18 guard column, HPLC–UV/VIS and The mobile phase: water: methanol (5:95), at a 1 30 min, – Plozza,
α-tocopherol extraction 2.1 × 150 mm, 5 µm HPLC–Fl mL/min flow rate (295–230 nm) 30 ◦ C. Craige
Trenerry,
and Caridi
9

(2012)
Human breast Retinol and HPLC LLE RP-18e, 100 mm × DAD In the mobile phase, 100% methanol was used at a 2 min – Plíšek et al.
milk α-tocopherol 4.6 mm monolithic 2.5 mL/min flow rate. The DAD detection of (2013)
column retinol and α-tocopherol was carried out at 325
and 295 nm, respectively.
Cow milk Vitamin E HPLC Saponification 4.0 × 125 mm Silica Fluorescence The mobile phase consisted of heptane containing 100 μL Jensen,
column detection 2-propanol (3.0 mL/l) and degassed with helium. Lashkari,
The flow rate was 3.0 mL/min. Excitation the and
wavelength of 290 nm and an emission Kristensen
wavelength of 327 nm. (2020)
Bovine milk Vitamins A, D2, D3, E HPLC LLE/SPE/ The column used was UV– VIS mobile phases, methanol–water (99 + 1, v/v) (A) – 5 µL Blanco et al.
and K1, retinyl acetate, Alkaline Extrasil ODS2, 150 × and methanol– tetrahydrofuran (70 + 30, v/v) (B) (2000)
retinyl palmitate, hydrolysis 2.1 mm id, 3 µm in gradient mode, the mobile phase is pumped at a
tocopherol acetate, and flow rate of 0.15 mL min21 to 5.5 min and then
7-dehydrocholesterol 0.2 mL min21 after to 6.3 min. The wavelength of
the detector was set at 325 nm for retinol, retinyl
acetate and retinyl palmitate, 264 nm for vitamin
D2 and vitamin D3, 280 nm for vitamin K1,
tocopherol, tocopherol acetate, and 7-

Food Chemistry 373 (2022) 131436

dehydrocholesterol
Bovine milk Vitamins A, D, D, E and HPLC LLE C18 BDS (150 × 0.3 UV–VIS [A5methanol–water (99:1); B5methanol– 15 min, 20 ◦ C 5 µL Gomis et al.
K, retinyl acetate, mm, 3 µm) tetrahydrofuran (70:30)] (2000)
retinyl palmitate,
tocopherol acetate,
ergosterol and 2 3 1 7-
dehydrocholesterol
All-trans-retinol, all- HPLC LLE C18/C30 tandem DAD-MS/M 19 ◦ C – Rocchi et al.
trans-retinal, all-trans- column; TSKgel Super- (2016)
(continued on next page)
 E. Karrar et al.
Table 3 (continued )
Matrix Analyte Technique Extraction Column Detector Mobile phase/flow rate Analysis time Injection References
(min) and volume
temperature

Raw milk (cow, retinoic acid, alltrans- ODS (4.6 mm × 100 Methanol was phase A and a mixture isopropanol/
buffalo, ewe, retinyl propionate, all- mm, 2 µm. C30 hexane 50:50 v/v was phase B. Flow rate of the
and goat). trans-retinyl palmitate column (4.6 × 250 mobile phase was 1 mL/min. 325 and 350 nm.
mm, 3 µm)
Bovine milk Cholcalciferol (D3) and HPLC–MS SPE/Silica (2.1 × 150 mm, 5 µm) UV The mobile phase consisted of methanol: water 30 ◦ C – Trenerry
ergocalciferol (D2) C18-A column (92:8 v/v) and the flow the rate was 0.2 mL/min. et al. (2011)
Breast milk Vitamins A, D, E, and K HPLC Saponification C18 UG120 RF-10AXL For the determination of α-Toc and vitamin K: 35 ◦ C 30 µL Kamao et al.
fluorescence methanol–H2O, 90:10, v/v). For the determination (2007)
of D and 25(OH)D: a mixture of acetonitrile and
H2O (30:70, v/v).
Humanmilk Vitamin K HPLC–MS/ LLE with hexane Two reversed-phase LC-F Methanol (phase A) and 2-propanol: hexane 20 µL Gentili et al.
MS columns C18, 4.6 × (50:50, v/v) solution (phase B) and flow rate of 1 (2016)
50 mm; 5 µm) and mL/min.
C18, 4.6 × 250 mm; 5
µm)
Sheep milk α-tocopherol RP- HPLC Saponification C18 column (5 μm, UV acetonitrile–methanol–dichloromethane – – Pulido et al.
250 × 4.6 mm) (40:50:10 vol/vol/vol) and at a flow rate of 1 mL/ (2019)
min. Wavelength set at 292 nm
Cow and goat Vitamins A and E HPLC LLP C18 column (5μ, 4.6 UV Methanol (100%) at a flow rate of 1.0 mL/min was 30 ◦ C 25 and 50 μL Guneser and
milk mm × 150 mm) used as a mobile phase for the separation of for vitamin E Karagul
vitamin E. Ultraviolet detection was performed at and A, Yuceer
292 nm. For vitamin A, the mobile phases were respectively (2012)
water and methanol (90:10), and the flow rate was
1.2 mL/min. Ultraviolet detection was performed
at 323 nm.
10

ilk Vitamin A and E HPLC DSPE C18 column (250 mm SPD-10 AD PDA 70% acetonitrile and 30% methanol. a flow rate of 50 ◦ C 20 μL Köseoğlu,
× 4.6 mm, 5 μm) 1 mL/min. 292 nm and 324 nm Ulusoy,
Yilmaz, and
Soylak
(2020)
Bovine milk vitamin D3 HPLC micro-SPE C18 column (250 mm UV A mixture of ACN: MeOH (80:20, v/v%). The flow 20 µL Sereshti
× 4.6 mm × 5 µm rate of 1 mL*min − 1. 264 nm et al. (2020)
Cow milk Vitamins A, D, and E HPLC Solvent C18 (3 μm, 250 mm × DAD The mobile phase selected was A 24 C◦
20 µL Ramalho,
extraction 4.6 mm) (acetonitrile–water-triethylamine, 95.5:4:0.5, vol/ Santos,
vol/vol) and B (ethyl acetate). The flow rate was Casal, Alves,
1.0 mL/min. Vitamin E was quantified using and Oliveira
fluorescence signals, with tocol as internal (2012)
standard. Retinoids (retinol, retinyl acetate, and
retinyl palmitate) were monitored at 325 nm,
vitamin D3 at 264 nm, provitamin D3 at 280 nm
Milk from all-trans-retinol, HPLC- Overnight cold C18 (4.6 mm × 50 UV − VIS Methanol (phase A) and an isopropanol/hexane 30 min, 30 ◦ C 5 μL Gentili et al.
different α-tocopherol, APCI-DAD- saponification mm, 5 μm) (50:50, v/v) solution (phase B). Flow rate of 1 mL (2013)
animal γ-tocopherol, MS/MS min− 1.(200–700 nm)
species δ-tocopherol,

Food Chemistry 373 (2022) 131436

(donkey’s, ergocalciferol,
cow’s, cholecalciferol,
buffalo’s, phylloquinone, and
goat’s, menaquinone-4
bovine,
sheep, and
ewe’s milk)
Bovine milk Retinol, retinyl acetate, HPLC/ Saponification RP C18 (150 × 4.6 PAD detector 210 and HPLC: mobile phase was a mix of 35 ◦ C HPLC:
δ-tocopherol, UPLC mm, 3-μm) RP C18 600 nm. acetonitrile–dichloromethane-ammonium acetate 36 min.
(continued on next page)
 E. Karrar et al.
Table 3 (continued )
Matrix Analyte Technique Extraction Column Detector Mobile phase/flow rate Analysis time Injection References
(min) and volume
temperature

γ-tocopherol, (250 × 4.6 mm, 5- 0.05 M in methanol–water 70–10–15–5and flow UPLC: 46 40 μL for Chauveau-
α-tocopherol, and μm). A 150 × 2.1 mm rate applied was 2 mL/min. UPLC: min. HPLC or 10 Duriot et al.
tocopheryl acetate Acquity UPLC HSS T3, acetonitrile–dichloromethane–methanol (A) μL for UPLC (2010)
1.8-μm column 75–10–15 and acetate ammonium 0.05 M in water
(B). The flow rate was 0.4 mL/min. Vitamins A and
vitamins E were detected at 325 and 292 nm,
respectively.
Ewe milk retinol and α-, and UPLC Alkaline A150 × 2.1 mm fluorometric detector The mobile phase for retinol was acetonitrile: 35 ◦ C – Revilla et al.
γ-tocopherol hydrolysis Acquity UPLC HSS T3, methanol (85:15)/isopropanol:water (50:50) 80/ (2017)
1.8 µm m column 20. with ʎexc = 325 and ʎem = 475 nm for
fluorometric detection. The mobile phase for the
different tocopherols analyzed was acetonitrile:
methanol (85:5)/isopropanol 90/10, with ʎexc =
295 and ʎem = 390 nm for fluorometric detection.
flow rate applied was 0.4 mL/min.
Human milk Vitamin D SFC-MS/ LLE Nine chromatographic Make-up solvent was 10 mM ammonium formate 45 ◦ C 3 μL Oberson
MS columns were chosen in methanol at a flow rate of 0.4 mL/min et al. (2020)
for screening
11

Donkey milk α-tocopherol, and GC Saponification Fusedsilica capillary Flameionization – 270 and – Martini et al.
γ-tocopherol column Zebron ZB- detector 300 ◦ C (2021)
5MSi (length = 30 m,
internal diameter =
0.25 mm, film
thickness = 0.25 µm
Human, Cow, Vitamin D LC-MS/MS Saponification/ C18 (150 × 2.1 mm, Jet Stream 0.1% formic acid in water (A) and 0.1% formic – 20 µL Gomes et al.
mare, goat LLE 2.7 µm) column and acid in methanol (B), and the flow rate was 0.2 (2015)
and sheep Pursuit PFP (150 × mL/min
milk 3.0 mm, 3 µm) column
Human milk Retinol, α-, LC-UV/FLD LLE Silica, 1.9 µm, 200 × Retinol and vitamin E: Retinol and vitamin E: Solvent (A) was n-hexane Retinol and 5 µL Levêques
γ-tocopherol, LC-MS/MS 2.1 mm column. PDA (326 nm for for chromatography and solvent (B) a mix of n- vitamin E: et al. (2019)
phylloquinone, and Vitamin K: C30, 2.6 retinol and 330 nm for hexane–dioxane (50 + 50, v/v) containing 0.01% 10 min
menaquinone-4 µm, 150 × 2.1 mm E) acetic acid. (35 ◦ C).
column Vitamin K: TQ-S Vitamin K: Solvent A was a mixture of water- Vitamin K:
Tandem Mass detector methanol (96 + 4) and solvent B was isopropanol 20 min
equipped with an (35 ◦ C)
APCI

Food Chemistry 373 (2022) 131436

E. Karrar et al.
such as immunoassay approaches have been developed. The use of im­
munoassays is justified when routine quality control of relatively simple
sample compositions is required. Immunoassays are suitable for daily
applications because of their high specificity, sensitivity, simplicity,
compactness, and cost-effectiveness (Zhang et al., 2018). These assays
are based on a specific interaction between an antibody and an antigen.
They may detect low levels of residues quickly without the need for
time-consuming extraction and clean-up processes. Enzyme-linked
immunosorbent assays (ELISA) are the most widely used immunoas­
says due to their high sample throughput. These approaches can reduce
the number of tests required to detect vitamins in diverse samples by a
substantial amount. The 25-OH vitamin D ELISA Assay kit was used by
Martin, Petrucka, and Buza (2014) to measure the quantity of vitamin D
in serum samples, and the technique performed well.
3.2. Detection techniques
3.2.1. UV detection
Ultraviolet and electrochemical detectors are the most widely used
detectors in detecting and quantifying vitamins (Perales et al., 2005).
Generally, the UV detector is used because all LSVs has a substantial UV
absorption range between 250 and 323 nm. Various studies used
wavelength within this range, specifically 254 nm (Holick et al., 1992),
264 nm (Blanco et al., 2000; Gomis et al., 2000; Sereshti, Toloutehrani,
& Nodeh, 2020), 265 nm (Barba et al., 2011), 292 nm (Pulido et al.,
2019), 296 nm (Sadrykia et al., 2019), and 323 nm (Guneser & Karagul
Yuceer, 2012) (see Table 3). UV detection proved to be sufficiently
sensitive, with limits of detection (LOD) of 0.82–1.57 ng/100 mL for
detecting α-, γ- and δ-tocopherol, as well as ergocalciferol and chole­
calciferol (Barba et al., 2011). For ergocalciferol (vitamin D2), chole­
calciferol (vitamin D3), and vitamin K, ergosterol, 7-dehydrocholesterol,
tocopherol, and tocopherol acetate a using narrow-bore column and a
UV detector (Blanco et al., 2000) achieved LOD of 0.80–4.1 ng/5 µL.
Although this method is appropriate for several fortified milk types, it
appears insufficient for determining vitamin levels in unfortified milk.
Capillary LC separation with UV detection was used to determine
vitamin D2, vitamin D3, vitamin K, ergosterol, 7-dehydrocholesterol
tocopherol, and tocopherol acetate in milk with a LOD of 0.02–46
ng/mL (Gomis et al., 2000). Vitamin E, vitamin D2, and vitamin D3
require using a more sensitive LC/UV for their efficient detection within
the range of 0.82 to 1.57 ng/100 mL (Barba et al., 2011). The LC-UV
approach solves vitamin E separation problems (tocopherols α, γ, and
δ) and D (vitamin D2 and vitamin D3), allowing all vitamins to be
measured and quantified. The photodiode array detector (PDA) detector
was used for the detection and quantification of vitamin D2. Various ʎmax
(228, 254, and 265 nm) were calculated for vitamin D2 analysis in milk.
During an HPLC study, the maximum peak area for vitamin D2 was
measured at 254 nm. This technique yields a LOD of 1 ng/100 µL for
vitamin D2 in milk (Kaushik et al., 2014).
3.2.2. MS
Over the last few years, there has been a surge in LC-MS use to
determine vitamin levels in biological samples (Trenerry et al., 2011).
As a routine analytical technique in clinical laboratories, this approach
is becoming more common, and it offers an appealing alternative to
conventional application techniques (van den Ouweland & Kema,
2012). One of the critical advantages of LC/MS over immunoassays is its
ability to measure multiple analytes in a single assay reliably (El-
Khoury, Reineks, & Wang, 2011). Both electrospray ionization (ESI) and
atmospheric pressure chemical ionization (APCI) methods have been
used in LC/MS to determine vitamins (Abernethy, 2012; Kamao et al.,
2007; Trenerry et al., 2011). Kamao et al. (2007) reported a technique
using stable isotope-labeled internal criteria for determining vitamin A,
vitamin D, vitamin E, and vitamin K in milk includes two extraction
techniques and a responsive liquid chromatography-atmospheric pres­
sure chemical ionization- tandem mass spectrometry (APCI-MS/MS)
12
Food Chemistry 373 (2022) 131436
detection. This technique can be used to identify the LSVs in milk.
Additionally, an excellent vitamin recovery of 96–105% was achieved
(Heudi et al., 2004). Trenerry et al. (2011) compared the LC/MS and
liquid chromatography-tandem mass spectrometry (LC/MS/MS) for
determining vitamin D3 in bovine milk. A better quantification limit
(LOQ) for vitamin D3 was achieved by LC-MS, at 0.01 µg/100 mL and
0.02 µg/100 mL, than by LC-MS/MS. The recovery was in the range of
61–86%. The disadvantages of these techniques were the low recovery
and analysis time, with the retention time being about 18 min. Aber­
nethy (2012) developed a faster analytical technique for vitamin D in
bovine milk with a comparable LOQ. In the mobile phase, ammonium
formate was added to water to boost the ionization in MS and used a
Diels-Alder derivatization reaction with 4-phenyl-1,2,4-triazoline-3,5-
dione (PTAD) to the determination of vitamin D3. Detection was ach­
ieved by electrospray ionization-tandem mass spectrometry (ESI-MS-
MS), and the retention time was 3.5 min. In this technique, the solvent
and waste were lower, about < 45 mL, including extraction and chro­
matography. Additionally, an excellent vitamin recovery of 103.2% was
achieved. LC/MS/MS is becoming a commonly widely used chromato­
graphic method for determining vitamins.
4. Current trends and future perspectives
Since 2010, significant progress has been achieved in vitamin anal­
ysis, accompanied by similarly significant improvements in sample
preparation and separation procedures. As a result, more precise con­
centration values for endogenous forms and vitamins added to fortified
meals have been discovered. Aside from this conventional method of
investigation, one of the most recent developments in this field is the
research of micronutrients present naturally in foods. Interfering
isomeric/isobaric chemicals co-extracted from the matrix can make
identifying complex vitamin profiles seen in nature. As we’ve seen, there
aren’t many articles on this subject yet; but, as this growing tendency
becomes more evident, more advanced analytical approaches based on
highly resolved separation and/or better detection systems will be
necessary to undertake unambiguous screening on a wide scale.
Although high-resolution MS offers appropriate selectivity and identi­
fication power, carotenoids and vitamins include numerous structural
and/or geometric isomers. In this regard, the ion mobility MS (IMMS)
appears to be an up-and-coming technical option. It may be used to
resolve isobaric and isomeric molecules and interferences from analytes
of interest.
5. Conclusions
Vitamins are a vital part of human nutrition, and analytical pro­
cedures for precise vitamin evaluation are significant concerns in
analytical chemistry. This review presents a thorough discussion of the
techniques for sample pretreatment and vitamin analysis in milk. We
looked at LSVs in different milk sources and analyzed the procedures
utilized to treat the samples. For example, because SPE is complicated
and has no substantial advantages over LLE, LLE is used to prepare milk
samples. Due to its enhanced sensitivity and accuracy, LC-MS is partic­
ularly helpful for measuring and quantifying vitamins. For example, to
enhance LC-MS detection, internal criteria that have been isotope-
labeled can be employed. The relevance of optimization in enhancing
sensitivity and decreasing matrix effects cannot be overstated. Advanced
sample preparation methods such as HPLC purification, SPE, and LLE
will replace automated or online sample preparations that achieve
identical outcomes. Automating the extraction step, whereby human
interaction may be lessened to eliminate operator mistakes and biolog­
ical risks, will be crucial in sample preparation. Furthermore, reducing
the number of samples and solvents used would save money and result in
less waste. Immunoassays are less expensive than chromatographic
methods and can thus be utilized for regular vitamin measurement. On
the other hand, the higher accuracy and specificity of LC make it one of
E. Karrar et al.
the fundamental approaches in vitamin research, so LC-MS will conse­
quently be the gold standard for assessing vitamins. The adoption of
current microprocessor technology, which decreases dead volume sub­
stantially, might also be beneficial.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgment
This work was supported by the National Natural Science Foundation
of China (31701558). We thank Dr. Alaa El-Din Bekhit (Department of
Food Science, University of Otago, New Zealand) for the assistance in
proofreading and English editing.
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