Journal of Chromatography A 1666 (2022) 462861

Contents lists available at ScienceDirect

Journal of Chromatography A
journal homepage: www.elsevier.com/locate/chroma

Simultaneous determination of fat-soluble vitamins and carotenoids in

human serum using a nanostructured ionic liquid based
microextraction method
Liyun Kong a,b, Jiaqi Wang a,b, Qingpeng Gao a,b, Xiaoqian Li a,b, Wenbin Zhang a,b,
Ping Wang a,b, Le Ma a,b,c,d,∗, Langchong He e,∗
a
School of Public Health, Xi’an Jiaotong University Health Science Center, Xi’an, Shaanxi 710061, China
b
Key Laboratory for Disease Prevention and Control and Health Promotion of Shaanxi Province, Xi’an, Shaanxi 710061, China
c
Key Laboratory of Shaanxi Province for Craniofacial Precision Medicine Research, College of Stomatology, Xi’an Jiaotong University, Xi’an, China
d
Key Laboratory of Environment and Genes Related to Diseases (Xi’an Jiaotong University), Ministry of Education of China, Xi’an, China
e
School of Pharmacy, Xi’an Jiaotong University Health Science Center, Xi’an, Shaanxi 710061, China

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

Article history: The determination of fat-soluble vitamins and carotenoids in human serum provides reliable information
Received 24 November 2021 for diagnosing malnutrition and for establishing appropriate intervention programs. Due to the complex
Revised 22 January 2022
composition of the biological samples, the efficient sample preparation is the key to the analysis. We
Accepted 26 January 2022
report here a surface active ionic liquid (SAIL)-based dispersive liquid-liquid microextraction (DLLME)
Available online 29 January 2022
method coupled with a high performance liquid chromatography (HPLC) to determine four fat-soluble
Keywords: vitamins and six carotenoids in human serum simultaneously. Liquid crystal structures were formed in
Surface-active ionic liquid the extract phase. And the enrichment factor of the analytes treated by DLLME was 4 to 26 times of
DLLME the traditional LLE method except lycopene. The limit of determination for these compounds was de-
Fat-soluble vitamins termined to be between 0.002 and 0.076 μg/mL. The accuracy was validated by the standard addition
Carotenoids method with recoveries ranging from 82.4 to 114.1%. The intra-day and inter-day relative standard de-
Human serum
viations were 2.76–12.63% and 4.01–13.54%, respectively. The proposed DLLME coupled with the HPLC
method was successfully applied in the determination of fat-soluble micronutrients in human serum.
© 2022 Elsevier B.V. All rights reserved.

1. Introduction ical fluids, the concurrence of numerous structural or functional

The presence of fat-soluble vitamins and carotenoids in the hu-
man body is of vital importance to maintain a vast array of phys-
iological and biochemical functions. Deficiencies of these micronu-
trients have been associated with many pathophysiological condi-
tions such as night blindness (vitamin A) [1], osteomalacia (vita-
min D) [2], prostate cancer (lycopene) [3] and age-related mac-
ular degeneration (lutein and zeaxanthin) [4]. Detection of these
biochemical markers is considered a reliable approach to be used
in groups to identify subjects at risk, diagnose subclinical mal-
nutrition at early age and establish appropriate intervention pro-
grams [5]. Due to the emerging perspectives and indications in
the clinical management and prevention of certain disorders, the
demand for the determination of these biomarkers in clinical set-
ting is increasing. However, the matrix interference of the biolog-
∗ significance for improving the efficiency and sensitivity of sample
Corresponding authors.
E-mail addresses: male@mail.xjtu.edu.cn (L. Ma), helc@mail.xjtu.edu.cn (L. He).
homologues within each family of vitamins and carotenoids as
well as their respective concentration ranges make the simulta-
neous analysis of these micronutrients in human serum difficult
[6,7]. Therefore, sample preparation procedures prior to high per-
formance liquid chromatography (HPLC) or less frequently used
liquid chromatography-tandem mass spectrometry (LC-MS/MS) are
required to remove potential interfering substances, isolate and
concentrate the analytes.
The liquid-liquid extraction (LLE) methods involving an initial
protein precipitation followed by solvents extraction, concentration
and re-dissolution steps were most widely used currently [8–12].
However, the traditional LLE method is characterized by the use
of hazardous organic solvents (n-hexane [8,10,11], heptane [12], or
dichloromethane [8,11]), complex and time-consuming operations,
which hampers the fast acquisition of quantitative information of
fat-soluble vitamins and carotenoids. Therefore, a simple, fast, effi-
cient and high-throughput sample preparation method is of great
analysis.

https://doi.org/10.1016/j.chroma.2022.462861
0021-9673/© 2022 Elsevier B.V. All rights reserved.
L. Kong, J. Wang, Q. Gao et al.
Alternative methods such as solid-phase extraction (SPE) [13],
supercritical fluid extraction (SFE) [14], solid phase microextraction
(SPME) [15] and dispersive liquid-liquid microextraction (DLLME)
[16–18] have also been applied in the extraction and isolation of
these micronutrients from different matrices. However, the SFE
requires a specific device operated under extreme conditions of
pressure and temperature. A multi-step process including activa-
tion, equilibration, adsorption and desorption is usually required
for SPE and SPME methods. Compared with the aforesaid tech-
niques, DLLME with advantages of simple operation, rapid extrac-
tion, high enrichment factor (EF) and low consumption of solvents
is extensively applied. Moreover, as ‘green’ solvents for the replace-
ment of hazardous volatile organic solvents, ionic liquids (ILs) have
been deeply explored in DLLME because of their properties in-
cluding negligible vapor pressure [19], good thermal stability [20],
variable and adjustable interactions such as electrostatic [21], hy-
drophobic [22], π –π [23,24] and hydrogen bonding [23,24]. The
extraction of bioactive vitamins and carotenoids from vegetables,
fruits, microbes or plasma using ILs can minimize the environ-
mental pollution and simultaneously improve the selectivity and
extraction yields [25–27]. Leonardo et al. employed the [C4 mim]
[BF4 ]-ethanolic solution to recover carotenoids from Bactris gasi-
paes fruits, this process decreased the carbon footprint by 50%
[28]. In another study, four times carotenoid from orange peel was
obtained by [BMIM] [Cl] relative to the conventional extraction
with acetone [29].
However, most previous studies on IL-based extraction focus
on non-amphiphilic ILs (NAILs), in which neither cation nor an-
ion is based on a classical surfactant structure. The surface ac-
tive ILs (SAILs) composed of long-chain cation or anion are in-
herently amphiphilic, and can self-assemble into nanostructures
such as micelles or liquid crystals [30–33]. EI-Hady et al. extracted
water-soluble vitamins B2 , B6 and C in human plasma by mi-
cellar solutions of SAIL. Then these micronutrients were detected
by electrochemical detection on protein/SAIL/glass carbon sensors,
and the sensitivity enhancement factor up to 50 0 0-fold compared
to conventional detection was observed [25]. Compared to NAILs,
the unusual nanostructure of SAILs is one of their most pecu-
liar properties, which has been applied on ion-conductors [34,35],
separation [31,33], synthesis [36,37] and electrochemistry [38,39].
Furthermore, the lyotropic liquid crystal structure featuring SAILs
has extremely high solubility for sparingly soluble drug molecules
[32], extraordinarily high extraction capacity and excellent separa-
tion selectivity for typical H-bond donor compounds [31], which
may lead to high extraction efficiency for fat-soluble vitamins and
carotenoids.
In this paper, we described a novel DLLME method with sur-
face active 1-hexadecyl-3-methylimidazolium hexafluorophosphate
[C16 mim] [PF6 ] as extractant. Lyotropic liquid crystal structure was
formed in the IL phase, which reduced consumption of toxic sol-
vents, and achieved efficient extraction and direct injection of the
extract layer. Then a reversed phase HPLC method using C30 sta-
tionary phase and multi-wave photo-diode-array (PDA) detection
was established for the simultaneous measurement of 10 kinds
of fat-soluble micronutrients in human serum. The throughput of
routine determination of serum fat-soluble micronutrients was im-
proved by the developed DLLME and HPLC method.
2. Materials and methods
2.1. Chemicals and materials
Retinol and 25-OH-Vitamin D3 standards were obtained from
Sigma Aldrich (Taufkirchen, Germany). a-Tocopherol, γ -tocopherol,
lutein and β -carotene were bought from Aladdin (Shanghai,
China). Zeaxanthin and lycopene were obtained from Macklin
2
Journal of Chromatography A 1666 (2022) 462861
(Shanghai, China). β -cryptoxanthin and α -carotene was obtained
from Extrasynthese (Genay, France) and Santa Cruz Biotechnol-
ogy (Santa Cruz, CA, USA), respectively. The internal standards
dl-tocol and echinenone were purchased from Tama Biochemical
(Tokyo, Japen) and CaroteNature (Lupsingen, Switzerland), respec-
tively. HPLC grade methanol, isopropanol, hexane and analytical
grade methanol, ethanol, acetonitrile, acetone, ethyl acetate, hex-
ane were all purchased from Kermel (Tianjin, China). All the ionic
liquids used in this work including [BMIm] [PF6 ], [HMIm] [PF6 ],
[OMIm] [PF6 ], [C10 MIm] [PF6 ], [C12 MIm] [PF6 ], [C14 MIm] [PF6 ],
[C16 MIm] [PF6 ], [BMIm] [NTf2 ], [BMIm] [BF4 ], [BPy] [PF6 ], [BPyr]
[PF6 ] and [P4444 ] [PF6 ] were bought from Lanzhou Greenchem ILs
(Lanzhou, China). BSA was purchased from Beijing Solarbio Science
& Technology Co., Ltd. Ultrapure water was prepared through a
Millipore Milli-Q RG system (Millipore, MA, USA).
2.2. Chromatographic analysis
HPLC analysis was carried out on a LC-20 Prominence HPLC-
PDA system (Shimadzu, Kyoto, Japan) equipped with a 20A3 de-
gasser, two LC-20AD pumps, a SIL-20A auto sampler, a CTO-20AC
column oven, a LC-PDA detector and a Lab-solution workstation.
Chromatographic separation was performed on an YMC C30 col-
umn (250 × 4.6 mm i.d., 5 μm) connected with a 10 × 4.0 mm
i.d. C18 guard column and kept at 25 ̊C. The mobile phase was
composed of methanol (A) and 50% (v/v) hexane/isopropanol (B).
The gradient program was as follows: 0–3 min 0% B, 3–20 min 0–
50% B, 20–24 min 50–100% B, 24–30 min 100%B and 30–31 min
100–0% B. The flow rate was set at 1.0 mL/min. The injection vol-
ume was 10 μL. The multi-wave PDA detector was adjusted at
254 nm for retinol, 263 nm for 25-OH-Vitamin D3 , 290 nm for a-
and γ -tocopherol, 450 nm for lutein, zeaxanthin, β -cryptoxanthin,
α -carotene and β -carotene, 470 nm for lycopene, respectively. The
sample molecules were identified by comparing the retention time
and UV–vis spectra to those known standards.
2.3. Serum samples collection and handling
The human serum samples were from participants aged 45 to
60 recruited from the local communities. All participants provided
written informed consent prior to catheterization for participation
in the study, and the study protocol was approved by the ethics
committees of Xi’an Jiaotong University Health Science Center. The
samples were drawn from volunteers into serum tubes, and left
to clot at room temperature. Then the samples were centrifuged,
and the serum were collected and stored at −80 °C until used. The
matrix serum sample was a blend of 300 checkup crowd serum
samples. Because the accurate content of the fat-soluble micronu-
trients in the matrix was unknown and no natural control serum
existed, control simulated serum was prepared by dissolving 2.5 g
BSA in 50 mL PBS.
2.4. SAIL-based dllme
All preparations were conducted in dim light in order to mini-
mize the photodegradation of the analytes. In a typical SAIL-based
DLLME, 250 μL serum was transferred to a 2.0 mL centrifuge
tube. 100 μL 0.4 mol/L [C16 MIm] [PF6 ] in ethyl acetate containing
30 mg/L BHT was added. Then 10 μL methanol solution of echi-
nenone (0.005 mg/mL) and dl-tocol (0.15 mg/mL) was added. The
solutions were vortex-mixed for 1 min followed by centrifugation
at 10,0 0 0 rpm for 20 min at 4 °C. The IL-rich phase was withdrawn
and analyzed by HPLC. To be in line with the actual situation, the
matrix serum sample obtained from the checkup crowd was em-
ployed to optimize the extraction performance. The peak areas of
L. Kong, J. Wang, Q. Gao et al.
the target compounds and the products of the areas and the ex-
traction volumes were used as the index of the enrichment factor
and extraction efficiency for screening of the extraction conditions.
While the enrichment factor (EF) and extraction efficiency (EE) of
the SAIL-based DLLME were determined in the spiked simulated
serum system containing known concentrations of fat-soluble vita-
mins and carotenoids. EF and EE were calculated by the following
equations, respectively:
CIL
EF =
C0
CIL × VIL
EE =
C0 × V0
where CIL and C0 are the concentration of the fat-soluble micronu-
trients in the extract phase after extraction and the spiked sim-
ulated serum before extraction, respectively. VIL and V0 represent
the volume of the extract phase and the original sample solution,
respectively.
2.5. POM characterization
Polarizing optical microscopy measurements for the extract
phase were carried out using an OLYMPUS BX51 microscope. The
images were captured by a CCD (charge-coupled device) camera.
2.6. Calibration and internal standard solutions
A quantity of fat-soluble vitamins and carotenoids stan-
dards were accurately weighted and dissolved in methanol
or chloroform depending on their solubility to make stock
solutions. The final concentration of the compounds in the
stock solution was as follows: retinol (0.5 mg/mL), 25-OH-
Vitamin D3 (0.24 mg/mL), a-tocopherol (5.5 mg/mL), γ -tocopherol
(4.0 mg/mL), lutein (0.34 mg/mL), zeaxanthin (0.4 mg/mL), β -
cryptoxanthin (0.1 mg/mL), α -carotene (0.36 mg/mL), β -carotene
(0.56 mg/mL) and lycopene (1.0 mg/mL). In order to test the abil-
ity for correcting the matrix effect of the internal standard, five
working standard solutions were prepared with the stock solution
by sequential dilution with methanol and simulated serum, respec-
tively. The internal standard solution containing 0.30 mg/mL dl-
tocol and 0.01 mg/mL echinenone was prepared in methanol. 10 μL
internal standard solution was added to every 250 μL standard so-
lution or sample solution. All solutions were protected from light,
stored at −80 °C and used within 6 months.
2.7. Method validation
The method was validated including an evaluation of matrix ef-
fects, linearity, limit of detection (LOD) and quantitation (LOQ), ac-
curacy and precision. The internal standard quantification method
was used. To evaluate the correction ability of the internal stan-
dards, a t-test statistical comparison of the slopes of the calibration
curves obtained with and without matrix was conducted. p<0.05
indicates statistically significant difference between the slopes. Ac-
curacy was determined using a recovery experiment from pooled
serum samples spiked with three different concentration levels of
standard solutions. Recovery was calculated as the percentage re-
covered in the spiked sample after subtraction of the basal con-
centration. Intra-day precision was tested by performing samples
with three different concentrations each in five replicates in one
day. Inter-day precision was determined with samples analyzed on
five different days within 1 week. The precision was evaluated by
the relative standard deviation (RSD).
3
Journal of Chromatography A 1666 (2022) 462861
2.8. Application of the RP-HPLC method
The developed HPLC method was applied to detect the fat-
soluble micronutrients in 50 human serum samples. The samples
were handled as Section 2.3 After the SAIL-based DLLME (accord-
ing to the procedures outlined in Section 2.4), the extract phase
was directly injected into the HPLC system. The data calculated by
the linear regression calibration were presented in Table S2.
3. Results and discussion
3.1. Optimization of the extraction parameters
To optimize extraction efficiency, the following factors were
studied: the structure of IL, the IL concentration, the amount of
the extract, the type of the dispersive solvent, the vortex and cen-
trifugation times. Each experiment was performed in triplicate.
3.1.1. Effect of the structure of il
The anion structure of ILs has significant effects on their water
miscibility, hydrogen bond acidity and basicity [40–42]. To achieve
phase separation, two hydrophobic anions ( [PF6 ]− and [Tf2 N]− )
were investigated. As shown in Fig 1(A) and Fig S1(A), [PF6 ]− had
higher enrichment effect and extraction efficiency than [Tf2 N]− , in-
dicating that the decrease of the hydrogen bond basicity is ben-
eficial to the distribution of the hydrophobic nutrients in the ILs
phase. Among the studied cations, the ring structured [BMIm]+ ,
[BPy]+ , [BPyr]+ and [BMPd]+ showed higher enrichment factor
than [P4444 ]+ (Fig 1(B)). This may be due to the π -π interaction
between the cations and the micronutrients [43]. In the middle
of the ring structured cations, higher extraction efficiency as a re-
sult of larger extract phase volume was obtained by [BMIm]+ and
[BPyr]+ (Fig S1(B)). [PF6 ]− and imidazolium cation were chosen for
further experiments.
The cation alkyl chain length greatly affects the hydrophobic-
ity of the ILs and the van der Waals interaction between ILs and
the extractant [44]. Otherwise, with the increment of the alkyl
chain length, the microstructure of the IL-rich phase changes from
isotropic to micelle and liquid crystal structures [33]. According
to the obtained results (Fig 1(C) and Fig S1(C)), both the enrich-
ment factor and the extraction efficiency declined towards decyl
alkyl (C6– C10 ), and then rose from dodecyl to cetyl (C12– C16 ). This
effect was also observed in the extraction of other natural com-
pounds [45], and could be accounted for by two opposing effects.
First, with increasing viscosity, extraction efficiency might decrease
owing to limited mass transfer. Second, the increasing chain length
might facilitate extraction with higher hydrophobicity and van der
Waals interaction. Meanwhile, polarizing optical micrograph (POM)
characterization demonstrated that liquid crystal structures were
formed when the alkyl chain length was larger than 12 (Fig 2).
Among the investigated ILs, surface active [C16 MIm] [PF6 ] with liq-
uid crystal structure showed the highest enrichment factor and ex-
traction efficiency. So [C16 MIm] [PF6 ] was chosen to extract and
concentrate vitamins and carotenoids from human serum.
3.1.2. Effect of dispersive solvent and other parameters
The selection of a dispersant is critical for DLLME method
[46,47]. Otherwise, dispersant was also required to dissolve the
solid SAIL. Several solvents such as methanol, ethanol, acetoni-
trile, acetone, ethyl acetate and n-hexane were explored. Among
the common solvents, acetonitrile and ethyl acetate showed good
solubility for this SAIL. Compared with strong polar acetonitrile,
[C16 MIm] [PF6 ] in medium polar ethyl acetate exhibited higher en-
richment factor and extraction efficiency. Therefore, ethyl acetate
with medium polarity and low toxicity was chosen as the suitable
dispersant. After the optimization of other parameters, we chose
L. Kong, J. Wang, Q. Gao et al. Journal of Chromatography A 1666 (2022) 462861

Fig. 1. The optimization of extraction parameters. (A) Effect of anion structure. (B) Effect of cation structure. (C) Effect of alkyl chain length. (D) Effect of dispersive solvent.
The mixed human serum was used a test sample. All the experiments were performed in triplicate.

Fig. 2. POM images of the extract phase with [C12 MIm] [PF6 ] (A), [C14 MIm] [PF6 ] (B), [C16 MIm] [PF6 ] (C) as extractant.

100 μL 0.4 mol/L [C16 MIm] [PF6 ] in ethyl acetate as the optimal
extractant, 1 min and 20 min as the suitable vortex and centrifu-
gation time, respectively. The detailed results were shown in the
supporting information.
3.2. Comparison with traditional lle and other methods from
literature
Under the modified conditions, the performance of SAIL-based
DLLME was compared with traditional LLE and other methods from
literature. Results in Table 1 showed that hazardous organic sol-
vents were widely used to concentrate typical micronutrients from
various biological fluids with satisfactory effect [48,49,51,52]. The
EF and EE of the SAIL-based DLLME were lower than data in
these references, indicating that further study was still needed to
develop green extractant with excellent extraction performance.
But for serum samples, protein precipitation was necessary when
organic solvents were utilized as the extract. However, IL-LLME
showed advantages of one-step extraction and direct injection of
the extract phase [50]. A classical LLE using n-hexane as the ex-

4
L. Kong, J. Wang, Q. Gao et al. Journal of Chromatography A 1666 (2022) 462861

Table 1
Comparison of SAIL-based DLLME with other extraction techniques for preparation of typical micronutrients from biological fluids.

Analyte Sample Method Type and volume of solvents Time consumption EF EE % Ref

Retinol 2 mL juice DLLME Extra : 100 μL tetrachloroethane; Sapoe : ∼ 16 h; Vortf : ∼1 min 50 – 48

Dispb : 2 mL methanol Centg : 2 min
25-OH-VD3 1 mL serum LDSc -DLLME Extra : 80 μL 1-octanol Dispb : 650 μL Prot prech : ∼7 min 180 – 49
methanol Vortf : 1 min
Centg : 5 min
α -Tocopherol 200 μL serum IL-LLME 110 μL [OMIm]OTf Vortf : 1 min – ∼100 50
Centg : 5 min
Lutein 1 mL egg yolk VA-HF-LPMEd 1-octanol:1-undecanol, 6:4, v/v Prot Prech : ∼10 min – ∼92 51
Fiber prepi : 5 min
Vortf : 5 min
β -Carotene 100 mg DLLME Extra : 115 μL chloroform Dispb : 2 mL Vortf : 1 min – ∼93 52
fruits/vegitables methanol Centg : 3 min
Retinol 250 μL serum LLE Extra : 2.4 mL n-hexane Prot prech : 6 min 0.39 16 This work
Vortf : 3 min
Centg : 35 min
Evapj : 30 min
25-OH-VD3 0.38 15
α -Tocopherol 0.40 16
Lutein 0.47 19
β -Carotene 0.24 10
Retinol 250 μL serum SAIL-based-DLLME Extra : [C16 MIm]PF6 Dispb : 100 ul ethyl Vortf : 1 min 3.3 53 This work
acetate Centg : 20 min
25-OH-VD3 3.7 59
α -Tocopherol 3.0 48
Lutein 2.1 34
β -Carotene 2.1 33
a
Extr: extraction solvent. b Disp: dispersing solvent. c LDS: low density solvent. d VA-HF-LPME: vortex-assisted hollow fiber liquid phase microextraction. e Sapo: saponifi-
cation. f Vort: vortex. g Cent: centrifugation. h Prot prec: protein precipitation. i Fiber prep: fiber preparation. j Evap: solvent evaporation.

tract was conducted to separate and concentrate the same ana-
lytes from human serum. The enrichment factor of the SAIL-based
DLLME was 4 to 26 times of the traditional LLE except lycopene
(Table 1, Table S1). The extraction efficiency except lycopene of the
DLLME was also higher than traditional LLE. Along with higher en-
richment factor and extraction efficiency, the nanostructured SAIL-
based DLLME required lesser organic solvents (100 μL vs 2.75 mL)
and simpler operation steps without complex protein precipita-
tion, concentration and re-dissolution. The SAIL-based DLLME was
demonstrated to be a simple, rapid, efficient and environmentally
friendly method for the isolation and concentration of fat-soluble
micronutrients from human serum.
3.3. Analytical performance
Under the chromatographic conditions used in this study, the
linear increase of hexane/isopropanol allowed gradual elution of
the more-polar compounds (retinol, 25-OH-VD3 and tocopherols)
to the less-polar molecules (lutein, zeaxanthin, cryptoxanthin and
carotenes). In the last separation phase, nonpolar compounds
(lycopene and its isomers) were separated with 100% of hex-
ane/isopropanol. All molecules were separated in a 35-min run
(Fig 3(A)). Among the carotenoids, the isomers lutein and zeax-
anthin were clearly separated with retention time differing about
1 min through the application of C30 stationary phase [53]. The
analysis of human serum demonstrated the presence of the con-
cerned micronutrients (Fig 3(B)). Otherwise, our results are consis-
tent with literature data that human serum contains both all-trans
and cis-isomers of lycopene [54]. The all-trans- and cis-lycopene
peaks were partly co-eluted, but could still be quantified sepa-
rately (Fig 3(B)). While only all-trans-lycopene was commercially
available, we quantify the all-trans-lycopene in this paper. As re-
ported by other authors, not only the above fat-soluble micronu-
trients, but also the following compounds were possibly present in
the extract phase: coenzyme Q [55], retinyl palmitate [56], water-
soluble Vitamin B and C [25] and so on. Several unidentified peaks
are also visible in Fig 3(B). The analytical specificity was confirmed
by the specific absorption wavelength, the retention time and the
standard addition method.
The internal standard method of quantitative was used and the
quantification was based on area ratios of compound over internal
standard versus concentration. dl-Tocol and echinenone were used
as the internal standard for fat-soluble vitamins and carotenoids,
respectively. Two linear regression equations were generated for
all analytes with regression coefficients in the range of 0.9990–
1.0 0 0 0 (Table 2). p values were obtained from t-test analysis of
the two types of slopes. The p values of retinol, 25-OH-VD3 , α -
tocopherol and lycopene were smaller than 0.05, indicating that
significant difference existed in these two types of calibration
curves. While the other slopes with p values larger than 0.05 could
not be considered statistically different. The matrix correction ef-
fect of echinenone for carotenoids was much better than dl-tocol
for fat-soluble vitamins. This may be caused by the high variation
in molecular structures between retinol, 25-OH-VD3 , α -tocopherol
with dl-tocol (Fig S2). Inversely, echinenone had similar molecular
structure with carotenoids except lycopene. Further research was
needed to study the matrix correction effect of the internal stan-
dard, especially for fat-soluble vitamins. Standard curves conducted
in simulated serum were used for the quantification of fat-soluble
micronutrients in human serum.
The linear calibration ranges and the limits of detection (LODs)
determined at signal-to-noise ratios (S/N) equal to 3 are shown in
Table 3. Compared with LODs obtained without DLLME prepara-
tion, lower LODs and higher sensitivity were obtained due to the
enrichment effect of the DLLME procedure. The LODs of analytes
extracted from simulated serum ranged from 0.002 to 0.076 μg/mL.
The LODs obtained in this work were in agreement with other
reports except that the present method was less sensitive for ly-
copene [12,55,56]. The precision was conducted in terms of the
intra-day repeatability and inter-day reproducibility using three
concentration levels of analytes spiked in human serum matrix.
The intra-day repeatability was evaluated as the relative stan-
dard deviations ranging from 2.76 to 12.63% and the inter-day re-
producibility was between 4.01 and 13.54% (Table 4). The accu-

5
L. Kong, J. Wang, Q. Gao et al. Journal of Chromatography A 1666 (2022) 462861

Fig. 3. Chromatograms of fat-soluble vitamins and carotenoids in standard solution (A) and in human serum (B). Peak identification: 1, Retinol; 2, 25-OH-Vitamin D3 ; 3,
γ -Tocopherol; 4. α -Tocopherol; 5, Lutein; 6, Zeaxanthin; 7, β -Cryptoxanthin; 8, α -Carotene; 9, β -Carotene; 10, Lycopene; 11, dl-Tocol; 12, Echinenone.
Table 2
Standard curves obtained from standards prepared in methanol or simulated serum and the t-test analysis of the slopes.

Compound Standard curvea R2,a Standard curveb R2,b p

Retinol y = 0.4169x-0.0106 0.9998 y = 0.3786x+0.0193 0.9990 0.015

25-OH-VD3 y = 1.3036x+0.0027 0.9996 y = 1.1536x-0.0010 0.9990 0.040
α -Tocopherol y = 10.1657x-0.3147 0.9991 y = 12.5757x+0.7744 0.9997 0.014
γ -Tocopherol y = 9.0455x-0.0352 0.9997 y = 8.6533x+0.1825 0.9996 0.938
Lutein y = 0.1509x-0.0095 0.9998 y = 0.1652x+0.0190 0.9992 0.456
Zeaxanthin y = 0.0910x+0.0008 1.0000 y = 0.0986x+0.0116 0.9997 0.563
β -Cryptoxanthin y = 0.1748x+0.0096 0.9990 y = 0.2196x+0.0128 0.9996 0.132
α -Carotene y = 0.0709x+0.0064 0.9997 y = 0.0915x+0.0078 0.9994 0.297
β -Carotene y = 0.1078x+0.0881 0.9997 y = 0.1324x+0.0120 0.9998 0.117
Lycopene y = 0.1982x+0.0481 0.9996 y = 0.6049x+0.0420 0.9999 0.006
a
with standards prepared in methanol.
b
with standard prepared in simulated serum.
p<0.05 indicates a significant difference.

Table 3
The concentration range of the standard curves and LODs obtained from standards prepared in methanol or simulated serum.

Compound Concentration range (μg/mL) LODa (μg/mL) LODb (μg/mL) LODc (μg/mL)

Retinol 0.075–1.5 0.004 0.002 0.007 [57]

25-OH-VD3 0.01–0.2 0.008 0.002 0.003 [58]
α -Tocopherol 1.25–25.0 0.102 0.055 0.078 [57]
γ -Tocopherol 0.25–5.0 0.082 0.046 0.018 [57]
Lutein 0.05–1.0 0.023 0.010 0.008 [12]
Zeaxanthin 0.025–0.5 0.015 0.007 0.010 [12]
β -Cryptoxanthin 0.025–0.5 0.028 0.017 0.009 [12]
α -Carotene 0.01–0.2 0.014 0.007 0.008 [12]
β -Carotene 0.25–5.0 0.014 0.007 0.010 [12]
Lycopene 0.05–1.0 0.052 0.076 0.009 [12]
a
with standards prepared in methanol.
b
with standards prepared in simulated serum.
c
data from references.

6
L. Kong, J. Wang, Q. Gao et al. Journal of Chromatography A 1666 (2022) 462861

Table 4
The intra-day, inter-day precision and recovery of fat-soluble vitamins and carotenoids from human serum.

Compound Nominal concentration (μg/mL) Precision Accuracy

Intra-day CV (%), n=5 Inter-day CV (%), n=5 Concentration calculated (μg/mL), n=5 Average (%), n=5

Retinol 0.1 6.22 6.3 0.099±0.006 99.4

0.3 2.87 8.66 0.325±0.009 108.4
0.5 2.76 7.4 0.549±0.015 109.7
25-OH-VD3 0.02 12.63 10.32 0.017±0.002 83.9
0.06 3.5 13.39 0.057±0.002 94.5
0.1 4.32 9.53 0.097±0.004 96.6
α -Tocopherol 1 5.76 7.06 1.019±0.059 101.9
3 2.82 10.72 3.024±0.085 100.8
5 2.84 8.02 5.195±0.147 103.9
γ -Tocopherol 0.5 5.65 6.92 0.483±0.027 96.5
1.5 6.87 8.68 1.632±0.112 108.8
2.5 2.81 7.25 2.675±0.075 107
Lutein 0.1 3.34 9.31 0.087±0.003 87.2
0.3 5.95 13.54 0.327±0.019 109.1
0.5 8.82 6.97 0.511±0.045 102.2
Zeaxanthin 0.05 3.62 4.01 0.042±0.002 84.8
0.15 5.9 8.88 0.144±0.009 96.1
0.25 4.2 11.3 0.236±0.010 94.4
β -Cryptoxanthin 0.05 3.08 7.45 0.043±0.001 86.8
0.15 7.92 10.57 0.163±0.013 108.7
0.25 4.75 10.21 0.279±0.013 111.7
α -Carotene 0.01 3.4 7.17 0.008±0.0003 83.6
0.03 5.11 10.83 0.034±0.002 114.1
0.05 8.8 9.2 0.0450±0.004 99.5
β -Carotene 0.1 3.57 8.22 0.082±0.003 82.4
0.3 6.48 12.32 0.312±0.020 103.9
0.5 4.35 12.74 0.519±0.023 103.8
Lycopene 0.1 3 9.15 0.089±0.003 88.7
0.3 8.08 6.94 0.299±0.024 99.5
0.5 6.36 11.84 0.493±0.031 98.5

Table 5
The minimum, maximum and average concentrations of the fat-soluble vitamins and carotenoids in 50 clin-
ical serum samples.

Compound Min concentration (μg/mL) Max concentration (μg/mL) Average concentration

(μg/mL) (μmol/L)

Retinol 0.227 1.708 0.693 2.42

25-OH-VD3 0.002 0.066 0.016 0.039
α -Tocopherol 3.515 46.82 17.787 42.687
γ -Tocopherol 0.08 10.977 1.371 3.184
Lutein 0.238 3.296 0.771 1.356
Zeaxanthin 0.017 0.259 0.084 0.147
β -Cryptoxanthin 0.046 1.009 0.265 0.479
α -Carotene 0.007 0.732 0.074 0.141
β -Carotene 0.026 2.111 0.518 0.966
lycopene 0.077 1.84 0.363 0.677

racy was estimated by the recovery of certain concentration lev-
els of analytes spiked in human serum samples. As can be seen in
Table 4, the recovery of the analytes ranged from 82.4 to 114.1%.
The precision and accuracy of the proposed method meet the an-
alytical requirements. The established SAIL-based DLLME coupled
with internal standard quantitative HPLC method provided a sim-
ple, fast, efficient and environmentally friendly sample preparation
method and an accurate, sensitive and fully validated analytical
method, which was suitable for simultaneous determination of fat-
soluble vitamins and carotenoids in mass clinical samples.
3.4. Application for analysis of real samples
To demonstrate the applicability of the developed technique,
the serum concentrations of fat-soluble vitamins and carotenoids
in participants recruited from the local communities were de-
termined. The content of fat-soluble micronutrients in 50 par-
ticipants were presented in Table S2. The range and average
value of these analytes are presented in Table 5. The ranges for
retinol were 0.227–1.708 μg/ml (mean 0.693 μg/ml, 2.420 μmol/l),
for 25-OH-VD3 0.002–0.066 μg/ml (mean 0.016 μg/ml, 0.039
μmol/l), for α -tocopherol 3.515–46.820 μg/ml (mean 17.787 μg/ml,
42.687 μmol/l), for γ -tocopherol 0.080–10.977 μg/ml (mean
1.371 μg/ml, 3.184 μmol/l), for lutein 0.238–3.296 μg/ml (mean
0.771 μg/ml, 1.356 μmol/l), for zeaxanthin 0.017–0.259 μg/ml
(mean 0.084 μg/ml, 0.147 μmol/l), for β -cryptoxanthin 0.046–
1.009 μg/ml (mean 0.265 μg/ml, 0.479 μmol/l), for α -carotene
0.007–0.732 μg/ml (mean 0.074 μg/ml, 0.141 μmol/l), for β -
carotene 0.026–2.111 μg/ml (mean 0.518 μg/ml, 0.966 μmol/l), and
for lycopene 0.077–1.840 μg/ml (mean 0.363 μg/ml, 0.677 μmol/l).
The ranges for these compounds are in good agreement with re-
ported values [10,55, 57, 58].
4. Conclusion
In this work, a SAIL-based DLLME followed by HPLC-PDA was
introduced to determine fat-soluble vitamins and carotenoids in
human serum. The SAIL-based DLLME provided advantages of sim-

7
L. Kong, J. Wang, Q. Gao et al.
pler extraction procedure, shorter extraction time, less organic sol-
vent consumption, higher enrichment factor and extraction effi-
ciency than traditional LLE method. However, further studies were
still needed to develop green extractant which can exhibit ex-
cellent extraction performance and avoid the consumption of or-
ganic dispersive solvent. The sensitivity, precision and accuracy of
the HPLC method meet the analytical requirements. The developed
DLLME coupled with HPLC-PDA analysis was also successfully ap-
plied to the analysis of clinical samples. This work offers insights
into the design principles for biological sample pretreatment based
on spontaneous self-assembly behavior. This fully validated work-
flow is well suited for both research and routine deployment of
quantitative profiling of fat-soluble vitamins and carotenoids in hu-
man serum.
Declaration of Competing Interest
The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared to
influence the work reported in this paper.
CRediT authorship contribution statement
Liyun Kong: Conceptualization, Methodology, Writing – origi-
nal draft. Jiaqi Wang: Methodology, Software, Validation, Data cu-
ration. Qingpeng Gao: Conceptualization, Data curation. Xiaoqian
Li: Conceptualization, Data curation. Wenbin Zhang: Conceptual-
ization, Data curation. Ping Wang: Conceptualization, Data cura-
tion. Le Ma: Supervision, Conceptualization, Writing – review &
editing. Langchong He: Supervision, Conceptualization, Writing –
review & editing.
Acknowledgments
This work was supported by National Natural Science Founda-
tion of China (21808178, 82022062, 81973025); Beijing Zhongy-
inghui Nutrition and Health Research Institute, the Chinese Nu-
trition Society Academy of Nutrition and Health (CNSCI2021043);
Nutrition Science Research Foundation of BY-HEALTH (TY0181101);
New-star Plan of Science and Technology of Shaanxi Province
(2015LJXX-07); the Nutrition Research Foundation Fund of the Chi-
nese Nutrition Society-DSM Special Research Foundation (CNSDSM
2016–041); and the Fundamental Research Funds for the Central
Universities (qngz2016004; xzy032019008).
Supplementary materials
Supplementary material associated with this article can be
found, in the online version, at doi:10.1016/j.chroma.2022.462861.
References
[1] J.E. Dowling, G. Wald, Vitamin a deficiency and night blindness, Proc. Natl.
Acad. Sci. U. S. A. 44 (7) (1958) 648–661, doi:10.1073/pnas.44.7.648.
[2] A. Bhan, A. Rao, S. Rao, Osteomalacia as a result of vitamin D deficiency, En-
docrin. Metab. Clin. 39 (2010) 321–331, doi:10.1016/j.ecl.2010.02.001.
[3] U. Singh, S. Devaraj, I. Jialal, Vitamin E, oxidative stress, and inflammation,
Annu. Rev. Nutr. 25 (1) (2005) 151–174, doi:10.1146/annurev.nutr.24.012003.
132446.
[4] The age-related eye disease study 2 research group, Secondary analyses of the
effects of lutein/zeaxanthin on age-related macular degeneration progression:
AREDS2 report No. 3, JAMA Ophthalmol 132 (2) (2014) 142–149, doi:10.1001/
jamaophthalmol.2013.7376.
[5] I. Elmadfa, A.L. Meyer, Developing suitable methods of nutritional status as-
sessment: a continuous challenge, Adv. Nutr. 5 (5) (2014) 590S–598S, doi:10.
3945/an.113.005330.
[6] A.A. Albahrani, R.F. Greaves, Fat-soluble vitamins: clinical indications and cur-
rent challenges for chromatographic measurement, Clin. Biochem. Rev. 37 (1)
(2016) 27–47 https://pubmed.ncbi.nlm.nih.gov/27057076.
[7] M. Eggersdorfer, A. Wyss, Carotenoids in human nutrition and health, Arch.
Biochem. Biophys. 652 (2018) 18–26, doi:10.1016/j.abb.2018.06.001.
8
Journal of Chromatography A 1666 (2022) 462861
[8] F. Granado-Lorencio, C. Herrero-Barbudo, I. Blanco-Navarro, B. Pérez-Sacristán,
Suitability of ultra-high performance liquid chromatography for the deter-
mination of fat-soluble nutritional status (vitamins A, E, D, and individual
carotenoids), Anal. Bioanal. Chem. 397 (3) (2010) 1389–1393, doi:10.1007/
s00216- 010- 3655- 2.
[9] B. Gleize, M. Steib, M. André, E. Reboul, Simple and fast HPLC method
for simultaneous determination of retinol, tocopherols, coenzyme Q10 and
carotenoids in complex samples, Food Chem 134 (4) (2012) 2560–2564, doi:10.
1016/j.foodchem.2012.04.043.
[10] D. Siluk, R.V. Oliveira, M. Esther-Rodriguez-Rosas, S. Ling, A. Bos, L. Ferrucci,
I.W. Wainer, A validated liquid chromatography method for the simultaneous
determination of vitamins A and E in human plasma, J. Pharmaceut. Biomed.
44 (4) (2007) 1001–1007, doi:10.1016/j.jpba.2007.03.033.
[11] A. Khan, M.I. Khan, Z. Iqbal, Y. Shah, L. Ahmad, D.G. Watson, An optimized and
validated RP-HPLC/UV detection method for simultaneous determination of
all-trans-Retinol (Vitamin A) and α -Tocopherol (Vitamin E) in human serum:
comparison of different particulate reversed-phase HPLC columns, J. Chro-
matogr. B 878(25) (2010) 2339–2347, doi:10.1016/j.jchromb.2010.07.009.
[12] J.N Eriksen, P.L Madsen, L.O Dragsted, E Arrigoni, Optimized, Fast-throughput
UHPLC-DAD based method for carotenoid quantification in spinach, serum,
chylomicrons, and feces, J. Agr. Food Chem. 65 (4) (2017) 973–980, doi:10.1021/
acs.jafc.6b04925.
[13] F. Priego Capote, J.R. Jiménez, J.M.M. Granados, M.D.L. de Castro, Identification
and determination of fat-soluble vitamins and metabolites in human serum
by liquid chromatography/triple quadrupole mass spectrometry with multi-
ple reaction monitoring, Rapid Commun. Mass Sp. 21 (11) (2007) 1745–1754,
doi:10.1002/rcm.3014.
[14] D.C. Murador, F. Salafia, M. Zoccali, P.L.G. Martins, A.G. Ferreira, P. Dugo,
L. Mondello, V.V. de Rosso, D. Giuffrida, Green extraction approaches for
carotenoids and esters: characterization of native composition from orange
peel, Antioxidants 8 (12) (2019) 613, doi:10.3390/antiox8120613.
[15] F.-F. Qi, T.-Y. Ma, Y.-M. Fan, L.-l. Chu, Y. Liu, Y. Yu, Nanoparticle-based polyacry-
lonitrile monolithic column for highly efficient micro solid-phase extraction
of carotenoids and vitamins in human serum, J. Chromatogr. A 1635 (2021)
461755, doi:10.1016/j.chroma.2020.461755.
[16] C Dal Bosco, V Di Lisio, P. D’Angelo, A Gentili, Hydrophobic eutectic sol-
vent with antioxidant properties: application for the dispersive liquid–liquid
microextraction of fat-soluble micronutrients from fruit juices, ACS Sustain.
Chem. Eng. 9 (24) (2021) 8170–8178, doi:10.1021/acssuschemeng.1c01473.
[17] P. Sricharoen, N. Limchoowong, S. Techawongstien, S. Chanthai, A novel ex-
traction method for β -carotene and other carotenoids in fruit juices using air-
assisted, low-density solvent-based liquid–liquid microextraction and solidi-
fied floating organic droplets, Food Chem 203 (2016) 386–393, doi:10.1016/j.
foodchem.2016.02.093.
[18] A. Rahimi, P. Hashemi, Development of a dispersive liquid-liquid microextrac-
tion method based on solidification of a floating organic drop for the determi-
nation of beta-carotene in human serum, J. Anal. Chem. 69 (4) (2014) 352–356,
doi:10.1134/S1061934814040078.
[19] V.N. Emel’yanenko, S.P. Verevkin, A. Heintz, The gaseous enthalpy of formation
of the ionic liquid 1-butyl-3-methylimidazolium dicyanamide from combustion
calorimetry, vapor pressure measurements, and ab Initio calculations, J. Am.
Chem. Soc. 129 (13) (2007) 3930–3937, doi:10.1021/ja0679174.
[20] F. Wu, N. Zhu, Y. Bai, L. Liu, H. Zhou, C. Wu, Highly safe ionic liquid electrolytes
for sodium-ion battery: wide electrochemical window and good thermal sta-
bility, ACS Appl. Mater. Inter. 8 (33) (2016) 21381–21386, doi:10.1021/acsami.
6b07054.
[21] T.G.A. Youngs, C. Hardacre, J.D. Holbrey, Glucose solvation by the ionic liquid
1,3-dimethylimidazolium chloride: a simulation study, J. Phys. Chem. B 111
(49) (2007) 13765–13774, doi:10.1021/jp076728k.
[22] K. Jumbri, H. Ahmad, E. Abdulmalek, M.B. Abdul Rahman, Binding energy and
biophysical properties of ionic liquid-DNA complex: understanding the role
of hydrophobic interactions, J. Mol. Liq. 223 (2016) 1197–1203, doi:10.1016/j.
molliq.2016.09.040.
[23] Y.-L. Wang, A. Laaksonen, M.D. Fayer, Hydrogen bonding versus π –π stacking
interactions in imidazolium–oxalatoborate ionic liquid, J. Phys. Chem. B 121
(29) (2017) 7173–7179, doi:10.1021/acs.jpcb.7b05564.
[24] R.P. Matthews, T. Welton, P.A. Hunt, Hydrogen bonding and π –π interactions
in imidazolium-chloride ionic liquid clusters, Phys. Chem. Chem. Phys. 17 (22)
(2015) 14437–14453, doi:10.1039/C5CP00459D.
[25] D Abd El-Hady, H.M. Albishri, Protein/ionic liquid/glassy carbon sensors fol-
lowing analyte focusing by ionic liquid micelle collapse for simultaneous de-
termination of water soluble vitamins in plasma matrices, Talanta 139 (2015)
150–158, doi:10.1016/j.talanta.2015.02.025.
[26] L.M. de Souza Mesquita, M. Martins, É. Maricato, C. Nunes, P.S.G.N. Quinteiro,
A.C.R.V. Dias, J.A.P. Coutinho, L.P. Pisani, V.V. de Rosso, S.P.M. Ventura, Ionic
liquid-mediated recovery of carotenoids from the bactris gasipaes fruit waste
and their application in food-packaging chitosan films, ACS Sustain. Chem. Eng.
8 (10) (2020) 4085–4095, doi:10.1021/acssuschemeng.9b06424.
[27] P.L.G. Martins, V.V. de Rosso, Thermal and light stabilities and antioxidant
activity of carotenoids from tomatoes extracted using an ultrasound-assisted
completely solvent-free method, Food Res. Int. 82 (2016) 156–164, doi:10.1016/
j.foodres.2016.01.015.
[28] L.M. de Souza Mesquita, S.P.M. Ventura, A.R.C. Braga, L.P. Pisani, A.C.R.V Dias,
V.V. de Rosso, Ionic liquid-high performance extractive approach to recover
carotenoids from Bactris gasipaes fruits, Green Chem 21 (9) (2019) 2380–2391,
doi:10.1039/C8GC03283A.
L. Kong, J. Wang, Q. Gao et al.
[29] D.C. Murador, A.R.C. Braga, P.L.G. Martins, A.Z. Mercadante, V.V. de Rosso, Ionic
liquid associated with ultrasonic-assisted extraction: a new approach to obtain
carotenoids from orange peel, Food Res. Int. 126 (2019) 108653, doi:10.1016/j.
foodres.2019.108653.
[30] B. Dong, N. Li, L. Zheng, L. Yu, T. Inoue, Surface adsorption and micelle forma-
tion of surface active ionic liquids in aqueous solution, Langmuir 23 (8) (2007)
4178–4182, doi:10.1021/la0633029.
[31] X. Liu, Q. Yang, Z. Bao, B. Su, Z. Zhang, Q. Ren, Y. Yang, H. Xing, Nonaque-
ous lyotropic ionic liquid crystals: preparation, characterization, and applica-
tion in extraction, Chem. Eur. J. 21 (25) (2015) 9150–9156, doi:10.1002/chem.
201500306.
[32] W. Jin, Q. Yang, Z. Zhang, Z. Bao, Q. Ren, Y. Yang, H. Xing, Self-assembly in-
duced solubilization of drug-like molecules in nanostructured ionic liquids,
Chem. Commun. 51 (67) (2015) 13170–13173, doi:10.1039/C5CC03463A.
[33] L. Kong, Q. Yang, H. Xing, B. Su, Z. Bao, Z. Zhang, Y. Yang, Q. Ren, A gen-
eral method for the separation of amphiphilic surface-active poly(ethylene gly-
col) mono- and di-esters with long-chain ionic liquid-based biphasic systems,
Green Chem 16 (1) (2014) 102–107, doi:10.1039/c3gc41804a.
[34] B. Soberats, E. Uchida, M. Yoshio, J. Kagimoto, H. Ohno, T. Kato, Macroscopic
photocontrol of ion-transporting pathways of a nanostructured imidazolium-
based photoresponsive liquid crystal, J. Am. Chem. Soc. 136 (27) (2014) 9552–
9555, doi:10.1021/ja5041573.
[35] B. Soberats, M. Yoshio, T. Ichikawa, S. Taguchi, H. Ohno, T. Kato, 3D anhydrous
proton-transporting nanochannels formed by self-assembly of liquid crystals
composed of a sulfobetaine and a sulfonic acid, J. Am. Chem. Soc. 135 (41)
(2013) 15286–15289, doi:10.1021/ja407883b.
[36] L. Yang, Q. Yang, J. Hu, Z. Bao, B. Su, Z. Zhang, Q. Ren, H. Xing, Metal nanoparti-
cles in ionic liquid-cosolvent biphasic systems as active catalysts for acetylene
hydrochlorination, AIChE J 64 (7) (2018) 2536–2544, doi:10.1002/aic.16103.
[37] J. Hu, Q. Yang, L. Yang, Z. Zhang, B. Su, Z. Bao, Q. Ren, H. Xing, S. Dai, Confining
noble metal (Pd, Au, Pt) nanoparticles in surfactant ionic liquids: active non-
mercury catalysts for hydrochlorination of acetylene, ACS Catal 5 (11) (2015)
6724–6731, doi:10.1021/acscatal.5b01690.
[38] P. Brown, C.P. Butts, J. Eastoe, D. Fermin, I. Grillo, H.-C. Lee, D. Parker,
D. Plana, R.M. Richardson, Anionic surfactant ionic liquids with 1-butyl-3-
methyl-imidazolium cations: characterization and application, Langmuir 28 (5)
(2012) 2502–2509, doi:10.1021/la204557t.
[39] X. Mao, P. Brown, C. Červinka, G. Hazell, H. Li, Y. Ren, D. Chen, R. Atkin,
J. Eastoe, I. Grillo, A.A.H. Padua, M.F. Costa Gomes, T.A. Hatton, Self-assembled
nanostructures in ionic liquids facilitate charge storage at electrified interfaces,
Nat. Mater. 18 (12) (2019) 1350–1357, doi:10.1038/s41563- 019- 0449- 6.
[40] S.P.M. Ventura, S.G. Sousa, L.S. Serafim, Á.S. Lima, M.G. Freire, J.A.P. Coutinho,
Ionic-liquid-based aqueous biphasic systems with controlled pH: the ionic
liquid anion effect, J. Chem. Eng. Data 57 (2) (2012) 507–512, doi:10.1021/
je2010787.
[41] D. Dupont, D. Depuydt, K. Binnemans, Overview of the effect of salts on bipha-
sic ionic liquid/water solvent extraction systems: anion exchange, mutual sol-
ubility, and thermomorphic properties, J. Phys. Chem. B 119 (22) (2015) 6747–
6757, doi:10.1021/jp002710x.
[42] J. Iqbal, N. Muhammad, A. Rahim, A.S. Khan, Z. Ullah, G. Gonfa, P. Ahmad,
COSMO-RS predictions, hydrogen bond basicity values and experimental evalu-
ation of amino acid-based ionic liquids for lignocellulosic biomass dissolution,
J. Mol. Liq. 273 (2019) 215–221, doi:10.1016/j.molliq.2018.10.044.
[43] L.-Z. Cheong, Z. Guo, Z. Yang, S.-C. Chua, X. Xu, Extraction and enrichment of
n-3 polyunsaturated fatty acids and ethyl esters through reversible π –π com-
plexation with aromatic rings containing ionic liquids, J. Agr. Food Chem. 59
(16) (2011) 8961–8967, doi:10.1021/jf202043w.
[44] M.M. Islam, S. Barik, M. Sarkar, Probing the interactions of 1-alkyl-3-
methylimidazolium tetrafluoroborate (Alkyl = Octyl, Hexyl, Butyl, and Ethyl)
Journal of Chromatography A 1666 (2022) 462861
ionic liquids with bovine serum albumin: an alkyl chain length-dependent
study, J. Phys. Chem. B 123 (7) (2019) 1512–1526, doi:10.1021/acs.jpcb.8b10795.
[45] T. Křížek, M. Bursová, R. Horsley, M. Kuchař, P. Tůma, R. Čabala, T. Hložek,
Menthol-based hydrophobic deep eutectic solvents: towards greener and ef-
ficient extraction of phytocannabinoids, J. Clean. Prod. 193 (2018) 391–396,
doi:10.1016/j.jclepro.2018.05.080.
[46] L. Ma, Y. Wang, H. Li, F. Peng, B. Qiu, Z. Yang, Development of QuEChERS-
DLLME method for determination of neonicotinoid pesticide residues in grains
by liquid chromatography-tandem mass spectrometry, Food Chem 331 (2020)
127190, doi:10.1016/j.foodchem.2020.127190.
[47] D.L. Kalschne, C. Canan, M.O. Beato, O.D. Leite, E.L. Moraes Flores, A new and
feasible analytical method using reversed-phase dispersive liquid-liquid mi-
croextraction (RP-DLLME) for further determination of Nickel in hydrogenated
vegetable fat, Talanta 208 (2020) 120409, doi:10.1016/j.talanta.2019.120409.
[48] P. Viñas, M. Bravo-Bravo, I. López-García, M. Hernández-Córdoba, An evalua-
tion of cis- and trans-retinol contents in juices using dispersive liquid–liquid
microextraction coupled to liquid chromatography with fluorimetric detection,
Talanta 103 (2013) 166–171, doi:10.1016/j.talanta.2012.10.027.
[49] A. Mollahosseini, M. Kamankesh, A. Mohammadi, Central composite design for
dispersive liquid–liquid microextraction of 25-hydroxy-cholecalciferol in hu-
man serum, J. Chromatogr. Sci. 57 (6) (2019) 575–581, doi:10.1093/chromsci/
bmz016.
[50] S. Yin, Y. Yang, J. Zhang, Y. Li, L. Wu, C. Sun, A novel ionic liquid-based liquid-
liquid microextraction combined with high performance liquid chromatogra-
phy for simultaneous determination of eight Vitamin E isomers in human
serum, J. AOAC Int. 103 (4) (2020) 989–996, doi:10.1093/jaoacint/qsz039.
[51] J. Wang, N. Wu, Y. Yang, Determination of carotenoids in egg yolk by high
performance liquid chromatography with vortex-assisted hollow fiber liquid-
phase microextraction using mixed extraction solvent, J. Chromatogr. Sci. 54
(10) (2016) 1834–1840, doi:10.1093/chromsci/bmw130.
[52] H. Sereshti, M. Ahmadvand, S. Asgari, A rapid quantification of β -carotene in
fruits and vegetables by dispersive liquid–liquid microextraction coupled with
UV–Vis spectrophotometry: optimized by response surface methodology, Food
Anal. Method. 7 (7) (2014) 1481–1488, doi:10.1007/s12161- 013- 9777-3.
[53] M. Dachtler, K. Kohler, K. Albert, Reversed-phase high-performance liquid chro-
matographic identification of lutein and zeaxanthin stereoisomers in bovine
retina using a C30 bonded phase, J. Chromatogr. B 720 (1) (1998) 211–216,
doi:10.1016/S0378-4347(98)004319.
[54] D. Thibeault, H. Su, E. MacNamara, H.M. Schipper, Isocratic rapid liquid chro-
matographic method for simultaneous determination of carotenoids, retinol,
and tocopherols in human serum, J. Chromatogr. B 877 (11) (2009) 1077–1083,
doi:10.1016/j.jchromb.2009.02.051.
[55] E.M. Paliakov, B.S. Crow, M.J. Bishop, D. Norton, J. George, J.A. Bralley, Rapid
quantitative determination of fat-soluble vitamins and coenzyme Q-10 in hu-
man serum by reversed phase ultra-high pressure liquid chromatography with
UV detection, J. Chromatogr. B 877 (1) (2009) 89–94, doi:10.1016/j.jchromb.
2008.11.012.
[56] F. Granado-Lorencio, B. Olmedilla-Alonso, C. Herrero-Barbudo, I. Blanco-
Navarro, S. Blázquez-García, B. Pérez-Sacristán, Simultaneous determination of
vitamins A, E and 25-OH-vitamin D: application in clinical assessments, Clin.
Biochem. 39 (2) (2006) 180–182, doi:10.1016/j.clinbiochem.20 05.11.0 04.
[57] H.-Y. Chung, A.L.A. Ferreira, S. Epstein, S.A. Paiva, C. Castaneda-Sceppa,
E.J. Johnson, Site-specific concentrations of carotenoids in adipose tissue: re-
lations with dietary and serum carotenoid concentrations in healthy adults,
Am. J. Clin. Nutr. 90 (3) (2009) 533–539, doi:10.3945/ajcn.2009.27712.
[58] A. Nishino, T. Ichihara, K. Sugimoto, T. Kuriki, H. Yasui, T. Maoka, Predicting
organ carotenoids levels from analysis of plasma could lead to errors: a study
in cynomolgus monkeys, Nutr. Res. 61 (2019) 95–101, doi:10.1016/j.nutres.2018.
10.001.