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Selective extraction and determination of vitamin B12in urine by ionic
liquid-based aqueous two-phase system prior to high-performance
Paula Bertona,b, Romina P. Monasteriob,c, Rodolfo G. Wuillouda,b,n
aLaboratory of Analytical Chemistry for Research and Development (QUIANID), Instituto de Ciencias Ba ´sicas, Universidad Nacional de Cuyo, Padre J. Contreras 1300,
Parque Gral. San Martı ´n, M5502JMA Mendoza, Argentina
bConsejo Nacional de Investigaciones Cientı ´ficas y Te ´cnicas (CONICET), Argentina
cDepartamento de Quı ´mica, Facultad de Ciencias Exactas y Naturales, Universidad Nacional de La Pampa, Santa Rosa, La Pampa, Argentina
a r t i c l e i n f o
Received 22 February 2012
Received in revised form
7 May 2012
Accepted 8 May 2012
Available online 15 May 2012
a b s t r a c t
A rapid and simple extraction technique based on aqueous two-phase system (ATPS) was developed for
separation and enrichment of vitamin B12in urine samples. The proposed ATPS-based method involves
the application of the hydrophilic ionic liquid (IL) 1-hexyl-3-methylimidazolium chloride and K2HPO4.
After the extraction procedure, the vitamin B12-enriched IL upper phase was directly injected into the
high performance liquid chromatography (HPLC) system for analysis. All variables influencing the
IL-based ATPS approach (e.g., the composition of ATPS, pH and temperature values) were evaluated. The
average extraction efficiency was 97% under optimum conditions. Only 5.0 mL of sample and a single
hydrolysis/deproteinization/extraction step were required, followed by direct injection of the IL-rich
upper phase into HPLC system for vitamin B12determination. A detection limit of 0.09 mg mL?1, a
relative standard deviation (RSD) of 4.50% (n¼10) and a linear range of 0.40–8.00 mg mL?1were
obtained. The proposed green analytical procedure was satisfactorily applied to the analysis of samples
with highly complex matrices, such as urine. Finally, the IL-ATPS technique could be considered as an
efficient tool for the water-soluble vitamin B12extraction.
& 2012 Elsevier B.V. All rights reserved.
Vitamin B12(cyanocobalamin) is an essential nutrient formed
by a tetrapyrrole complex, which contains a cobalt (Co) atom in
its molecule . Vitamin B12(VB12) promotes growth and cell
development, helps in maintenance of the myelin sheath, and
mammals . Moreover, its deficiency may result in anemia,
while its prolonged deficiency leads to nerve degeneration and
irreversible neurological damage . The human requirements of
VB12 are about 0.40–2.80 mg per day . The non-vegetarian
average diet generally contains adequate daily intake of VB12.
However, since plants cannot synthesize VB12, strict vegetarians
(vegans) have a greater risk of developing VB12deficiency and,
hence, depend on VB12-fortified foods or VB12-containing dietary
supplements to meet the requirements .
of fatandcarbohydrate in
Consequently, a high demand for rapid, specific, and simple
methodologies to determine vitamins is growing because of their
importance for health . It is clear that determination of trace
amounts of VB12in biological samples plays an important role in
the fields of medicine and toxicology. In the simplest case, VB12is
determined as total Co, assuming that no free Co exists. However,
the applicability of these methods for VB12quantitation in real
samples is somewhat limited since these methods cannot distin-
guish between free inorganic Co and Co bonded to VB12forms.
More specific, cumbersome and time-consuming analytical sys-
tems for VB12detection, including microbiological-, radioisotopic
dilution- or enzyme-linked immunosorbent-assays and different
methods such as chromatographic, electrochemical, spectroscopic
and chemiluminescence were critically reviewed recently by
Kumar et al. . Although these methods show some advantages,
at the same time they present certain drawbacks such as being
laborious, time-consuming, nonspecific, less safe, too expensive
and limited sensitivity . Moreover, most of these methods have
not been tested in samples with complex matrices, such as
urine . One of the most widely used instrumental techniques
for VB12separation is high performance liquid chromatography
(HPLC) with various detection methods, including UV/Vis [8–13],
Contents lists available at SciVerse ScienceDirect
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0039-9140/$-see front matter & 2012 Elsevier B.V. All rights reserved.
nCorresponding author at: Laboratory of Analytical Chemistry for Research and
Development (QUIANID), Instituto de Ciencias Ba ´sicas, Universidad Nacional de
Cuyo, Padre J. Contreras 1300, Parque Gral. San Martı ´n, M5502JMA Mendoza,
Argentina. Tel.: þ54 261 5244064; fax: þ54 261 5244001.
E-mail address: firstname.lastname@example.org (R.G. Wuilloud).
Talanta 97 (2012) 521–526
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atomic absorption spectrometry , fluorescence [15,16], or
mass spectrometry [17–21]. However, and despite UV/Vis is the
most common detector for LC, it shows limited sensitivity and
selectivity, making it unsuitable for determining low levels of
VB12in presence of complex matrices . Therefore, an extraction
technique is usually needed for trace analysis prior to HPLC.
Conventional liquid–liquid extractions (LLE) can effectively
decrease detection limits and eliminate matrix interference.
However, as an attempt to miniaturize and to overcome some
shortcomings originated from LLE, such as limited enrichment
factors, slow and tedious procedures, and the use of large volumes
of organic solvents; several liquid phase microextraction (LPME)
techniques have recently emerged [22,23]. Furthermore, in order
to solve safe and environmental problems related to regular
organic solvents, their replacement by ionic liquids (ILs) has been
proposed. ILs have beneficial properties compared to organic sol-
vents like nonflammability and no detectable vapor pressure .
During extraction processes, hydrophobic ILs can lead to obtain IL/
water biphasic systems. Compared with hydrophilic ILs, hydrophobic
ILs are more expensive, and their number is much more limited than
the former ones. Moreover, the high viscosity of the IL phase could
induce possible denaturation during extraction/separation of biomo-
lecules using simple IL/water biphasic systems . To overcome
these limitations, hydrophilic ILs have been employed in aqueous
two-phase systems (IL-ATPSs) for extraction of analytes in the
presence of inorganic salts . During ATPS, a dispersion occurs,
thus generating a high interfacial contact area between the two
phases for efficient mass transfer. Thus, and due to the high water
content within the phases (70–90%) and low surface tension
between them, ATPS offers a mild and biocompatible method for
biomolecules purification . State-of-the-art techniques based on
IL-ATPS have been recently proposed as an attractive alternative to
conventional extraction procedures for high recovery and purifica-
tion of several biomolecules . To the best of our knowledge, IL-
ATPS technique has never been applied for extraction of water-
soluble vitamins during sample preparation steps.
In the present work, a fast and simple clean-up and separation
method for selective and accurate VB12 determination at trace
levels is proposed. VB12was extracted from pre-treated samples
by application of ATPS technique based on the IL 1-hexyl-
3-methylimidazolium chloride ([C6mim][Cl]) and an inorganic
salt. The upper IL enriched phase was directly injected into HPLC
for VB12determination. The study focused on the development of
an easy, inexpensive, fast, and accurate method for quantitative
determination of VB12in biological samples, such as urine.
2. Material and methods
Absorbance at 300–600 nm of the stock solutions was scanned
with a Lambda 35 UV/Vis spectrometer (Perkin Elmer, Shelton, CT,
USA), equipped with 1 cm quartz cuvette. Samples were run in a
HPLC system (200LC series, Perkin Elmer), composed by quatern-
ary pump, column oven and UV–VIS detector. The column
temperature was maintained at 25 1C and an injection volume
of 20 mL was used in all experiments. Chromatographic separa-
tions were performed on a Zorbax-SB-Aq column (4.6?150 mm,
particle size 5 mm), purchased to Agilent Technologies (Santa
Clara, CA, USA). The column was run with different mobile phases
at a flow rate of 1 mL min?1for 20 min and monitored at 360 nm.
Instrumental conditions are summarized in Table 1.
A vortex model Bio Vortex V1 (Boeco, Hamburg, Germany) was
used for mixing the phases.
A 1000 mg L?1VB12stock standard solution was prepared by
dissolving 10 mg of VB12(Sigma, Milwaukee, WI, USA) in 10 mL of
ultrapure water. Lower concentrations were prepared by diluting
the stock solution with ultrapure water. Salts evaluated for ATPS
occurrence included: KH2PO4, KOH, K2HPO4, K2SO4and K2CO3and
were purchased from Sigma. Different ILs including, [Cnmim][Cl]
(n¼4, 6, 8) were synthesized according to a method proposed by
J.G. Huddleston and coworkers . Qualitative analysis of
synthesized IL was performed by comparison of infrared spectra
Instrumental and experimental conditions for vitamin B12determination.
Zorbax SB-Aq (5 mm?4.6 mm i.d. x 150 mm)
Zorbax Realiance Analytical Cartridge
1 mL min?1
B: 5 mmol L?1phosphate buffer (pH 5)
Pre-treated sample volume
Salt amount (K2HPO4)
HPLC gradient program
Final composition of
mobile phase (linear gradient)
90% B; 10% D
10% B; 90% D
10% B; 90% D
P. Berton et al. / Talanta 97 (2012) 521–526
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with commercially available [Cnmim][Cl] (Solvent Innovation
GmbH, K¨ oln, Germany).
Ultrapure water (18 MO cm) was obtained from a Millipore
Continental Water System (Bedford, MA, USA). All glassware was
washed with 0.1 mol L?1HNO3 solution at least for 24 h and
thoroughly rinsed 5 times with ultrapure water before any use.
2.3. Sample collection and conditioning
The urine samples used in the analysis were the first-voided
morning specimens collected from volunteers. The samples were
collected in clean, acid-washed, amber glass bottles. The speci-
mens were centrifuged for 5 min at 2500 rpm (503 g) to remove
any insoluble material. The supernatant was transferred to a 20-
mL vial for immediate analysis.
2.4. IL-ATPS procedure
An amount of 0.2 g of [C6mim][Cl] was added and fully dissolved
into 5 mL of the pre-treated sample. After addition of 3.0 g of K2HPO4,
the mixture was vortexed and the homogeneous solution became
cloudy, extracting VB12into the IL phase. After 5 min without vortex-
assisted stirring, two well-defined phases were formed, without the
need of centrifugation. The upper IL-enriched phase was then directly
injected into the HPLC column for VB12separation and determination.
Calibration was performed by spiking the samples with known
concentrations of VB12. Same procedure as described above for
samples was applied for calibration standards. Optimized instrumen-
tal and experimental conditions are shown in Table 1.
3. Results and discussion
3.1. [C6mim][Cl]-salt IL-ATPS extraction
Several Kþion-containing compounds, including K2SO4, KOH,
K2CO3, K2HPO4 and KH2PO4 have been evaluated for their
suitability to form [C6mim][Cl]-salts IL-ATPS. Results show that
IL-ATPS can be formed only by adding appropriate amounts of
K2CO3, KOH or K2HPO4to water–[C6mim][Cl] homogeneous solu-
tion. As expected, the observed order follows the Hofmeister series
about the strength of kosmotropic salts: K2HPO44K2CO34KOH.
The kosmotropic ions, e.g. HPO4
which are usually small and highly charged, exhibit stronger
interaction with water molecules than that between water mole-
cules, being beneficial to ATPS formation. However, the chaotropic
ions, e.g. Cl?, H2PO4
species, have the opposite effect because of their weaker interac-
tions with water. This theory leads to the statement that ion
specificity is mainly determined by the ion’s polarizability in
water, and the generally observed greater and dominating Hof-
meister effects of anions over that of cations can be explained by
the larger polarizability values of anions with respect to cations
[30,31]. Although SO4
second one (11 vs. 150 g/100 mL H2O). Therefore, SO4
tration can never be high enough to allow the formation of ATPSs,
even in saturated solutions. As expected, strongly kosmotropic
ions favor IL-ATPS formation, thus less salt amount was needed as
more kosmotropic was the assayed salt. However, the mechanism
through which the salt influences phase separation is still poorly
understood. An appropriate explanation for phase separation in
IL-ATPS correlates the observed behavior to the tendency of
chaotropic salts to be salted-out by kosmotropic salts. Since ILs
are designed to have depressed melting points, a result of low
2?, OH?, CO3
?, which are large-size and lowly charged
2?has almost the same kosmotropic level as
2?, the solubility of the former anion is much lower than the
intermolecular interactions, most ILs would be classified as chao-
tropic salts . The phase separation process can thus be
hypothesized as follows: after addition of an inorganic salt to an
IL solution, the ions will compete with each other for the solvent
molecules. The more kosmotropic inorganic ions have a stronger
affinity for the solvent. Consequently, a ‘‘migration’’ of solvent
molecules away from the ions of the IL to those of the inorganic
salt takes place, which in turn decreases the hydration and hence
the solubility of the ions of the IL. As a consequence, an IL-rich
phase separates from the rest of the solution. Therefore, the
resulting salting-out effect could be directly correlated to hydra-
tion strength of the different ions of inorganic salt .
After studying the effect of different salts on IL-ATPS forma-
tion, recovery of VB12in the top phase of [Cnmim]Cl–salt IL-ATPS
was evaluated. The results indicated that K2HPO4led to achieving
the highest extraction efficiency. When K2HPO4was employed,
a final pH of 11 was obtained. Since VB12is stable within the
pH interval of 4.0–12.0 , its extraction efficiency could be
considered as pH-independent under this interval. Therefore, as
expected, the highest extraction efficiency was obtained with
K2HPO4 because less salt amount was needed for a complete
phase separation. Thus, K2HPO4was selected as the phase form-
ing salt. Phase diagrams of IL-K2HPO4systems based on the three
alkylimidazolium chloride ILs with different alkyl groups were
not determined in this work, since this phase-forming studies
were already published in a previous work by Cao et al. .
The effect of the amount of K2HPO4on phase behavior of ATPS
was also investigated. Different amounts of K2HPO4were added
(between 2.0 and 4.0 g) into 5.0 mL water containing 0.2 g IL for
the phase separation. The higher was K2HPO4concentration, more
[C6mim]Cl was driven into the upper phase resulting in the
decreasing of phase ratio. As a result, the phase separation step
was easier and the enrichment factor was optimal. However,
when the amount of K2HPO4was higher than 3.0 g, the reached
top phase volume was constant at 0.25 mL as the salting-out
ability of K2HPO4was maximum. Therefore, 3.0 g of K2HPO4were
chosen for further experiments.
Alkyl-imidazolium-ILs with different carbon chains were evalu-
ated in this work. In good agreement with a previous work , the
phase-forming ability follows the order: [C6mim]þ4[C4mim]þ4
[C8mim]þ. In traditional ATPS, when increasing the molar mass of
polyethylene glycol (PEG), the binodal curves became closer to the
origin . A suitable explanation could be that the higher hydro-
phobic character of PEG, accounted for its larger molar mass,
increases the incompatibility between the phase-forming compo-
nents. However, the phase-forming ability of the ILs with different
alkyl-chain lengths was not in agreement with the order of their
hydrophobicity, i.e. [C6mim]þ
showed the best phase-forming
ability. Although this anomalous behavior was previously reported
[30,37], the reason has not been fully understood yet.
Since extraction efficiency and analyte detection in HPLC can
be remarkably affected by IL amount, this is a critical parameter
to be optimized in order to yield the highest VB12extraction while
getting the best analytical sensitivity. Recovery of VB12 upon
different [C6mim]Cl amounts was examined within the range of
0.1–0.7 g. As shown in Fig. 1, the highest recovery was achieved
when 0.1–0.2 g of [C6mim]Cl was employed. Higher amounts of IL
did not improve extraction efficiency, while led to a decrease in
the enrichment factor. Therefore, in order to economize IL and
gain a higher enrichment factor with minimal matrix effect, 0.2 g
was used for subsequent experiments in this work.
The effect of temperature on VB12extraction efficiency was also
investigated. Within 10–70 1C, the extraction efficiency remains
practically constant, indicating that temperature has little influence
on the distribution and kinetic behavior of VB12between the two
phases formed. This new extraction system can afford a relatively
P. Berton et al. / Talanta 97 (2012) 521–526
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wide range of temperature for the study on the extraction behavior of
VB12. The following studies were developed at room temperature.
3.2. Optimization of separation conditions
Initial studies demonstrated that isocratic elution was suitable
for VB12elution from the column when an aqueous standard was
injected into a mobile phase containing 40% of 0.005 mol L?1
phosphate buffer (pH 5) prepared in high purity water (mobile
phase B) and 60% of methanol (mobile phase D) at a flow rate of
1 mL min?1. However, when a VB12-containing IL-matrix stan-
dard was injected into the column under the isocratic conditions,
retention times were significantly modified and VB12was eluted
at a time corresponding to the column dead-volume (Fig. 2a).
Therefore, different conditions for gradient elution were studied
in this work. Improved resolution was achieved when a gradient
program was employed (Fig. 2b). After 0.5 min the isocratic run
(90% B and 10% D) was started, solvent B was decreased linearly
(increasing D) and reached 10% at 15 min. The final composition
(10% B and 90% D) was kept constant for 5 min (until 20 min).
After the acquisition time, 5 min post time was set for the
equilibration of the initial solvent composition. The selected
column, a Zorbax SB-Aq, has an alkyl reversed bonded phase
designed to retain hydrophilic and other compounds, while it is
compatible with the most common mobile phases, including
highly aqueous ones. Under these conditions, VB12was eluted
within 10 min (Fig. 2b) and no significant interfering peaks were
observed at this retention time.
3.3. Analytical performance
The partition of VB12between the phases was characterized by
several parameters, including extraction efficiency and enrichment
factor (EF). The EF was defined as the ratio of the calibration curve
slopes before and after the preconcentration step . Hence, at
optimal experimental conditions, the obtained extraction efficiency
was 97% and the EF for a sample volume of 5 mL was 25. The
relative standard deviation (RSD) resulting from the analysis of 10
replicates of 5 mL solution containing 0.50mg mL?1VB12was 4.5%.
The calibration graph was linear with a correlation coefficient of
0.9979 within a concentration range between 0.40 mg mL?1and up
to at least 8.00mg mL?1. The limit of detection (LOD) of the
proposed methodology, calculated based on the signal at intercept
and three times the standard deviation about regression of the
calibration curve, was 0.09mg mL?1.
Extraction efficiency (%)
IL ammount (g)
0.2 0.40.6 0.8
Fig. 1. Effect of IL amount on VB12recovery. Other experimental conditions are
illustrated in Table 1.
810 12 14
8 10 1214
Fig. 2. Chromatogram obtained for (a) VB12standard of 100mg mL?1in an IL matrix
with isocratic elution; (b) VB12standard of 100mg mL?1in an IL matrix with gradient
elution; and (c) IL without VB12. Other experimental conditions are illustrated in
P. Berton et al. / Talanta 97 (2012) 521–526
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Furthermore, capacity factor, a chromatographic parameter
which characterize the performance of the method, was calculated
as k0¼(tR?t0)/t0; where tRis the migration time and t0is the dead
time. A capacity factor of 6.68 was obtained with the proposed
method. It is widely accepted, that capacity factors between 2 and
10 are optimum in practice for a mixture of few components.
Moreover, reproducible retention times were observed throughout
a regular working day (8–12 h of analysis).
3.4. Application of IL-ATPS-HPLC method for vitamin B12
determination in complex samples
In order to demonstrate the applicability of the proposed method
to matrices where VB12 determination is highly significant, the
proposed method was applied to urine samples (Table 2). Blood is
the ideal matrix for most chemicals due to its contact with the
whole organism and its equilibrium with organs and tissues where
chemicals are stored . However, blood requires an invasive
extraction method to be obtained and collected amounts are limited.
On the other hand, urine can be collected in larger amounts and by
non-invasive methods. Moreover, urine is the second most common
matrix for the biomonitoring of water-soluble compounds . The
representative chromatogram of urine after IL-ATPS-HPLC analysis is
shown in Fig. 3. No evident interference was observed on the
separation of VB12. The results showed excellent selectivity of the
method for the determination of VB12. Furthermore, high reprodu-
cibility of retention time for VB12was obtained in presence of the
sample matrix, which might allow the application of the proposed
method to samples different than urine.
The analytical recovery of VB12 in urine (Table 2) was also
studied. The proposed method was applied to six portions of
different matrices and average concentrations of VB12were taken
as base values. Then, 1.00 and 3.00 mg mL?1VB12were added to
samples and the same procedure was followed. VB12recoveries
were highly satisfactory in all cases.
A rapid microextraction method based on [C6mim]Cl IL for
selective vitamin B12determination is presented in this work. Due
to the low solvent consumption and the selection of IL as organic
phase, the developed method is in good agreement with green
chemistry principles. Compared with conventional ATPS, the
method presents many advantages such as low viscosity, quick
phase separation and high extraction efficiency. Moreover, the
proposed IL-ATPS is simpler and faster, compared to conventional
solid-phase extraction (SPE) (which usually includes a number of
laborious steps, such as sorbent conditioning, rinsing the sample,
washing and elution of the analytes).
All in all, the results shown in this work indicate that the
proposed procedure is simple, fast, interference-free, selective and
Determination of vitamin B12in urine samples (95% confidence interval; n¼6).
SampleAdded (mg mL?1)a
Found (mg mL?1) Recovery (%)b
Urine 1 0.00
Fig. 3. Determination of VB12 in urine sample with IL-ATPS-HPLC method:
(a) urine sample 1 without VB12standard addition; (b) urine sample 1 spiked at
2 mg mL?1of VB12. Experimental conditions are illustrated in Table 1.
Performance data of the proposed method compared to other methods with
HPLC–UV detection for VB12determination.
P. Berton et al. / Talanta 97 (2012) 521–526
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environment-friendly, and it can be used for VB12 selective
preconcentration and determination in urine. IL-ATPS technique
combined with HPLC chromatography shows a good limit of
detection and a wide calibration range with a reduced amount
of sample, while using low cost and widely spread instrumenta-
tion. The method detection limit is comparable to, or better than,
others extraction methods prior HPLC–UV analysis reported for
VB12(Table 3). Furthermore, most of the extraction methodologies
previously proposed, employed larger volumes of sample and
longer extraction times, when informed. The simplicity of the
process and the low cost of phase-forming materials make the
proposed method feasible for large-scale vitamin purification
using appropriate scale-up techniques. The present study demon-
strates that IL-ATPS can be an excellent and green extraction
technique for separation and preconcentration of water-soluble
vitamins, even from complex matrices like urine.
This work was supported by Consejo Nacional de Investigaciones
Cientı ´ficas y Te ´cnicas (CONICET), Agencia Nacional de Promocio ´n
Cientı ´fica y Tecnolo ´gica (FONCYT) (PICT-BID) and Universidad
Nacional de Cuyo (Argentina).
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