Food Chemistry 141 (2013) 14331437

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

Analytical Methods

A rapid HPLC column switching method for sample preparation

and determination of b-carotene in food supplements
Ivana Brabcov, Markta Hlavckov, Dalibor atnsky , Petr Solich
Department of Analytical Chemistry, Faculty of Pharmacy, Charles University, Heyrovskho 1203, Hradec Krlov 500 05, Czech Republic

a r t i c l e

i n f o

Article history:
Received 20 December 2011
Received in revised form 8 November 2012
Accepted 19 April 2013
Available online 30 April 2013
Keywords:
b-carotene
On-line sample preparation
Column-switching
HPLC
Food supplements

a b s t r a c t
A simple and automated HPLC column-switching method with rapid sample pretreatment has been
developed for quantitative determination of b-carotene in food supplements. Commercially samples of
food supplements were dissolved in chloroform with help of saponication with 1 M solution of sodium
hydroxide in ultrasound bath. A 20-min sample dissolution/extraction step was necessary before chromatography analysis to transfer b-carotene from solid state of food supplements preparations (capsules, tablets) to chloroform solution. Sample volume 3 lL of chloroform phase was directly injected
into the HPLC system. Next on-line sample clean-up was achieved on the pretreatment precolumn Chromolith Guard Cartridge RP-18e (Merck), 10  4.6 mm, with a washing mobile phase (methanol:water,
92:8, (v/v)) at a ow rate of 1.5 mL/min. Valve switch to analytical column was set at 2.5 min in a
back-ush mode. After column switching to the analytical column Ascentis Express C-18, 30  4.6 mm,
particle size 2.7 lm (Sigma Aldrich), the separation and determination of b-carotene in food supplements
was performed using a mobile phase consisting of 100% methanol, column temperature at 60 C and ow
rate 1.5 mL/min. The detector was set at 450 nm. Under the optimum chromatographic conditions standard calibration curve was measured with good linearity correlation coefcient for b-carotene
(r2 = 0.999014; n = 6) between the peak areas and concentration of b-carotene 20200 lg/mL. Accuracy
of the method dened as a mean recovery was in the range 96.66102.40%. The intraday method precision was satisfactory at three concentration levels 20, 125 and 200 lg/mL and relative standard deviations were in the range 0.901.02%. The chromatography method has shown high sample throughput
during column-switching pretreatment process and analysis in one step in short time (6 min) of the
whole chromatographic analysis.
2013 Elsevier Ltd. All rights reserved.

1. Introduction
b-carotene is an important biological compound that is widely
distributed in fruits and vegetables. It is converted in the human
body to retinol (vitamin A) that is essential for proper function of
the retina, skin and mucous membranes. Biological studies have
shown that the dietary intake of b-carotene may reduce the risk
of cancer and infectious diseases. b-carotene protects plant, human
and animal cells (most mucous membranes and skin) from the
destructive effects of UV radiation. As an antioxidant deactivates
harmful free radicals, thus slows down the ageing process.
Traditionally, the separation and determination of carotenoids
(including b-carotene) was often performed using high performance liquid chromatography (HPLC) with a reversed phase C-18
columns and detection at visible part of spectra (Iwase, 2002; Karppi, Nurmi, Alonso, Lorencio, & Nyyssnen, 2008; Pupin, Dennis, &
Toledo, 1999). However, most HPLC methods employ time con Corresponding author. Tel.: +420 495067228; fax: +420 495518718.
E-mail address: satinsky@faf.cuni.cz (D. atnsky).
0308-8146/$ - see front matter 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodchem.2013.04.063

suming and laborious sample preparation steps such as pressurized liquid extraction (PLE) (Plaza et al., 2011; Sanagi, See,
Ibrahim, & Naim, 2005), liquidliquid extraction (LLE) (Burgos
et al., 2009; Jackson, Lien, White, Bruns, & Kuhlman, 1998; Karppi
et al., 2008; Pupin et al., 1999), supercritical uid extraction (SFE)
(Birtigh, Johannsen, Brunner, & Nair, 1995; Chuang & Brunner,
2006), solid phase extraction (SPE) (Iwase, 2002; Shen et al.,
2009; Snchez, Granados, Serrana, & Martnez, 2010), ultrasoundassisted extraction (UAE) (Plaza et al., 2011), or matrix solid-phase
dispersion (MSPD) (Gentili & Caretti, 2011; Putzbach et al., 2005),
which are carried out in off-line mode before sample injection into
the chromatography system. In addition, using above mentioned
sample pretreatment methods could be accompanied with risk of
loss of analytes during sample manipulation and greater
biohazard.
Nowadays the application of on-line extraction on special SPE
sorbents is the growing trend in sample preparation procedures
(Sadlek, atnsky, & Solich, 2007). The introduction of special
extraction sorbents, such as restricted access materials (RAM), allows the direct and repetitive injection of complex biological

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I. Brabcov et al. / Food Chemistry 141 (2013) 14331437

matrices onto these supports. The coupling of RAM into the column-switching HPLC systems represents a very attractive approach in biological sample preparation. Use of this technique
leads to the automation, simplication, and speeding up of the
sample preparation process. The term RAM sorbents is shared by
materials used mainly for the analysis of substances with low
molecular mass (e.g. drugs, endogenous substances, xenobiotics)
in complex matrices containing high-molecular substances (most
frequently proteins). These materials enable direct injection of
the biological samples into the ow analytical systems without
previous sample treatment. They have several different structures,
but their mechanism of separation is identical: a hydrophilic barrier enables the small molecules to permeate through the hydrophobic part of the stationary phase, and, at the same time, it
excludes the macromolecules (by physical or chemical means, or
a combination). Column-switching technique using these special
sorbents for complex matrix samples can work without the laborious and time-consuming clean-up protocols. From this perspective, column-switching technique for direct injection, on-line
preparation and simultaneously analysis of samples is modern approach in implementation of all of analytical process to one step. In
case of analysis of simple matrix such as food supplements samples usual chromatography precolumns can be coupled to column switching system without time-consuming clean-up
protocols and without sorbent destruction.
The main advantage of this technique is strong reduction of
time and sample manipulation during sample preparation steps.
One sample pretreatment step is necessary ltration before injection to chromatography system. On the other side method development process is not simple in some cases and it requires
special attention mainly during the rst chromatographic dimension for sample clean up elution power of mobile phase for interferences removal and sufcient recovery of analytes of interest,
pretreatment column choice, ow rate of mobile phase and time
of column switch. Moreover, the next complication of this technique is requirement for 2-D chromatography system two
pumps, high pressure column switching valve and control software
(Sadlek et al., 2007; Souverain, Rudaz, & Veuthey, 2004).
This paper presents for the rst time the development of a new
HPLC method for the determination of b-carotene in food supplements employing column switching technique using on-line sample preparation and separation in one step. Developed method was
validated and successfully applied for the determination of b-carotene in food supplements available on local market.
2. Materials and methods
2.1. Chemicals and materials
Standard of b-carotene and organic solvents (HPLC grade)
methanol and acetonitrile were obtained from SigmaAldrich Chemie GmbH, Germany. The ultra-pure water used for mobile phase
preparation was puried through a Milli-Q (Millipore, Bedford, MA,
USA). Chloroform and sodium hydroxide were from Merck, Darmstadt, Germany. All other materials were of analytical grade. The
food supplements with content of b-carotene (capsules, tablets
and oil suspension) were purchased on local market in Czech
Republic.
2.2. Equipment and chromatographic system
HPLC chromatographic apparatus consisted of a Shimadzu
Prominence system (Shimadzu Corporation, Kyoto, Japan),
equipped with solvent delivery systems LC-20AD, with a SIL20AC autosampler, DGU-AS on-line degasser, SPD-M20A DAD

detector, CTO 20AC column oven with FCV-12AH high pressure

six-port switching valve and CBM-20A communication module.
The system control, data acquisition and data evaluation were performed by Shimadzu LC Lab-Solution software (Shimadzu Corporation, Kyoto, Japan).
2.3. Preparation of stock solution and food supplements sample
preparation
Standard stock solution of b-carotene was prepared by dissolving of substance in chloroform in concentration 2000 lg/mL. Standard stock solution was stored at 18 C in dark for 1 month.
Standard stock solution was further diluted with chloroform to obtain working standard solution in concentration 100 lg/mL for
method development and validation. The calibration standard
solutions were prepared over the concentration range of 20
200 lg/mL, using seven calibration points. All solutions were
stored in amber laboratory glassware.
Food supplements samples (capsule, homogenised tablet powder or 2 g of oil suspension) were dissolved in 20 ml of 1 M sodium
hydroxide with help of ultrasound bath. Twenty millilitre of chloroform was added to sample solution and the mixture was extracted for 20 min. Closed PTFE centrifugation tubes were used
for sample dissolution and extraction to avoid evaporation of chloroform. Three millilitre of chloroform layer was injected directly
into the HPLC system. Samples with higher concentration of b-carotene were diluted with pure chloroform to obtain concentration
inside calibration range. All food supplement samples and working
standard solutions were prepared fresh daily.
2.4. HPLC column switching analysis
The simultaneous sample pretreatment and b-carotene determination was performed using a two column HPLC system. The
sample pretreatment column was precolumn Chromolith Guard
Cartridge RP-18e (Merck, Darmstadt, Germany) 10  4.6 mm, with
a washing mobile phase (methanol:water; 92:8) at a ow rate of
1.5 mL/min. The chromatographic separation and determination
of b-carotene was performed on an analytical column Ascentis Express C-18 30  4.6 mm, particle size 2.7 lm (Sigma Aldrich, Germany), using a mobile phase consisting of 100% methanol and
ow rate 1.5 mL/min. The column oven temperature was kept constant at 60 C for both columns. The detector was set at 450 nm.
On-line sample pretreatment step (02.5 min) 3 lL chloroform sample solution of food supplement was directly injected
onto
precolumn
Chromolith
Guard
Cartridge
RP-18e
10 mm  4.6 mm where polar interferences and (tablets, capsules)
sample matrix were removed to the waste. Used washing mobile
phase consisted of mixture methanol:water; 92:8, (v/v) was
pumped for 2.5 min at ow rate of 1.5 mL min 1. The analytical
column was equilibrated using the analytical mobile phase at this
time. Valve switch for column was set at 2.5th min.
Separation step and determination of b-carotene was performed in interval range from 2.5th to 6th min. Zone of preconcentrated sample was transported in back-ush direction from
pretreatment column onto analytical column Ascentis Express C18 30  4.6 mm, particle size 2.7 lm, where the analysis was performed in isocratic mode. The analytical mobile phase used for
determination of b-carotene consisted of 100% methanol pumped
at ow rate 1.5 mL/min.
3. Results and discussion
No method for determination of b-carotene in food, food
supplements and in another plant material with HPLC column

I. Brabcov et al. / Food Chemistry 141 (2013) 14331437

switching technique is reported in the literature so far. Therefore,

prior to the method development and validation, the parameters
of sample preparation, conditions of mobile phases and stationary
phases in rst and second chromatography dimension were
optimised.
3.1. Optimisation of rst chromatography dimension
The rst chromatography dimension was used for sample pretreatment and preconcentration of b-carotene in our case. Sample
solution dissolved in chloroform was injected to rst column,
where b-carotene adsorption and matrix component interference
elution were carried out. Choice of the preconcentration column
and pretreatment mobile phase composition had to fulll requirements on low interferences absorption on column sorbent and
strong retention of b-carotene. The following preconcentration columns were tested in our study: Precolumn sorbent lter Optiguard C-18-1 mm in tting screw, (Sigma Aldrich, USA), Chromolith Guard Cartridge RP-18e 10  4.6 mm and Chromolith Guard
Cartridge RP-18e 5  4.6 mm (Merck, Darmstadt, Germany).
Different pretreatment mobile phases with methanol ranging
from 90% to 100% (v/v) with water and different ow rates ranging
from 1.0 to 3.0 ml min 1 were tested and optimised for all pretreatment columns. Washing times for rst column dimension
ranging from 30 s to 3 min were tested. Volume of methanol lower
than 90% resulted in decreasing of recovery (decreasing of peak
area) due to poor solubility of b-carotene in mobile phase. On the
other side, 100% of methanol was not found to be suitable for bcarotene preconcentration on pretreatment column due to the
weak retention of b-carotene and washing out it with ballast matrix of food supplements. Optimisation of column-switch time
and ow rate were estimated as the time and ow rate sufcient
for washing ballast matrix components for each food supplement
preparation. Optimal pretreatment mobile phase was found as ratio methanol water (92:8, v/v) pumped for 2.5 min at ow rate
1.5 mL min 1 through the preconcetration column Chromolith
Guard Cartridge RP-18e 10  4.6 mm. Mentioned conditions were
found to be optimal for sample preparation and preconcentration
prior to b-carotene separation in the analytical column. Precolumn
sorbent lter Opti-guard C-18-1 mm was found to be sorbent
without sufcient adsorption capacity. Partially elution of b-carotene after the sample zone injection was observed. This effect
was caused by chloroform (strong eluent) in sample solution and
small amount of sorbent (1 mm length) in tting screw. On the
other hand, there was possible to say that monolithic precolumns
were suitable for the desired purpose of sample pretreatment. Both
monolithic precolumns showed ability of sample pretreatment and
b-carotene preconcentration under the tested and optimised conditions. Chromolith Guard Cartridge RP-18e 10  4.6 mm was nally chosen for validation of the method.
3.2. Optimisation of second chromatography dimension
Two analytical chromatographic columns with totally different
structure of stationary phases core shell particle column Ascentis
Express C-18 30  4.6 mm, particle size 2.7 lm (Sigma Aldrich,
Germany) and monolithic column Chromolith SpeedRod RP-18e
50  4.6 mm (Merck, Darmstadt, Germany) were tested. The isocratic separation of b-carotene was optimised with methanol/
water mobile phases ranging from 95% to 100% (v/v) of methanol
in water. Addition of water to mobile phase resulted in b-carotene
retention times increasing and peak symmetry started to be out of
validation range (higher than 1.5) for both columns. Successful
peak symmetry and faster analysis was achieved on Ascentis Express C-18 column. The column temperature optimisation was carried out in the range 3060 C for both columns (precolumn and

1435

analytical column) together. Decreasing time of analysis and better

peak symmetry was observed with increasing temperature of column oven. Therefore the nal separation conditions for method
validation on Ascentis Express C-18 column were chosen at 60 C
and ow rate of mobile phase 1.5 mL/min. The chromatogram of
b-carotene determination in food supplement sample with using
of column switching technique is given in Fig. 1.
3.3. Validation of the method
Validation of the method includes the evaluation of following
performance parameters such as linearity, precision, accuracy
and selectivity in order to evaluate the reliability of the results provided by the method. Within the method validation the parameters
of the HPLC system suitability test (SST) were measured and evaluated in accordance with ICH guidelines International Conference on harmonisation (ICH, 2005). The samples of standard
solution were six times injected into the chromatographic system.
Mean values and standard deviations of retention time, number of
theoretical plates, peak asymmetry, resolution, and repeatability of
analytical run were calculated according to the European Pharmacopoeia recommendations (European Pharmacopoeia, 2007). Obtained validation results and chromatography system suitability
parameters are summarized in Tables 1 and 2.
3.3.1. Linearity, selectivity and sensitivity
Seven test solutions ranging 20200 lg/mL of b-carotene were
prepared by dilution of stock solution of 2000 lg/mL and tested
for linearity test. The detector response at 450 nm was found linear
in whole range of calibration curve. The linear regression curve
was obtained by plotting peak area count of b-carotene at each level against the concentration of each injection. The detailed
descriptions of regression curve are depicted in Table 2. Statistical
data of calibration curve parameters were computed from seven
concentrations levels in mentioned range (200, 175, 150, 100,
50, 20 lg/mL). Good linearity (coefcient of correlation
r2 = 0.999014) was achieved in the investigated range for b-carotene. Linear regression parameters were described by the following
equation: A = (7726.6 153.6)c (24263.5 20244.9), where A is
the peak area at 450 nm and c b-carotene concentration. All samples were measured in triplicates.
The limit of detection (LOD) and limit of quantication (LOQ)
were calculated by the comparison of the threefold (3r) and 10fold (10r) variation, respectively, of base-line noise and signals
of b-carotene. The detection limit of b-carotene determination
was 0.50 lg/mL. The limit of quantication was estimated to be
1.67 lg/mL.
The selectivity of the method was evaluated by conducting the
experiments using batches of different food supplements samples
to check the peak purity of b-carotene onto the chromatogram.
No interfering peaks in retention time of b-carotene were observed
and peak purity index reached the values greater than 0.9999 for
all food supplement samples. Ballast matrix components from capsules and tablets dissolved in chloroform were eluted in the front
of the chromatogram of the rst dimension to the waste.
3.3.2. Repeatability, precision and accuracy
Repeatability of the proposed method was characterised by relative standard deviation (RSD, (%)), which was calculated for six
consecutive measurements at three concentration levels 20, 125
and 200 lg/mL of b-carotene standard solutions at the same day.
The results in form of RSD were determined in the range 0.90
1.02%.
To validate the precision of the method a number of 6 food supplement sample solutions were used, which were prepared from
the same batch and analysed consecutively. This approach

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I. Brabcov et al. / Food Chemistry 141 (2013) 14331437

Fig. 1. Representative chromatogram of b-carotene determination in food supplement sample (capsule) with using of column switching technique.

Table 1
HPLC column switching system suitability parameters.
Retention Retention time Repeatability Number of Peak
time (min) repeatability
of peak areas theoretical symmetry
RSD (%)a
RSD (%)a
plates
b-Carotene 4.86
a

0.17

1.01

2630

1.374

Made in six replicates at one concentration level 125 lg/mL.

Table 2
Analytical characteristics of the validated HPLC column switching method.
Parameters

b-carotene

Standard calibration range (lg/mL)a

Correlation coefcient
LOD (lg/mL)
LOQ (lg/mL)
Repeatability RSD (%)b
Method precision intra-day RSD (%)c
Accuracy spike recovery (%) SDd

20200
0.9990
0.50
1.67
0.901.02
1.432.59
96.66102.40 (1.712.92)

Each concentration of calibration standard was measured in triplicate.

Relative standard deviation (RSD) was calculated from six injections of b-carotene standard at three concentration levels 20, 125 and 200 lg/mL.
c
Relative standard deviation (RSD) for repeated injections of multiple preparations of different food supplement samples (n = 6), three injections of each sample
preparation.
d
Accuracy was determined as a method recovery using spiked food supplement
samples at one concentration level (added amount 100% of b-carotene content for
each food supplement) in six samples from one batch (minimal and maximal
standard deviation of recovery determination).
b

provides a means of covering the precision of the entire method,

from sample preparation to data handling. The intra-day precision
values of measured concentration of b-carotene in different food
supplement samples were calculated as RSD values which were
in the range 1.432.59%. In both situations (parameters of repeatability and precision), the relative standard deviation values were
in the range recommended in accordance with European Pharmacopoeia recommendations.
The accuracy of the method was carried out measuring of the
food supplement samples fortied with known quantity of b-carotene (addition of 100% amount of the standard to food supplement

preparation). Fortied sample solutions and un-fortied sample

solutions were compared for recovery evaluation. The method
accuracy results were tested for different food supplement preparations and values of the recoveries were found in the range 96.66
102.40% (1.712.92%) for b-carotene. Assay values of recoveries
show that the HPLC column switching method allows direct determination of b-carotene in commercial food supplement preparations in the presence of other antioxidants and excipients.
3.3.3. Robustness and stability
The robustness of the method was determined by measuring
the effect of small and deliberated changes in the analytical parameters on retention time and peak area counts. The parameters that
were taken into consideration were ow rate of mobile phase and
temperature in column oven. Mobile phase composition in second
chromatography dimension was kept constant at 100% of methanol. At a time only one parameter was changed while the second
was kept constant. The relative standard deviations of peak area
counts of b-carotene were calculated for both parameters. Differences in peak area due to the changes in column temperature were
2.00% for 50 C and 7.90% for 70 C. Variations in peak area due to
the changes in ow rate (tested range 1.0 and 2.0 mL/min) were
higher than 10%. The higher variations of peak areas are in agreement that the optimised conditions of the method must be strictly
kept to avoid impression during analysis.
Stability of b-carotene working standard solution was tested at
4 C and 20 C in autosampler for 24 h. Variations (RSD (%)) in peak
areas of b-carotene (range 1st24th h) were at 4 C and 20 C 2.88%
and 1.60%, respectively.
3.3.4. Determination of b-carotene in food supplements
The method developed has been applied to the determination of
b-carotene in capsules, tablets and oil suspension. The samples
were commercially available on the local market. The disturbing
effect of excipients (saccharine, gelatin, starch, shellac, matrix of
oil suspension and other antioxidants) was not observed. Peak purity in sample run and high accuracy of the analysis showed that no
interference was observed in the quantitative analysis of b-carotene. The optimal extraction procedure was reached due to column
switching technique. The suggested procedure of sample
preparation was simple, fast and achieving high precision and

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I. Brabcov et al. / Food Chemistry 141 (2013) 14331437

Table 3
Determination of b-carotene content in various food supplement samples (n = 4).

Food supplement

Type

Declared amount of b-carotene in

of food supplement (mg)a

Found amount in (%) RSD

Walmark Beta carotene

GS Betacarotene Forte
Bioactive carotene
Betacarotene farmax
Max Beta carotene
Carovite
Selzink plus
Primrose oil

Capsules
Capsules
Capsules
Capsules
Capsules
Tablets
Tablets
Oil suspension

6.00
15.00
9.00
6.00
6.00
6.00
6.00
30.00

98.76 1.77
100.07 1.41
97.00 1.91
96.66 1.71
111.67 3.28
127.17 2.72
128.83 4.40
73.20 1.19

Declared amount in one capsule; in one tablet; or in 100 g of oil suspension in mg.

low sample and reagent consumption. The average amounts of bcarotene determined in food supplement preparations are given
in Table 3. Obtained results in range 95.0105.0% for the active
compound content in preparations were fullled for four commercially products only.
4. Conclusion
The HPLC column switching methodology described in this paper provides a simple, rapid and rugged approach for qualitative
and quantitative analysis of b-carotene in food supplements samples. The determination of b-carotene with using HPLC column
switching method as sample pretreatment is reported here for
the rst time. The sample preparation procedure involved sonication followed by dissolution in chloroform (20 min in ultrasound
bath) and direct injection into HPLC system eliminated usual time
consuming sample preparation steps such as off-line solid phase
extraction or liquidliquid extraction. Real application of the new
method was carried out and the procedure was applied for the
quantitation of b-carotene in different commercial food supplements with high recovery. It can be concluded that because of
the simple column switching extraction procedure, high precision,
accuracy, simplicity and short chromatography analysis time of
6 min, the method may be useful for fast screening or quality control testing of b-carotene content in different food supplements
such as tablets, capsules or oil suspensions.
Acknowledgements
The authors acknowledge the nancial support of the Charles
University in Prague project SVV 267 002 and project UNCE No.
204026/2012.
References
Birtigh, A., Johannsen, M., Brunner, G., & Nair, N. (1995). Supercritical-uid
extraction of oil-palm components. The Journal of Supercritical Fluids, 8, 4650.
Burgos, G., Salas, E., Amoros, W., Auqui, M., Munoa, L., Kimura, M., & Bonierbale, M.
(2009). Total and individual carotenoid proles in Solanum phureja of
cultivated potatoes: I. Concentrations and relationships as determined by

spectrophotometry and HPLC. Journal of Food Composition and Analysis, 22,

503508.
Chuang, M. H., & Brunner, G. (2006). Concentration of minor components in crude
palm oil. The Journal of Supercritical Fluids, 37, 151156.
Council of Europe European (EDQM). (2007), European Pharmacopoeia, sixth ed.,
Strasbourg Cedex, 72.
Gentili, A., & Caretti, F. (2011). Evaluation of a method based on liquid
chromatographydiode array detectortandem mass spectrometry for a rapid
and comprehensive characterization of the fat-soluble vitamin and carotenoid
prole of selected plant foods. Journal of Chromatography A, 1218, 684697.
ICH (Q2). (2005). In: Proceeding of the international conference on harmonization.
IFPMA, Geneva, November, September 4 Version.
Iwase, H. (2002). Simultaneous sample preparation for high-performance liquid
chromatographic determination of vitamin A and b-carotene in emulsied
nutritional supplements after solid-phase extraction. Analytica Chimica Acta,
463, 2129.
Jackson, J. G., Lien, E. L., White, S. J., Bruns, N. J., & Kuhlman, C. F. (1998). Major
carotenoids in mature human milk: Longitudinal and diurnal patterns. Journal
Nutritional Biochemistry, 9, 27.
Karppi, J., Nurmi, T., Alonso, B. O., Lorencio, F. G., & Nyyssnen, K. (2008).
Simultaneous measurement of retinol, a-tocopherol and six carotenoids in
human plasma by using an isocratic reversed-phase HPLC method. Journal of
Chromatography B, 867, 226232.
Plaza, M., Santoyo, S., Jaime, L., Avalo, B., Cifuentes, A., Reglero, G., Reina, G. B.,
Seorns, F. J., & Ibez, E. (2011). Comprehensive characterization of the
functional activities of pressurized liquid and ultrasound-assisted extracts from
Chlorella vulgaris. LWT Food Science and Technology, 3, 19.
Pupin, A. M., Dennis, M. J., & Toledo, M. C. F. (1999). HPLC analysis of carotenoids in
orange juice. Food Chemistry, 64, 269275.
Putzbach, K., Krucker, M., Grynbaum, M. D., Hentschel, P., Webb, A. G., & Albert, K.
(2005). Hyphenation of capillary high-performance liquid chromatography to
microcoil magnetic resonance spectroscopy determination of various
carotenoids in a small-sized spinach sample. Journal of Pharmaceutical and
Biomedical Analysis, 38, 910917.
Sadlek, P., atnsky, D., & Solich, P. (2007). Using restricted-access materials and
column switching in high-performance liquid chromatography for direct
analysis of biologically active compounds in complex matrices. Trac-Trends in
Analytical Chemistry, 26, 375384.
Sanagi, M. M., See, H. H., Ibrahim, W. A. W., & Naim, A. A. (2005). Determination of
carotene, tocopherols and tocotrienols in residue oil from palm pressed ber
using pressurized liquid extraction-normal phase liquid chromatography.
Analytica Chimica Acta, 538, 7176.
Snchez, C. S., Granados, J. J. Q., Serrana, H. L. G., & Martnez, L. M. C. (2010). BCarotene, squalene and waxes determined by chromatographic method in
picual extra virgin olive oil obtained by a new cold extraction system. Journal of
Food Composition and Analysis, 23, 671676.
Shen, Y., Hu, Y., Huang, K., Yin, S., Chen, B., & Yao, S. (2009). Solid-phase extraction of
carotenoids. Journal of Chromatography A, 1216, 57635768.
Souverain, S., Rudaz, S., & Veuthey, J. L. (2004). Restricted access materials and large
particle supports for on-line sample preparation: an attractive approach for
biological uids analysis. Journal of Chromatography B, 801, 141156.