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N. Furusawa / Chemistry Journal (2013), Vol. 03, Issue 04, pp. 112-116
ISSN 2049-954X
Research Paper
An Easy and Small-Scale Sample Preparation Technique
Followed by HPLC-PDA Analysis for Determining
Canthaxanthin in Chicken Liver, Fat, and Egg Yolk
Naoto Furusawa
Graduate School of Human Life Science, Osaka City University, Osaka 558-8585, Japan
E-Mail: [email protected]
Abstract
This paper described a fast, simple, and small scale method of sample preparation followed by High-Performance Liquid
Chromatography (HPLC) coupled Photo-Diode Array (PDA) detector for quantification of canthaxanthin in chicken liver
and fat, and hen’s egg yolk. The HPLC-PDA was performed on a C18 column with an isocratic mobile phase. Analyte was
extracted from the sample using a handheld ultrasonic homogenizer, and purified by MonoSpin®-SI, a centrifugal
monolithic silica spin mini-columns, and quantified <15 min. The proposed method obtained average recoveries for the
analyte in the range of 93.5-101.0% with relative standard deviations ≤ 2.7%. The quantification limits in chicken liver, fat,
and egg yolk were 0.48, 0.47, and 0.5 μg g-1, respectively.
Keywords: Canthaxanthin, Chicken, HPLC, CM Silica Spin Mini-Column, International Harmonized Analytical Method
1. Introduction
Canthaxanthin (CX) (Figure 1), colouring agent, xanthophyll, is frequently used as feed additives to pigment the eggs
or meat of poultry. The permitted CX is red pigment and
commonly added to feeds for chickens in order to achieve
the desired chicken product colour. Although the European
Union (EU) permits eight xanthophylls to be added to the
feed of chickens, the use of chemically synthesized CX is
nearly the norm in the EU and Japan (JMAFF, 1953;
EFSA, 2007 and NFCA, 2011).
CX is highly potent red colorant available to poultry Industry. In comparison to other red xanthophylls, it is in a high
bioavailability form and has a higher deposition rate in
chicken tissues and egg yolk (DSM, 2004; LTL, n.d. and
Na et al, 2004). Notably, colour of egg yolks greatly affects the purchasing behaviour of consumer (Santos-Bocanegra et al, 2004). CX is used to brighten the yolks of chicken eggs in response to consumer demands. Since consumers associate bright product colouration with health and
quality, CX is particularly important in the poultry-farming
industry as their pigments for egg yolks (EC, 2002 and
Beardsworth & Hernandez, 2004).
CX has been known to cause liver injury and an eye
disorder, which is the formation of yellow deposits on the
retina (FDA, 2003). Following scientific assessments establishing a link between high CX intake and eyesight
problems in humans (due to an accumulation of pigment in
the retina), the European Commission adopted a directive
in 2003 to reduce the authorised level of CX in animal feed
(EC, 2002). The European Food Safety Authority (EFSA)
(EFSA, 2007) has set maximum residue limits (MRLs) for
the CX in several xanthophylls in chicken products as 30
μg g-1 in egg yolk; 15 μg g-1 in liver and 2.5 μg g-1 in fat,
to ensure the safety and appropriateness of chicken products for human consumption. Monitoring CX in chicken
products is, thus, an important means of guaranteeing food
safety.
In answer to the present expansion in the internal food
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N. Furusawa / Chemistry Journal (2013), Vol. 03, Issue 04, pp. 112-116
trade, the development of international standardized methods to determine chemical contents in foods is essential in
order to guarantee equitable international trade in these foods and ensure food safety for consumers. Without rega-rd
for industrial nations and developing countries, the optimal
harmonized method for chemical monitoring in foods must
be simple, small scale, economical in time and cost and
must cause negligible harm to the environment and analysts. Several techniques based on High-Performance Liquid
Chromatography (HPLC) for the quantification of xanthophylls such as CX in food samples have been reported
(Hamilton, 1992; Bononi et al, 2002; Breithaupt, 2004; Li
et al, 2005; Schlatterer & Breithaupt, 2006; Rajesha et al,
2009; Wenzel et al, 2010 and Furusawa, 2011). The drawback of these methods, however, is that these involve several analytical steps in the sample preparation, which are
time-and cost-consuming and do not permit the determination of large number of samples or some methods are
based on LC-MS. The facility is available are limited to
part of industrial nations because these are hugely expensive, and the methodologies use complex and specific.
These are unavailable in a lot of laboratories for routine
analysis, particularly in developing countries. No optimal
method that satisfies the aforementioned requirements has
yet been identified.
ISSN 2049-954X
ation: handheld ultrasonic-homogenizer (model HOM-100,
2 mm ID probe, Iwaki Glass Co., Ltd., Funabashi, Japan);
micro-centrifuge (Biofuge® fresco, Kendo Lab. Products,
Hanau, Germany); a MonoSpin® as centrifugal monolithic
silica spin mini-column (sample throughput volume ≤ 300
μL), MonoSpin-SI (silica gel, bonded Si-OH, normalphase mode) (GL Sciences, Inc., Tokyo, Japan). An Inertsil® ODS-4 (C18) (5 μm dp, 150×4.6 mm) (Pore diameter,
10 nm; Pore volume, 1.05 mL g-1; Surface area, 450 m2 g-1;
Carbon load, 11%) column for HPLC was used (GL Sciences, 2011).
The HPLC system, used for method development, included
a model PU-980 pump and DG-980-50-degasser (Jasco
Corp., Tokyo, Japan) equipped with a model CO-810 column oven (Thosoh Corp., Tokyo, Japan), as well as a model
SPD-M10A VP Photodiode-Array (PDA) detector (Shimadzu Scientific Instruments, Kyoto, Japan).
2.2. HPLC Operating Conditions
The analytical column was an Inertsil® ODS-4 (5 μm, 150
× 4.6 mm) column using an acetone-water (90:10, v v-1)
mobile phase at a flow rate of 1.0 mL min-1 at 25 oC. PDA
detector was operated at 190-600 nm: the monitoring wav-
Figure 1. Chemical Structure of CX
The present method was developed in such a way that, in
idiotproof and small-scale with minimized organic solvent
consumption, CX contents in chicken liver, fat and egg
yolk can be determined with higher accuracy and precision.
2. Material and Method
2.1. Reagents and Apparatus
All chemicals including Canthaxanthin (CX) (β,β-carotene-4,4’-dione, ≥98% purity) standard were purchased from
Wako Pure Chem. Ltd. (Osaka, Japan). Acetone and distilled water was of HPLC grade.
The following apparatuses were used in the sample prepar-
elength was adjusted to 470 nm which represents a maximum for the analyte. The injection volume was 10 μL.
2.3. Preparation of Stock Standard & Working Solutions
A stock standard solution of CX was prepared by dissolveng CX in acetone to a concentration of 100 μg mL-1. The
solution was put into cryo-vial, sealed and stored at -20 oC
and protected from light until further use. Working mixed
standard solutions were freshly prepared by suitably diluteing the stock solution with acetone on the day of analysis
2.4. Blank Samples
To obtain blank chicken liver, fat and egg yolk samples, 5
adult chickens (laying hens aged 30 weeks) were kept in
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N. Furusawa / Chemistry Journal (2013), Vol. 03, Issue 04, pp. 112-116
individual cages and given pigment-free basal diet and tap
water ad libitum. After the feeding basal diet for 3 weeks,
all of the eggs laid by hens were collected for 2 days. The
albumens and egg yolk of each egg were separated immediately. They were then killed and liver and intraperitoneal
adipose tissues were collected. The collected chicken tissues and yolks were uniformed fully and used as blank
samples.
ISSN 2049-954X
can provide the quantitation and identification of the analyte.
2.5. Preparation of Calibration Standards and Quality
Control Samples
For method validation studies, calibration standards and
Quality Control samples (QCs), terms defined in the FDA
guideline (FDA/CDER/CVM, 2001) were prepared by spiking appropriate aliquots of the standard solution in blank
samples. Calibration standards were used to construct calibration curves from which the concentrations of an analyte
in unknown monitoring samples are determined practically. QCs used to evaluate the performance of the proposed
method. In this study, the standards were prepared in the
range of 1-50 μg g-1 for CX. Three QC levels (QC1 = 1 μg
g-1; QC2= 10 μg g-1; QC3 = 30 μg g-1) were prepared.
Figure 2. Typical Chromatograms Obtained from the HPLC System
for an Egg Yolk Sample Spiked with CX (10 μg g-1) (Upper
Profile) and a Blank Egg Yolk Sample (Lower Profile). The
PDA Detector was set at 470 nm. Peak, 1= CX (retention
time, Rt= 4.08 min)
2.6. Sample Preparation
3.1. Method Validation
An accurate 0.2 g sample was taken into a 1.5 mL microcentrifuge tube and homogenized with 0.6 mL of 80% (v v1
) acetone solution (in water) with a handheld ultrasonichomogenizer for 30 s. After being homogenized, the capped tube was centrifuged at 10,000 g for 5 min. A 0.1 mL of
supernatant liquid was poured to a MonoSpin-SI and, immediately after, the capped mini-column was centrifuged
at 3,000 g for 1 min. The eluate was injected into the
HPLC system.
2.7. Method Validation
The performance of the developed method was validated
in terms of some parameters from the international guidelines for bio-analytical procedure (ICH, 1994; Huber, 1998;
Codex Alimentarius Commission, 2001; FDA/CDER/
CVM, 2001 and AOAC, 2002).
3.1.1. Main Method Validation Data
Table 1 summarizes the method validation data for the main performance parameters (linearity, range, accuracy, precision, and sensitivity). The accuracy and precision are
well within the international method acceptance criteria
(Huber, 1998; Codex Alimentarius Commission, 2001 and
AOAC, 2002). The decision limit (CCα) and detection
capability (CCβ) values calculated according to the EU
regulation decision (EC, 2002) are described in Table 1.
The other validation findings are as follows:
3.1.2. Specificity and Selectivity
The application of the proposed procedure to 30 blank chicken liver, fat, or egg yolk samples demonstrated that no
interference peak was presented around the retention times
for CX in any of the sample examined.
3. Results and Discussion
Figure 2 displays examples of typical HPLC traces of blank and spiked (CX 10 μg g-1) egg yolk samples obtained
under the established procedure. The resulting chromatograms were free of interfering compounds for quantitation
and identification of CX by HPLC with PDA detector.
Similar chromatograms were obtained from chicken liver
and fat samples. The present HPLC analysis accomplished
good separations without the need for a gradient system to
improve the separation and pre-column washing after
analysis. This figure demonstrates that the present method
The present HPLC-PDA system easily confirmed the peak
identity of target compound. The analyte was identified in
a sample by its retention times and absorption spectrum.
The CX spectrum obtained from the sample was practically identical to that of the standard. Because of the complete separations and the high absorbance of the analyte, PDA
detection at trace levels is fully available. It is, therefore,
instructive to demonstrate purification effectiveness of the
sample preparation. The system did not require the use of
MS, which is very expensive and is not available in a lot of
laboratories for routine analysis.
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N. Furusawa / Chemistry Journal (2013), Vol. 03, Issue 04, pp. 112-116
Table1. The Main Method Validation Data
Liver
Linearity (ra))
0.995
-1
Range (μg g )
Accuracyb)
93.5-100.6
Precisionc)
≤ 1.9
d)
Sensitivity (QL )
0.48
CCαe)
15.73
CCβf)
16.46
ion times, respectively.
Fat
Egg Yolk
3.1.5. Cost and Time Performance
0.998
1-50
94.9-101.0
≤ 1.8
0.47
2.59
2.68
0.991
94.5-99.0
≤ 2.7
0.50
31.17
33.08
The total time and budget required for the analysis of a
single sample was <15 min and approximately € 3.41 (i.e.
£ 2.91 or $ 4.38) as of 23 May (13:56), 2013, respectively.
For sequential analyses, a batch of 24 samples could be
analyzed in < 2.5 h. These findings became term required
for the routine assay. The short analytical time not only
increased the sample throughput for analysis but also
positively affected the cost.
a
r is the correlation coefficient (P< 0.01): mean of three determinations
using spiked samples for calibration curves
b
Average recoveries (%) from six replicates at three QC levels (1, 10
and 30 μg g-1 for CX)
c
Values are relative standard deviations (RSDs, %, n=6 of each level)
d
Quantitation limit (μg g-1), QL as the concentration of analyte giving a
signal-to-noise ratio > 10
e
ISSN 2049-954X
Decision limit (α = 5 %, μg g-1) by the EU regulation 2002/657/EC
f
Detection capability (β = 5 %, μg g-1) by the EU regulation
2007/657/EC
4. Conclusions
A simplified sample preparation followed by HPLC-PDA
method for determination of CX in chicken tissues has
been successfully developed and validated. The present procedure provided an easy-to-use, rapid and space-saving. It
gave high recovery and repeatability with considerable
saving of analysis time/cost. The procedure may be proposed as an international harmonized method for determining
CX in the domestic/import chicken products.
Acknowledgement
3.1.3. Robustness
The author wishes to thank Mr. Yoshimura and Kuwahara,
GL Sciences, for wholeheartedly supporting this work.
Some HPLC parameters were performed using a spiked
(10 μg g-1 of CX) egg yolk sample obtained under the established procedure.
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