Chromatographia (2013) 76:523–528
DOI 10.1007/s10337-013-2405-y
ORIGINAL
Determination of Carbadox and Olaquindox Residues in Chicken
Muscles, Chicken Liver, Bovine Meat, Liver and Milk by MLC
with UV Detection: Application to Baby Formulae
Jenny Jeehan Nasr • Shereen Shalan
Fathalla Belal
•
Received: 29 October 2012 / Revised: 17 January 2013 / Accepted: 21 January 2013 / Published online: 9 February 2013
Ó Springer-Verlag Berlin Heidelberg 2013
Abstract A simple and sensitive method was optimized
and validated for the analysis of carbadox and olaquindox
residues in chicken muscles, chicken liver, bovine meat,
liver and milk. Analytical separation was performed in less
than 4 min using a C18 column with UV detection at 373 nm
and a micellar solution of 0.1 M sodium dodecyl sulphate,
10 % acetonitrile and 0.3 % triethylamine in 0.02 M phosphoric acid buffered at pH 4 as the mobile phase. The
method was fully validated in accordance with ICH guidelines. The micellar method was successfully applied to
quantitatively determine carbadox and olaquindox residues
in spiked chicken muscles, chicken liver, bovine meat, liver
and milk. It was also extended to the determination of
carbadox and olaquindox residues in baby formulae. The
recoveries obtained were in the 89.2–93.6 and 93.0–107.2 %
ranges for carbadox and olaquindox, respectively. High
extraction efficiency for carbadox and olaquindox was
obtained without matrix interference in the extraction process and in the subsequent chromatographic determination.
No organic solvent was used during the pretreatment step.
Keywords Micellar liquid chromatography Carbadox Olaquindox Chicken muscles Liver Milk
Introduction
Carbadox
(methyl-3-(2-quinoalinylmethylene)carbazateN1,N4-dioxide) (CBX) and olaquindox (2-(N-2-hydroxyPresented at: 29th International Symposium on Chromatography,
Torun´, Poland, September 9–13, 2012
J. J. Nasr (&) S. Shalan F. Belal
Department of Analytical Chemistry, Faculty of Pharmacy,
University of Mansoura, Mansoura 35516, Egypt
e-mail: [email protected]
ethylcarbamonyl)-3-methyl-quinoxaline-N 1,N4-dioxide)
(OLQ) are the best known members of quinoxaline-1,4dioxides (Fig. 1), a group of synthetic antibacterial drugs
which are often used as growth promoters as well as to
prevent a number of diseases in animals [1]. Due to health
concerns over possible carcinogenic, mutagenic and photoallergenic effects of the drugs and their metabolites
[2, 3], the use of CBX and OLQ has been banned in Europe
since 1998 [4]. Recently, the health concerns and the
ongoing use of these compounds in some countries have
been stated at the 18th session of the Codex Committee on
Residues of Veterinary Drugs in Foods (2009) [5]. In
Australia, the maximum residue limit (MRL) for OLQ in
pig and poultry meat has been set at 300 lg kg-1 [6]. The
literature reveals several HPLC methods for the determination of CBX and OLQ residues in muscle tissues. CBX
was determined together with its metabolites and OLQ
metabolites in porcine and bovine muscle tissues using
tandem-mass spectrometric detection [7]. It was also
determined with some of its metabolites in swine tissues
using UV–Vis detection [8, 9]. OLQ residues were also
determined in swine tissues using UV-detection [10]. CBX
and OLQ residues were determined together with CBX
metabolites in chicken muscles using a UV-diode array
detector [11]. CBX and OLQ were determined in animal
feeds using UPLC [12], LC–MS-MS [13] and HPLC with
UV-detection [14–16].
To control the compliance of the ban of carbadox and
olquindox, a simple and effective analytical technique for
the monitoring of these drugs in animal feeds is of great
significance. Most protocols dealing with the determination
of carbadox and olaquindox in biological samples involve
liquid–liquid extraction followed by a clean-up step, which
make them time consuming and expensive to perform
when many samples must be analyzed [12].
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J. J. Nasr et al.
Reagents and Materials
Fig. 1 Chemical structures of the drugs investigated
Micellar liquid chromatography (MLC) allows complex
matrices to be analyzed without the aid of extraction and
with direct injection of the samples [17]. Micelles tend to
bind proteins competitively, thereby releasing proteinbound drugs and proteins, rather than precipitating into the
column. Proteins are solubilized and washed harmlessly
away, eluting with the solvent front. This means that costs
and analysis times are considerably reduced [18]. Micellar
mobile phases usually need smaller quantities of organic
modifier and generate smaller amounts of toxic waste in
comparison to aqueous-organic solvents, so that they are
less toxic, non-flammable, biodegradable and relatively
inexpensive [18]. MLC has proved to be a useful technique
in the determination of diverse groups of compounds in
several matrices [19–21], including food samples [22–24].
The aim of this work was to develop a rapid, simple,
sensitive and selective LC method with UV detection for
determining CBX and OLQ residues in chicken muscles,
chicken liver, bovine meat, liver and milk. The proposed
procedure benefits from the two main advantages associated to the use of micellar mobile phases, namely, the use
of environment-friendly eluents and fast and easy sample
preparation. The procedure could also be extended to the
analysis of these residues in baby formulae.
All reagents and solvents used were of HPLC grade.
Carbadox and Olaquindox analytical standards were of 100 %
purity from VetranalÒ, Sigma-Aldrich (Seelze, Germany).
Methanol, 1-propanol, acetonitrile and sodium dodecyl
sulphate (SDS) were from Sigma-Aldrich. Dimethyl sulfoxide, triethylamine and phosphoric acid were from
Riedel-deHae¨n (Seelze, Germany). Nylon filters and syringe filters were from Sartorius-Stedium (Goettingen,
Germany). Chicken muscles, chicken liver, bovine meat
and liver samples were purchased from the local market.
Milk was from JuhaynaÒ (Cairo, Egypt) and Infant Formula Milk was from HeroÒ (Spain).
Preparation of Solutions
Stock solutions of 0.2 mg mL-1 of each CBX and OLQ
were prepared by dissolving CBX in the least amount of
dimethyl sulfoxide then completing to volume with methanol, whereas OLQ was prepared in methanol. Working
solutions were prepared by diluting the stock solutions with
the mobile phase. Stock solutions were found to be stable
for 1 week if kept in the refrigerator protected from light.
Preparation of Calibration Curves
Working solutions containing 0.5–40 lg mL-1 of CBX
and OLQ were prepared by serial dilutions of aliquots of
the stock solutions. Then, 20 lL aliquots were injected
(triplicate) and eluted with the mobile phase under the
reported chromatographic conditions. The average peak
areas of each drug were plotted versus the concentrations
of drug in lg mL-1. Alternatively, the corresponding
regression equations were derived.
Experimental
Analysis of Bulk Substance
Apparatus
The method mentioned under the previous section was
applied to the determination of the purity of CBX and OLQ
raw materials. The percentage recoveries were calculated
by referring to the calibration graphs previously prepared
or by applying the regression equations.
Chromatographic analyses were carried out using a Shimadzu Prominence HPLC system (Shimadzu, Japan) with
a LC-20 AD pump, DGU-20 A5 degasser, CBM-20A
interface, and SPD-20A UV–Vis detector with 20 lL
injection loop. The columns used were Supelco DiscoveryÒ HS C18 column (250 mm 9 4.6 mm i.d., 5 lm particle size; Supelco, Pennsylvania, USA) and Nucleodur
MN-C18 column (150 mm 9 4.6 mm i.d., 5 lm particle
size; Macherey–Nagel, Du¨ren, Germany). Centrifugation
was carried out using a TDL-60B Centrifuge (Anke, Taiwan).
A BHA-180T Sonicator (Abbotta, USA) was used. Tissue
homogenization was made using Tissue Master-125 Homogenizer (Omni International, GA, USA).
123
Samples Preparation
Amounts of 2.5 g of each of the solid samples were
accurately weighed and 5 mL of milk were spiked with
aliquots of CBX and OLQ solutions. The spiked samples
were mixed with 25 mL of 0.1 M SDS solution of pH 4.
The samples (except milk samples) were then homogenized at 5,000 rpm for 5 min, then the homogenate was
sonicated for 15 min, then centrifuged at 3,000 rpm for
Determination of Carbadox and Olaquindox
5 min. Milk samples were only sonicated for 2 min without
centrifugation. The supernatant of all samples was filtered
through 0.45-lm membrane filters using a vacuum pump
and diluted with the mobile phase. Aliquots of 20 lL were
injected (triplicate) and eluted with the mobile phase under
the reported chromatographic conditions.
525
Choice of Column
Two different columns were used for performance investigations, including: Supelco DiscoveryÒ HS C18 column,
and Nucleodur MN-C18 column.
The experimental studies revealed that the first column
was more suitable, since it produced well-resolved peaks
with a very high sensitivity.
Results and Discussion
Mobile Phase Composition
The proposed method permits the quantitation of CBX and
OLQ in raw material, in chicken muscles, chicken liver,
bovine meat, liver, milk and baby formula milk. Figure 2a
shows a chromatogram indicating good resolution of OLQ
(tR = 2.5 min) and CBX (tR = 3.1 min). The proposed
method offers high sensitivity: as low as 0.07 lg mL-1 of
CBX and 0.16 lg mL-1 of OLQ could be detected
accurately.
Optimization of Chromatographic Performance
A well-defined symmetrical peak was obtained upon
measuring the response of eluent under the optimized
conditions after thorough experimental trials that could be
summarized as follows:
Several modifications in the micellar mobile phase composition were performed in order to study the possibilities
of changing the selectivity of the chromatographic system.
These modifications included the change of the surfactant
concentration, the concentration and type of cosurfactant,
the pH, and the flow rate. The results obtained are shown in
Table 1. The mobile phase was prepared using 0.3 % triethylamine and 0.02 M phosphoric acid. Triethylamine
was used to adjust the pH and it has a role in improving
the efficiency [25]. The effect of changing the pH of the
mobile phase on the selectivity and retention time of CBX
and OLQ was investigated using mobile phases of pH
ranging from 3.0 to 6.0 with 0.1 M SDS concentration and
10 % 1-propanol. Table 1 shows that a pH of 4.0 was most
appropriate, giving well-resolved peaks and the highest
Fig. 2 Chromatograms showing: (a) 5 lg mL-1 OLQ, (b) 5 lg mL-1 CBX in: a CBX and OLQ standards, b bovine meat, c chicken liver,
d bovine liver
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J. J. Nasr et al.
Table 1 Optimization of experimental factors affecting the chromatographic performance of the proposed method
Parameter
Number
of theoretical plates
Tailing factor
Resolution
CBX
OLQ
CBX
OLQ
CBX/OLQ
3.0
30,247
21,564
1.18
1.04
1.83
4.0
33,985
25,611
1.17
1.03
1.89
4.5
29,136
20,389
1.18
1.03
1.84
5.0
26,830
21,349
1.19
1.04
1.80
5.5
25,504
18,733
1.20
1.06
1.80
8,931
1.26
1.14
1.72
Optimal chromatographic separation was achieved using
a Supelco DiscoveryÒ HS C18 column. A solution containing 0.1 M sodium dodecyl sulphate and 10 % acetonitrile and 0.3 % triethylamine in 0.02 M phosphoric acid
of pH 4.0 was used as mobile phase with a flow rate of
1 mL min-1. The detector wavelength was 373 nm.
pH
SDS concentration (M)
0.02
27,300
0.05
27,917
13,584
1.25
1.08
1.71
0.075
31,137
23,565
1.26
1.01
1.67
0.1
34,266
24,973
1.19
0.99
1.81
0.12
30,111
21,834
1.19
1.05
1.76
1-Propanol concentration (%)
6
8
27,627
29,919
11,501
21,916
1.25
1.20
1.22
1.12
1.82
1.91
10
33,580
24,702
1.17
0.99
1.79
12
30,958
20,107
1.18
0.98
1.81
Organic modifier nature
Methanol
27,063
20,178
1.24
1.18
1.23
1-Propanol
31,979
23,611
1.17
0.96
1.78
Acetonitrile
37,081
33,599
1.18
0.91
1.89
number of theoretical plates. Values of pH higher than 6.0
resulted in a very low numbers of theoretical plates and low
resolution. The effect of changing the concentration of
surfactant on the selectivity and retention times of CBX and
OLQ was investigated using mobile phases containing SDS
concentrations in the range 0.02–0.12 M and containing
10 % 1-propanol and buffered at pH 4. Table 1 shows that
0.1 M SDS was the best, giving well-resolved peaks and the
highest number of theoretical plates. Retention times
increased when concentration of surfactant decreased. The
effect of changing the concentration of organic modifier on
the selectivity and retention times of CBX and OLQ was
investigated using mobile phases containing concentrations
of 6–12 % 1-propanol and containing 0.1 M SDS and buffered at pH 4. Table 1 shows that 10 % of 1-propanol was
the best, giving well-resolved peaks and the highest number
of theoretical plates. Hence, a small amount of 1-propanol is
added to accelerate and control the elution of the drug. The
effect of changing the type of cosurfactant on the selectivity
and retention times of CBX and OLQ was investigated using
mobile phases containing 10 % methanol, 1-propanol or
acetonitrile. Table 1 shows that 10 % acetonitrile was the
best, giving well-resolved peaks and the highest number of
theoretical plates.
123
Method Validation
Concentration Ranges and Calibration Graphs
Under the above-described experimental conditions, linear
relationships were established by plotting peak areas
against CBX and OLQ concentrations. The concentration
range was found to be 0.5–40 lg mL-1 for each of CBX
and OLQ. Linear regression analysis of the data gave the
following equations:
PCBX ¼ 17; 392 þ 12; 158 CCBX
ðr ¼ 0:9999Þ
POLQ ¼ 10; 775 þ 47; 495 COLQ
ðr ¼ 0:9999Þ
where C is the concentration of drug in lg mL-1 and P is
the peak area.
The high values of the correlation coefficient (r values
[0.999) indicate good linearity of the calibration graphs.
Statistical analysis of the data gave small values of the %
relative error (% Er) 0.7 % for both CBX and OLQ,
respectively [26].
Limit of Quantitation (LOQ) and Limit of Detection
(LOD)
The limit of quantitation (LOQ) was determined by
establishing the lowest concentration that can be measured
according to ICH Q2B recommendations [27] below which
the calibration graph is non-linear and was found to be 2.5
and 5.2 lg g-1 for CBX and OLQ, respectively. The limit
of detection (LOD) was determined by establishing the
minimum level at which the analyte can be reliably
detected (S/N = 3); it was found to be 0.7 lg g-1
(2. 7 9 10-7 M) and 1.6 lg g-1 (6.1 9 10-7 M) for CBX
and OLQ, respectively.
Accuracy and Precision
The proposed method was evaluated by studying the accuracy
as % Er and precision as percent relative standard deviation
(% RSD) using three preparations with suitable concentrations, as shown in Table 2. The intra-day (n = 3) and interday (n = 3) accuracy, calculated as % Er, was found to be
within 0.1 and 0.2 % for CBX, respectively and 0.1–0.2 and
0.2–0.3 % for OLQ, respectively. The repeatability of the
Determination of Carbadox and Olaquindox
Table 2 Accuracy and
precision data for carbadox and
olaquindox using the proposed
method
527
Analyte
Concentration
(lg mL-1)
Carbadox
Intra-daya
Inter-dayb
Recovery
(mean ± SD)
RSD
(%)
Er
(%)
Recovery
(mean ± SD)
RSD
(%)
Er
(%)
5.0
100.4 ± 0.2
0.2
0.1
100.6 ± 0.4
0.4
0.2
10.0
100.6 ± 0.2
0.2
0.1
100.8 ± 0.4
0.4
0.2
20.0
100.0 ± 0.2
0.2
0.1
100.1 ± 0.4
0.4
0.2
5.0
100.9 ± 0.3
0.3
0.2
100.9 ± 0.6
0.6
0.3
Intra-day: within the day
10.0
100.6 ± 0.3
0.3
0.2
100.9 ± 0.3
0.3
0.2
Inter-day: three consecutive
days
20.0
100.3 ± 0.2
0.2
0.1
99.8 ± 0.3
0.3
0.2
Each result is the average of
three separate determinations
a
Olaquindox
b
assay (intra-day) was found to be within 0.2 and 0.2–0.3
(n = 3) at 5, 10, and 20 lg ml-1 for CBX and OLQ,
respectively. The reproducibility of the assay (inter-day) at
the same concentration levels was found to be within 0.4 and
0.3–0.6 (n = 3) for CBX and OLQ, respectively.
The results of the proposed method were favorably compared with those obtained using the reference method [14].
The reference method depends on the RP-HPLC determination of CBX and OLQ with UV detection using a C18 column and a mobile phase composed of acetonitrile:water
containing 25 mmol L-1 ammonium acetate (10:90 v/v)
using gradient elution at a flow rate of 1 mL min-1. Detection was carried out at 373 nm using solid-phase extraction
for sample clean-up [14]. Statistical analysis of the results
obtained by the proposed and reference methods showed no
significant difference in the performance of the two methods
using Student’s t test and the variance ratio F test. The proposed procedure offers additional advantages over the reference procedure in that the proposed is more sensitive with
good accuracy and precision.
Applications
The applicability of the procedure developed here to
determine CBX and OLQ was tested by analyzing it in
spiked chicken muscles, liver, bovine meat, liver and milk
in addition to spiked baby formula milk. All samples were
bought at a local supermarket. Table 3 shows the results of
the analysis of CBX and OLQ determined in all samples
after homogenization with micellar solution, sonication,
centrifugation and filtration. Samples were spiked at the
following concentration levels: 5, 10 and 15 ppm. Three
replicates of each concentration were injected into the
chromatograph. The data obtained (Table 3) show satisfactory recoveries for CBX and OLQ in all samples, and
the results fall in the range of 89.2–93.6 and 93.0–107.2 %
for CBX and OLQ, respectively.
Figure 2 depicts the chromatograms obtained from different spiked samples of CBX and OLQ analyzed with the
optimum mobile phase. These chromatograms reveal how a
surveillance programme for CBX and OLQ residues can be
Table 3 Assay of carbadox and olaquindox in food samples using the proposed and reference methods
Method
CBX
OLQ
Prop.
Ref.
CBX
Prop.
Ref.
OLQ
Prop.
Ref.
CBX
Prop.
Ref.
Chicken liver
OLQ
Prop.
Ref.
Prop.
Ref.
91.2
Sample type
Chicken muscles
Bovine meat
a
Mean (X)
89.4
88.6
107.2
105.5
90.8
89.8
93.8
91.9
89.9
90. 6
93.0
±SD
2.2
2.4
2.1
2.5
1.8
2.6
1.3
2.5
1.5
2.9
2.9
4.0
Variance
4.8
5. 6
4.4
6.4
3.3
6.6
1.8
6.5
2.3
8.4
8.2
16.3
Student’s t valueb
0.42
0.93
0.55
1.15
0.32
0.62
Variance ratio F valueb
1.15
1.46
2.00
3.65
3.65
1.99
Sample type
Bovine liver
a
Mean (X)
Milk
Baby formula milk
89.2
91.6
95.9
94.0
89.3
87.7
95.9
89.3
93.6
91.3
±SD
1.4
3.2
1.3
1.9
1.7
2.1
2.9
1.7
1.4
2.6
2.8
3.0
Variance
2.1
10.4
1.7
3.7
2.9
4.5
8.7
9.1
1.9
6.5
7.7
8.8
Student’s t valueb
Variance ratio F valueb
1.21
4.90
1.41
2.23
0.99
1.55
a
Number of experiments = 3
b
Tabulated t and F values at p = 0.05 are 2.78 and 19.00, respectively
0.22
1.05
1.35
3.50
102. 9
101.8
0.46
1.14
123
528
performed under the proposed chromatographic conditions.
The low detection limits of the proposed method are useful
for the determination of any traces of CBX and OLQ residues that are prohibited in baby formula milk.
Conclusion
The proposed procedure is useful for food quality testing
and control areas to determine the content of CBX and
OLQ in chicken muscles, liver, bovine meat, liver and milk
samples. Moreover, it allows the detection of traces of
CBX and OLQ residues in baby formula milk with high
sensitivity. One advantage of this procedure is the possibility of injecting the samples directly into the chromatographic system with no previous treatment other than
homogenization, dilution and filtration, thus avoiding
tedious extractions from matrices. Validation according to
ICH regulation provides satisfactory results in terms of
sensitivity, linearity, accuracy and recoveries and at the
ppm level. It is noteworthy that the use of micellar mobile
phases endows the procedure advantages such as nontoxicity, non-flammability, biodegradability, and low cost.
Acknowledgments This work was supported by L’Ore´al-UNESCO
Pan Arab 2010 Fellowship ‘For Women in Science’, in partnership
with the Arab Science and Technology Foundation (ASTF).
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