Food Chemistry 176 (2015) 219–225
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Analytical Methods
Determination of enrofloxacin and ciprofloxacin in foods of animal origin by capillary electrophoresis with field amplified sample stacking–sweeping technique Xiaoying Xu, Lihong Liu ⇑, Zhimin Jia, Yang Shu School of Pharmaceutical Sciences, Southern Medical University, 1838 North Guangzhou Avenue, Guangzhou 510515, China
a r t i c l e
i n f o
Article history: Received 31 July 2014 Received in revised form 7 December 2014 Accepted 13 December 2014 Available online 23 December 2014 Keywords: Enrofloxacin Ciprofloxacin c-CD Field-amplified sample stacking Sweeping Animal origin food
a b s t r a c t A simple on-line preconcentration method combining field-amplified sample stacking (FASS) with sweeping was developed and validated for the determination of enrofloxacin (ENRO) and ciprofloxacin (CIP) in multiple foods. To improve the efficiency of sweeping, gamma-cyclodextran (c-CD) was introduced to the sample matrix. The optimal experimental conditions were as follows: running buffer (pH 8.8), (a) 20 mM Tris and 80 mM NaDC with 300 s sample injection time, (b) 20 mM Tris and 120 mM NaDC with 240 s sample injection time; sample matrix, 300 mM Tris and 5 mM c-CD at pH 10.0; applied voltage, 15 kV; detection wavelength, 270 nm. With buffer condition (a), the limits of detection (LODs) for ENRO and CIP were 1.87 and 2.21 ng/mL. The sensitivity was improved 376 and 406-fold for ENRO and CIP compared to conventional capillary electrophoresis (CE) method, respectively. The buffer with condition (b) has been successfully applied to the analysis of six kinds of animal foodstuffs by capillary electrophoresis with UV detector. The recoveries (85–102%) were achieved with relative standard deviations of 0.1–4.4%. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Fluoroquinolones (FQs) are antibacterial agents related to nalidixic acid. They are used to treat a variety of infections in both human and veterinary medicine. Their therapeutic actions are based on inhibition of DNA gyrase in Gram-negative species and of topoisomerase IV in Gram-positive species (Drlica, 1999). Among them, ENRO (Fig. 1) was developed for exclusive use in animals. In many countries, ENRO is approved for the treatment of some infectious diseases in cattle, swine, chickens, dogs and cats (Giguère, Prescott, & Dowling, 2013, Chap. 17). CIP (Fig. 1) is a major, active metabolite of ENRO in different species and is formed by the de-ethylation of ENRO. CIP is one of the most widely used clinical antibiotics in the world. However, once antibiotic treatments are applied to cure livestock diseases, undesirable antibiotic residues will be remained in animal tissues and biofluids, which badly endanger the health of humans via the food chain (Blasco, Torres, & Picó, 2007). To protect consumers’ health, the European Agency for the Evaluation of Medicinal Products in Europe, the Food and Drug Administration in United States, and the Chinese Ministry of Agriculture have set maximum residue limits (MRLs) for monitoring the levels of FQs ⇑ Corresponding author. Tel.: +86 20 61648595. E-mail address: [email protected] (L. Liu). http://dx.doi.org/10.1016/j.foodchem.2014.12.054 0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.
in animal-originated foodstuffs. ENRO and CIP are allowed at concentrations below their MRLs, which fall in the range of 100– 500 lg/kg for the sum of ENRO and CIP in different tissues (EMEA/ MRL/574/99-FINAL, 1999; U.S. Food and Drug Administration, 2012). Given this, effective analytical methodologies are essential to determine ENRO and CIP in foods of animal origin. Hitherto, most analytical methodologies for ENRO and CIP fell into two main categories: screening and confirmatory. Screening methods include immunoassays (Hu, Huang, Jiang, Fang, & Yang, 2010; Zhu et al., 2008), microbiological tests (Ashwin et al., 2009), biosensors (Huang et al., 2013) and spectrometry (Chen & Li, 2013). These methods can detect an analyte or a family at the level of interest, but they usually only provide qualitative or semi-quantitative results (Cháfer-Pericás, Maquieira, & Puchades, 2010). On the contrary, confirmatory methods provide full or complementary information, enabling unequivocal identification and quantification of the analytes at levels of interest (Blasco et al., 2007). In most studies, high performance liquid chromatography (HPLC) is chosen for analyses of ENRO and CIP in foodstuffs samples (Cinquina et al., 2003; González, Moreno, Small, Jones, & Bruni, 2006). However, expensive chromatographic columns are used and a large amount of reagent is consumed in the HPLC process. Compared to HPLC, CE needs minimal sample and solvent consumption, and inexpensive capillary columns are used (Lombardo-Agui, Garcia-Campana, Gamiz-Gracia, & Blanco, 2010;
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Pinero, Garrido-Delgado, Bauza, Arce, & Valcarcel, 2012; Zhou, Xing, Zhu, Tang, & Jia, 2008). Unfortunately, most CE methods require a complex extraction and cleanup procedure. This additional step is tedious and slows down the analysis process. What’s more, it suffers from poor concentration sensitivity when on-line UV detection is utilized, due to the small loaded sample volume (nL) and the narrow optical path length (as defined by the diameter of the capillary). In response to the sensitivity problem, several on-line preconcentration methods (Wen, Li, Ma, & Chen, 2012) have been developed to preconcentrate analytes inside the capillary before separation and detection. This can be done by injecting a large volume of sample solution and focusing the analyte into a narrow band inside the capillary. Sweeping utilizes the phenomenon that hydrophobic analytes tend to be incorporated into the micelle (Quirino, 1998), and is used substantially for pharmaceutical and herbal products applications. Field-amplified sample stacking (FASS), normally capable of 100-fold sensitivity enhancement for charged analytes, is widely used in food and food contaminant analysis. The technique is based on the use of low-conductivity sample matrix and high-conductivity background solution (BGS) (Lagarrigue et al., 2008). FASS–sweeping technique has been used for determination of varied compounds in micellar electrokinetic chromatography (Dziomba, Kowalski, & Baczek, 2012). The goal of this study is to propose a highly sensitive on-line concentration method for the determination of ENRO and CIP in animal-originated samples. Based on the method developed by Dziomba et al. (2012), we improved the sensitivity of the sweeping technique by adding c-CD to sample matrix. That was because c-CD could increase the affinity between the analytes and the pseudostationary phase of BGS which led to remarkably enhance the determination sensitivities of ENRO and CIP. 2. Materials and methods 2.1. Chemicals and reagents ENRO was obtained from Dr. Ehrenstorfer GmbH (Augsburg, Germany). CIP was obtained from National Institute for Food and Drug Control (Beijing, China). The standards were used as received. Milk, milk powder, chicken (chicken muscle), pork (swine muscle), swine liver and swine kidney were purchased from a local supermarket. a-CD, b-CD and c-CD were from Duly Biotech Co., Ltd. (Nanjing, China). Sodium deoxycholate (NaDC) was from Acros Organics (Brussels, Belgium). Tris(hydroxymethyl)aminomethane (Tris) was from Kehao Bioengineering Co., Ltd. (Xian, China). Acetonitrile and methylene chloride were from Tianjin Fuyu Fine Chemical Co., Ltd. (Tianjin, China). All chemicals were of analytical reagent grade and were used as received. All solutions were made in distilled water. 2.2. Apparatus All CE experiments were performed on a CL1030 high-performance CE apparatus with a UV detector (Beijing Cailu Instrumental Co., Beijing, China). Uncoated fused-silica capillaries purchased
Fig. 1. The structure of enrofloxacin and ciprofloxacin.
from Yongnian Optical Fiber Factory (Hebei, China) were used. The dimensions of the capillary were 65 cm 75 lm I.D. 375 lm O.D. The effective length of the capillary was 57 cm from the injection end of the capillary. The data acquisition was carried out with an HW-2000 Chromatography Workstation (Shanghai Qianpu Software Company, Shanghai, China). A PH-3C acidity meter (Shanghai Hongyi Instrument Co., Ltd., Shanghai, China) was used for the pH measurement. UV detection was carried out at 270 nm. Samples were introduced into the capillary by gravity, where the sample vial was raised by 15.5 cm. Polyvinylidene fluoride (PVDF) syringe filters (Shanghai Peninsula Instrumental Co. Purification Equipment Factory, Shanghai, China) were used for filtering solutions. The new capillary was flushed with 1 M NaOH for 1 h and then with distilled water for 10 min before use. At the beginning of each working day, the capillary was flushed sequentially with distilled water (5 min), 100 mM NaOH (5 min), distilled water (5 min), and the running BGS (5 min). Simultaneously the CE instrument was warmed up until a stable baseline was achieved. Between consecutive analyses, the capillary was rinsed with distilled water (2 min), 100 mM NaOH (2 min), distilled water (2 min), and the running BGS (3 min). The capillary was left filled with distilled water overnight. 2.3. Solution preparation Standard stock solutions (1 mg/mL) of ENRO and CIP were prepared in methanol. Work solutions were prepared at various concentrations by appropriate dilution of the stock solutions with standard sample matrix. The standard sample matrix contained 300 mM Tris and 5 mM c-CD. The composition of running BGS was as follows: a. 20 mM Tris and 80 mM NaDC at pH 8.8; b. 20 mM Tris and 120 mM NaDC at pH 8.8. The running BGS was prepared daily from stock solutions of 100 mM Tris and 200 mM NaDC. All solutions were filtered through 0.45 lm syringe filters before use. 2.4. Sample preparation The method has been applied to six kinds of animal origin foods: milk, milk powder, pork, chicken, swine liver and swine kidney. 2.4.1. Preparation of milk samples An aliquot (1.0 g) of milk, spiked at different concentration levels using the standard stock solutions, was placed in a 15 mL glass centrifuge tube. After shaking the mixture briefly (about 30 s) on a vortex-mixer, 2 mL of acetonitrile was added. The mixture was vortexed for about 30 s at high speed and then centrifuged for 10 min at 4000 rpm (2840g). The supernatant was decanted into another 15 mL glass centrifuge tube, and 1 mL of 50 mM phosphate buffer (pH 7.4) and 3 mL of methylene chloride was added. The mixture was vortexed for about 30 s at high speed and then centrifuged for 10 min at 4000 rpm. The upper, aqueous layer was transferred into a waste bottle. The nonaqueous layer was evaporated under vacuum in a rotary evaporator at room temperature. The residue was reconstituted in 1.0 mL of standard sample matrix and filtered through a 0.45 lm membrane filter. 2.4.2. Preparation of other samples 0.5 g of milk powder or minced chicken and swine tissues was accurately weighed and placed in a 15 mL glass centrifuge tube, then spiked at different concentration levels using the standard stock solutions. After shaking the mixture briefly (about 30 s) on a vortex-mixer, 2 mL of acetonitrile was added. The mixture was
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vortexed for about 30 s at high speed and extracted in an ultrasonic bath (250 W, 28 °C, Yuhua Instrument Co., Ltd., Henan, China) for 30 min, and then centrifuged for 10 min at 4000 rpm (2840g). The supernatant was decanted into another 15 mL glass centrifuge tube, and 1 mL of 50 mM phosphate buffer (pH 7.0) and 3 mL of methylene chloride was added. The mixture was vortexed for about 30 s at high speed and then centrifuged for 10 min at 4000 rpm. The upper, aqueous layer was transferred into a waste bottle. The nonaqueous layer was evaporated under vacuum in a rotary evaporator at room temperature. The residue was reconstituted in 0.5 mL of standard sample matrix and filtered through a 0.45 lm membrane filter. 2.5. Calculation of LOD and sensitivity enhancement factor (SEF) The LOD was calculated as the peak height at a signal-to-noise ratio of 3 (S/N = 3). The SEF were calculated by comparing the LOD to that obtained with the conventional method (using 5 s of hydrodynamic injection with the BGS as the sample matrix). 3. Results and discussion 3.1. FASS–sweeping mechanism FASS is a concentration technique based on a mismatch between the electric conductivity of the sample and that of the running buffer (Zhang & Thormann, 1996). If the sample conductivity is lower than that of the BGS, the higher electric field results in a higher velocity of the analytes in the sample plug than that in the BGS. And this produces a sharpening of the analytes zone at the boundary with the BGS. Sweeping utilizes the interactions between the analytes and a pseudostationary phase or a complexing agent that is present only in the BGS (Quirino, 1998). In this paper, the NaDC, a kind of anionic surfactant is added to the BGS as a pseudostationary phase for sweeping, and provides a higher conductivity of the BGS. ENRO and CIP, with pI values of 6.33 and 7.37, respectively, are negatively charged at pH 8.8, which weakens the analytes affinity to NaDC because two like electric charges repel each other. To solve this problem, c-CD is introduced to the sample matrix. It is well known that cyclodextrans (CDs) can form water soluble inclusion complexes with many hydrophobic drugs (Abe, Ogawa, Nagase, Endo, & Ueda, 2010). It goes without saying that they can interact with ENRO and CIP. The formation of inclusion complexes increases the hydrophobicity of the analytes and reduces the effect of charges, thus enhances the sweeping efficiency. Schematic mechanism of separation is shown in Fig. 2. The process is as follows. At first, the capillary is filled with BGS, and a long sample plug is injected by gravity into the capillary (Fig. 2A). Then a high voltage is applied. The analytes migrate rapidly toward the anode and stack on the boundary between BGS and sample zones (Fig. 2B). Meanwhile, BGS containing micelles is introduced into the capillary from the inlet end due to the presence of electroosmotic flow (EOF). Because the electrophoretic mobility of charged micelles at the front boundary of sample plug was opposite to and lower than that of EOF. Therefore, the micelles traverse the sample plug and sweep the analytes at the behind boundary of the sample plug (Fang et al., 2014) (Fig. 2C). Subsequently, ENRO and CIP were separated according to the micellar electrokinetic capillary chromatography (MEKC) mode (Fig. 2D). 3.2. Optimization of the FASS–sweeping experimental conditions 3.2.1. Effect of Tris concentration In CE analysis, the selectivity was adjusted by changing the pH and BGS concentration. Therefore, in our studies, the effect of Tris
Fig. 2. Schematic illustration of the FASS–sweeping procedure.
concentration was observed in a range from 5 to 60 mM. Fig. 3A showed that the peak heights of the two analytes, as well as the resolution between them, reached the largest values with a concentration of 20 mM. This could be explained by the fact that the increase in Tris concentration could reduce the absorption of the analytes to the capillary wall, which slowed down the EOF. A slow EOF was helpful to the sweeping process. At the same time, the ionic strength of the BGS increased, which resulted in an enhancement in the conductance of the BGS. If the specific conductance of the BGS was too high, the Joule heat production would increase, and high system temperatures led to extra peak broadening and a decrease in resolution (Beckers & Bocˇek, 2003). Therefore, in consideration of the resolution, peak shapes, and peak heights of the analytes, the optimal concentration of Tris was chosen as 20 mM. 3.2.2. Effect of sample matrix and pH value FQs existed as cations in acidic pH, as zwitterions in neutral pH, or as anions in basic pH (Chen & Li, 2013). The influence of different pH values in sample matrix on signals intensities had also been investigated. The subject preliminary research showed that an alkaline sample matrix was beneficial to the FASS. Therefore, several sample matrixes differing in pH values (in the range from 9.1 to 11.0) were tested. As shown in Fig. 3B, the most significant changes in signals intensities of the analytes occurred when the pH value of sample matrix was 10.0. The reason was as follows: NaOH was added to increase pH and the ionic strength of the sample resulting in improving matching the conductivity ratio between the BGS and the sample with pH increasing from 9.1 to 10.0. The efficiency of FASS increasing was observed. However, with further pH increasing from 10.0 to 11.0, the mismatch between the local electroosmotic velocities in the sample and in the BGE generated laminar flows and inducing the broadening of the stacked zone (Burgi & Chien, 1991). For FASS, the concentrating effect basically relied on the change in electrophoretic velocity when the analyte molecules reach the interface between the high-resistance sample solution or water zone and low-resistance background solution zone. The higher difference in resistance or conductance, the greater concentrating effect was obtained (Dziomba et al., 2012). Tris solution with low conductance was chosen as the sample matrix. Our further study showed that the best result was obtained by using 300 mM Tris, which could maintain the pH of sample matrix at 10.0 without other pH regulators.
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Fig. 3. Effect of the FASS–sweeping experimental conditions on the peak heights and the resolution of the peaks. (A) Tris in BGS; (B) sample pH; (C) c-CD; (D) applied voltage; (E) NaDC; (F) injection time (80 mM NaDC). s Resolution of ENRO and CIP; d resolution of ENRO and the unknown substance; h ENRO; 4 CIP.
3.2.3. Effect of c-CD CDs can generate inclusion complexes with a wide variety of hydrophobic organic compounds in aqueous solution. The CDs, which serve as media enhancing the interaction between NaDC and the analytes, could greatly increase the sample injection volume. And it didn’t result in deterioration of the peak. To observe the effect of different kinds of CDs, a-, b- and c-CD were used. Under the same conditions, a lower sensitivity was obtained by applying a-CD compared to applying b-CD or c-CD. The corresponding LODs were 13.7, 12.3, 12.2 ng/mL for ENRO and 15.1,
12.9, 13.0 ng/mL for CIP. Although the addition of b-CD presented a good sensitivity as high as the addition of c-CD, poorer separation efficiency was also observed. c-CD is composed of eight glucose units and forms the shape of a hollow truncated cone with a hydrophylic exterior and hydrophobic interior. Due to the larger number of glucose units, c-CD is more favored than a- and b-CD. Therefore, c-CD was considered optimal. Different concentrations of c-CD in sample matrix (ranging from 0 to 10 mM) were tested. As shown in Fig. 3C, a continuous increase in peak heights with increasing c-CD concentration was
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observed. On the contrary, resolution between the ENRO and the unknown peak decreased through all the tested range. Resolution between the ENRO and CIP increased before decreasing. Taking peak heights and resolution into account, a concentration of 5 mM was chosen. 3.2.4. Effect of applied voltage The effect of the applied voltage on concentration efficiency was investigated in the range from 12 to 24 kV. As shown in Fig. 3D, peak widths progressively became narrower as the applied voltage decreased until 15 kV. For sweeping, analytes with a high affinity for the micellar carrier (i.e., very hydrophobic) spend a greater amount of time complexed with the micelles and hence have a velocity lower than EOF, while those with less affinity (i.e., less hydrophobic) have velocity more dependent on EOF. Most analytes fall between these extremes, with their velocity a function of affinity for the micelles, micelle velocity, and EOF velocity (Palmer, Munro, & Landers, 1999). Sweeping was also found to work better under suppressed or low EOF compared to high EOF conditions (Quirino & Terabe, 1999). Decreasing voltage can slow down the EOF. As a result, high sweeping efficiency was obtained. At lower applied voltage, the peaks broadened gradually and the resolution decreased. Thus, 15 kV was selected as the optimal applied voltage. 3.2.5. Effect of NaDC To determine the impact of the amounts of surfactant on the sweeping efficiency, different concentrations (40–140 mM) of NaDC in the BGS were tested. The concentration of NaDC had a dual effect on the analytes electrophoresis. First, it affected the sweeping of analytes which manifested itself in peaks height and separation efficiency. Too low concentration of NaDC (40 mM) in the BGS not only caused an overlap of ENRO and an unknown peak, but also resulted in a poor enrichment of CIP. As shown in Fig. 3E, the resolution of ENRO and the unknown peak and that of ENRO and CIP were improved with increasing NaDC concentration from 60 to 140 mM. Meanwhile, the peak heights of the two analytes decreased for peaks broadening. The level of NaDC also had a significant effect on the baseline noise. Too high concentration of NaDC (140 mM) in the BGS led to an awful baseline. 3.2.6. Effect of injection time Injection time had a great influence on signal amplification. In our study, different injection times varying from 150 to 330 s were evaluated. The result showed in Fig. 3F. Both the peak heights and areas of the analytes obviously increased without a deterioration of peak shapes with increasing injection time from 150 to 300 s. These could be explained by the fact that the on-line preconcentration technique decreased the width of the injected sample band, so that there is neither breakdown in resolution nor efficiency despite the larger-than-usual injection volumes. However, with a longer injection time, remarkable broadening peaks were observed, which led to the decrease of separation efficiency and a failure to achieve baseline separation. A 300 s injection time was suitable for the standard. However, it wasn’t nice to apply to the real samples. The optimization of injection time for real samples was given in Sections 3.3 and 3.4.
Table 1 LOD, RSD of peak area, regression equation, correlation coefficient (r), linear range, SEF for CE system with FASS–sweeping method (n = 6). Validation parameters
Regression equationa a b r LOD (S/N = 3) (ng/mL) Linear range (ng/mL) SEF %RSD (intra-day) %RSD (inter-day) a
80 mM NaDC CIP
ENRO
CIP
70.768 383.96 0.9998 1.87 10–500 376 1.46 2.46
104.51 147.24 0.9999 2.21 10–500 406 1.79 2.95
61.876 260.95 0.9994 5.70 10–500 124 1.99 2.44
66.119 263.3 0.9997 7.39 10–500 122 2.40 2.14
y = ax + b; y, peak area; x, standard concentration (ng/mL).
analysis. Further study showed that injection time of 240 s was more suitable for highly complex matrices analysis. According to these facts, validation parameters have been designated and applied in this paper for both methods (80 mM with 300 s and 120 mM with 240 s). Under the optimal conditions, the ENRO and CIP analysis by CE-UV using the FASS–sweeping technique was validated in terms of its linearity, repeatability, and LOD. The results were summarized in Table 1. Standard calibration curves were prepared by the injection of mixed-standard solutions at seven concentration
Table 2 Results for determination of the two components in real samples (n = 4). Sample
Ingredient Content (ng/g)
Spiked (ng/mL)
Found (ng/mL)
Recovery (%)
RSD (%)
Milk
ENRO
19.08
CIP
–a
50 100 150 50 100 150
40.8 88.9 125.5 46.3 96.5 137.1
82 89 84 93 96 91
1.0 2.1 2.7 0.1 3.1 1.6
ENRO
13.25
CIP
–
50 100 150 50 100 150
47.0 90.5 125.9 47.5 92.6 145.8
94 91 84 95 93 97
2.0 2.7 0.6 2.1 2.1 2.9
ENRO
29.90
CIP
–
50 100 150 50 100 150
43.4 87.3 121.3 52.6 107.9 139.7
87 87 81 105 108 93
1.8 4.1 0.1 1.8 1.2 3.3
ENRO
34.97
CIP
–
50 100 150 50 100 150
47.7 93.6 134.7 45.3 95.7 146.7
95 94 90 91 96 98
2.8 1.9 2.7 3.8 0.8 3.8
ENRO
–
CIP
17.37
150 200 250 150 200 250
141.1 177.2 241.3 134.7 175.1 235.8
94 89 97 90 88 94
4.4 1.2 2.5 3.4 3.4 2.5
ENRO
12.53
CIP
11.35
250 300 350 250 300 350
238.3 276.3 327.4 241.1 287.7 330.1
95 92 94 96 96 94
3.1 1.6 2.2 4.3 1.2 0.8
Milk powder
Chicken
Pork
Swine liver
3.3. Method validation Investigation of NaDC concentration has shown that the optimal concentration could be 80 mM, which provides about 1.5- to 2-fold better result in comparison with that of 120 mM. However, when it was applied to the real sample, a baseline separation between the tested compounds and unknown substances wasn’t achieved for the lower separation efficiency. As a compromise for selectivity and sensitivity, 120 mM was chosen for real sample
120 mM NaDC
ENRO
Swine kidney
a The substance was not found, or its content below the linearity range of the method.
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same) were presented for the analysis of ENRO and CIP. The methods had the advantages of simplicity, good precision and accuracy. High sensitivity was obtained with this preconcentration method. The LODs were lower than the MRLs of the tested compounds in animal origin foods. Furthermore, the method used simple reagents, with minimum sample preparation procedures, encouraging its application in routine analysis. This preconcentration method (120 mM NaDC with 240 s injection time) was applied to analyzing six kinds of animal foodstuffs. The proposed method was easy to carry out, and suited for assaying complex samples such as tissues, milk etc. intended for human consumption. Conflicts of interest The authors declare no competing financial interests. Fig. 4. Electrochromatograms of ENRO and CIP extracted from pork. (A) 300 ng/mL mixed standard; (B) blank; (C) spiked 150 ng/mL. Peaks: 1 = ENRO, 2 = CIP.
Acknowledgements levels (10, 50, 100, 200, 300, 400 and 500 ng/mL). The repeatability of the FASS–sweeping method was determined by repeated (n = 6) injections of the standard mixture solutions at concentrations of 300 ng/mL for all analytes. The peak areas were employed for quantification. Good linearity (r > 0.999) and repeatability (the %RSD values of peak area were 1.46–2.40% and 2.14–2.95% for intra-day and inter-day, respectively) were obtained for the analytes. The LODs obtained using the 120 mM NaDC with 240 s injection time method were 5.70 and 7.39 ng/mL for ENRO and CIP, respectively. Lower LODs were achieved (1.87 and 2.21 ng/mL for ENRO and CIP, respectively) by using the 80 mM NaDC with 300 s injection time method. 3.4. Application Investigation of NaDC concentration and injection time had shown that a higher sensitivity was obtained by using 80 mM NaDC with 300 s injection time method. Unfortunately, the lower separation efficiency hampered its application in complex sample analysis. Animal origin food samples are very complex mixtures of organic and inorganic compounds. Therefore, the 120 mM NaDC with 240 s injection time method was chosen for the animal foodstuffs analysis. The practical applicability of the system was demonstrated by separating ENRO and CIP and determining their respective contents in milk, milk powder, pork, chicken, swine liver and swine kidney. Actual sample matrices are complex and varying viscosity with standard solutions. The electrophoretic mobility of the analytes can be affected by solution conditions such as pH, ionic strength and viscosity (Paull & King, 2003). So, the analytes peak position shifts in actual sample occur. And there are many unknown peaks appearing on the electrophoretogram. These factors lead to difficult to identify peaks of ENRO and CIP according to migration time in real sample. Therefore, the peaks were identified using the standard addition methods. The analyte contents found in the different samples are given in Table 2. The electropherograms of standard sample solution and the pork samples detected was shown in Fig. 4. The accuracy of the methods and the potential matrix effects were established by analyzing spiked samples. The adaptability of the method used was shown by the acceptable recoveries of 85–102%. 4. Conclusions An on-line preconcentration method using FASS–sweeping was developed in this paper. Two kinds of conditions ((a) 80 mM NaDC with 300 s injection time for standards; (b) 120 mM NaDC with 300 s injection time for real samples. Other conditions were the
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Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Analytical Methods
Determination of enrofloxacin and ciprofloxacin in foods of animal origin by capillary electrophoresis with field amplified sample stacking–sweeping technique Xiaoying Xu, Lihong Liu ⇑, Zhimin Jia, Yang Shu School of Pharmaceutical Sciences, Southern Medical University, 1838 North Guangzhou Avenue, Guangzhou 510515, China
a r t i c l e
i n f o
Article history: Received 31 July 2014 Received in revised form 7 December 2014 Accepted 13 December 2014 Available online 23 December 2014 Keywords: Enrofloxacin Ciprofloxacin c-CD Field-amplified sample stacking Sweeping Animal origin food
a b s t r a c t A simple on-line preconcentration method combining field-amplified sample stacking (FASS) with sweeping was developed and validated for the determination of enrofloxacin (ENRO) and ciprofloxacin (CIP) in multiple foods. To improve the efficiency of sweeping, gamma-cyclodextran (c-CD) was introduced to the sample matrix. The optimal experimental conditions were as follows: running buffer (pH 8.8), (a) 20 mM Tris and 80 mM NaDC with 300 s sample injection time, (b) 20 mM Tris and 120 mM NaDC with 240 s sample injection time; sample matrix, 300 mM Tris and 5 mM c-CD at pH 10.0; applied voltage, 15 kV; detection wavelength, 270 nm. With buffer condition (a), the limits of detection (LODs) for ENRO and CIP were 1.87 and 2.21 ng/mL. The sensitivity was improved 376 and 406-fold for ENRO and CIP compared to conventional capillary electrophoresis (CE) method, respectively. The buffer with condition (b) has been successfully applied to the analysis of six kinds of animal foodstuffs by capillary electrophoresis with UV detector. The recoveries (85–102%) were achieved with relative standard deviations of 0.1–4.4%. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Fluoroquinolones (FQs) are antibacterial agents related to nalidixic acid. They are used to treat a variety of infections in both human and veterinary medicine. Their therapeutic actions are based on inhibition of DNA gyrase in Gram-negative species and of topoisomerase IV in Gram-positive species (Drlica, 1999). Among them, ENRO (Fig. 1) was developed for exclusive use in animals. In many countries, ENRO is approved for the treatment of some infectious diseases in cattle, swine, chickens, dogs and cats (Giguère, Prescott, & Dowling, 2013, Chap. 17). CIP (Fig. 1) is a major, active metabolite of ENRO in different species and is formed by the de-ethylation of ENRO. CIP is one of the most widely used clinical antibiotics in the world. However, once antibiotic treatments are applied to cure livestock diseases, undesirable antibiotic residues will be remained in animal tissues and biofluids, which badly endanger the health of humans via the food chain (Blasco, Torres, & Picó, 2007). To protect consumers’ health, the European Agency for the Evaluation of Medicinal Products in Europe, the Food and Drug Administration in United States, and the Chinese Ministry of Agriculture have set maximum residue limits (MRLs) for monitoring the levels of FQs ⇑ Corresponding author. Tel.: +86 20 61648595. E-mail address: [email protected] (L. Liu). http://dx.doi.org/10.1016/j.foodchem.2014.12.054 0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.
in animal-originated foodstuffs. ENRO and CIP are allowed at concentrations below their MRLs, which fall in the range of 100– 500 lg/kg for the sum of ENRO and CIP in different tissues (EMEA/ MRL/574/99-FINAL, 1999; U.S. Food and Drug Administration, 2012). Given this, effective analytical methodologies are essential to determine ENRO and CIP in foods of animal origin. Hitherto, most analytical methodologies for ENRO and CIP fell into two main categories: screening and confirmatory. Screening methods include immunoassays (Hu, Huang, Jiang, Fang, & Yang, 2010; Zhu et al., 2008), microbiological tests (Ashwin et al., 2009), biosensors (Huang et al., 2013) and spectrometry (Chen & Li, 2013). These methods can detect an analyte or a family at the level of interest, but they usually only provide qualitative or semi-quantitative results (Cháfer-Pericás, Maquieira, & Puchades, 2010). On the contrary, confirmatory methods provide full or complementary information, enabling unequivocal identification and quantification of the analytes at levels of interest (Blasco et al., 2007). In most studies, high performance liquid chromatography (HPLC) is chosen for analyses of ENRO and CIP in foodstuffs samples (Cinquina et al., 2003; González, Moreno, Small, Jones, & Bruni, 2006). However, expensive chromatographic columns are used and a large amount of reagent is consumed in the HPLC process. Compared to HPLC, CE needs minimal sample and solvent consumption, and inexpensive capillary columns are used (Lombardo-Agui, Garcia-Campana, Gamiz-Gracia, & Blanco, 2010;
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Pinero, Garrido-Delgado, Bauza, Arce, & Valcarcel, 2012; Zhou, Xing, Zhu, Tang, & Jia, 2008). Unfortunately, most CE methods require a complex extraction and cleanup procedure. This additional step is tedious and slows down the analysis process. What’s more, it suffers from poor concentration sensitivity when on-line UV detection is utilized, due to the small loaded sample volume (nL) and the narrow optical path length (as defined by the diameter of the capillary). In response to the sensitivity problem, several on-line preconcentration methods (Wen, Li, Ma, & Chen, 2012) have been developed to preconcentrate analytes inside the capillary before separation and detection. This can be done by injecting a large volume of sample solution and focusing the analyte into a narrow band inside the capillary. Sweeping utilizes the phenomenon that hydrophobic analytes tend to be incorporated into the micelle (Quirino, 1998), and is used substantially for pharmaceutical and herbal products applications. Field-amplified sample stacking (FASS), normally capable of 100-fold sensitivity enhancement for charged analytes, is widely used in food and food contaminant analysis. The technique is based on the use of low-conductivity sample matrix and high-conductivity background solution (BGS) (Lagarrigue et al., 2008). FASS–sweeping technique has been used for determination of varied compounds in micellar electrokinetic chromatography (Dziomba, Kowalski, & Baczek, 2012). The goal of this study is to propose a highly sensitive on-line concentration method for the determination of ENRO and CIP in animal-originated samples. Based on the method developed by Dziomba et al. (2012), we improved the sensitivity of the sweeping technique by adding c-CD to sample matrix. That was because c-CD could increase the affinity between the analytes and the pseudostationary phase of BGS which led to remarkably enhance the determination sensitivities of ENRO and CIP. 2. Materials and methods 2.1. Chemicals and reagents ENRO was obtained from Dr. Ehrenstorfer GmbH (Augsburg, Germany). CIP was obtained from National Institute for Food and Drug Control (Beijing, China). The standards were used as received. Milk, milk powder, chicken (chicken muscle), pork (swine muscle), swine liver and swine kidney were purchased from a local supermarket. a-CD, b-CD and c-CD were from Duly Biotech Co., Ltd. (Nanjing, China). Sodium deoxycholate (NaDC) was from Acros Organics (Brussels, Belgium). Tris(hydroxymethyl)aminomethane (Tris) was from Kehao Bioengineering Co., Ltd. (Xian, China). Acetonitrile and methylene chloride were from Tianjin Fuyu Fine Chemical Co., Ltd. (Tianjin, China). All chemicals were of analytical reagent grade and were used as received. All solutions were made in distilled water. 2.2. Apparatus All CE experiments were performed on a CL1030 high-performance CE apparatus with a UV detector (Beijing Cailu Instrumental Co., Beijing, China). Uncoated fused-silica capillaries purchased
Fig. 1. The structure of enrofloxacin and ciprofloxacin.
from Yongnian Optical Fiber Factory (Hebei, China) were used. The dimensions of the capillary were 65 cm 75 lm I.D. 375 lm O.D. The effective length of the capillary was 57 cm from the injection end of the capillary. The data acquisition was carried out with an HW-2000 Chromatography Workstation (Shanghai Qianpu Software Company, Shanghai, China). A PH-3C acidity meter (Shanghai Hongyi Instrument Co., Ltd., Shanghai, China) was used for the pH measurement. UV detection was carried out at 270 nm. Samples were introduced into the capillary by gravity, where the sample vial was raised by 15.5 cm. Polyvinylidene fluoride (PVDF) syringe filters (Shanghai Peninsula Instrumental Co. Purification Equipment Factory, Shanghai, China) were used for filtering solutions. The new capillary was flushed with 1 M NaOH for 1 h and then with distilled water for 10 min before use. At the beginning of each working day, the capillary was flushed sequentially with distilled water (5 min), 100 mM NaOH (5 min), distilled water (5 min), and the running BGS (5 min). Simultaneously the CE instrument was warmed up until a stable baseline was achieved. Between consecutive analyses, the capillary was rinsed with distilled water (2 min), 100 mM NaOH (2 min), distilled water (2 min), and the running BGS (3 min). The capillary was left filled with distilled water overnight. 2.3. Solution preparation Standard stock solutions (1 mg/mL) of ENRO and CIP were prepared in methanol. Work solutions were prepared at various concentrations by appropriate dilution of the stock solutions with standard sample matrix. The standard sample matrix contained 300 mM Tris and 5 mM c-CD. The composition of running BGS was as follows: a. 20 mM Tris and 80 mM NaDC at pH 8.8; b. 20 mM Tris and 120 mM NaDC at pH 8.8. The running BGS was prepared daily from stock solutions of 100 mM Tris and 200 mM NaDC. All solutions were filtered through 0.45 lm syringe filters before use. 2.4. Sample preparation The method has been applied to six kinds of animal origin foods: milk, milk powder, pork, chicken, swine liver and swine kidney. 2.4.1. Preparation of milk samples An aliquot (1.0 g) of milk, spiked at different concentration levels using the standard stock solutions, was placed in a 15 mL glass centrifuge tube. After shaking the mixture briefly (about 30 s) on a vortex-mixer, 2 mL of acetonitrile was added. The mixture was vortexed for about 30 s at high speed and then centrifuged for 10 min at 4000 rpm (2840g). The supernatant was decanted into another 15 mL glass centrifuge tube, and 1 mL of 50 mM phosphate buffer (pH 7.4) and 3 mL of methylene chloride was added. The mixture was vortexed for about 30 s at high speed and then centrifuged for 10 min at 4000 rpm. The upper, aqueous layer was transferred into a waste bottle. The nonaqueous layer was evaporated under vacuum in a rotary evaporator at room temperature. The residue was reconstituted in 1.0 mL of standard sample matrix and filtered through a 0.45 lm membrane filter. 2.4.2. Preparation of other samples 0.5 g of milk powder or minced chicken and swine tissues was accurately weighed and placed in a 15 mL glass centrifuge tube, then spiked at different concentration levels using the standard stock solutions. After shaking the mixture briefly (about 30 s) on a vortex-mixer, 2 mL of acetonitrile was added. The mixture was
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vortexed for about 30 s at high speed and extracted in an ultrasonic bath (250 W, 28 °C, Yuhua Instrument Co., Ltd., Henan, China) for 30 min, and then centrifuged for 10 min at 4000 rpm (2840g). The supernatant was decanted into another 15 mL glass centrifuge tube, and 1 mL of 50 mM phosphate buffer (pH 7.0) and 3 mL of methylene chloride was added. The mixture was vortexed for about 30 s at high speed and then centrifuged for 10 min at 4000 rpm. The upper, aqueous layer was transferred into a waste bottle. The nonaqueous layer was evaporated under vacuum in a rotary evaporator at room temperature. The residue was reconstituted in 0.5 mL of standard sample matrix and filtered through a 0.45 lm membrane filter. 2.5. Calculation of LOD and sensitivity enhancement factor (SEF) The LOD was calculated as the peak height at a signal-to-noise ratio of 3 (S/N = 3). The SEF were calculated by comparing the LOD to that obtained with the conventional method (using 5 s of hydrodynamic injection with the BGS as the sample matrix). 3. Results and discussion 3.1. FASS–sweeping mechanism FASS is a concentration technique based on a mismatch between the electric conductivity of the sample and that of the running buffer (Zhang & Thormann, 1996). If the sample conductivity is lower than that of the BGS, the higher electric field results in a higher velocity of the analytes in the sample plug than that in the BGS. And this produces a sharpening of the analytes zone at the boundary with the BGS. Sweeping utilizes the interactions between the analytes and a pseudostationary phase or a complexing agent that is present only in the BGS (Quirino, 1998). In this paper, the NaDC, a kind of anionic surfactant is added to the BGS as a pseudostationary phase for sweeping, and provides a higher conductivity of the BGS. ENRO and CIP, with pI values of 6.33 and 7.37, respectively, are negatively charged at pH 8.8, which weakens the analytes affinity to NaDC because two like electric charges repel each other. To solve this problem, c-CD is introduced to the sample matrix. It is well known that cyclodextrans (CDs) can form water soluble inclusion complexes with many hydrophobic drugs (Abe, Ogawa, Nagase, Endo, & Ueda, 2010). It goes without saying that they can interact with ENRO and CIP. The formation of inclusion complexes increases the hydrophobicity of the analytes and reduces the effect of charges, thus enhances the sweeping efficiency. Schematic mechanism of separation is shown in Fig. 2. The process is as follows. At first, the capillary is filled with BGS, and a long sample plug is injected by gravity into the capillary (Fig. 2A). Then a high voltage is applied. The analytes migrate rapidly toward the anode and stack on the boundary between BGS and sample zones (Fig. 2B). Meanwhile, BGS containing micelles is introduced into the capillary from the inlet end due to the presence of electroosmotic flow (EOF). Because the electrophoretic mobility of charged micelles at the front boundary of sample plug was opposite to and lower than that of EOF. Therefore, the micelles traverse the sample plug and sweep the analytes at the behind boundary of the sample plug (Fang et al., 2014) (Fig. 2C). Subsequently, ENRO and CIP were separated according to the micellar electrokinetic capillary chromatography (MEKC) mode (Fig. 2D). 3.2. Optimization of the FASS–sweeping experimental conditions 3.2.1. Effect of Tris concentration In CE analysis, the selectivity was adjusted by changing the pH and BGS concentration. Therefore, in our studies, the effect of Tris
Fig. 2. Schematic illustration of the FASS–sweeping procedure.
concentration was observed in a range from 5 to 60 mM. Fig. 3A showed that the peak heights of the two analytes, as well as the resolution between them, reached the largest values with a concentration of 20 mM. This could be explained by the fact that the increase in Tris concentration could reduce the absorption of the analytes to the capillary wall, which slowed down the EOF. A slow EOF was helpful to the sweeping process. At the same time, the ionic strength of the BGS increased, which resulted in an enhancement in the conductance of the BGS. If the specific conductance of the BGS was too high, the Joule heat production would increase, and high system temperatures led to extra peak broadening and a decrease in resolution (Beckers & Bocˇek, 2003). Therefore, in consideration of the resolution, peak shapes, and peak heights of the analytes, the optimal concentration of Tris was chosen as 20 mM. 3.2.2. Effect of sample matrix and pH value FQs existed as cations in acidic pH, as zwitterions in neutral pH, or as anions in basic pH (Chen & Li, 2013). The influence of different pH values in sample matrix on signals intensities had also been investigated. The subject preliminary research showed that an alkaline sample matrix was beneficial to the FASS. Therefore, several sample matrixes differing in pH values (in the range from 9.1 to 11.0) were tested. As shown in Fig. 3B, the most significant changes in signals intensities of the analytes occurred when the pH value of sample matrix was 10.0. The reason was as follows: NaOH was added to increase pH and the ionic strength of the sample resulting in improving matching the conductivity ratio between the BGS and the sample with pH increasing from 9.1 to 10.0. The efficiency of FASS increasing was observed. However, with further pH increasing from 10.0 to 11.0, the mismatch between the local electroosmotic velocities in the sample and in the BGE generated laminar flows and inducing the broadening of the stacked zone (Burgi & Chien, 1991). For FASS, the concentrating effect basically relied on the change in electrophoretic velocity when the analyte molecules reach the interface between the high-resistance sample solution or water zone and low-resistance background solution zone. The higher difference in resistance or conductance, the greater concentrating effect was obtained (Dziomba et al., 2012). Tris solution with low conductance was chosen as the sample matrix. Our further study showed that the best result was obtained by using 300 mM Tris, which could maintain the pH of sample matrix at 10.0 without other pH regulators.
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Fig. 3. Effect of the FASS–sweeping experimental conditions on the peak heights and the resolution of the peaks. (A) Tris in BGS; (B) sample pH; (C) c-CD; (D) applied voltage; (E) NaDC; (F) injection time (80 mM NaDC). s Resolution of ENRO and CIP; d resolution of ENRO and the unknown substance; h ENRO; 4 CIP.
3.2.3. Effect of c-CD CDs can generate inclusion complexes with a wide variety of hydrophobic organic compounds in aqueous solution. The CDs, which serve as media enhancing the interaction between NaDC and the analytes, could greatly increase the sample injection volume. And it didn’t result in deterioration of the peak. To observe the effect of different kinds of CDs, a-, b- and c-CD were used. Under the same conditions, a lower sensitivity was obtained by applying a-CD compared to applying b-CD or c-CD. The corresponding LODs were 13.7, 12.3, 12.2 ng/mL for ENRO and 15.1,
12.9, 13.0 ng/mL for CIP. Although the addition of b-CD presented a good sensitivity as high as the addition of c-CD, poorer separation efficiency was also observed. c-CD is composed of eight glucose units and forms the shape of a hollow truncated cone with a hydrophylic exterior and hydrophobic interior. Due to the larger number of glucose units, c-CD is more favored than a- and b-CD. Therefore, c-CD was considered optimal. Different concentrations of c-CD in sample matrix (ranging from 0 to 10 mM) were tested. As shown in Fig. 3C, a continuous increase in peak heights with increasing c-CD concentration was
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observed. On the contrary, resolution between the ENRO and the unknown peak decreased through all the tested range. Resolution between the ENRO and CIP increased before decreasing. Taking peak heights and resolution into account, a concentration of 5 mM was chosen. 3.2.4. Effect of applied voltage The effect of the applied voltage on concentration efficiency was investigated in the range from 12 to 24 kV. As shown in Fig. 3D, peak widths progressively became narrower as the applied voltage decreased until 15 kV. For sweeping, analytes with a high affinity for the micellar carrier (i.e., very hydrophobic) spend a greater amount of time complexed with the micelles and hence have a velocity lower than EOF, while those with less affinity (i.e., less hydrophobic) have velocity more dependent on EOF. Most analytes fall between these extremes, with their velocity a function of affinity for the micelles, micelle velocity, and EOF velocity (Palmer, Munro, & Landers, 1999). Sweeping was also found to work better under suppressed or low EOF compared to high EOF conditions (Quirino & Terabe, 1999). Decreasing voltage can slow down the EOF. As a result, high sweeping efficiency was obtained. At lower applied voltage, the peaks broadened gradually and the resolution decreased. Thus, 15 kV was selected as the optimal applied voltage. 3.2.5. Effect of NaDC To determine the impact of the amounts of surfactant on the sweeping efficiency, different concentrations (40–140 mM) of NaDC in the BGS were tested. The concentration of NaDC had a dual effect on the analytes electrophoresis. First, it affected the sweeping of analytes which manifested itself in peaks height and separation efficiency. Too low concentration of NaDC (40 mM) in the BGS not only caused an overlap of ENRO and an unknown peak, but also resulted in a poor enrichment of CIP. As shown in Fig. 3E, the resolution of ENRO and the unknown peak and that of ENRO and CIP were improved with increasing NaDC concentration from 60 to 140 mM. Meanwhile, the peak heights of the two analytes decreased for peaks broadening. The level of NaDC also had a significant effect on the baseline noise. Too high concentration of NaDC (140 mM) in the BGS led to an awful baseline. 3.2.6. Effect of injection time Injection time had a great influence on signal amplification. In our study, different injection times varying from 150 to 330 s were evaluated. The result showed in Fig. 3F. Both the peak heights and areas of the analytes obviously increased without a deterioration of peak shapes with increasing injection time from 150 to 300 s. These could be explained by the fact that the on-line preconcentration technique decreased the width of the injected sample band, so that there is neither breakdown in resolution nor efficiency despite the larger-than-usual injection volumes. However, with a longer injection time, remarkable broadening peaks were observed, which led to the decrease of separation efficiency and a failure to achieve baseline separation. A 300 s injection time was suitable for the standard. However, it wasn’t nice to apply to the real samples. The optimization of injection time for real samples was given in Sections 3.3 and 3.4.
Table 1 LOD, RSD of peak area, regression equation, correlation coefficient (r), linear range, SEF for CE system with FASS–sweeping method (n = 6). Validation parameters
Regression equationa a b r LOD (S/N = 3) (ng/mL) Linear range (ng/mL) SEF %RSD (intra-day) %RSD (inter-day) a
80 mM NaDC CIP
ENRO
CIP
70.768 383.96 0.9998 1.87 10–500 376 1.46 2.46
104.51 147.24 0.9999 2.21 10–500 406 1.79 2.95
61.876 260.95 0.9994 5.70 10–500 124 1.99 2.44
66.119 263.3 0.9997 7.39 10–500 122 2.40 2.14
y = ax + b; y, peak area; x, standard concentration (ng/mL).
analysis. Further study showed that injection time of 240 s was more suitable for highly complex matrices analysis. According to these facts, validation parameters have been designated and applied in this paper for both methods (80 mM with 300 s and 120 mM with 240 s). Under the optimal conditions, the ENRO and CIP analysis by CE-UV using the FASS–sweeping technique was validated in terms of its linearity, repeatability, and LOD. The results were summarized in Table 1. Standard calibration curves were prepared by the injection of mixed-standard solutions at seven concentration
Table 2 Results for determination of the two components in real samples (n = 4). Sample
Ingredient Content (ng/g)
Spiked (ng/mL)
Found (ng/mL)
Recovery (%)
RSD (%)
Milk
ENRO
19.08
CIP
–a
50 100 150 50 100 150
40.8 88.9 125.5 46.3 96.5 137.1
82 89 84 93 96 91
1.0 2.1 2.7 0.1 3.1 1.6
ENRO
13.25
CIP
–
50 100 150 50 100 150
47.0 90.5 125.9 47.5 92.6 145.8
94 91 84 95 93 97
2.0 2.7 0.6 2.1 2.1 2.9
ENRO
29.90
CIP
–
50 100 150 50 100 150
43.4 87.3 121.3 52.6 107.9 139.7
87 87 81 105 108 93
1.8 4.1 0.1 1.8 1.2 3.3
ENRO
34.97
CIP
–
50 100 150 50 100 150
47.7 93.6 134.7 45.3 95.7 146.7
95 94 90 91 96 98
2.8 1.9 2.7 3.8 0.8 3.8
ENRO
–
CIP
17.37
150 200 250 150 200 250
141.1 177.2 241.3 134.7 175.1 235.8
94 89 97 90 88 94
4.4 1.2 2.5 3.4 3.4 2.5
ENRO
12.53
CIP
11.35
250 300 350 250 300 350
238.3 276.3 327.4 241.1 287.7 330.1
95 92 94 96 96 94
3.1 1.6 2.2 4.3 1.2 0.8
Milk powder
Chicken
Pork
Swine liver
3.3. Method validation Investigation of NaDC concentration has shown that the optimal concentration could be 80 mM, which provides about 1.5- to 2-fold better result in comparison with that of 120 mM. However, when it was applied to the real sample, a baseline separation between the tested compounds and unknown substances wasn’t achieved for the lower separation efficiency. As a compromise for selectivity and sensitivity, 120 mM was chosen for real sample
120 mM NaDC
ENRO
Swine kidney
a The substance was not found, or its content below the linearity range of the method.
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same) were presented for the analysis of ENRO and CIP. The methods had the advantages of simplicity, good precision and accuracy. High sensitivity was obtained with this preconcentration method. The LODs were lower than the MRLs of the tested compounds in animal origin foods. Furthermore, the method used simple reagents, with minimum sample preparation procedures, encouraging its application in routine analysis. This preconcentration method (120 mM NaDC with 240 s injection time) was applied to analyzing six kinds of animal foodstuffs. The proposed method was easy to carry out, and suited for assaying complex samples such as tissues, milk etc. intended for human consumption. Conflicts of interest The authors declare no competing financial interests. Fig. 4. Electrochromatograms of ENRO and CIP extracted from pork. (A) 300 ng/mL mixed standard; (B) blank; (C) spiked 150 ng/mL. Peaks: 1 = ENRO, 2 = CIP.
Acknowledgements levels (10, 50, 100, 200, 300, 400 and 500 ng/mL). The repeatability of the FASS–sweeping method was determined by repeated (n = 6) injections of the standard mixture solutions at concentrations of 300 ng/mL for all analytes. The peak areas were employed for quantification. Good linearity (r > 0.999) and repeatability (the %RSD values of peak area were 1.46–2.40% and 2.14–2.95% for intra-day and inter-day, respectively) were obtained for the analytes. The LODs obtained using the 120 mM NaDC with 240 s injection time method were 5.70 and 7.39 ng/mL for ENRO and CIP, respectively. Lower LODs were achieved (1.87 and 2.21 ng/mL for ENRO and CIP, respectively) by using the 80 mM NaDC with 300 s injection time method. 3.4. Application Investigation of NaDC concentration and injection time had shown that a higher sensitivity was obtained by using 80 mM NaDC with 300 s injection time method. Unfortunately, the lower separation efficiency hampered its application in complex sample analysis. Animal origin food samples are very complex mixtures of organic and inorganic compounds. Therefore, the 120 mM NaDC with 240 s injection time method was chosen for the animal foodstuffs analysis. The practical applicability of the system was demonstrated by separating ENRO and CIP and determining their respective contents in milk, milk powder, pork, chicken, swine liver and swine kidney. Actual sample matrices are complex and varying viscosity with standard solutions. The electrophoretic mobility of the analytes can be affected by solution conditions such as pH, ionic strength and viscosity (Paull & King, 2003). So, the analytes peak position shifts in actual sample occur. And there are many unknown peaks appearing on the electrophoretogram. These factors lead to difficult to identify peaks of ENRO and CIP according to migration time in real sample. Therefore, the peaks were identified using the standard addition methods. The analyte contents found in the different samples are given in Table 2. The electropherograms of standard sample solution and the pork samples detected was shown in Fig. 4. The accuracy of the methods and the potential matrix effects were established by analyzing spiked samples. The adaptability of the method used was shown by the acceptable recoveries of 85–102%. 4. Conclusions An on-line preconcentration method using FASS–sweeping was developed in this paper. Two kinds of conditions ((a) 80 mM NaDC with 300 s injection time for standards; (b) 120 mM NaDC with 300 s injection time for real samples. Other conditions were the
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