- Email: [email protected]
Simultaneous Determination of 7 Quinolones Residues in Animal Muscle Tissues by High Performance Liquid Chromatography
CHINESE JOURNAL OF ANALYTICAL CHEMISTRY Volume 35, Issue 6, June 2007 Online English edition of the Chinese language journal
Cite this article as: Chin J Anal Chem, 2007, 35(6), 786–790.
RESEARCH PAPER
Simultaneous Determination of 7 Quinolones Residues in Animal Muscle Tissues by High Performance Liquid Chromatography Zhao Si-Jun, Li Cun, Jiang Hai-Yang, Li Bing-Yu, Shen Jian-Zhong* College of Veterinary Medicine, China Agricultural University, Beijing 100094, China
Abstract: A rapid and robust multiresidue method was developed for the analysis of seven quinolones (ciprofloxacin, danofloxacin, enrofloxacin, sarafloxacin, difloxacin, oxolinic acid, and flumequine) in animal muscle tissues by HPLC-FLD. The samples were extracted with phosphate aqueous solution and cleaned with HLB-SPE. The analytes were separated using formic acid aqueous solution-acetontrile system as mobile phase with a linear gradient elution program, and determined by programmable fluorescence detection. The linear range was 0.3–1000 µg l–1 with correlation coefficients (r) greater than 0.9989. The limits of detection were 0.1–0.3 µg kg–1, and limits of quantification were 0.3–1.0 µg kg–1. The mean recoveries for each analyte in chicken and pig muscles ranged from 70.4% to 105.8% with relative standard deviation below 9.3% at 1–100 µg kg–1 fortification levels. The method is convenient, fast, safe, sensitive, and accurate for determining residual quinolones in actual samples. Key Words: Quinolones; Animal muscle tissues; High performance liquid chromatography (HPLC); Programmable fluorescence detection; Multi-residue determination
1
Introduction
Quinolones (QNs) are a class of broad-spectrum synthetic bactericidal medicines. QNs have been widely used in both food-producing animals and humans to treat bacterial infections, because of their broad antimicrobial spectrum and effectiveness in treating bacterial infections[1]. It would result in residues in edible tissue, which was potentially harmful to consumer health, and inducing pathogen resistance in humans[2,3]. Therefore, concerns increased on their residues in edible tissues and the maximum residue limits (MRLs) for certain QNs have been established by the Food and Agriculture Organization of the United Nations (JECFA) and the European Union (EU). The United States Food and Drug Administration dose not approve enrofloxacin for use in poultry[4]. The MRL for the seven QNs (ciprofloxacin, danofloxacin, enrofloxacin, sarafloxacin, difloxacin, oxolinic acid, and flumequine) had been set at 10–500 µg kg–1 in animal muscle tissues by China in 2002[5]. However, there is still no reported method for simultaneous determination of
these seven drugs in domestic China. Analytical methods for the determination of QNs antibiotics in foods of animal origin were based on enzyme-linked immunosorbent assay (ELISA)[6], high performance liquid chromatography (HPLC)[7–10], or liquid chromatography-mass spectrometric (LC-MS)[11–13] detection. Dong et al.[7] determined four kinds of QNs in edible tissue using phosphate aqueous solution as extraction, and the quantification limits (LOQ) were 4–20 μg kg–1. Bailac et al.[9] determined seven kinds of QNs in chicken muscles, which were extracted with dichlormethane. Verdon et al.[14] detected 10 kinds of QNs in chicken muscle by HPLC-fluorescence detection (FLD) and the samples were extracted with trichloracetic acid. However, both of the latter methods use chlorine compound as the extraction. In this study, a LC method with programmable fluorescence was developed for determination of seven QNs in animal muscle tissue. The samples were extracted with an aqueous solution and cleaned by hydrophile-lipophile balance (HLB) solid-phase extraction (SPE). The method is
Received 22 November 2006; accepted 10 January 2007 Corresponding author. Email: [email protected]; Tel: 010-62732803; Fax: 010-62731032 This work was supported by the Foundation of Distinguished Young Project of China (No. 30325032). Copyright © 2007, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences. Published by Elsevier Limited. All rights reserved.
ZHAO Si-Jun et al. / Chinese Journal of Analytical Chemistry, 2007, 35(6): 786–790
convenient, safe, sensitive, accurate, and available for determining residual quinolones in actual samples.
2
Experimental
2.1
Instruments and reagents
A Waters 2695 chromatographic system with a 2475 fluorescence detector was used. Nitrogen evaporation system (N-EVAPTM-111) was from Organomation Associates (USA). Centrifuge (TDL-40B) was from Shanghai An-ting (China).
HLB SPE cartridge (60 mg, 3 cc) was purchased from Waters Co. (USA). Ciprofloxacin HCl (CIP), danofloxacin (DAN), enrofloxacin (ENR), sarafloxacin (SAR), oxolinic acid (OXO), flumequine (FLU) were from National Control Institute of Veterinary Drugs (Beijing, P.R. China). Difloxacin (DIF) was purchased from Sigma (USA). The chemical structures of these QNs are shown in Fig.1. Methanol and acetonitrile were HPLC grade (Fisher, USA). Water was purified using a Milli-Q Plus water purification system (Millipore, USA). The other reagents were analytical grade or better.
Fig.1 Chemical structures of the quinolones
2.2 2.2.1
Standards solution
each drug versus the concentration of standard solutions. 2.3
Preparation of standards solution
Stock standard solution (100 mg l–1) - Accurately weighed appropriate CIP, DAN, ENR, SAR, DIF, OXO, and FLU standard and then dissolved with 2 ml 0.03% NaOH and diluted to a final volume of 100 ml with methanol. The multiquinolone working standard solution (1000 μg l–1) was prepared by mixing each of the seven stock standard solution and diluted to a final volume of 100 ml. 2.2.2
Calibration curve
Accurately pipette appropriate multiquinolone working standard solution was evaporated to dryness under a nitrogen stream, diluted with phosphate solution (PB, 0.1 M, pH 7.0) to 0.1, 0.3, 1.0, 10, 20 and 100 μg l–1 standard solutions. 100 μl each working standard solution was injected and calibration curve was constructed by plotting the absolute peak area of
Sample preparation
2.00 g ± 0.02 g control chicken and swine muscle were weighted into a 50 ml centrifuge tube and fortified with appropriate standard solution. After the sample was allowed to stand for 15 min in the dark at room temperature, 10 ml of PB solution was added to the sample. The sample was mixed for 10 sec and then centrifuged at 3000 rpm for 5 min. Supernatant was decanted into a clean tube. The sample was extracted repeatedly. The cartridge was conditioned with 2 ml of methanol followed by 2 ml of water. Ten ml of extraction was percolated into the cartridge. After the cartridge was washed with 3 ml of methanol/water (1:4, v/v), the analytes were eluted with 2 ml methanol. The collected eluent was evaporated to dryness under a nitrogen stream and reconstituted in 1 ml of PB solution. The sample was filtered through a 0.22 µm nylon membrane filter into an HPLC autosampler vial before injection into the HPLC system.
Table 1 HPLC mobile phase gradient program for the analysis of QNs Time (min) Formic acid aqueous solution (%) Acetontrile (%)
2.4
0.00
8.00
9.00
13.00
17.00
22.00
22.10
32.00
91 9
91 9
89 11
89 11
55 45
55 45
91 9
91 9
Liquid chromatography condition
The reverse phase column was Symmetry C18 (250 mm × 4.6 mm, i.d., 5 μm) from Waters Co. The injection volume was 100 μl and the column temperature was 30℃. The
fluorescence excitation/emission wavelength were set at 280/450 nm from 0 to 16.5 min, and then 320/365 nm from 16.5 to 32.0 min. A gradient program in HPLC system was used with solvent A (0.02% formic acid aqueous solution, pH 2.8) and solvent B (acetonitrile) as shown in Table 1.
ZHAO Si-Jun et al. / Chinese Journal of Analytical Chemistry, 2007, 35(6): 786–790
3
Results and discussion
3.1
The optimization of the chromatography conditions
Piperazinyl quinolones (CIP, DAN, ENR, SAR and DIF), which have carboxyl group and piperazinyl group in their structures, are amphoteric compounds. Reversed-phase liquid chromatography determined QNs would lead to tailing peaks. Acid solution was usually applied to the determination of QNs and tail-suppressing compounds (triethylamine)[6,7] were sometimes added to the mobile phase to improve peak shape. Phosphate buffer[7,8,10,15,16], citric acid buffer[9,17], and ion pair agent[18,19] were usually used as the mobile phase. However, crystallization of salts may occur when mixing the salt buffer with methanol and/or acetonitrile, which may result in blocking the chromatography system. The pH of the mobile phase was a critical factor in achieving the chromatographic separation of the QNs due to their similar structure. In this study, formic acid-aqueous solution was used as the ingredient of the mobile phase, and its pH was studied. The result indicates that the retention time of the QNs extends with the decrease of the pH in the same proportion of the organic solvent. The chromatographic peak separation of SAR and DIF could not be obtained at low pH (< 2.0), even though the proportion of formic acid-aqueous solution and acetonitril was adjusted. Meanwhile, the lower pH also affected the life of the chromatographic column. In this study, formic acid-aqueous solution (0.02%, v/v, pH 2.8)-acetonitrile was used as the mobile phase. Piperazinyl QNs (CIP, DAN, ENR, SAR, and DIF) and acid QNs (OXO and FLU) have different fluorescence wavelength. For Piperazinyl QNs, the optimal excitation (λex)
and emission (λem) wavelengths are set at 280 nm and 450 nm, respectively. And for the QNs, λex = 320 nm, λem = 365 nm. The analytical method was proposed in this study for determination of the seven QNs by HPLC with programmable wavelength fluorescence detector with a linear gradient elution. Not only is the chromatographic separation of the seven QNs favorable, but also each drug was determined with optimum wavelength to obtain higher sensibility. The chromatographic peak of each drug is shown in Fig.2.
Fig.2 Chromatogram of quinolones standard solution (100 μg l–1 for CIP, ENR, SAR, DIF, OXO, FLU, and 30 μg l–1 for DAN)
3.2 Calibration curve and the limit of detection and quantitation (LOD and LOQ) The linearity of the method was determined with a serial working reference standard by the above-mentioned method. The standard curve was linear over the range of 0.1–100 μg l–1. The related parameters are shown in Table 2. The LOD and the LOQ were based on a signal-to-noise ratio of 3:1 and 10:1, respectively. The LOD and LOQ for the method of determination of seven QNs in the animal muscle tissue were 0.1–0.3 µg kg–1 and 0.3–1.0 µg kg–1, respectively.
Table 2 Regression analysis and limits of detection and quantitation Correlation coefficient (n = 3)
Detection limits (µg kg–1)
Quantitation limits (µg kg–1)
Y = 5.3 × 103X – 6.2 × 102 Y = 3.9 × 104X – 1.4 × 103
0.9998 0.9998
0.3 0.1
1.0 0.3
0.3–100
Y = 1.0 × 104X – 5.4 × 102
0.9999
0.3
1.0
SAR
0.3–100
Y = 4.4 × 103X – 1.2 × 103
0.9989
0.3
1.0
DIF
0.3–100
Y = 7.4 × 103X – 3.1 × 103
0.9999
0.3
1.0
OXO FLU
0.3–100 0.3–100
Y = 2.9 × 103X – 1.2 × 103 Y = 3.7 × 103X – 1.5 × 103
0.9993 0.9994
0.3 0.3
1.0 1.0
Drug
Linear range (μg l–1)
CIP DAN
0.3–100 0.1–30
ENR
Calibration equation
3.3 Recovery and precision 3.4 The optimization of the extraction conditions The accuracy and precision of the method were studied by assaying control muscle tissue fortified at the levels of 1–100 μg kg–1. The mean recoveries for every seven QNs in chicken and pig muscles were in the range of 70.4%–102.1% and 72.6%–105.8%, respectively. Relative standard deviations ranged 1.1%–8.9% and 0.7%–9.3%, respectively. The good stability and reproducibility of the method obtained are shown in Table 3 and Fig.3. The method could be used to simultaneously analyze residual quinolones in animal samples.
The satisfactory recovery was observed when the extraction of quinolones from biological matrices was usually performed with organic solvents (such as ethanol[16], acetonitrile[10,11,16], trichloracetic acid[19], dichloromethane[9,15]) and mixed solution with acetic acid, phosphonic acid, ammoniae aqua [10,11,16]. However, the usage of organic solvents (especially chlorine compound usage) had generated considerable concern due to the growing problem of the pollution to the environment[20].
ZHAO Si-Jun et al. / Chinese Journal of Analytical Chemistry, 2007, 35(6): 786–790
In this study, the method of extraction of seven QNs from animal tissue was optimized by the reported means of Liu[7] and Dong[8]. The different pH of PB solution (pH = 2, 7, 11), the time of vortex and centrifuge were specially investigated. A satisfactory recovery was obtained when the sample was extracted with the different pH of PB solution. However, there were interfering peaks when the sample was extracted with acid and/or alkaline PB solution. The less interfering peaks and the higher sensibility were obtained with PB solutions of pH 7 as extraction solvents. The recovery of each analyte was did not show any significant change using the longer time of vortex (> 10 s) and centrifuge (> 5 min). The preparation procedure of the sample described in this study as extracted twice with PB buffer, less time of vortex and centrifuge and with lower centrifuge velocity. The pre-procedure (extraction and clean-up step) of 32 tissue samples could be fulfilled within 3 h. Less organic solvents (at most 5 ml of methanol) were used in the extraction and clean-up procedure. The average recoveries for each analyte in chicken and pig muscles ranged from 70.4% to 105.8% with
relative standard deviation below 9.3%. The LOQ was 1.0 µg kg–1 far lower than the MRL in P. R. China. A method was developed in this study for simultaneous determination of seven QNs (CIP, DAN, ENR, SAR, DIF, OXO and FLU) in swine and chicken muscle tissues by HPLC-FLD. The higher precision and lower LOD (LOQ) were obtained with less organic solvents and short analysis time. The method could meet the request of the residue analysis in China and used to determine analyze residual QNs in animal food.
Fig.3 Chromatograms of blank swine muscle (a) and spiked tissue (b)
Table 3 Recoveries and RSD of QNs in fortified animal muscle tissues Chicken muscle
Spiked concentration( μg kg–1)
QNs
1 10 100 0.3 10 100 1 10 100 1 10 100 1 10 100 1 10 100 1 10 100
CIP
DAN
ENR
SAR
DIF
OXO
FLU
3.5 Analysis of incurred sample Ten swine muscle samples and 12 chicken muscle samples were determined from the market in Beijing, China. Five of the
Swine muscle
Recovery (%)
RSD (%)
Recovery (%)
RSD (%)
70.4 76.3 75.2 87.9 89.0 86.2 99.5 99.8 96.7 73.7 80.3 76.1 91.8 97.1 97.2 88.0 95.1 99.2 93.1 102.1 96.6
5.1 2.1 2.9 2.5 5.2 3.2 5.2 1.1 2.5 6.7 2.6 3.0 5.1 3.2 2.2 8.3 1.9 1.7 8.9 3.6 1.7
84.8 87.9 79.9 94.1 90.3 79.6 105.8 104.6 92.2 85.9 83.3 82.8 96.4 98.4 92.0 72.6 86.9 86.0 94.9 93.1 86.5
8.7 2.9 2.1 5.8 3.4 1.6 8.6 9.3 1.4 4.3 1.4 1.9 1.4 4.3 1.5 2.6 0.7 1.3 4.1 1.7 1.3
22 samples (ZR06001, ZR06008, JR06005, JR06006 and JR06012) were found to contain one or multiple QNs in swine and chicken samples. The results are shown in Table 4.
Table 4 Quinolone residues in market muscle samples in Beijing Sample
Detection concentration (μg kg–1) CIP+ENR
DAN
SAR
DIF
OXO
FLU
JR06005
45.5
ND
ND
4.3
ND
ND
JR06006 JR06012 ZR06001 ZR06018
22.3 ND ND ND
8.3 ND ND 37.9
ND ND 5.7 ND
ND ND 21.3 ND
ND 14.2 ND ND
ND ND ND ND
ZHAO Si-Jun et al. / Chinese Journal of Analytical Chemistry, 2007, 35(6): 786–790
References
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[11] Lin Feng, Lin H D, Wu Y X, Wang F. J. Instrumental Anal, 2004, 5(23): 43–47 [12] Toussaint B, Chedin M, Vincent U, Bordin G, Rodriguez A. J. Chromatogr. A, 2005, 1088: 40–48 [13] Peng T, Yong W, An J, Chu X G, Tang Y Z, Li C J. Chinese J. Anal. Chem., 2006, 34: s10–s14 [14] Verdon E, Couedor P, Roudaut B, Sandérs P. J. AOAC. Int., 2005, 88: 1179–1192 [15] Gigosos P, Revesado P, Cadahía O, Fente C, Vazquez B, Franco C, Cepeda A. J. Chromatogr. A, 2000, 871: 31–36 [16] Du L M, Wei H Q, Zhang J Y, Zhang Q P. Chinese J. Anal. Chem., 2003, 5(31): 637 [17] Zeng Z, Dong A, Yang G, Chen Z, Huang X. J. Chromatogr. B, 2005, 821: 202–209 [18] Ramos M, Aranda A, Garcia E, Reuvers T, Hooghuis H. J. Chromatogr. B, 2003, 789: 373–381 [19] Shervington L, Abba M, Hussain B, Donnelly J. J. Pharm. Biomed. Anal., 2005, 397: 769–775
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Cite this article as: Chin J Anal Chem, 2007, 35(6), 786–790.
RESEARCH PAPER
Simultaneous Determination of 7 Quinolones Residues in Animal Muscle Tissues by High Performance Liquid Chromatography Zhao Si-Jun, Li Cun, Jiang Hai-Yang, Li Bing-Yu, Shen Jian-Zhong* College of Veterinary Medicine, China Agricultural University, Beijing 100094, China
Abstract: A rapid and robust multiresidue method was developed for the analysis of seven quinolones (ciprofloxacin, danofloxacin, enrofloxacin, sarafloxacin, difloxacin, oxolinic acid, and flumequine) in animal muscle tissues by HPLC-FLD. The samples were extracted with phosphate aqueous solution and cleaned with HLB-SPE. The analytes were separated using formic acid aqueous solution-acetontrile system as mobile phase with a linear gradient elution program, and determined by programmable fluorescence detection. The linear range was 0.3–1000 µg l–1 with correlation coefficients (r) greater than 0.9989. The limits of detection were 0.1–0.3 µg kg–1, and limits of quantification were 0.3–1.0 µg kg–1. The mean recoveries for each analyte in chicken and pig muscles ranged from 70.4% to 105.8% with relative standard deviation below 9.3% at 1–100 µg kg–1 fortification levels. The method is convenient, fast, safe, sensitive, and accurate for determining residual quinolones in actual samples. Key Words: Quinolones; Animal muscle tissues; High performance liquid chromatography (HPLC); Programmable fluorescence detection; Multi-residue determination
1
Introduction
Quinolones (QNs) are a class of broad-spectrum synthetic bactericidal medicines. QNs have been widely used in both food-producing animals and humans to treat bacterial infections, because of their broad antimicrobial spectrum and effectiveness in treating bacterial infections[1]. It would result in residues in edible tissue, which was potentially harmful to consumer health, and inducing pathogen resistance in humans[2,3]. Therefore, concerns increased on their residues in edible tissues and the maximum residue limits (MRLs) for certain QNs have been established by the Food and Agriculture Organization of the United Nations (JECFA) and the European Union (EU). The United States Food and Drug Administration dose not approve enrofloxacin for use in poultry[4]. The MRL for the seven QNs (ciprofloxacin, danofloxacin, enrofloxacin, sarafloxacin, difloxacin, oxolinic acid, and flumequine) had been set at 10–500 µg kg–1 in animal muscle tissues by China in 2002[5]. However, there is still no reported method for simultaneous determination of
these seven drugs in domestic China. Analytical methods for the determination of QNs antibiotics in foods of animal origin were based on enzyme-linked immunosorbent assay (ELISA)[6], high performance liquid chromatography (HPLC)[7–10], or liquid chromatography-mass spectrometric (LC-MS)[11–13] detection. Dong et al.[7] determined four kinds of QNs in edible tissue using phosphate aqueous solution as extraction, and the quantification limits (LOQ) were 4–20 μg kg–1. Bailac et al.[9] determined seven kinds of QNs in chicken muscles, which were extracted with dichlormethane. Verdon et al.[14] detected 10 kinds of QNs in chicken muscle by HPLC-fluorescence detection (FLD) and the samples were extracted with trichloracetic acid. However, both of the latter methods use chlorine compound as the extraction. In this study, a LC method with programmable fluorescence was developed for determination of seven QNs in animal muscle tissue. The samples were extracted with an aqueous solution and cleaned by hydrophile-lipophile balance (HLB) solid-phase extraction (SPE). The method is
Received 22 November 2006; accepted 10 January 2007 Corresponding author. Email: [email protected]; Tel: 010-62732803; Fax: 010-62731032 This work was supported by the Foundation of Distinguished Young Project of China (No. 30325032). Copyright © 2007, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences. Published by Elsevier Limited. All rights reserved.
ZHAO Si-Jun et al. / Chinese Journal of Analytical Chemistry, 2007, 35(6): 786–790
convenient, safe, sensitive, accurate, and available for determining residual quinolones in actual samples.
2
Experimental
2.1
Instruments and reagents
A Waters 2695 chromatographic system with a 2475 fluorescence detector was used. Nitrogen evaporation system (N-EVAPTM-111) was from Organomation Associates (USA). Centrifuge (TDL-40B) was from Shanghai An-ting (China).
HLB SPE cartridge (60 mg, 3 cc) was purchased from Waters Co. (USA). Ciprofloxacin HCl (CIP), danofloxacin (DAN), enrofloxacin (ENR), sarafloxacin (SAR), oxolinic acid (OXO), flumequine (FLU) were from National Control Institute of Veterinary Drugs (Beijing, P.R. China). Difloxacin (DIF) was purchased from Sigma (USA). The chemical structures of these QNs are shown in Fig.1. Methanol and acetonitrile were HPLC grade (Fisher, USA). Water was purified using a Milli-Q Plus water purification system (Millipore, USA). The other reagents were analytical grade or better.
Fig.1 Chemical structures of the quinolones
2.2 2.2.1
Standards solution
each drug versus the concentration of standard solutions. 2.3
Preparation of standards solution
Stock standard solution (100 mg l–1) - Accurately weighed appropriate CIP, DAN, ENR, SAR, DIF, OXO, and FLU standard and then dissolved with 2 ml 0.03% NaOH and diluted to a final volume of 100 ml with methanol. The multiquinolone working standard solution (1000 μg l–1) was prepared by mixing each of the seven stock standard solution and diluted to a final volume of 100 ml. 2.2.2
Calibration curve
Accurately pipette appropriate multiquinolone working standard solution was evaporated to dryness under a nitrogen stream, diluted with phosphate solution (PB, 0.1 M, pH 7.0) to 0.1, 0.3, 1.0, 10, 20 and 100 μg l–1 standard solutions. 100 μl each working standard solution was injected and calibration curve was constructed by plotting the absolute peak area of
Sample preparation
2.00 g ± 0.02 g control chicken and swine muscle were weighted into a 50 ml centrifuge tube and fortified with appropriate standard solution. After the sample was allowed to stand for 15 min in the dark at room temperature, 10 ml of PB solution was added to the sample. The sample was mixed for 10 sec and then centrifuged at 3000 rpm for 5 min. Supernatant was decanted into a clean tube. The sample was extracted repeatedly. The cartridge was conditioned with 2 ml of methanol followed by 2 ml of water. Ten ml of extraction was percolated into the cartridge. After the cartridge was washed with 3 ml of methanol/water (1:4, v/v), the analytes were eluted with 2 ml methanol. The collected eluent was evaporated to dryness under a nitrogen stream and reconstituted in 1 ml of PB solution. The sample was filtered through a 0.22 µm nylon membrane filter into an HPLC autosampler vial before injection into the HPLC system.
Table 1 HPLC mobile phase gradient program for the analysis of QNs Time (min) Formic acid aqueous solution (%) Acetontrile (%)
2.4
0.00
8.00
9.00
13.00
17.00
22.00
22.10
32.00
91 9
91 9
89 11
89 11
55 45
55 45
91 9
91 9
Liquid chromatography condition
The reverse phase column was Symmetry C18 (250 mm × 4.6 mm, i.d., 5 μm) from Waters Co. The injection volume was 100 μl and the column temperature was 30℃. The
fluorescence excitation/emission wavelength were set at 280/450 nm from 0 to 16.5 min, and then 320/365 nm from 16.5 to 32.0 min. A gradient program in HPLC system was used with solvent A (0.02% formic acid aqueous solution, pH 2.8) and solvent B (acetonitrile) as shown in Table 1.
ZHAO Si-Jun et al. / Chinese Journal of Analytical Chemistry, 2007, 35(6): 786–790
3
Results and discussion
3.1
The optimization of the chromatography conditions
Piperazinyl quinolones (CIP, DAN, ENR, SAR and DIF), which have carboxyl group and piperazinyl group in their structures, are amphoteric compounds. Reversed-phase liquid chromatography determined QNs would lead to tailing peaks. Acid solution was usually applied to the determination of QNs and tail-suppressing compounds (triethylamine)[6,7] were sometimes added to the mobile phase to improve peak shape. Phosphate buffer[7,8,10,15,16], citric acid buffer[9,17], and ion pair agent[18,19] were usually used as the mobile phase. However, crystallization of salts may occur when mixing the salt buffer with methanol and/or acetonitrile, which may result in blocking the chromatography system. The pH of the mobile phase was a critical factor in achieving the chromatographic separation of the QNs due to their similar structure. In this study, formic acid-aqueous solution was used as the ingredient of the mobile phase, and its pH was studied. The result indicates that the retention time of the QNs extends with the decrease of the pH in the same proportion of the organic solvent. The chromatographic peak separation of SAR and DIF could not be obtained at low pH (< 2.0), even though the proportion of formic acid-aqueous solution and acetonitril was adjusted. Meanwhile, the lower pH also affected the life of the chromatographic column. In this study, formic acid-aqueous solution (0.02%, v/v, pH 2.8)-acetonitrile was used as the mobile phase. Piperazinyl QNs (CIP, DAN, ENR, SAR, and DIF) and acid QNs (OXO and FLU) have different fluorescence wavelength. For Piperazinyl QNs, the optimal excitation (λex)
and emission (λem) wavelengths are set at 280 nm and 450 nm, respectively. And for the QNs, λex = 320 nm, λem = 365 nm. The analytical method was proposed in this study for determination of the seven QNs by HPLC with programmable wavelength fluorescence detector with a linear gradient elution. Not only is the chromatographic separation of the seven QNs favorable, but also each drug was determined with optimum wavelength to obtain higher sensibility. The chromatographic peak of each drug is shown in Fig.2.
Fig.2 Chromatogram of quinolones standard solution (100 μg l–1 for CIP, ENR, SAR, DIF, OXO, FLU, and 30 μg l–1 for DAN)
3.2 Calibration curve and the limit of detection and quantitation (LOD and LOQ) The linearity of the method was determined with a serial working reference standard by the above-mentioned method. The standard curve was linear over the range of 0.1–100 μg l–1. The related parameters are shown in Table 2. The LOD and the LOQ were based on a signal-to-noise ratio of 3:1 and 10:1, respectively. The LOD and LOQ for the method of determination of seven QNs in the animal muscle tissue were 0.1–0.3 µg kg–1 and 0.3–1.0 µg kg–1, respectively.
Table 2 Regression analysis and limits of detection and quantitation Correlation coefficient (n = 3)
Detection limits (µg kg–1)
Quantitation limits (µg kg–1)
Y = 5.3 × 103X – 6.2 × 102 Y = 3.9 × 104X – 1.4 × 103
0.9998 0.9998
0.3 0.1
1.0 0.3
0.3–100
Y = 1.0 × 104X – 5.4 × 102
0.9999
0.3
1.0
SAR
0.3–100
Y = 4.4 × 103X – 1.2 × 103
0.9989
0.3
1.0
DIF
0.3–100
Y = 7.4 × 103X – 3.1 × 103
0.9999
0.3
1.0
OXO FLU
0.3–100 0.3–100
Y = 2.9 × 103X – 1.2 × 103 Y = 3.7 × 103X – 1.5 × 103
0.9993 0.9994
0.3 0.3
1.0 1.0
Drug
Linear range (μg l–1)
CIP DAN
0.3–100 0.1–30
ENR
Calibration equation
3.3 Recovery and precision 3.4 The optimization of the extraction conditions The accuracy and precision of the method were studied by assaying control muscle tissue fortified at the levels of 1–100 μg kg–1. The mean recoveries for every seven QNs in chicken and pig muscles were in the range of 70.4%–102.1% and 72.6%–105.8%, respectively. Relative standard deviations ranged 1.1%–8.9% and 0.7%–9.3%, respectively. The good stability and reproducibility of the method obtained are shown in Table 3 and Fig.3. The method could be used to simultaneously analyze residual quinolones in animal samples.
The satisfactory recovery was observed when the extraction of quinolones from biological matrices was usually performed with organic solvents (such as ethanol[16], acetonitrile[10,11,16], trichloracetic acid[19], dichloromethane[9,15]) and mixed solution with acetic acid, phosphonic acid, ammoniae aqua [10,11,16]. However, the usage of organic solvents (especially chlorine compound usage) had generated considerable concern due to the growing problem of the pollution to the environment[20].
ZHAO Si-Jun et al. / Chinese Journal of Analytical Chemistry, 2007, 35(6): 786–790
In this study, the method of extraction of seven QNs from animal tissue was optimized by the reported means of Liu[7] and Dong[8]. The different pH of PB solution (pH = 2, 7, 11), the time of vortex and centrifuge were specially investigated. A satisfactory recovery was obtained when the sample was extracted with the different pH of PB solution. However, there were interfering peaks when the sample was extracted with acid and/or alkaline PB solution. The less interfering peaks and the higher sensibility were obtained with PB solutions of pH 7 as extraction solvents. The recovery of each analyte was did not show any significant change using the longer time of vortex (> 10 s) and centrifuge (> 5 min). The preparation procedure of the sample described in this study as extracted twice with PB buffer, less time of vortex and centrifuge and with lower centrifuge velocity. The pre-procedure (extraction and clean-up step) of 32 tissue samples could be fulfilled within 3 h. Less organic solvents (at most 5 ml of methanol) were used in the extraction and clean-up procedure. The average recoveries for each analyte in chicken and pig muscles ranged from 70.4% to 105.8% with
relative standard deviation below 9.3%. The LOQ was 1.0 µg kg–1 far lower than the MRL in P. R. China. A method was developed in this study for simultaneous determination of seven QNs (CIP, DAN, ENR, SAR, DIF, OXO and FLU) in swine and chicken muscle tissues by HPLC-FLD. The higher precision and lower LOD (LOQ) were obtained with less organic solvents and short analysis time. The method could meet the request of the residue analysis in China and used to determine analyze residual QNs in animal food.
Fig.3 Chromatograms of blank swine muscle (a) and spiked tissue (b)
Table 3 Recoveries and RSD of QNs in fortified animal muscle tissues Chicken muscle
Spiked concentration( μg kg–1)
QNs
1 10 100 0.3 10 100 1 10 100 1 10 100 1 10 100 1 10 100 1 10 100
CIP
DAN
ENR
SAR
DIF
OXO
FLU
3.5 Analysis of incurred sample Ten swine muscle samples and 12 chicken muscle samples were determined from the market in Beijing, China. Five of the
Swine muscle
Recovery (%)
RSD (%)
Recovery (%)
RSD (%)
70.4 76.3 75.2 87.9 89.0 86.2 99.5 99.8 96.7 73.7 80.3 76.1 91.8 97.1 97.2 88.0 95.1 99.2 93.1 102.1 96.6
5.1 2.1 2.9 2.5 5.2 3.2 5.2 1.1 2.5 6.7 2.6 3.0 5.1 3.2 2.2 8.3 1.9 1.7 8.9 3.6 1.7
84.8 87.9 79.9 94.1 90.3 79.6 105.8 104.6 92.2 85.9 83.3 82.8 96.4 98.4 92.0 72.6 86.9 86.0 94.9 93.1 86.5
8.7 2.9 2.1 5.8 3.4 1.6 8.6 9.3 1.4 4.3 1.4 1.9 1.4 4.3 1.5 2.6 0.7 1.3 4.1 1.7 1.3
22 samples (ZR06001, ZR06008, JR06005, JR06006 and JR06012) were found to contain one or multiple QNs in swine and chicken samples. The results are shown in Table 4.
Table 4 Quinolone residues in market muscle samples in Beijing Sample
Detection concentration (μg kg–1) CIP+ENR
DAN
SAR
DIF
OXO
FLU
JR06005
45.5
ND
ND
4.3
ND
ND
JR06006 JR06012 ZR06001 ZR06018
22.3 ND ND ND
8.3 ND ND 37.9
ND ND 5.7 ND
ND ND 21.3 ND
ND 14.2 ND ND
ND ND ND ND
ZHAO Si-Jun et al. / Chinese Journal of Analytical Chemistry, 2007, 35(6): 786–790
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