Food Control 31 (2013) 359e365. Contents lists available at SciVerse ScienceDirect. Food Control. journal homepage:

Food Control 31 (13) 359e365 Contents lists available at SciVerse ScienceDirect Food Control journal homepage: www.elsevier.com/locate/foodcont Highly sensitive and simultaneous determination of sixteen sulphonamide antibiotics, four acetyled metabolites and trimethoprim in meat by rapid resolution liquid chromatography-tandem mass spectrometry Hui Li a,b, Hanwen Sun a, *, Jingxuan Zhang b, Kun Pang b a College of Chemistry and Environmental Science, Hebei University, Key Laboratory of Analytical Science of Hebei Province, Wusi East Road 188, Baoding 071002, China b Hebei Institute of Food Quality Supervision Inspection and Research, Shijiazhuang 050051, China article info abstract Article history: Received 14 June 12 Received in revised form 17 September 12 Accepted 18 September 12 Keywords: Sulphonamides Acetyled metabolites Trimethoprim Solid phase extraction RRLC-MS/MS Meat A novel multiresidue analysis method is developed for highly sensitive and simultaneous determination of 16 sulphonamides (SAs), 4 acetyled metabolites, and trimethoprim in pork and mutton by rapid resolution liquid chromatography-tandem mass spectrometry (RRLC-MS/MS). The sample was extracted with acetonitrile under ultrasonication incubation, followed by solid phase extraction (SPE). The calibration curves showed good linearity with correlation coefficient (r) more than 0.998. The limit of quantification (LOQ) was 0.35e1.0 mg/kg, which can ensure to detect studied drugs at the maximum residue level (MRL) of 10 mg/kg. The mean recoveries at addition level of 1.0, 5.0 and 50 mg/kg were in the range of 68.3e104% with the relative standard deviation (RSD) of 3.5e9.2%. The intra-day precision (as RSD) for six determinations at 50 mg/kg spiked level within a day was in the range of 4.2e8.9%. The method is sensitive, accurate, convenient and rapid, and can be used for the qualitatively and quantitatively determination of multiresidue of the studied drugs in meat. Ó 12 Elsevier Ltd. All rights reserved. 1. Introduction The residues of sulphonamides (SAs) in foods of animal origin are a major concern because they are harmful to the consumer s health, and could induce pathogens to develop resistance. SAs can be acetylated at the N 4 -position. N 4 -acetyl metabolite of SAs in foodproducing animals may cause the renal toxicity as a result of precipitation in the kidney (Vree & Hekster, 1985), and may affect their excretion rates due to higher plasma protein binding than the parent compound (Vree, Hekster, Nouws, & Dorrestein, 1987). Trimethoprim is another antibiotic agent, which is often coadministered with sulfamethoxazole to enhance treatment against a variety of bacterial infections. In humans and animals, it can cause changes in the bone marrow and significant effects on some organ weights. To ensure food safety for consumers, the European Community, the U.S. Food and Drug Administration (FDA), and China Agriculture Department have laid down the maximum residue level (MRL) of 100 mg/kg for SAs in foodstuffs of animal origin and 50 mg/ kg for trimethoprim in muscle, fat/skin, liver and kidney tissues of pigs (China Agriculture Ministry, 02; European Union Regulation, * Corresponding author. Tel.: þ86 312 5079719; fax: þ86 312 5079739. E-mail address: hanwen@hbu.edu.cn (H. Sun). 10; Food and Drug Regulation, 1991, pp. 1478e1480). Korea set an MRL of 100 mg/kg as sum of the 14 SAs in meat and milk (Korea Food Code, 08). The Japan s Positive List System presents an MRL of 50 mg/kg for sulfaquinoxaline, mg/kg for sulfamerazine, 40 mg/kg for sulfadimethoxypyrimidine, and 10 mg/kg for sulfamethazine. For other SAs the MRL takes 10 mg/kg (Ministry of Health, Labour and Welfare of Japan, 06). In order to better assess the occurrence of SAs in food, their metabolites should also be considered. These early conclusions evidenced the need for analytical methods capable to simultaneously determine parent compound as well as their metabolites at the low concentrations normally present in food. Therefore, it is urgently in need of developing rapid and effective method for the simultaneous determination of SAs residues and their metabolites in meats. High-performance liquid chromatography (HPLC) as separation and analytical technology has been used widely to detect veterinary residues in food. A series of analytical methods were reported for the determination of SAs residues in food (See Table 1). Analytical detection limits of HPLC-UV method are generally limited by significant signal interference associated with UV spectral overlaps with other food constituents. Recently, several HPLC- UV/DAD methods combined with effective extraction and cleanup were reported for the determination of residual SAs in food (Huang et al., 12; Kowalski, Plenis, Oledzka, & Konieczna, 11; Shi et al., 0956-7135/$ e see front matter Ó 12 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodcont.12.09.028

360 Table 1 References for the determination of SAs, acetyled metabolites and trimethoprim in food. Analyte Matrix Extraction Cleanup Final method LOD/recovery/RSD Ref. Ref. no. 6 SAs c Shrimp, fish Ultrasonication, 1% acetic acid Molecularly imprinted LCeUV 8.4e10.9 mg/kg, 85.5e106.1%, 1.2e7.0% Shi et al., 11 1 polymers 7 SAs Milk Cloud point extraction, n-butyl LCeUV 2.23e9.79 mg/l, 67.0e105.7%, 93e8.31% Zhang et al., 11 2 alcohol 7 SAs Poultry tissue Ultrasonication, acetonitrile C18 cartridges MECC a 1.3e7.8 mg/kg, >77.2%, 9.5%, 11.2% Kowalski et al., 11 3 4 SAs Milk, chicken Acetonitrileewater Micro-solid phase extraction LC 4.52e15.63 mg/kg, 43.11e83.81% Huang et al., 12 4 muscle 2 SAs, etc. Porcine tissues TwogramsC18, EDTA-Na 2 eoxalic Matrix solid-phase LCeDAD LOQ: 7e34 mg/kg, 80.6e99.2%, <6.1%, Yu et al., 12 5 acid dispersion 6 SAs Cattle meats Acetonitrile LCeFLD 8e15 mg/kg, 44.67e81%, <6% Mor, Kocasari, Ozdemir, 6 & Oz, 12 6 SAs Milk Water, dichloromethane PCSFC b ems 41e181 mg/kg Dost, Jones, & Davidson, 7 00 14 SAs Milk, egg Distilled water Solid-phase extraction LCeMS 1e10 mg/kg, 76e112%, <13% Cavaliere et al., 03 8 13 SAs Raw meat, infant Accelerated hot water extraction LCeMS/MS 2.6 mg/kg, 70e101% Gentili et al., 04 9 foods 16 SAs Poultry/meat Acetonitrile LCeMS/MS 1. 0e12.0 mg/kg, 75.4e97.3%, Pang et al., 05 10 3.48e14$09% 12 SAs Pork meat Pressurized liquid extraction, water Oasis HLB cartridge CEeMS/MS <12.5 mg/kg, 76e98%, <14%) Font et al., 07 11 8 SAs Pork Ultrasonication, water Solid-phase microextraction LCeMS 16 39 mg/kg below 15% Lu et al., 07 12 17 SAs, etc. Fish tissue Ultrasonication, MeOH:CAN: formic LCeMS/MS 5.65e24.0 mg/kg, <% Dasenaki & Thomaidis, 13 acid 10 5 SAs Honey Acetate buffereoctanol:pentanol Hollow fiber renewal liquid LCeMS/MS 5.1e27.4 mg/kg, 80.9e103.1%, <15% Bedendo et al., 10 14 membrane 18 SAs Muscles, livers, Pressurized liquid extraction with Hydrophilicelipophilic LC, LCeMS/MS 3 mg/kg, 71.1e118.3%, <13% Yu et al., 11 15 kidneys acetonitrile balance cartridge 14 SAs Marine products Ultrasonication, acetonitrile C18 powder LCePDA, LCeMS/MS 3e6 mg/kg, 51.8e89.7%, 5.6e8.6% Won et al., 11 16 13 SAs Grass, carp tissues C18-bonded silica Matrix solid-phase LCeMS/MS 0.75e3.0 mg/kg, 69.0e96.3%, 13.2% Lu et al., 11 17 dispersion 6 SAs Milk, milk powder Hydrochloric acid (ph 2) 15% NaCl C18-stir bar sorptive LCeMS/MS 2.7e31.5 mg/kg, 68e115% Yu & Hu, 12 18 extraction SMM, d AcSMM, e Eggs Ultrasonication saturated ammonium HPLC-DAD,30 mg/kg, >91%,within 4%. Kishida, 07 19 SDM, f AcSDM g sulphate SMM, AcSMM Chicken plasma, Ultrasonication, ethanol HPLC-DAD <30 mg/kg, >90% within 4%. Kishida & Furusawa, SDM, AcSDM tissues, eggs 05 SMM, AcSMM, Milk Ultrasonication, ethanoleacetic HPLC-DAD LOQ: < mg/l, >81%, within 5% Kishida & Furusawa, 21 SDM, AcSDM Acid (97:3, v/v) 04 SDM, AcSDM Chicken meat Ultrasonication % (w/v) perchloric HPLC-DAD LOQ: 0.1 mg/kg, 84%, 6% Furusawa, 07 22 acid SDA, h TMP i Broiler tissues Ethyl acetate or dichloromethane Hexaneechloroform HPLC 15e100 mg/kg SDA, e mg/kg TMP Dagorn & Delmas, 1994 23 in an acidic buffer SDA, TMP Swine tissues 3 ml of 0.1 M acetic acid in water SCX column HPLC-DAD LCeMS/MS 15 mg/kg SDA, mg/kg TMP De Baere et al., 00 24 H. Li et al. / Food Control 31 (13) 359e365 a MECC d micellar electrokinetic capillary chromatograp. b PCSFC d packed column supercritical fluid chromatography. c SAs d sulfonamides. d SMM d sulfamonomethoxine. e AcSMM d N4-acetyl sulfamonomethoxine. f SDM d sulfadimethoxine. g AcSDM d N4-acetyl sulfadimethoxine. h SDA d sulfadiazine. i TMP d trimethoprim.

H. Li et al. / Food Control 31 (13) 359e365 361 11; Yu, Mun, & Hu, 12; Zhang, Duan, & Wang, 11). HPLC coupled to mass spectrometry (MS) or tandem MS (MS/MS) is the most effective method because of their high sensitivities and accuracy for compounds confirmation. A numbers of LC-MS or LC- MS/MS methods have been used for the determination of SAs residues in food (Bedendo, Jardim, & Carasek, 10; Cavaliere, Curini, Corcia, Nazzari, & Samperi, 03; Dasenaki & Thomaidis, 10; Gentili et al., 04; Lu, Chen, & Lee, 07; Lu, Shen, Dai, Zhang, & Wang, 11; Pang et al., 05; Won et al., 11; Yu & Hu, 12; Yu et al., 11 in Table 1). Since the concentration of SAs drugs commonly found in food samples is extremely low, pretreatment is necessary for their sensitive detection and quantification. Extraction and cleanup are the most challenging parts for SAs analysis in foods, which are often the critical steps in deciding the levels of detection limits of the overall methods. A series of LC-MS or LC-MS/MS methods combined with deferent cleanup technique for the determination of SAs residues in food were reported. C 18 SPE cartridges were used for cleanup of 7 SAs in poultry tissue (Kowalski et al., 11), 14 SAs in marine products (Won et al., 11), and 6 SAs in milk and milk powder (Yu & Hu, 12). Oasis HLB cartridges with reversed-phase sorbents were used for cleanup of 12 SAs in pork meat (Font, Juan-Garcia, & Pico, 07). For SAs, acetyled metabolites and trimethoprim with strong polarity and ionogenous character, positive SPE column or ion exchange SPE column should be selected for cleanup. From 11 year to date, several higher throughout methods were developed for simultaneous determination of 5e18 SAs residues in different food matrix by LC-MS or LC-MS/MS (see De Baere et al., 00; Lu et al., 11; Won et al., 11; Yu & Hu, 12; Yu et al., 11 in Table 1). However, up to date, only a few LC methods were reported for the analysis of the metabolites of SAs in food. For the analysis of acetyled metabolites, such as N 4 -acetyl sulfamonomethoxine, N 4 -acetyl sulfadimethoxine in eggs (Kishida, 07), chicken tissues (Kishida & Furusawa, 05), and milk (Kishida & Furusawa, 04) aswellasn 4 -acetyl sulfadimethoxine in chicken meat (Furusawa, 07), the sensitivity was not high enough for trace residue analysis. Thermal pinch analyses were performed by using reverse phase-hplc, with LOD ranging from to 50 mg/kg (Dagorn & Delmas, 1994), which are not low enough to fulfil the EU requirements for residue analysis. An HPLCeUV detection method for quantitative determination of sulfadiazine and trimethoprim in swine tissues was developed with LOQ of 50 and mg/kg, respectively (De Baere et al., 00). Table 1 shows that some methods reported can be used for the determination of SAs residues in food at the MRL level (100 mg/ kg). Among them, LCeMS/MS method combined with matrix solid-phase dispersion (Lu et al., 11) and pressurized liquid extraction (Yu et al., 11) have high sensitivity, which can be used for determination of SAs residues in food at the MRL level (10 mg/ kg). Recently, an isotope dilution-lc-ms/ms method for the determination of six SAs, two metabolites and trimethoprim in wastewater (Le-Minh, Stuetz, & Khan, 12). Development of selective multiresidue method for high throughput simultaneous determination of SAs, acetyled metabolites and trimethoprim in food is required. This paper develops a high sensitive approach enabling simple and rapid multiresidue analysis of 21 analytes including sixteen SAs, four acetyled metabolites, and trimethoprim. The analytes were extracted with ethyl acetate from pork and mutton samples under ultrasonic vibration, and then the extract was cleaned up through SPE, followed by LC-MS/MS detection. The evaluated LOD and LOQ values ranged from 0.1 to 0.3 mg/kg, and from 0.35 to 1.0 mg/kg in pork and mutton, respectively. These values are far lower than the MRLs set by several control authorities. 2. Materials and methods 2.1. Chemicals, reagents and solutions Seventeen SAs (purity: 99%) were purchased from DR.HIELSCHER Co. (Germany), and N 4 -acetylsulfamethazine, N 4 - acetylsulfamerazine, N 4 -acetylsulfamerazine, and N 4 -acetylsulfadiazine (purity: 98%) were purchased from Toronto Research Chemicals Inc. (Canada). Methanol, acetonitrile, ethyl acetate, and formic acid (chromatographic grade) were obtained from Alfa Aesar (Tianjin, China). Other reagents are of analytical grade. Mixed stock solution of twenty one analytes at concentrations of 1 mg/l of each compound was prepared in methanol. The stock and working standard solutions were stored at 4 C in a refrigerator. Water used in solution preparation was purified on an MYQ-sub-boiling distilling water purification system (Changsha, China). 2.2. Instrument An Agilent 10-6410 series rapid resolution liquid chromatography-triple quadrupole tandem mass spectrometry system (Agilent, USA) was used. Data acquisition and evaluation were carried out using ChemStation software (Agilent Technologies). The following instruments were used for sample preparation and cleanup: An SPE-30 Four Chunnel Full Aut SPE instrument (Tianjin BNAJE Science and Technique Co. Ltd.), Ultrasonic cleaner (Ultrasonic Instrument Co., Kunshan, China), RE3000A rotary vacuum evaporator (Shanghai Yarong biochemical instrument factory, China), CT15RT high-speed refrigerated centrifuge (Shanghai Tianmei Com. China), T Basic homogenizer (IKACom. Germany), and Nitrogen concentrator (Organomation Com. USA). An XGJ-30 highly pure water machine (Yongcheng purification Science & Technology Co. Ltd., Beijing, China) was used. 2.3. Extraction and cleanup Five gram of homogenized pork and mutton samples was weighed into a 50 ml conical flask, and ml of ethyl acetate was added. The sample was homogenized with a vortex stirrer for 30 s, and then extracted by ultrasonic vibration for 10 min, followed by centrifuging at 15,000 rpm for 10 min. The supernatant was taken, and the residue was extracted again with 5 ml acetone. The supernatant obtained two times was moved in a separatory funnel with 10 g of anhydrous sodium sulphate, and filtered. Antifoaming agent isopropanol of 1 ml was added in the collected filtrate, and then concentrated in vacuo to dryness at 30 C, the residue was dissolved with 2 ml methanol, and the solution was used for cleanup further. After the AccuBONDII SCX cartridge (100 mg, 3 ml, Agilent, USA) was preconditioned by adding 5 ml of acetic acid (4%, v/v) in acetonitrile, the sample solution was passed through the cartridge at a flow rate of 1 ml/min. After sample loading, the column was washed sequentially with 5 ml methanol and 5 ml acetonitrile. The analytes were eluted with 8 ml ammonia solution/acetonitrile (1:19, v/v); the eluate was collected in a 10 ml glass flask, and then evaporated to dryness under a stream of nitrogen at 30 C. The residue was dissolved in 1 ml mobile phase (0.1% formic acid 10 mm ammonium formateeacetonitrile). The final solution was used for LC determination. 2.4. CLeMS/MS conditions Agilent-Plus C 18 analytical column (100 mm 2.1 mm, 1.8 mm) was used for the separation of the 21 analytes. The flow rate of the mobile phase was set at 0. ml/min. A ml volume of sample solution was injected in the column. The mobile phase consisted of

362 H. Li et al. / Food Control 31 (13) 359e365 10 mm ammonium formatee0.1% formic acid (A) and acetonitrile (B). Gradient elution program was as follows: B increased linearly from 5% to 35% for 0 / 7 min, retained at 35% for 5 min, decreased from 35 % to 5% for 12 / 12.1 min, then retained at 5% for 12.1 / 16 min. Matrix-matched calibration curve was measured on RRLC-MS/MS within the concentration range of 1e0 mg/kg. The electrospray ionization source and multiple reactions monitoring (MRM) were applied where the parent ions and fragment ions were monitored at Q1 and Q3, respectively. Positive ionization mode was employed. The optimization of MS parameters (precursor ions, collision energy, collision cell exit potential, and quantification and confirmation transitions) was performed by flow injection analysis for each compound dissolved in mobile phase. Table 2 shows the values of the parameters optimized and the MRM transitions selected. 2.5. Validation method Quantitative analysis was performed in multi-selected reaction monitoring (MRM). Identification of the 21 analytes was accomplished by comparing the retention time (within 2%) and the ratio (within %) of the two selected precursor ion-production ion transitions with those of standards. The selectivity of the method was evaluated in triplicate using pork and mutton matrices. Linearity was studied from matrix-matched calibration, spiking blank extracts at 1, 5, 50, 100, and 0 mg/kg. Calibration curve was performed in triplicate using a least-square linear regression analysis at different concentrations. The regression equations were obtained using the 5- points concentration of standard as abscissa and area of chromatogram peak as vertical coordinate. The method limit of detection (LOD) was determined as the sample concentration that produces a peak with a height three times the level of the baseline noise, and the limit of quantification (LOQ) was calculated that produced a peak with 10 times the signal-to-noise ratio. Recovery and precision were assessed by performing test on spiked samples at three concentration levels 1.0, 5.0 and 50 mg/kg. The intra-day precision (as RSD) was investigated for six determinations at 50 mg/kg spiked level within aday. 3. Results and discussion 3.1. Choice of extraction conditions SAs are sparingly soluble in water, but easily dissolved in dilute acid, dilute base, and organic solvent. So methanol, acetonitrile, ethyl acetate, and dichloromethane were selected as possible extraction solvents. The extraction recoveries of the analytes in spiked pork samples were investigated. The result showed that Table 2 MS parameters for the detection of 21 compounds. No. Compound Molecular weight Retention time (min) Quantification transition (m/z) Confirmation transition (m/z) 1 Sulacetanide 214 0.471 215/156 215/156 215/92 2 Sulfadiazine 0 0.67 1/156 1/156 1/108 3 Sulfapyriline 249 0.961 0/156 0/156 0/92 4 Sulfamerazine 264. 1.2 265/156 265/156 265/172 5 Sulfamethoxazole 3 2.413 4/156 4/156 4/108 6 Sulfameter 280 2.603 281/156 281/156 281/108 7 Sulfisoxazole 267 2.961 268/156 268/156 268/113 8 Trimethoprim 290 3.241 291/230 291/230 291/123 9 Sulfamethoxypyridazine 280 4.134 281/156 281/156 281/108 10 Sulfamonomethoxine 280 4.219 281/156 281/156 281/108 11 Sulfadimethoxypyrimidine 310 5.316 311/156 311/156 311/108 12 Sulfamethazine 278 5.500 279/156 279/156 279/123 13 Sulfadimethoxine 310 6.042 311/156 311/156 311/108 14 Sulfadoxine 310 8.463 311/156 311/156 311/108 15 N 4 -acetylsulfadiazine 292 8.614 293/198 293/198 293/134 16 Sulfaphenazole 314 8.837 315/160 315/160 315/158 17 N 4 -acetylsulfamethoxazole 295 9.212 296/198 296/198 296/134 18 Sulfaquinoxaline 300 9.575 301/156 301/156 301/108 19 Sulfamethylthiazole 270 9.751 271/156 271/156 271/108 N 4 -acetylsulfamerazine 306 11.816 307/172 307/172 307/198 21 N 4 -acetylsulfamethazine 3 11.904 321/5 321/5 321/186 Fragment (V) Collision energy (ev) 1 10 110 30 110 10 15 1 10 15 100 10 1 15 1 10 10 130 130 15 1 15 110 1 15 15 1 15 110 10 110 30 10 110 15 110 30 10 110 10 1 10 110 35 35 1 10 30

H. Li et al. / Food Control 31 (13) 359e365 363 acetonitrile or ethyl acetate as an extraction solvent gave higher recoveries than the other solvents for the extraction of the 16 SAs, four acetyled metabolites, and trimethoprim in spiked pork samples. After the extraction with ethyl acetate, the extract was extracted again with acetone, the recovery increased further, preponderating over 78% for all 21 analytes, because acetone could destroy the interaction between protein and amido in SAs molecules. So ethyl acetate and acetone were selected for the extraction of the 21 analytes from the matrix. 3.2. Optimization of cleanup The cleanup procedure of the analyte extracts in blank pork sample spiked with 5 mg/kg for each analyte was investigated by using different SPE columns (AccuBONDII ODS-C 18, Oasis HLB and AccuBONDII SCX). The SPE columns were preconditioned by adding 5 ml of acetic acid (4%, v/v) in acetonitrile. After sample loading, the column was washed sequentially with 5 ml methanol and 5 ml acetonitrile, and the analytes were eluted with 3 ml ammonia solution/acetonitrile (1:19, v/v). Both C 18 column and HLB column with reversed-phase sorbents allowed to achieving the recoveries from 65% to 113%. SCX column with strong cation exchange sorbents can extract positively charged basic compounds. It allowed to achieving the recoveries from 81% to 109%, less interferent peaks and shortest time requirements, so that SCX column was selected in later work. acetonitrile. The result showed that the last mobile phase presented better separation effect than the others. Formic acid and ammonium formate could make the peak shapes to be sharp and symmetrical. It was observed that the recoveries of the analytes decreased notably when the ph of the mobile phase went down below 3.0. The mobile phase used in this work was a mixture solution of 10 mm ammonium formatee0.1% formic acid (A)e acetonitrile (B). The ph of the mobile phase was 3.21 and 3.72 for AeB of 95: 5 (v/v) and 65:35 (v/v), respectively. The satisfactory recoveries of the 21 analytes were achieved. The amount of acetonitrile in the mobile phase greatly affected the retentions of the 21 analytes, and acetonitrile at low concentration gave good separation of the SAs with lower molecular weight, but higher concentrations were required for the separation of the SAs with higher molecular weight. The result showed that 35% of acetonitrile in the mobile phase could not be exceeded. Good separation of the 21 SAs was obtained by using the gradient elution procedure given in Table 1 and flow rate of 0. ml/min. The total ion chromatograms of 21 standards (each ng/ml) and spiked blank matrix (0.5 mg/kg for each analyte) were observed, as shown in Fig. 1. Fig. 1 shows that the retention time of each analyte in blank matrix and standard solution is basically consistent, showing without matrix interference. Twenty one analytes including the isomers (sulfameter, sulfamethoxypyridazine and 3.3. Optimization of mobile phase The effect of different mobile phases on the separation of the 21 analytes was investigated, such as 10 mm ammonium acetatee methanol, 10 mm ammonium acetateeacetonitrile, 0.1% formic acidemethanol, and 0.1% formic acide10 mm ammonium formatee Fig. 1. TIC chromatogram of 21 analytes in standard solution of each ng/ml (above) and spiked blankpork matrixwitheach0.5 mg/kg (below).1. Sulacetanide, 2. Sulfadiazine, 3. Sulfapyriline, 4. Sulfamerazine, 5. Sulfamethoxazole, 6. Sulfameter, 7. Sulfisoxazole, 8. Trimethoprim, 9. Sulfamethoxypyridazine, 10. Sulfamonomethoxine, 11. Sulfadimethoxypyrimidine, 12. Sulfamethazine, 13. Sulfadimethoxine, 14. Sulfadoxine, 15. N 4 -acetylsulfadiazine, 16. Sulfaphenazole, 17. N 4 -acetylsulfamethoxazole, 18. Sulfaquinoxaline, 19. Sulfamethylthiazole,. N 4 -acetylsulfamerazine, 21. N 4 -acetylsulfamethazine. Fig. 2. Production process of parent ion and some fragment ions of sulfonamides, N 4 -acetyled metabolites and trimethoprim.

364 H. Li et al. / Food Control 31 (13) 359e365 Table 3 Analytical performance of the method. Analyte Linearity range (mg/kg) Regression equation r LOD (mg/kg) LOQ (mg/kg) Sulacetanide 1 0 y ¼ 5802.104835x þ 1682.265413 0.9993 0.1 0.35 Sulfapyriline 1 0 y ¼ 67.042628x 685.2685429 0.9991 0.1 0.35 Sulfadiazine 1 0 y ¼ 1598.351x þ 1126.07521 0.9986 0.3 1.0 Sulfamethoxazole 1 0 y ¼ 1017.557418xþ7.04527 0.9995 0.3 1.0 Sulfamerazine 1 0 y ¼ 5710.811402x 11.322346 0.9993 0.3 1.0 Sulfisoxazole 1 0 y ¼ 4666.437752x 3685.542141 0.9984 0.1 0.35 Sulfamethylthiazole 1 0 y ¼ 5303.406183x þ 132.2454 0.9991 0.1 0.35 Sulfamethazine 1 0 y ¼ 3896.726719x 635.21454 0.9983 0.1 0.35 Sulfamonomethoxine 1 0 y ¼ 1642.883770x þ 165.3412 0.9996 0.2 0.67 Sulfamethoxypyridazine 1 0 y ¼ 2867.822242x þ 6523.227232 0.9994 0.2 0.67 Sulfameter 1 0 y ¼ 3994.050807x 227.268247 0.9991 0.2 0.67 Trimethoprim 1 0 y ¼ 3241.4003x 426.110246 0.9985 0.2 0.67 Sulfaquinoxaline 1 0 y ¼ 4664.976055x 3075.4217 0.9991 0.2 0.67 Sulfadimethoxypyrimidine 1 0 y ¼ 4387.722268x 2365.4375 0.9996 0.1 0.35 Sulfadimethoxine 1 0 y ¼ 5340.845660x þ 826.547459 0.9991 0.1 0.35 Sulfadoxine 1 0 y ¼ 2875.430953x þ 129.352462 0.9986 0.1 0.35 Sulfaphenazole 1 0 y ¼ 2790.800343x 652.352419 0.9995 0.2 0.67 N 4 -acetylsulfadiazine 1 0 y ¼ 1627.983990x 169.354716 0.9992 0.3 1.0 N 4 -acetylsulfamethoxazole 1 0 y ¼ 1483.906632x 162.3652874 0.9996 0.3 1.0 N 4 -acetylsulfamerazine 1 0 y ¼ 1166.894347x 1685.39571 0.9993 0.3 1.0 N 4 -acetylsulfamethazine 1 0 y ¼ 2855.198508x þ 2286.2267521 0.9997 0.3 1.0 sulfamonomethoxine; sulfadimethoxypyrimidine, sulfadimethoxine and sulfadoxine) could be baseline separated within 12 min. 3.4. Mass spectrum resolution Based on chemical ionization character of sulphonamides with a secondary amino group or tertiary amino groups, ESI source and positive ionization mode were selected. MRM was applied where the parent ions and fragment ions were monitored at Q1 and Q3, respectively. The parent ion [M þ H] þ with higher abundant was obtained, The selected product ions constituted the ion pair for qualitative analysis, and the ion pair with parent ions for quantification (see Table 2). Production process of the parent ions and some fragment ions is shown in Fig. 2. Typical fragment ions [MeRNH 2 ] þ (m/z ¼ 156)for most SAs were observed, except for sulfaphenazole{[c 9 N 3 H 10 ] þ (m/z 160)}. Typical fragment ions of N 4 -acetylsulfadiazine and N 4 -acetylsulfamethoxazole were [MeRNH 2 ] þ (m/z 198), and typical fragment ions of N 4 -acetylsulfamerazine and N 4 -acetyl-sulfamethazine were [M þ HeC 8 NOH 8 ] þ (m/z 172) and [MeNH 2 eso 2 ] þ (m/z 5), respectively. Typical fragment ion of trimethoprim was [Mþ2He C 2 O 2 H 6 ] þ (m/z 230). 3.5. Validation study We have mainly considered the items related to the estimation of the well known performance characteristics parameters. The following criteria were considered: selectivity, sensitivity (limits of detection and quantification), and linearity of the response function, precision, and recovery. The selectivity of the method was evaluated in triplicate using pork and mutton matrices; no interference peaks were found at the retention times of the analytes studied. Linearity was studied from matrix-matched calibration, spiking blank extracts at the five concentration levels. The regression equations were obtained using the 5-points concentration of Table 4 Determination of each analyte in pork and mutton spiked at three concentration levels. Analyte Added 1.0 mg/kg Added 5.0 mg/kg Added 50.0 mg/kg Recovery (%) RSD a (%) Recovery (%) RSD a (%) Recovery (%) RSD a (%) Intra-day RSD a (%) Sulacetanide 80.8 5.4 90.3 6.9 97.4 8.0 8.4 Sulfapyriline 74. 1 8.6 87.6 7.2 103 4.8 5.2 Sulfadiazine 70.1 3.7 83.2 5.7 96.2 5.1 5.3 Sulfamethoxazole 73.2 3.8 92.6 7.2 103 6.3 6.0 Sulfamerazine 77.5 5.1 87.1 6.6 93.5 5.5 6.1 Sulfisoxazole 74.4 5.4 88.5 4.8 93.7 3.9 4.2 Sulfamethylthiazole 81.4 9.0 104 5.4 82.8 5.7 6.7 Sulfamethazine 69.1 6.2 101 5.6 83.1 7.3 8.0 Sulfamonomethoxine 83.8 7.4 103 7.9 77.5 4.2 5.0 Sulfamethoxypyridazine 86.7 3.5 101 9.2 98.0 4.8 5.0 Sulfameter 74.2 8.3 99.6 4.8 88.6 4.3 4.9 Trimethoprim 84.1 6.9 79.8 4.6 83.1 6.8 7.2 Sulfaquinoxaline 68.8 6.8 104 6.5 85.6 8.5 8.0 Sulfadimethoxypyrimidine 80.6 7.5 99.4 5.5 97.1 7.0 7.5 Sulfadimethoxine 68.3 7.5 92.1 8.3 89.2 5.2 6.1 Sulfadoxine 85.4 5.3 89.5 5.3 85.9 5.7 6.7 Sulfaphenazole 80.5 7.3 89.3 8.7 101 4.9 5.6 N 4 -acetylsulfadiazine 79.2 6.8 91.4 7.9 79.5 9.0 9.2 N 4 -acetylsulfamethoxazole 73.8 7.2 95.0 4.2 87.3 7.6 8.4 N 4 -acetylsulfamerazine 69.5 4.4 103 6.1 103 4.7 5.6 N 4 -acetylsulfamethazine 77.9 4.9 86.4 4.7 94.7 8.4 8.9 a n ¼ 6.

H. Li et al. / Food Control 31 (13) 359e365 365 standard. Table 3 shows that the good linearity was obtained for the 21 analytes with the correlation coefficient of >0.998. The LODs and LOQs of this method were listed Table 3. The LODs of 21 analytes were in the range of 0.1e0.3 mg/kg, and the LOQs were in the range from 0.35 to 1.0 mg/kg. The LOD values of the method are better than those reported by literature in Table 1, and can ensure to detect studied drugs at the MRL of 10 mg/kg. The whole procedure was applied to 10 blank samples of pork to verify the specificity of the method showing that no interference was detected around the retention times of the analytes in any of the samples analysed. Recovery and precision (Table 4) were assessed by performing test on spiked samples at three concentration levels 1.0, 5.0 and 50 mg/kg. The recovery of 21 analytes was obtained to be 68.3e104%, with RSD (n ¼ 6) of 3.5e9.2%. The intraday precision (as RSD) for six determinations at 50 mg/kg spiked level within a day was in range of 4.2e8.9%. 3.6. Application In this study, the applicability of this extraction method to real samples including pork and mutton was investigated at the optimized extraction conditions. No target analyte was detected in pork and mutton samples taken from different local markets. Hence, the spiked pork and mutton samples were studied. None of the commercial samples analysed yielded a positive result for the analytes. The contents of 16 SAs, 4 acetyled metabolites, and trimethoprim in pork and mutton were all below the LOQ of the proposed method. 4. Conclusion The LOD values of the proposed method are lower than those reported in the literature, it can ensure to detect studied drugs at the MRL of 10 mg/kg. 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