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Jun 5, 2017 - ABSTRACT: The purpose of this study was to develop and validate a simple, fast, and specific extraction method for the analysis of 64 an...
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A Simple and Fast Extraction Method for the Determination of Multiclass Antibiotics in Eggs Using LC-MS/MS Kun Wang,† Kunde Lin,† Xinwen Huang,‡ and Meng Chen*,† †

State Key Laboratory of Marine Environmental Science, Key Laboratory of the Ministry of Education for Coastal and Wetland Ecosystem, College of the Environment and Ecology, Xiamen University, Xiamen 361102, China ‡ College of Environment, Zhejiang University of Technology, Hangzhou 310032, China S Supporting Information *

ABSTRACT: The purpose of this study was to develop and validate a simple, fast, and specific extraction method for the analysis of 64 antibiotics from nine classes (including sulfonamides, quinolones, tetracyclines, macrolides, lincosamide, nitrofurans, β-lactams, nitromidazoles, and cloramphenicols) in chicken eggs. Briefly, egg samples were simply extracted with a mixture of acetonitrile−water (90:10, v/v) and 0.1 mol·L−1 Na2EDTA solution assisted with ultrasonic. The extract was centrifuged, condensed, and directly analyzed on a liquid chromatography coupled to tandem mass spectrometry. Compared with conventional cleanup methods (passing through solid phase extract cartridges), the established method demonstrated comparable efficiencies in eliminating matrix effects and higher or equivalent recoveries for most of the target compounds. Typical validation parameters including specificity, linearity, matrix effect, limits of detection (LODs) and quantification (LOQs), the decision limit, detection capability, trueness, and precision were evaluated. The recoveries of target compounds ranged from 70.8% to 116.1% at three spiking levels (5, 20, and 50 μg·kg−1), with relative standard deviations less than 14%. LODs and LOQs were in the ranges of 0.005−2.00 μg·kg−1 and 0.015−6.00 μg·kg−1 for all of the antibiotics, respectively. A total of five antibiotics were successfully detected in 22 commercial eggs from local markets. This work suggests that the method is suitable for the analysis of multiclass antibiotics in eggs. KEYWORDS: antibiotics, eggs, extraction optimization, multiclass analysis, LC-MS/MS

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INTRODUCTION Antibiotics are antibacteria drugs, which were originally used in human or animals to cure microbial infection diseases or as food additives in animal feeding to improve productive efficiency.1,2 However, inappropriate or abusive use of antibiotics in farm animals might provoke their residues in food of animal origin and cause allergic reactions and bacterial resistance.3,4 The amount of manufactured antibiotics in China has increased rapidly in recent years.5 About 18,100 tons of antibiotics were applied in bird breeding in 2013, accounting for more than 10% of the total usage. Studies have shown that antibiotics may be transferred and accumulated in chicken eggs,6−8 which are wholesome, cheap, and easily available foodstuff. To ensure human consumers’ safety, several international organizations, such as the European Union, have established maximum residue limits (MRLs) of antibiotics in eggs.9 For instance, MRLs of tetracycline, chlorotetracycline, and tylosin in eggs have been set at 200 μg·kg−1, while the acceptable level of lincomycin was 50 μg·kg−1.9 In the past decade, an emerging trend in antibiotic analysis is to develop novel and reliable methods for the simultaneous determination of a wide range of compounds.10,11 However, antibiotics comprise multiclass compounds with various chemical structurals and amphoteric properties and polarity, which greatly challenge the methods used for extraction, cleanup, and instrumental analysis. Liquid chromatography coupled to mass spectrometry in tandem (LC-MS/MS) has unique advantages in the analysis of multiresidue antibiotics in food,12−19 for both screening and quantitative methods. Several © 2017 American Chemical Society

extraction procedures, including solid-phase extraction (SPE),20,18 matrix solid-phase dispersion (MSPD),14,21 pressurized liquid extraction,15 and Quick, Easy, Cheap, Effective, Rugged and Safe (QuEChERS) approach,14,22 have been applied to remove or to reduce the interference and suppression resulting from the high lipid and protein content in eggs (Table S1). However, SPE and MSPD are laborintensive, time-consuming, and inefficient in sample throughput. QuEChERS is a fast and inexpensive method, but failed to extract the very important antibiotics of tetracyclines and quinolones.14 Frenich et al.14 compared four common methods (SPE, MSPD, QuEChERS, and solvent extraction) and found that the solvent extraction procedure was the most suitable approach for the simultaneous extraction of antibiotics from eggs. In recent years, solvent extraction was further purified by passing the extract through SPE cartridges.23−25 For example, HLB,14 silica,12 and Hybrid26 SPE cartridges have been employed in analyzing more than 100 compounds belonging to more than 10 different classes of antibiotics in eggs. However, the recoveries of target antibiotics generally varied with different SPE cartridges used for the purification. For example, silica cartridges failed to extract tetracyclines and βlactams.12 Therefore, there is a great need to establish a more Received: Revised: Accepted: Published: 5064

April 18, 2017 June 2, 2017 June 5, 2017 June 5, 2017 DOI: 10.1021/acs.jafc.7b01777 J. Agric. Food Chem. 2017, 65, 5064−5073

Article

Journal of Agricultural and Food Chemistry

Figure 1. Effect of Na2EDTA on the recoveries of target compounds in egg samples spiked with 20 μg·kg−1 of antibiotics. Conditions: the extraction mixture was 7.5 mL of ACN−H2O (90:10, v/v) with 0.1% formic acid (v/v); the concentration of 0.5 mL of Na2EDTA varied from 0 to 0.1 mol·L−1; the extraction solvent did not pass through any SPE cartridge. The abbreviations of antibiotics are the same as given in Table 1. 1 to 5 mg·L−1. The standard solutions were all stored at −20 °C prior to use. Other chemicals used were of analytical or high performance liquid chromatography (HPLC) grade. Methanol (>95%) and acetonitrile (ACN, >95%) of HPLC grade were purchased from Tedia (Fairfield, IA, USA). Disodium ethylenediaminetetraacetate (Na2EDTA) was purchased from Xilong Chemical (Shantou, Guangdong, China). Formic acid was purchased from Sinopharm Chemical (Shanghai, China). Reagent water (18.2 MΩ resistivity) was prepared with a Milli-Q water purification system (Millipore, Bedford, MA, USA). Oasis HLB SPE cartridges (60 mg, 3 mL) and PRiME HLB cartridges (60 mg, 3 mL) were purchased from Waters (Milford, MA, USA). C18 cartridges (200 mg, 6 mL) were purchased from SigmaAldrich. Sample Extraction. Blank egg samples for the method development and validation tests were previously analyzed to confirm that they were free from target antibiotics. To do extraction, six fresh eggs (yolk and albumen combined) were homogenized at room temperature under continuous agitation for 5 min and were stored at 4 °C before extraction. Two grams of homogenized sample was weighed into a 50 mL polypropylene centrifuge tube and spiked with 20 μL of the working standard solution. After continuous vortexing for 30 s, the tube was

universal extraction and cleanup method for the analysis of multiclass antibiotics in eggs. The objective of the present work was to develop a simple, fast, and specific extraction method for simultaneous determination of multiclass antibiotics in eggs. The extraction method involved a simple extraction using a mixture followed by a high-speed centrifugation. The composition of the extraction mixture was optimized. Results obtained from the present work were also compared with conventional methods of passing through SPE cartridges. Finally, the established method was applied in the analysis of antibiotics in chicken eggs from local supermarkets.

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MATERIALS AND METHODS

Chemicals and Reagents. Analytical standards of sulfonamides, quinolones, tetracyclines, macrolides, lincosamide, nitrofurans, βlactams, nitromidazoles, cloramphenicols, sulfathiazole-13C, and chloramphenicol-d5 were purchased from Dr Ehrenstorfer GmbH (Ausgburg, Germany) and Sigma-Aldrich (St. Louis, MO, USA) (Table S2). Individual stock solutions were prepared by dissolving each standard in methanol at 1000 mg·L−1, and the concentrations of each individual standard in the working standard solution varied from 5065

DOI: 10.1021/acs.jafc.7b01777 J. Agric. Food Chem. 2017, 65, 5064−5073

Article

Journal of Agricultural and Food Chemistry

Figure 2. Effect of formic acid on the recoveries of target compounds in egg samples spiked with 20 μg·kg−1 of antibiotics. Conditions: the extraction mixture was 7.5 mL of ACN−H2O (90:10, v/v) with different percentage of formic acid (0, 0.1%, 1%, v/v); the concentration of 0.5 mL of Na2EDTA was 0.1 mol·L−1; the extraction solvent did not pass through any SPE cartridge. The abbreviations of antibiotics are the same as given in Table 1. column (C18; particle size 2.7 μm) at 30 °C. The mobile phase for the negative ionization mode (ESI−) detection consisted of H2O and ACN, while a mobile phase consisting of H2O and ACN containing 0.1% formic acid was used for the positive ionization mode (ESI+) detection. The injection volume was 10 μL. Other major LC parameters for the two separate runs (ESI+ and ESI−) are given in Tables S3 and S4. For MS/MS detection, the ESI source was performed in both ESI− and ESI+ under the following conditions: dry gas (nitrogen) temperature, 160 °C; dry gas flow, 16 L·min−1; sheath gas (nitrogen) temperature, 350 °C; sheath gas flow, 12 L·min−1; nebulizer pressure, 35 psi; nozzle voltage, 1500 V; capillary voltage, 4000 V; fragmentation voltage, 380 V. The values of collision energy, transitions, and data acquisition time segments for the multiple-reaction monitoring (MRM) mode are given in Table S2. Validation and Quantification of the Analytical Method. The analytical method was validated according to CAC/GL 71-2009 and EU Commission Decision 2002/657/EC.27,28The validation parameters of the solvent extraction procedure involved specificity, linearity, matrix effect, limits of detection (LODs), limits of quantification (LOQs), the decision limit (CCα), detection capability (CCβ), and

allowed to stay at room temperature for 2 h. Then, 7.5 mL of ACN− H2O (90:10, v/v) and 0.5 mL of 0.1 mol·L−1 Na2EDTA solutions were added to the sample. The sample was subsequently homogenized by a high-speed dispersion rotor (IKA T18 digital ULTRA TURRAX, Germany) at 7000 rpm for 90 s, ultrasonically oscillated for 15 min, and centrifuged at 6000g and at 4 °C for 10 min. Five milliliters of the supernatant was collected and evaporated under a gentle stream of nitrogen to dryness at 45 °C. The resulting residue was redissolved in 1 mL of ACN−H2O (20:80, v/v) and centrifuged at 10000g for 15 min. The supernatant was filtered through a PVDF syringe filter (0.2 μm pore size), and the filtrate was collected for LC-MS/MS analysis. To evaluate cleanup effect of SPE cartridges, 5 mL of the supernatant from extraction was passed through SPE cartridges (preconditioned with 3 mL of methanol and 3 mL of extraction mixture) before evaporation to dryness at 45 °C. The other operation was identical to the procedures described above. Instrumentation and Parameters. Antibiotic analysis was carried out on an Agilent 1290 Infinity LC system coupled to an Agilent 6490 triple quadrupole mass spectrometer equipped with an electrospray ionization (ESI) source (Agilent, Palo Alto, CA, USA). Separation was achieved on a Proshell 120 EC-C18 column (100 mm × 2.1 mm i.d., particle size 2.7 μm, Agilent) protected with a guard 5066

DOI: 10.1021/acs.jafc.7b01777 J. Agric. Food Chem. 2017, 65, 5064−5073

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Journal of Agricultural and Food Chemistry

Figure 3. Recoveries of samples with/without cleanup by passing through SPE cartridges. Blank eggs were spiked with 20 μg·kg−1 of antibiotics. Conditions: the extraction mixture was 7.5 mL of ACN−H2O (90:10, v/v) with 0.1% formic acid (v/v); the concentration of 0.5 mL of Na2EDTA was 0.1 mol·L−1; the extraction solvent passed through different SPE cartridges (no SPE, HLB, C18, PRiME). The abbreviations of antibiotics are the same as given in Table 1. trueness and precision (repeatability and within-laboratory reproducibility). All the samples were subjected to strict quality control procedures. The concentrations of target compounds were calculated based on a matrix-matched calibration curve method by spiking corresponding amounts of target analytes into 1.00 mL of extract from blank egg matrices. Internal standards were also spiked to the samples prior to LC-MS/MS analysis to eliminate the fluctuation of the instrument.

compound should count for 4 identification points (IPs, one IP could be earned from a precursor ion and 1.5 IPs could be earned from a daughter ion), which confirms positive findings by MRM. For most of the target compounds, the daughter ion with the highest intensity was selected for quantification. However, only one daughter ion was selected for oxolinic acid because of the lack of other intensive daughter ions. Two gradient elution programs were optimized to enhance the separation of analytes. Formic acid (0.1%) was added into the mobile phase to fortify the ionization efficiency of antibiotics and improve chromatographic separation in the positive mode.29 Special attention should be paid to the structural isomers of sulfonamides and quinolone drugs, because these compounds could not be distinguished by selecting different precursor or daughter ions. Four subgroups of compounds, i.e., sulfameter and sulfamonomethoxine and sulfamethoxypyridazine, sulfacetamide and sulfaguanidine, sulfamethazine and sulfaisodimidine, and flumequine and oxolinic acid, each had identical precursor and daughter ions. Therefore, gradient elution programs must be employed to

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RESULTS AND DISCUSSION LC-MS/MS Optimization. To select and tune the precursor and daughter ions of target compounds, direct infusion of individual antibiotics was performed at the concentrations of 1−5 mg·L−1. The precursor ions for the major compounds were monitored as the protonated ([M + H]+) or deprotonated ([M − H]−) molecular ions because of their relatively high intensity. The compounds of sulfonamides, quinolones, tetracyclines, macrolides, lincomcycin, β-lactams, nitrofurans, and nitromidazoles were detected in the positive mode, whereas cloramphenicols were detected in the negative mode. According to EU Commission Decision 2002/657/EC,27 a 5067

DOI: 10.1021/acs.jafc.7b01777 J. Agric. Food Chem. 2017, 65, 5064−5073

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Journal of Agricultural and Food Chemistry Table 1. Matrix Effects, Recovery, and Precision of the Developed Method recovery ± RSDra (%, n = 4)

interday RSDRb (%, n = 6)

compounds

abbreviation

matrix effects (%)

5 μg·kg−1

20 μg·kg−1

50 μg·kg−1

5 μg·kg−1

20 μg·kg−1

50 μg·kg−1

sulfamethoxazole sulfamethizole sulfapyridine sulfathiazole sulfaquinoxaline sulfachlorpyridazine sulfamerazine sulfadimethoxine sulfameter sulfamonomethoxine sulfamethazine sulfadiazine sulfaisodimidine sulfacetamide sulfisoxazole sulfanitran sulfamethoxypyridazine sulfaguanidine trimethoprim ciprofloxacin norfloxacin enrofloxacin ofloxacin danofloxacin flumequine marbofloxacin sarafloxacin oxolinic acid lomefloxacin nalidixic acid difloxacin orbifloxacin fleroxacin enoxacin sparfloxacin pefloxacin oxytetracycline chlortetracycline tetracycline doxycycline demeclocycline anhydrotetracycline erythromycin tylosin tilmicosin spiramycin kitasamycin lincomycin amoxicillin ceftiofur penicillin G dicloxacillin oxacillin cloxacillin ampicillin penicillin V nafcillin furazolidone furaltadone

SMOZ SMTZ SPD STZ SQA SCPZ SMZ SDX SMT SMMX SMA SDZ SMD STM SXZ SNT SMPZ SGD TTP CIP NOR ENR OFX DNFX FLQ MFX SFX OA LFX NA DFX OBFX FRX EX SPX PFX OTC CTC TTC DCC DCTC ADTC ETM TYL TMS SPI KIT LIN AMO CFT PG DIC OXA CLOX AMP PV NAF FZD FTD

−5.6 8.7 10.5 −4.4 −28.0 −44.1 17.2 −23.1 −6.0 4.5 −8.9 −14.0 −25.6 −39.0 −2.3 −14.7 −13.5 −33.2 13.8 96.5 344.3 45.5 278.8 121.6 14.9 536.6 −38.4 26.0 9.5 5.2 119.1 0.0 19.5 96.0 −59.5 261.4 14.9 −5.0 24.1 −72.2 −29.7 −12.8 −66.6 −77.4 −50.1 −23.3 −65.0 10.1 −2.9 −0.9 −23.6 −33.4 −36.6 −34.4 16.7 −23.9 51.2 −48.9 −33.6

102.4 ± 6.8 103.2 ± 3.5 108.9 ± 5.3 101.8 ± 4.0 103.7 ± 10.5 91.5 ± 10.5 103.3 ± 6.3 100.5 ± 13.3 112.3 ± 3.2 105.4 ± 4.5 105.6 ± 2.7 107 ± 3.6 100.5 ± 2.2 103.7 ± 0.9 101.3 ± 5.9 96 ± 7.5 104.6 ± 2.9 107.7 ± 5.7 103.7 ± 2.7 106.4 ± 9.1 103.5 ± 3.9 104.3 ± 7.3 102.1 ± 6.0 99.3 ± 7.5 102.7 ± 6.6 107.9 ± 5.0 95.9 ± 12.9 109.4 ± 6.8 96.9 ± 3.0 106.4 ± 10.7 99.9 ± 13.1 105.6 ± 4.1 102 ± 3.9 98.2 ± 9.3 91.8 ± 11.8 106 ± 4.7 86 ± 3.1 88.5 ± 5.8 106.9 ± 4.6 98.9 ± 3.6 80.4 ± 12.4 99.5 ± 4.6 103.7 ± 12 107.3 ± 7.2 93 ± 11.3 88.9 ± 12.5 103.9 ± 8.2 97.1 ± 4.8 84.8 ± 3.9 82.2 ± 11.9 104.3 ± 5.2 108.2 ± 4.4 98 ± 5.4 103.3 ± 2.8 94.6 ± 7.0 −c 104.9 ± 5.4 108 ± 2.7 99.3 ± 2.4

103.2 ± 6.7 99.3 ± 5.2 100.3 ± 4.6 102.2 ± 8.2 94.6 ± 5.6 85.2 ± 8.1 108.4 ± 6.0 99.8 ± 4.7 101.5 ± 8.4 102.2 ± 4.2 106.4 ± 5.2 98.5 ± 4.0 104.4 ± 4.1 99.7 ± 4.8 102.3 ± 3.2 93.8 ± 5.0 111.8 ± 8.1 90.5 ± 2.7 102.4 ± 3.3 95.5 ± 4.0 94.2 ± 6.3 97.7 ± 4.3 100.3 ± 3.9 100.3 ± 5.6 110.2 ± 9.6 97 ± 4.1 92.4 ± 1.4 107.1 ± 4 92.6 ± 3.4 114.9 ± 5.9 103.8 ± 4.4 97.4 ± 6.5 97 ± 4.4 90.1 ± 9.8 84.6 ± 2.4 101.8 ± 3.4 79.9 ± 5.0 86.5 ± 6.0 101.6 ± 3.3 95.1 ± 6.9 75.4 ± 4.0 98.3 ± 4.4 98 ± 5.4 103.3 ± 4.3 84.8 ± 8.2 87.4 ± 7.4 107.8 ± 6.8 97.9 ± 5.4 81.8 ± 4.7 80.4 ± 5.7 102.4 ± 5.9 82.2 ± 7.6 97.3 ± 2.1 103.4 ± 4.3 96.7 ± 2.5 113.4 ± 10.6 100.6 ± 5.4 102.5 ± 6.4 93.5 ± 6.6

94.5 ± 2.3 91.9 ± 2.7 96.1 ± 4.8 93.8 ± 2.2 95 ± 1.4 89.1 ± 5.2 94.3 ± 4.4 90.8 ± 5.3 89.4 ± 3.6 93 ± 4.3 93.6 ± 3.8 88.6 ± 7.0 96.4 ± 0.8 90.5 ± 5.4 88.5 ± 5.3 84 ± 8.1 85.9 ± 1.3 79.6 ± 9.0 90 ± 2.0 80.1 ± 2.0 78.2 ± 5.3 91.2 ± 3.0 85.1 ± 6.0 92.5 ± 3.6 97.7 ± 2.9 91.5 ± 2.4 78.8 ± 10.0 93.7 ± 2.6 84.3 ± 1.7 87.6 ± 6.0 87.3 ± 4.9 84.8 ± 2.4 94.4 ± 3.9 73.4 ± 4.9 78.1 ± 5.6 91.3 ± 2.0 72.5 ± 3.8 72.7 ± 2.5 84.4 ± 3.5 79.4 ± 5.6 73.7 ± 4.4 85.9 ± 4.0 88.9 ± 4.0 88.5 ± 3.7 80.9 ± 10.6 79.4 ± 5.6 96.2 ± 7.2 79.8 ± 3.5 70.8 ± 1.0 71.5 ± 9.6 86.2 ± 2.4 86.4 ± 4.4 87.1 ± 0.8 89.7 ± 4.8 79.3 ± 3.0 89.7 ± 6.8 93.1 ± 3.0 94.2 ± 3.1 87.7 ± 6.1

6.8 8.4 7.4 7.2 4.9 4.8 9.0 7.2 9.1 6.3 7.5 7.6 9.1 2.3 8.8 8.8 8.8 2.0 8.7 7.4 6.0 6.9 5.1 4.8 9.7 5.6 7.2 7.3 4.7 8.6 12.3 6.5 6.0 5.0 6.2 6.9 4.9 9.2 7.4 4.7 9.0 6.0 5.9 11.2 5.3 4.8 10.4 8.3 14.2 9.6 4.4 8.3 2.4 5.4 4.9 8.6 5.0 4.4

9.2 12 10.5 14.0 6.4 5.7 11.8 8.9 11.5 10.3 10.9 9.2 10.4 8.4 9.5 11.2 9.7 9.9 9.5 12.4 11.8 11.4 10.6 13.0 7.1 4.7 14.9 9.7 11.1 9.4 15.9 10.2 11.4 11.8 11.7 11.7 10.2 11.7 13.0 11.3 10.5 9.7 7.1 8.7 8.6 10.0 9.8 11.6 14.3 14.2 10.8 8.7 13.7 11.0 8.9 12.7 14.4 13.9 10.0

3.6 3.8 4.0 5.1 2.2 6.2 7.7 5.5 5.2 4.5 3.0 5.9 2.9 4.6 5.4 6.9 4.7 6.1 3.5 10.9 5.9 3.3 4.0 3.2 7.0 2.2 10.2 2.3 2.5 4.7 12.2 2.4 3.6 5.3 10.3 2.9 3.8 5.4 4.0 5.8 10.8 5.4 6.7 5.7 10.6 10.4 3.8 4.2 7.3 9.2 5.2 3.9 5.4 6.5 7.9 6.6 5.8 4.5 4.7

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DOI: 10.1021/acs.jafc.7b01777 J. Agric. Food Chem. 2017, 65, 5064−5073

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Journal of Agricultural and Food Chemistry Table 1. continued recovery ± RSDra (%, n = 4) compounds chloramphenicol thiamphenicol florfenicol metronidazole dimetridazole a

abbreviation CAP TAP FF MNZ DMZ

matrix effects (%)

5 μg·kg−1

20 μg·kg−1

−18.4 2.2 −22.5 −35.2 −19.2

116.1 ± 2.0 101.3 ± 3.2 98.2 ± 1.1 97.5 ± 2.4 95.2 ± 3.2

95.2 ± 1.8 104.6 ± 3.3 113.5 ± 2.1 98.1 ± 4.6 89.9 ± 7.1

interday RSDRb (%, n = 6)

50 μg·kg−1

5 μg·kg−1

20 μg·kg−1

50 μg·kg−1

± ± ± ± ±

9.5 5.6 11.3 3.7 8.2

9.3 9.3 5.4 11.0 14.7

1.3 2.4 4.5 2.5 8.9

90.8 95.7 96.7 86.5 80.7

1.3 2.2 1.4 3.6 3.6

RSDr: repeatability values. bRSDR: intraday reproducibility values. cThe LOQ of PV was higher than 5 μg·kg−1.

monitoring lists,5 the extraction mixture was chosen to be a combination of 0.1 mol·L−1 Na2EDTA and ACN−H2O without the addition of formic acid. The extract of egg samples by solvent extraction method is generally cleaned up with SPE cartridges prior to LC-MS/MS analysis.14,26 In this study, extracts were centrifuged at 10000g rather than cleanup with SPE cartridges. This approach simplified the extraction procedures and shortened the time of sample treatment. The recoveries of the compounds in the samples cleaned up with SPE cartridges are illustrated in Figure 3, and their corresponding matrix effects are listed in Table S5. Most of the antibiotics (except some sulfonamides) displayed close recoveries in samples cleaned up with HLB and PRiME HLB cartridges. In comparison, samples cleaned up with C18 cartridges showed poor recoveries. Although passing the extract through SPE cartridges greatly reduced visual pigment and turbidity, no reduction was observed in matrix effects in LCMS/MS analysis (Table S5). Our results clearly demonstrated that higher or equivalent extraction efficiencies were achieved by centrifuge (Figure 3), suggesting some compound loss in the SPE purification process. Overall, solvent extraction and highspeed centrifuge provided satisfactory recoveries (64−119%) for most of the target antibiotics except erythromycin. Method Validation. Selectivity and Identification. Six blank egg samples from different brands were analyzed to evaluate the selectivity of the method. All the blank samples did not show any signals at the corresponding retention time, indicating the absence of chemical or matrix interferences. Identification of the antibiotics was first carried out by searching target compounds in appropriate retention time windows, which were determined by the mean retention time ± three standard deviations of the retention times from seven blank samples spiked with 50 μg·kg−1 of antibiotics.14,17 In this study, two internal standards (one in ESI+ mode and the other in ESI− mode) were spiked in the samples and the ratio of the relative retention time of analytes to that of its internal standard should be within 2.5% tolerance (Table S2). Furthermore, the acquisition parameters of most target compounds except oxolinic acid were identified with at least 4 IPs. Matrix Effects. Method matrix effects, or the ionization signals of suppression or enhancement for each compound during LC-MS/MS analysis, especially in the ESI mode, may result from various physical and chemical processes and are difficult to eliminate. To estimate matrix effects, the slope of the matrix-matched calibration curves was compared with those obtained in solvent without matrices, and matrix effect (ME%) was evaluated as

improve the separation of their four epimeric pairs. All of the four pairs were successfully separated under the elution program on a traditional C18 column (Figure S1). The optimal MS parameters (e.g., precursor ion, daughter ions, and collision energy) and retention times for the 64 targeted analytes and the two internal standards are summarized in Table S2. Optimization of Extraction Method. The composition of the extraction mixture played an irreplaceable role in solvent extraction. Both organic solvents and aqueous buffers have been widely used in solvent extraction. ACN and methanol are the most representative organic solvents due to their capacity to precipitate proteins and extract target compounds. Compared with methanol, ACN generally exhibited a higher extraction efficiency and better deproteinization ability.8,17,18,30 However, ACN could not efficiently extract polar analytes such as βlactams.31,32 Because aqueous solvents are more suitable for the extraction of polar compounds, a number of modified extraction methods adopted the combination of water or aqueous buffer with organic solvents.15,16 Thus, the mixture of ACN and H2O was chosen as extraction solvent in this study. During the extraction procedure, some antibiotics such as tetracyclines have a strong tendency to form complexes with metal ions in egg matrices,33,34 causing low extraction efficiencies of these compounds. It was therefore necessary to add Na2EDTA in the extract to improve the recovery of these antibiotics by masking the interfering metal ions. However, excessive Na2EDTA might also chelate with tetracyclines.35 It is apparent that a higher concentration of Na2EDTA enhanced the recovery of tetracyclines, quinolones, nitrofurans, β-lactams, and marcrolides, but had negligible effect on the other analytes (Figure 1). Using 0.5 mL of 0.1 mol·L−1 Na2EDTA for the extraction, the recoveries were 80.7% for oxytetracycline, 81.6% for chlortetracycline, 109.3% for tetracycline, 109.0% for doxycycline, 90.1% for demeclocycline, and 107.8% for anhydrotetracycline. However, none of these compounds was extracted without the addition of Na2EDTA. The recoveries might also be influenced by pH values, because some of the analytes are amphoteric compounds. A certain amount of formic acid (no formic acid, 0.1%, 1%, v/v) was added into 7.5 mL of ACN−H2O (90:10, v/v) to evaluate the possible pH effect (Figure 2). The recoveries of sulfonamides, tetracyclines, chloramphenicols, and β-lactams in the extract without formic acid were consistently higher than those fortified with 0.1% or 1% of formic acid. Similar results were also obtained in previous studies.17,36 On the contrary, quinolones and major macrolides exhibited higher recoveries when the extraction mixture was fortified with 0.1% of formic acid. It should be noted that the recoveries of erythromycin markedly decreased when extracted with mixtures in the presence of 0.1% or 1% of formic acid, likely due to the instability of erythromycin under acidic conditions.8 Given the priority of sulfonamides, tetracyclines, and β-lactams on

⎞ ⎛k ME% = 100 × ⎜ a − 1⎟ ⎠ ⎝ kb 5069

DOI: 10.1021/acs.jafc.7b01777 J. Agric. Food Chem. 2017, 65, 5064−5073

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Journal of Agricultural and Food Chemistry

Table 2. Limits of Detection (LODs), Limits of Quantification (LOQs), R2, Linear Range, CCα, and CCβ for the Analytical Instrument and Method (μg·kg−1) instrumental performance

method performance

compoundsa

MRLs

LODs

LOQs

R2

linear range

LODs

LOQs

R2

linear range

CCα

CCβ

SMOZ SMTZ SPD STZ SQA SCPZ SMZ SDX SMT SMMX SMA SDZ SMD STM SXZ SNT SMPZ SGD TTP CIP NOR ENR OFX DNFX FLQ MFX SFX OA LFX NA DFX OBFX FRX EX SPX PFX OTC CTC TTC DCC DCTC ADTC ETM TYL TMS SPI KIT LIN AMO CFT PG DIC OXA CLOX AMP PV NAF FZD

b

0.02 0.15 0.06 0.03 0.02 0.08 0.06 0.01 0.02 0.05 0.03 0.02 0.006 0.30 0.009 0.30 0.03 0.60 0.03 0.44 1.50 0.14 0.19 0.60 0.03 2.50 0.06 0.05 0.15 0.03 0.26 0.06 0.07 0.60 0.005 1.00 3.00 2.50 1.50 0.45 0.50 1.88 0.002 0.26 7.50 1.50 0.02 0.05 0.81 1.00 0.004 0.03 0.08 0.02 0.02 3.00 0.03 0.68

0.07 0.50 0.21 0.10 0.06 0.26 0.21 0.04 0.06 0.16 0.10 0.07 0.020 1.00 0.031 1.00 0.10 2.00 0.09 1.47 5.00 0.46 0.62 2.00 0.11 7.50 0.18 0.15 0.50 0.10 0.88 0.21 0.22 2.00 0.016 3.33 10.00 7.50 5.00 1.49 1.50 6.25 0.005 0.88 25.00 5.00 0.06 0.17 2.70 3.33 0.012 0.09 0.25 0.08 0.07 10.00 0.09 2.25

0.9981 0.9987 0.9981 0.9989 0.9984 0.9985 0.9970 0.9989 0.9985 0.9990 0.9996 0.9988 0.9995 0.9990 0.9989 0.9984 0.9974 0.9998 0.9984 0.9991 0.9986 0.9996 0.9997 0.9971 0.9990 0.9986 0.9987 0.9993 0.9977 0.9983 0.9991 0.9990 0.9947 0.9944 0.9991 0.9988 0.9989 0.9984 0.9981 0.9981 0.9988 0.9979 0.9993 0.9993 0.9981 0.9943 0.9998 0.9999 0.9984 0.9981 0.9998 0.9999 0.9993 0.9996 0.9998 0.9973 0.9990 0.9998

0.5−100 0.5−100 0.5−100 0.5−100 0.5−100 0.5−100 0.5−100 0.5−100 0.5−100 0.5−100 0.5−100 0.5−100 0.5−100 1−100 0.5−100 0.5−100 0.5−100 2−100 0.5−100 2−100 2−100 0.5−100 0.5−100 1−100 0.5−100 10−500 0.5−100 0.5−100 0.5−100 0.5−100 1−100 0.5−100 0.5−100 2−100 0.5−100 5−100 10−500 10−500 5−500 2.5−500 2−500 10−500 0.5−100 0.5−100 25−500 5−100 0.5−100 0.5−100 4−200 10−500 1−200 1−200 1−200 1−200 1−200 10−200 1−200 2.5−500

0.03 0.13 0.08 0.03 0.07 0.07 0.10 0.02 0.02 0.04 0.03 0.09 0.06 0.60 0.03 0.72 0.02 1.13 0.01 0.11 0.17 0.04 0.02 0.04 0.007 0.10 0.11 0.02 0.08 0.06 0.08 0.01 0.02 0.09 0.007 0.05 0.31 1.00 0.28 0.55 0.26 0.33 0.005 0.07 0.75 0.75 0.15 0.02 1.20 1.00 0.005 0.01 0.04 0.02 0.010 2.00 0.02 0.45

0.11 0.43 0.26 0.11 0.23 0.22 0.33 0.07 0.06 0.13 0.11 0.28 0.18 2.00 0.11 2.50 0.08 3.76 0.04 0.37 0.56 0.13 0.07 0.14 0.025 0.30 0.38 0.05 0.28 0.20 0.25 0.03 0.07 0.30 0.024 0.17 1.03 3.33 0.94 1.84 0.87 1.11 0.016 0.24 2.50 2.50 0.50 0.07 4.00 3.00 0.015 0.04 0.13 0.07 0.033 6.66 0.06 1.50

0.9983 0.9985 0.9994 0.9987 0.9994 0.9992 0.9984 0.9992 0.9999 0.9990 0.9991 0.9999 0.9990 0.9996 0.9988 0.9992 0.9984 0.9999 0.9994 0.9982 0.9990 0.9990 0.9992 0.9967 0.9991 0.9990 0.9991 0.9998 0.9991 0.9996 0.9996 0.9991 0.9993 0.9986 0.9994 0.9990 0.9998 0.9988 0.9989 0.9994 0.9997 0.9989 0.9995 0.9995 0.9997 0.9980 0.9990 0.9992 0.9986 0.9977 0.9999 0.9989 0.9992 0.9987 0.9995 0.9992 0.9990 0.9995

0.5−100 0.5−100 0.5−100 0.5−100 0.5−100 0.5−100 0.5−100 0.5−100 0.5−100 0.5−100 0.5−100 0.5−100 0.5−100 2−100 0.5−100 5−100 0.5−100 5−100 0.5−100 0.5−100 0.5−100 0.5−100 0.5−100 0.5−100 0.5−100 2.5−500 0.5−100 0.5−100 0.5−100 0.5−100 0.5−100 0.5−100 0.5−100 0.5−100 0.5−100 0.5−100 2.5−500 5−500 2.5−500 2.5−500 1−500 2.5−500 0.5−100 0.5−100 2.5−500 5−100 0.5−100 0.5−100 4−200 5−500 1−200 1−200 1−200 1−200 1−200 10−200 1−200 2.5−500

2.29 2.37 1.29 1.77 1.52 1.70 2.42 1.45 1.10 1.31 2.33 0.85 2.58 1.56 1.16 2.62 2.37 2.65 1.77 3.62 2.35 2.46 2.14 3.28 1.83 1.80 2.99 1.17 1.73 1.21 3.13 1.98 1.91 2.72 1.67 2.43 204.21 205.35 203.92 10.67 9.00 7.66 150.98 201.11 2.58 4.40 2.59 50.62 5.99 5.54 0.64 4.11 2.79 3.53 0.71 25.30 3.61 1.90

2.72 3.07 1.60 2.27 1.86 1.99 2.80 1.76 1.26 1.48 2.81 1.04 2.98 1.75 1.44 4.68 3.01 4.39 2.01 4.21 2.59 2.72 2.40 3.68 2.21 2.00 3.43 1.44 1.84 1.45 3.81 2.14 2.10 3.17 1.94 2.60 222.60 222.27 215.85 11.85 16.78 8.79 160.88 213.31 3.77 5.97 2.98 57.27 7.07 6.08 0.76 5.24 3.27 4.05 0.78 30.67 4.23 2.08

F F F F F F F F F F F F F F F F F F F F

F F

F

200 200 200 F

150 200 F F 50 F F F F F F 25 F F

5070

DOI: 10.1021/acs.jafc.7b01777 J. Agric. Food Chem. 2017, 65, 5064−5073

Article

Journal of Agricultural and Food Chemistry Table 2. continued instrumental performance compounds

a

FTD CAP TAP FF MNZ DMZ

MRLs F F F F F

2

method performance

LODs

LOQs

R

linear range

LODs

LOQs

R2

linear range

0.23 0.01 0.03 0.004 0.20 0.09

0.76 0.04 0.09 0.014 0.68 0.30

0.9990 0.9989 0.9994 0.9984 0.9997 0.9994

2.5−500 0.5−100 2.5−500 2.5−500 0.5−100 0.5−100

0.11 0.03 0.25 0.04 0.12 0.08

0.35 0.08 0.85 0.15 0.40 0.25

0.9996 0.9994 0.9984 0.9985 0.9993 0.9994

2.5−500 0.5−100 2.5−500 2.5−500 0.5−100 0.5−100

CCα

CCβ

3.21 2.21 9.24 7.00 4.44 1.83

3.70 2.46 11.10 8.59 5.02 2.09

a The abbreviations of antibiotics are the same as given in Table 1. bForbidden to be used in animals from which eggs are produced for human consumption.

Table 3. Determination of Antibiotics in Egg Samples (n = 22) from Local Supermarkets retention time (min) samples

analyte detected

daughter ion

incurred samples

standard

concn (μg·kg−1)

MRLs (μg·kg−1)

EGG 3 EGG 21

orbifloxacin sulfaquinoxaline ciprofloxacin enrofloxacin doxycycline ciprofloxacin enrofloxacin doxycycline

295.0/352.1 156.0/208.0 314.4/288.1 342.1/316.1 428.1/410.1 314.4/288.1 342.1/316.1 428.1/410.1

14.363 19.149 11.819 14.388 16.975 11.819 14.376 16.987

14.351 19.161 11.793 14.328 16.963 11.793 14.328 16.963

0.26