Article pubs.acs.org/JAFC
Antibiotic Residue Monitoring Results for Pork, Chicken, and Beef Samples in Vietnam in 2012−2013 Takahiro Yamaguchi,† Masahiro Okihashi,† Kazuo Harada,*,‡ Yoshimasa Konishi,† Kotaro Uchida,† Mai Hoang Ngoc Do,§ Huong Dang Thien Bui,§ Thinh Duc Nguyen,§ Phuc Do Nguyen,§ Vien Van Chau,# Khanh Thi Van Dao,# Hue Thi Ngoc Nguyen,# Keiji Kajimura,† Yuko Kumeda,† Chien Trong Bui,# Mai Quang Vien,# Ninh Hoang Le,§ Kazumasa Hirata,‡,⊥ and Yoshimasa Yamamoto†,⊥ †
Osaka Prefectural Institute of Public Health, 1-3-69 Nakamichi, Higashinari-ku, Osaka 537-0025, Japan Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan § Institute of Public Health, 159 Hung Phu, District 8, Ward 8, Ho Chi Minh City, Vietnam # Pasteur Institute, 8-10 Pasteur Xuong Huan Ward, Nha Trang City, Vietnam ⊥ Global Collaboration Center, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan ‡
ABSTRACT: A monitoring plan of residual antibiotics in food of animal origin was conducted in Vietnam from 2012 to 2013. Meat samples were collected from slaughterhouses and retail stores in Ho Chi Minh City and Nha Trang. A total of 28 antibiotics were analyzed using a LC-MS/MS screening method. Sulfonamides, fluoroquinolones, and tilmicosin were detected in some of the samples. Sulfaclozine and fluoroquinolones were mainly detected in chicken samples, and sulfamethazine was mainly detected in pork samples. High levels of sulfonamide residues, ranging between 2500 and 2700 μg/kg sulfaclozine and between 1300 and 3600 μg/kg sulfamethazine, were present in two chicken and three pork samples, respectively. Tilmicosin was detected at ranges of 150−450 μg/kg in 10 chicken samples. Positive percentages were 17.3, 8.8, and 7.4% for chicken, pork, and beef, respectively, for an average of 11.9%. The results suggest an appropriate withdrawal period after drug administration had not been observed in some livestock. KEYWORDS: residual antibiotics, Vietnam, food, liquid chromatography−tandem mass spectrometry
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INTRODUCTION
stockbreeding farms, and it is difficult to regulate the use of antibiotics on the many small farms. Resistant bacteria carriers are rapidly increasing in Asia,9,10 particularly in Vietnam, where there are higher carriage ratios compared with other countries. Currently, 42% of the population carry drug-resistant bacteria, which leads to growing concerns over further diffusion.11 The threat of resistant bacteria has been caused by the abuse of antibiotics in medicines and in stockbreeding and aqua farming.12−14 Residual antibiotics in food and the spread of resistant bacteria are a cause for concern in human health and the environment.15,16 Therefore, we investigated residual antibiotics in meats at Ho Chi Minh City (HCM) and Nha Trang (NT). All samples were transferred to the Institute of Public Health, Ho Chi Minh City, for analysis using a liquid chromatography−tandem mass spectrometry (LCMS/MS) method. We discuss the risk to human health from residual antibiotics in meat from Vietnam.
Antibiotics have been widely used in the treatment and prevention of infectious diseases to maintain and improve quality in livestock and fisheries.1,2 Advanced nations and national organizations set limits, using risk-based assessments,3,4 for residual levels of veterinary drugs so that consumers are not exposed to unacceptable levels of residues in foods of animal origin. The European Union (EU) establishes maximum residue limits (MRLs) for antibiotics in food, and the use of these compounds as feed additives for growth promotion is banned.5 Production of livestock and fisheries is increasing with rapid population growth in developing countries. It is suggested that the management of antibiotics is insufficient in advancing countries. Vietnam’s economic influence in Asia is emerging as its GDP growth has exceeded 5% over the past 25 years.6 There are nearly 90 million inhabitants, and it is the world’s 13th most populated country.6 The production quantity of indigenous meats (pork, beef, and chicken) increased by 90% from 2002 to 2012.7 Pork is the main meat food in Vietnam: in 2012, it accounted for about 80% of the production quantity. There is a complex farming style in Vietnam, the so-called “VAC (Vuon, Ao, Chuong = field, pond, stockyard) system” that consists of agriculture, aqua farming, and stockbreeding.8 In this system, each byproduct of one farming method is used for the other farming methods, such as for feed or fertilizer. This style has promoted many small © XXXX American Chemical Society
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MATERIALS AND METHODS
Chemicals. Sulfachlorpyridazine, sulfaclozine (sulfachlorpyrazine), sulfadimethoxine, sulfamerazine, sulfamethazine (sulfadimidine), sulfaSpecial Issue: 51st North American Chemical Residue Workshop Received: October 31, 2014 Revised: January 12, 2015 Accepted: January 20, 2015
A
DOI: 10.1021/jf505254y J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry Table 1. Retention Times (RT) and MS/MS Parameters RT (min)
precursor ion (m/z)
product ion (m/z)a
DP (V)
CE (V)
CXP (V)
4.7
350
aspoxicillinb
4.2
494
ciprofloxacinc
4.9
332
danofloxacinc
5.0
358
difloxacinc
5.4
400
enrofloxacinc
5.1
360
marbofloxacinc
4.7
363
norfloxacinc
4.8
320
ofloxacinc
4.8
362
orbifloxacinc
5.2
396
oxacillinb
9.0
402
penicillin Gb
8.2
335
penicillin Vb
8.7
351
sarafloxacinc
5.4
386
spiramycind
5.3
422
sulfachlorpyridazinee
6.8
285
sulfaclozinee
7.6
285
sulfadimethoxinee
7.7
311
sulfadoxinee
7.0
311
sulfamethazinee
6.2
279
sulfamerazinee
5.7
265
sulfamethoxazolee
7.0
254
sulfamethoxypyridazinee
6.2
281
sulfamonomethoxinee
6.6
281
sulfapyridinee
5.5
250
tilmicosind
5.8
435
trimethoprim
4.7
291
tylosind
6.7
917
106 160 250 349 314 231 283 82 356 299 316 245 72 345 302 276 318 261 352 295 243 160 176 160 160 114 299 342 174 101 156 92 92 156 156 92 156 92 186 156 108 92 156 92 156 92 156 92 156 92 88 174 230 261 174 83
51 51 40 40 46 46 56 56 44 44 44 44 41 41 46 46 36 36 41 41 51 51 61 61 65 65 52 52 40 40 21 21 21 21 41 41 41 41 64 64 41 41 36 36 41 41 46 46 41 41 100 100 61 61 90 90
30 17 20 20 25 49 35 61 25 34 24 37 43 25 25 21 23 36 25 32 19 21 17 17 17 47 35 25 27 69 19 39 39 19 23 43 23 43 24 44 33 41 21 37 23 39 23 43 21 37 47 47 31 33 54 111
15 15 15 15 6 4 4 4 4 4 4 4 4 6 6 4 4 4 4 4 15 15 15 15 15 15 4 4 15 15 4 4 4 4 4 4 4 4 15 15 4 4 4 4 4 4 4 4 4 4 4 14 14 4 15 15
compound ampicillin
a
b
Upper, quantification ion; lower, identification ion. bβ-Lactam. cQuinolone. dMacrolide. eSulfonamide.
doxine, sulfamethoxazole, sulfamethoxypyridazine, sulfamonomethoxine, sulfapyridine, ciprofloxacin, danofloxacin, difloxacin, enrofloxacin,
marbofloxacin, norfloxacin, ofloxacin, orbifloxacin, sarafloxacin (Dr. Ehrenstorfer GmbH, Augsburg, Germany), aspoxicillin, ampicillin, B
DOI: 10.1021/jf505254y J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry Table 2. Detected Compounds and Percentages at Sampling Sites HCM
a
MRL (μg/kg)
type
compound
store
slaughterhouse
total
NT
total
EU
Japan
chicken
sulfaclozine sulfamonomethoxine enrofloxacin difloxacin norfloxacin tilmicosin sub total
15.6%a 0% 3.1% 3.1% 0% 0% 7/32b 21.9%
12.5% 0% 5.0% 0% 0% 12.5% 10/40 25.0%
13.9% 0% 4.2% 1.4% 0% 6.9% 17/72 23.6%
0% 1.2% 1.2% 0% 3.6% 6.0% 10/84 11.9%
6.4% 0.6% 2.6% 0.6% 1.9% 6.4% 27/156 17.3%
100 100 100 300 −d 75
−d 100 50 −d 20 70
pork
sulfamethazine sub total
17.9% 10/56 17.9%
6.7% 2/30 6.7%
14.0% 12/86 14.0%
3.5% 3/85 3.5%
8.8% 15/171 8.8%
100
100
beef
sulfamethazine difloxacin sub total
10.5% 5.3% 5/38 13.2%
0% 0% 0/30 0%
5.9% 2.9% 5/68 7.4%
−c −c −c −c
5.9% 2.9% 5/68 7.4%
100 400
100 −d
all
total
22/126 17.5%
12/100 12.0%
34/226 15.0%
13/169 7.7%
47/395 11.9%
Detected percentage. bPositive/sample number. cNo data available. dNo MRL.
oxacillin, penicillin G, penicillin V, spiramycin, tilmicosin, trimethoprim, and tylosin were purchased from Fluka (St. Louis, MO, USA). A dispersive solid-phase extraction (SPE) BONDESIL-C18 (C18) was purchased from Agilent Technologies. (Santa Clara, CA, USA). Highperformance liquid chromatography (HPLC) grade acetonitrile was purchased from Scharlau (Barcelona, Spain), and formic acid was from J. T. Baker (Avantor Performance Materials, Center Valley, PA, USA). Membrane filter (PTFE, 3 mm ⌀, 0.2 μm) was obtained from Agilent. Ultrapure water was made using an EASY pure II system (Thermo Fisher Scientific Inc., Waltham, MA, USA). Samples. About 50 g−1 kg of samples was obtained from slaughterhouses and retail stores in HCM from October 2012 to December 2013 and a central market of NT, which contained about 400 small shops including over 50 butchers in July 2013. A total of 68 beef samples consisting of 65 meat and 3 internal organs (heart, nerve, stomach), 171 pork samples consisting of 160 meat and 11 liver, and 156 chicken samples consisting of 148 meat and 8 liver were analyzed. Samples were minced, mixed, and stored in polypropylene bags at −20 °C until analysis. Stock Standard Solutions. Individual stock standard solutions (200 μg/mL) of sulfonamides, fluoroquinolones, and macrolide antibiotics and trimethoprim were prepared in methanol using polypropylene volumetric flasks and stored in the freezer at −20 °C until use. The same concentration of β-lactam antibiotics was prepared using distilled water and stored in the refrigerator at 4 °C for no more than 2 weeks. Sample Preparation. Samples were prepared according to our method for LC-MS/MS33 and previous analytical reports.17,18 A 2 g aliquot of each sample was weighed in a 50 mL polypropylene tube before 10 mL of an acetonitrile/water (4:1) solution was added and homogenized for 30 s at high- speed (20000 rpm) using a blender (PT1300 D, Kinematica AG, Luzern, Switzerland) at room temperature. The sample was centrifuged (Universal 320R, Hettich GmbH & Co. KG, Tuttlingen, Germany) at 1500g for 5 min, and the supernatant was poured into a 15 mL polypropylene tube containing 0.3 g of dispersive C18. The supernatant was shaken for 1 min and centrifuged at 1500g for 5 min. A 1 mL aliquot was taken and diluted with 1 mL of distilled water. The extract was then filtered through a PTFE membrane filter. This final extract corresponded to a 0.1 g/mL sample equivalent. Instruments and Analytical Methods. LC-MS/MS analysis was performed using a Prominence UFLC (Shimadzu, Kyoto, Japan)
interfaced with an API-5500QTRAP (AB SCIEX, Framingham, CA, USA). The antibiotics were separated in Luna C18 columns (100 × 3 mm i.d., 2.5 μm) (Phenomenex, Torrance, CA, USA) with Alltima HP C18 precolumns (5 × 4.6 mm i.d., 5 μm, Thermo Fisher Scientific Inc.), using a binary gradient system (solvent A, 0.1% formic acid in water; solvent B, acetonitrile), at a flow rate of 0.4 mL/min. Analytes were quantified by multiple reaction monitoring (MRM) using positive electrospray ionization mode. Column temperature was maintained at 40 °C, and the sample injection volume was 5 μL. The antibiotic analytes were resolved using a gradient program, as follows: 5% solvent B at 0.2 mL/min increased to 95% over 10 min and maintained at 95% solvent B for 8 min. Details of the ions monitored are presented in Table 1. LOQs were confirmed to be >10 times the signal-to-noise ratio for the peak intensity of each compound at 10 μg/kg except tilmicosin, which was confirmed to be 10 times more signal-to-noise at 100 μg/kg. The instrument was calibrated every 3 months, and a blank meat sample and six fortified samples were analyzed with every batch of analysis.
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RESULTS AND DISCUSSION Residual antibiotics were detected with an average of 11.9% in the samples analyzed, particularly 17.3, 8.8, and 7.4% in chicken, pork, and beef, respectively (Table 2). Sulfaclozine was detected in chicken samples from HCM but not NT and was not detected in beef or pork samples from either city. Sulfamethazine, enrofloxacin, and tilmicosin were detected in samples from both cities, but difloxacin and norfloxacin were found only in one particular region, suggesting an isolated use of these antibiotics. In the case of chicken, detected percentages (number of detects/total number of samples analyzed) ranged from 11.9 to 25.0% from each site. The detected percentage of sulfaclozine was comparable between retail stores and slaughterhouses in HCM, and nearly a fourth of chicken samples contained antibiotics. Tilmicosin was detected in 10 samples, 5 each from HCM and NT. Three types of fluoroquinolones were detected in chicken, and its detected percentages were
