Article pubs.acs.org/JAFC
Monitoring of Antibiotic Residues in Aquatic Products in Urban and Rural Areas of Vietnam Kotaro Uchida,†,‡ Yoshimasa Konishi,† Kazuo Harada,*,‡,# Masahiro Okihashi,†,‡,# Takahiro Yamaguchi,†,‡ Mai Hoang Ngoc Do,Δ Long Thi Bui,Δ Thinh Duc Nguyen,Δ Phuc Do Nguyen,Δ Diep Thi Khong,⊥ Hoa Thi Tran,⊥ Thang Nam Nguyen,⊥ Ha Viet Le,⊥ Vien Van Chau,⊗ Khanh Thi Van Dao,⊗ Hue Thi Ngoc Nguyen,⊗ Keiji Kajimura,† Yuko Kumeda,† Khanh Tran Pham,Π Khai Ngoc Pham,⊥ Chien Trong Bui,⊗ Mai Quang Vien,⊗ Ninh Hoang Le,Δ Chinh Van Dang,Δ 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 # Global Collaboration Center, Osaka University, 2-7 Yamadaoka, Suita, Osaka 565-0871, Japan Δ Institute of Public Health, Ho Chi Minh City, 159 Hung Phu, District 8, Ward 8, Ho Chi Minh City, Vietnam ⊥ Thai Binh University of Medicine and Pharmacy, 373 Ly Bon Street, Thai Binh, Vietnam ⊗ Nha Trang Pasteur Institute, 8-10 Tran Phu, Xuong Huan Ward, Nha Trang City, Vietnam Π Vietnam Food Administration, Viet Nam Ministry of Health, 138A Giang Vo Street, Ba Dinh District, Ha Noi, Vietnam ‡
S Supporting Information *
ABSTRACT: Antibiotic residues in aquatic products in Vietnam were investigated. A total of 511 fish and shrimp samples were collected from markets in Ho Chi Minh City (HCMC), Thai Binh (TB), and Nha Trang (NT) from July 2013 to October 2015. The samples were extracted with 2% formic acid in acetonitrile and washed with dispersive C18 sorbent. Thirty-two antibiotics were analyzed by LC-MS/MS. Of the 362 samples from HCMC, antibiotic residues were found in 53 samples. Enrofloxacin was commonly detected, at a rate of 10.8%. In contrast, samples from TB and NT were less contaminated: only 1 of 118 analyzed samples showed residues in TB and only 1 of 31 showed residues in NT. These differences were attributed to the local manufacturing/distribution systems. To understand the current status of antibiotic use and prevent adverse effects that may be caused by their overuse, continual monitoring is required. KEYWORDS: antibiotic residue, enrofloxacin, ciprofloxacin, trimethoprim, sulfamethazine, fish, shrimp, aquaculture, LC-MS/MS, Vietnam
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INTRODUCTION In 2012, Vietnam was one of the largest producers of aquatic products and the fourth largest exporter worldwide.1 Annual production reached 6 million tons, and approximately half that amount was due to aquaculture. Aquaculture in Vietnam has increased rapidly, from 845 thousand tons in 2002 to 3115 thousand tons in 2012, although the area devoted to aquaculture remained nearly the same.2 Thus, the nature of aquaculture in Vietnam is changing to an intensive production type. High-density aquaculture is easily prone to disease outbreaks that result in huge economic damages to farmers. To prevent such problems, a wide range of veterinary drugs such as antibiotics, hormones, and additives have been used.3 Among these, antibiotic usage is of major concern because of the adverse effects due to antibiotic resistance.4,5 Although antibiotics are vital for the treatment of bacterial diseases, there is copious evidence that drug-resistant bacteria are generated by their overuse, resulting in infections that are more difficult to treat.4 In addition, the environment can also suffer adverse effects. Antibiotics used in aquaculture can be discharged with the wastewater or runoff from aquaculture © XXXX American Chemical Society
ponds and may persist in the aquatic environment for several months.6−8 Sulfamethazine, sulfamethoxazole, and trimethoprim have frequently been detected in surface water in the Mekong Delta.9,10 High concentrations of sulfamethoxazole, trimethoprim, norfloxacin, and oxolinic acid were found in sediments in mangrove-based shrimp-farming areas.11 These sublethal concentrations of antibiotics are suspected to play an important role in the development and spread of antibioticresistant bacteria.12,13 Although antibiotic concentrations in the natural environment have been well documented, information specific to aquatic products in Vietnam is limited and is even less available for products intended for the domestic market.14−16 To ascertain the prevalence of antibiotic residues in Vietnamese Special Issue: 52nd North American Chemical Residue Workshop Received: January 7, 2016 Revised: May 6, 2016 Accepted: May 9, 2016
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DOI: 10.1021/acs.jafc.6b00091 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry Table 1. LOQs, Accuracies, and Precisions of the Analysis Method fish class
a
analyte
shrimp
LOQ (μg/kg)
recovery (%)
CVa (%)
recovery (%)
CV (%)
1.2 6.5 1.7 0.5 1.2 6.1 0.9 2.2 1.4 0.6 4.0 1.8
76 75 76 72 71 78 67 83 70 73 70 73
9 16 13 12 15 12 12 9 14 14 12 15
66 78 72 71 78 63 74 65 66 81 72 75
7 11 13 10 10 16 10 10 11 11 14 9
quinolone
ciprofloxacin danofloxacin difloxacin enrofloxacin flumequine marbofloxacin nalidixic acid norfloxacin ofloxacin orbifloxacin oxolinic acid sarafloxacin
sulfonamide
sulfabenzamide sulfacetamide sulfachlorpyridazine sulfaclozine sulfadimethoxine sulfadoxine sulfamerazine sulfamethazine sulfamethoxazole sulfamethoxypyridazine sulfamonomethoxine sulfapyridine sulfathiazole sulfisozole
0.6 17.4 2.1 4.9 0.8 0.5 0.4 0.9 2.1 0.7 3.3 2.2 2.0 8.3
74 70 93 92 85 85 78 85 74 90 85 88 85 70
13 16 10 10 11 12 14 12 11 12 14 14 13 13
76 73 83 71 76 78 83 71 79 83 68 78 72 71
11 12 12 10 11 12 9 8 12 5 6 9 11 13
β-lactam
amoxicillin ampicillin cefapirin cefoperazone cefquinome
8.4 3.1 10.3 6.6 6.8
73 87 68 69 55
21 16 15 14 20
38 48 52 76 59
17 17 18 10 17
diaminopyrimidine
trimethoprim
0.4
74
13
64
9
CV, coefficient of variation. Ciprofloxacin was dissolved in 50% acetonitrile, and the others were dissolved in acetonitrile. They were protected from light and stored at −20 °C when not in use. Mixtures of standards for calibration curves and recovery tests were prepared from these stock solutions. They were mixed and filled with acetonitrile until the concentration of each compound was 4.0 mg/L. Reagents and Materials. HPLC grade acetonitrile was purchased from Scharlab S.L. (Barcelona, Spain). HPLC grade methanol and formic acid were obtained from J. T. Baker (Center Valley, PA, USA), and Bondesil C18 bulk sorbent and Econofilter PTFE filters (13 mm diameter, 0.2 μm pore size) were from Agilent Technologies (Santa Clara, CA, USA). An Easypure System II (Thermo Fisher Scientific, Waltham, MA, USA) was used to produce deionized water. Samples. Sampling activities were carried out from July 2013 to October 2015. Aquatic (freshwater or brackish) products were purchased at eight supermarkets (chain stores) and a wholesale market in HCMC and at three local markets in TB and one local market in NT. The samples of HCMC were harvested at a variety of aquaculture ponds in many provinces, and they were transported to markets through wholesale markets. The samples of TB and NT were raised in household aquaculture ponds, and some of them were natural products. After sampling, the samples were transported to our laboratories near the sampling sites. The edible tissues were chopped and kept in polyethylene plastic bags at −20 °C. Then, they were
domestic markets, we analyzed aquatic products (fish and shrimp) from local urban and rural markets in Ho Chi Minh City (HCMC), Thai Binh (TB), and Nha Trang (NT). Four classes of antibioticsquinolones, sulfonamides, β-lactams, and trimethoprimwere analyzed. Samples included products from aquaculture of the household-like Vuon Ao Chuong (VAC, = garden pond pigpen) type,17 but did not include products intended for export.
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MATERIALS AND METHODS
Preparation of Standard Solutions. Analytical grade amoxicillin, ampicillin, cefapirin, cefoperazone, cefquinome, sulfaclozine, and trimethoprim were purchased from Sigma-Aldrich (St. Louis, MO, USA). Ciprofloxacin, danofloxacin, difloxacin, enrofloxacin, flumequine, marbofloxacin, nalidixic acid, norfloxacin, ofloxacin, orbifloxacin, oxolinic acid, sarafloxacin, sulfabenzamide, sulfacetamide, sulfachlorpyridazine, sulfadimethoxine, sulfadoxine, sulfamerazine, sulfamethazine, sulfamethoxazole, sulfamethoxypyridazine, sulfamonomethoxine, sulfapyridine, sulfathiazole, and sulfisozole were obtained from Dr. Ehrenstorfer GmbH (Augsburg, Germany). Standard stock solutions of 4.0 g/L were prepared for each compound. Amoxicillin and ampicillin were dissolved in deionized water. Cefapirin, cefoperazone, and cefquinome were prepared in 20% methanol, respectively. B
DOI: 10.1021/acs.jafc.6b00091 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry Table 2. Percentage of Samples That Contained Antibioticsa Fish HCMC store no. no. tested ciprofloxacin enrofloxacin norfloxacin ofloxacin oxolinic acid sulfamethazine sulfamethoxazole trimethoprim all
wholesale market
ratio (%)
no.
92 5 11 nd 1 nd 2 2 4 16
ratio (%)
total no.
110 5.4 12.0 nd 1.1 nd 2.2 2.2 4.3 17.4
10 16 1 5 nd 2 nd 2 23
ratio (%)
no.
202 9.1 14.5 0.9 4.5 nd 1.8 nd 1.8 20.9
15 27 1 6 nd 4 2 6 39
no. no. tested ciprofloxacin enrofloxacin norfloxacin ofloxacin oxolinic acid sulfamethazine sulfamethoxazole trimethoprim all a
wholesale market
ratio (%)
no.
62
7.4 13.4 0.5 3.0 nd 2.0 1.0 3.0 19.3 Shrimp
4 8 nd nd nd 1 nd nd 8
ratio (%)
no.
6.5 12.9 nd nd nd 1.6 nd nd 12.9
nd 4 nd nd 1 1 nd nd 6
ratio (%)
no.
160 nd 4.1 nd nd 1.0 1.0 nd nd 6.1
4 12 nd nd 1 2 nd nd 14
ratio (%)
nd nd nd nd nd nd nd nd nd
total
98
NT store no.
62
HCMC store
TB store
nd nd nd nd nd nd 1 1 1
ratio (%)
no.
nt nd nd nd nd nd nd nd nd nd
nt nt nt nt nt nt nt nt nt
TB
NT store
ratio (%)
no.
15 27 1 6 nd 4 2 6 39
nd nd nd nd nd nd 1 nd 1
5.7 10.2 0.4 2.3 nd 1.5 0.8 2.3 14.8 total
ratio (%)
no.
31 nd nd nd nd nd nd 1.8 1.8 1.8
ratio (%) 264
nt nt nt nt nt nt nt nt nt
store 56 2.5 7.5 nd nd 0.6 1.3 nd nd 8.8
total
ratio (%) 247
nd nd nd nd nd nd 3.2 nd 3.2
4 12 nd nd 1 2 2 1 16
1.6 4.9 nd nd 0.4 0.8 0.8 0.4 6.5
nd, not detected; nt, not tested.
transported to the laboratory in HCMC, where the materials were minced using a food processor and applied to the following treatments. Sample Preparation. The samples were prepared by a slight modification of our previously reported methods.18,19 To analyze many kinds of antibiotics, the method consisted of a one-step cleanup using a dispersive sorbent, which has recently become popular.20−22 An aliquot of sample (2.0 g) was transferred to a 50 mL polypropylene tube. Then, 2% formic acid in acetonitrile (8 mL) was added, and the mixture was homogenized for 30 s. After centrifugation, the supernatant was poured into a 15 mL polypropylene tube. Deionized water was added, and the volume was fixed at 10 mL. The dispersive sorbent (Bondesil C18, 0.3 g) was added, and the mixture was mixed vigorously for 30 s using a vortex mixer. After centrifugation, an aliquot (1 mL) of the supernatant was diluted with deionized water (3 mL). The extract was filtered through an Econofilter membrane, and an aliquot (5 μL) of the eluent was injected into the LC-MS/MS. LC-MS/MS Analysis. Liquid chromatography was carried out using a Prominence ultrafast liquid chromatograph (UFLC, Shimadzu, Kyoto, Japan) interfaced with an API-5500 QTRAP mass spectrometer (AB SCIEX, Framingham, MA, USA). A Luna C18 column (3 mm × 100 mm, 2.5 μm, Phenomenex, Torrance, CA, USA) with an Alltima HP C18 precolumn filter (4.6 mm × 5 mm, 2.5 μm, Thermo Fisher Scientific) was used for separation. Gradient elution was carried out using a binary gradient composed of solvents A (0.1% formic acid in deionized water) and B (acetonitrile) according to the following program: a linear gradient from 5 to 95% B over 0−10 min, returning to the initial conditions at 15 min and maintaining this condition for 5 min. The flow rate was set to 0.2 mL/min. The column temperature was kept at 40 °C. The LC-MS/MS data are presented in the Supporting Information (Table S1). Calibration Method. Matrix-matched calibration curves were used by adding standard solutions to blank extracts. These blank extracts
were made from representative matrices of catfish and shrimp, which were previously confirmed to be free of the analytes. All fish samples were screened with the catfish calibration curves, and all shrimp samples were compared with the shrimp calibration curves. Recovery experiments were performed to check the performance of the method by comparing the analytical results for the catfish and shrimp in which antibiotics were spiked at 100 μg/kg before the extraction procedure. Two sets of experiments were performed daily for 5 days. Statistical Analysis. Data management was performed on Excel (Microsoft, Redmond, WA, USA). Chi-squared tests were performed to examine the potential differences of detection rates of antibiotics on R (R Foundation, Vienna, Austria).
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RESULTS AND DISCUSSION Analytical Performance. Table 1 shows the analyzed antibiotics, their limits of quantitation (LOQs), average recoveries, and coefficients of variation. The LOQ of each analyte was estimated from spiked blank samples at the lowest calibration level at which the signal-to-noise ratio (S/N) was >10. The LOQs were low enough to detect the maximum residue limits (MRLs) of the antibiotics. The correlation coefficients exceeded 0.99 for calibration levels between 0.4 and 100 μg/kg. The average recoveries were mostly >70%, and the coefficients of variation (CV) were
