Food Chemistry 113 (2009) 297301

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Analytical Methods

Analysis of sulfonamide and quinolone antibiotic residues in Korean milk using microbial assays and high performance liquid chromatography
Hyun-Hee Chung a, Jung-Bin Lee b, Yun-Hee Chung a, Kwang-Geun Lee b,*
a b

Korea Consumer Agency, 300-4, Yumgok-dong, Sucho-gu, Seoul, Republic of Korea Department of Food Science and Technology, Dongguk University, 26, 3-Ga, Pil-dong, Chung-gu, Seoul 100-715, Republic of Korea

a r t i c l e

i n f o

a b s t r a c t
A new protocol to analyse sulfonamides and quinolones from cows and goats milk was developed. Seven antibiotics, sulfamonomethoxine (SMM), sulfadimethoxine (SDM), sulfamethazine (SMZ), sulfamerazine (SMR), sulfaquinoxaline (SQX), enrooxacin (ERF), and ciprooxacin (CIP), were analysed by microbial assays and HPLC. In microbial assays, 21 of 269 samples were screened to have possible antibiotic residues. In HPLC analysis 4 samples were detected to contain SMR (3 samples) and CIP (1 sample). The concentration of SMR in the cows milk was 12.2 lg kg1, which is higher than the LOQ (limit of quantication) and LOD (limit of detection). The levels of SMR and CIP in the other 3 samples were between their LOD and LOQ. Based on this study, we were able to establish an analytical protocol for accurate and robust detection of sulfonamide and quinolone-type antibiotics in milk using two assays. 2008 Elsevier Ltd. All rights reserved.

Article history: Received 31 March 2008 Received in revised form 7 May 2008 Accepted 10 July 2008

Keywords: Antibiotic residues Sulfonamide Quinolone Milk Microbial assays High performance liquid chromatography (HPLC)

1. Introduction Antibiotics are widely used in dairy farms for the treatment of diseases involving bacterial infections, such as mastitis (Knecht et al., 2004). Their use may produce residues in milk, and subsequently induce allergic reactions in humans (Wang, Leung, & Lenz, 2006). In addition, antibiotics give rise to an increase in the antibiotic resistance of pathogenic bacteria, which may result in health problems (Choma, Grenda, Malinowska, & Suprynowics, 1999; Schenck & Callery, 1998). In many countries, governmental authorities have established monitoring programmes to determine the antibiotic levels in food and set a maximum residual level (MRL) for these antibiotics (Ramrez et al., 2003). In Table 1, MRLs of sulfonamide and quinolone-type antibiotics in milk are shown. Synthetic antibiotics such as sulfonamide and quinolone types have a similar function to natural antibiotics (Kishida, 2007; Malintan & Mohd, 2006; Rubies Vaquerizo, Centrich, Compa, Granados & Prat, 2007). Commonly used sulfonamide-type antibiotics are sulfamonomethoxine (SMM), sulfadimethoxine (SDM), sulfamethazine (SMZ), sulfamerazine (SMR), and sulfaquinoxaline (SQX). The quinolone-type antibiotics include ciprooxacin (CIP) and enrooxacin (ERF). They are all used in veterinary medicine to treat livestock diseases, such as gastrointestinal and respiratory tract infections (Hormazabal, Steffenak, & Yndestad, 1993). If antibiotic
* Corresponding author. Tel.: +82 2 2260 3370; fax: +82 2 2285 3370. E-mail address: kwglee@dongguk.edu (K.-G. Lee). 0308-8146/$ - see front matter 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2008.07.021

treatments are inappropriately applied to livestock without monitoring safety recommendations, antibiotic residues can remain in the animal tissues after slaughter (Zayas-Blanco, Garcia-Falcon, & Simal-Gandara, 2004). These residues may also result in a decrease in the quality of fermented milks (Yoon, Kim, Shin, & Beak, 2003). The presence of antibiotic residues in milk gives rise to public health concerns due to the development of drug resistance in intestinal bacteria populations (Zayas-Blanco et al., 2004). Various analytical methods have been described to determine antibiotic residues in milk (Ramrez et al., 2003; Yoon et al., 2003; Zayas-Blanco et al., 2004). They include microbial assays and instrumental analysis. Microbial assay can be used as a screening technique and instrumental techniques such as high performance liquid chromatography (HPLC), thin layer chromatography (TLC), and electrophoresis, are conrmatory methods (Ramrez et al., 2003). Though methods of instrumental analysis such as HPLC produce accurate data of the level of antibiotic residues, expensive instruments and skillful researchers are needed for this technique. Microbial assays are time- and labour-efcient because they involve prescreening of possible antibiotic residues. However, undesirable false-positive and false-negative results can occur if only the microbial assay is used. For detection of sulfonamide and quinolone type antibiotics in milk, two microbial assays, STAR protocol and TTC (2,3,5-triphenyltetrazolium chloride reduction) test, were compared in the present study. STAR protocol presented by Gaudin et al. (2004) is a plate culture method. TTC test, registered for Korean poultry and livestock products, is a tube culture method (Korea

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Table 1 Maximum residual levels (MRLs) of sulfonamide and quinolenes in milk Antibiotics Sulfonamide Sulfamonomethoxine Sulfadimethoxine Sulfamethazine Sulfamerazine Sulfaquinoxaline Quinolone Enrooxacin Ciprooxacin
a b c

EU (lg kg1)a

CODEX (lg kg1)b 25

Republic of Korea (lg kg1)c 10 25

100 The combined total residues of all substances within the sulfonamide group should not exceed 100 lg kg1

100 Sum of enrooxacin and ciprooxacin

EU Council regulation (EEC) No. 2377/90 and amendments. Codex Alimentarius Commission; maximum residue limits for veterinary drugs in foods (2006). Korean Food Code (2007).

national veterinary research, 2006). Sulfonamides, inhibiting the synthesis of folic acid compounds needed to grow microorganisms, compete with para-aminobenzoic acid (PABA) and synergistically act with trimethoprim (Stockstad & Jukes, 1987). The TTC test uses PABA acid and trimethoprim to detect sulfonamide residues. In this study, we developed a protocol that combined microbial assays and HPLC for analysis of sulfonamide and quinolone-type antibiotics in Korean milk products.

assay. For the HPLC analysis of antibiotics, the samples were kept at 45 C. 2.5. Microbial assay I: 2,3,5-triphenyltetrazolium chloride reduction test (TTC test) For the detection of sulfonamide residues in milk the TTC test proposed by Cho et al. was used (Cho, Kim, Sohn, Jin, & Park, 1993; Lee, Lee, Lee, Do, & Park, 1999). S. thermophilus ATCC 14485 was incubated in the 10% skim milk medium at 37 C for 12 h. Yellow supernatant liquid in the incubated medium was transferred to an equivalent volume of 10% skim milk medium. An 8 ml aliquot of milk known not to contain antibiotics was added to each of two tubes marked control and trimethoprim (TMP) control. Sample milk (8 ml) was then added to a sample tube. One millilitre of trimethoprim solution (50 lg ml1) was added to the trimethoprim control tube and the sample tube. The control, trimethoprim control, and the sample tube were placed in a water bath at 82 2 C for 2.5 min. Prepared S. thermophilus inoculum (1 ml) was added to each tube and incubated at 37 0.5 C for 2 h. Then, 0.3 ml of 4% 2,3,5-triphenyltetrazolium chloride (TTC) solution was added to each tube and incubated at 37 0.5 C for 3060 min. If the pink colour of an incubated sample tube did not reach that of the trimethoprim tube, the sample was judged to be presumed positive. The next procedure was performed for the presumed positive samples only. Presumed positive sample (8 ml) was transferred to each of three tubes marked as trimethoprim (TMP), penase and PABA. One millilitre of trimethoprim solution (50 lg ml1) was added to both the trimethoprim and penase tubes; 1 ml of para-aminobenzoic acid solution (500 lg ml1) was added to the PABA tube. The TMP, penase and PABA tubes were placed in a water bath at 82 2 C for 2.5 min and then immediately dipped into tap water to cool below 37 C. One hundred microlitres of penicillinase solution (1000 IU ml1) was added to the penase tube. One millilitre of prepared S. thermophilus inocula was added to each tube and incubated at 37 0.5 C for 2 h. Then, 0.3 ml of 4% TTC solution was added to each tube and incubated at 37 0.5 C for 3060 min. When the pink colour of the TMP tube reached to that of the negative control tube, the reaction was considered to be complete. The possible antibiotic type was identied presumptively by combination of the results for each tube. Interpretation according to the combination of tubes is shown in Table 2. 2.6. Microbial assay II: screening test for antibiotic residues (STAR) protocol In the STAR protocol, ve test organisms were prepared according to the method previously published (Gaudin et al., 2004). The B.

2. Materials and methods 2.1. Chemicals Sulfamerazine (SMR), sulfamethazine (SMT), sulfamonomethoxine (SMM), sulfaquinoxaline (SDM), sulfadimethoxine (SQX), ciprooxacin (CIP), enrooxacin (ERF), and trimethoprim were bought from SigmaAldrich corporation (Seoul, Republic of Korea). Potassium phosphate (KH2PO4), phosphoric acid, and sodium sulfate were purchased from Dae Jung Co. (Seoul, Republic of Korea). HPLC-grade solvents such as acetonitrile, methanol, water, n-hexane, and ethyl acetate were purchased from J.T. Baker & Co. (Phillipsburg, NJ). 2.2. Culture media Skim milk and diagnostic sensitivity test (D.S.T) agar were bought from Oxoid Limited (Cambridge, UK). Mueller Hinton agar at pH 7.4, medium test agar at pH 6.0 and medium test agar at pH 8.0 were purchased from Merck Korea Ltd. (Seoul, Republic of Korea). 2.3. Microorganisms for the microbial assays Streptococcus thermophilus ATCC 14485 was obtained from Korea National Veterinary Research & Quarantine Service (Seoul, Republic of Korea) and Bacillus subtilis BGA was purchased from Merck Korea Ltd. Kocuria rhizophila ATCC 9341, Bacillus cereus ATCC 11778, Escherichia coli ATCC 11303, and Bacillus stearothermophilus ATCC 10149 were obtained from Korean Culture Center of Microorganisms (Seoul, Republic of Korea). 2.4. Milk samples Milk samples including cows milk (n = 239) and goats milk (n = 30) were purchased from various markets located in 13 Korean cities: Seoul, Suwon, Osan, Busan, Daegu, Gwangju, Iksan, Jeonju, Cheongju, Daejeon, Chuncheon, Gangneung, and Wonju. Purchased samples were stored at 4 C after collection and tested by microbial

H.-H. Chung et al. / Food Chemistry 113 (2009) 297301 Table 2 Possible antibiotic type deduced from TTC test Trimethoprim (TMP) tube a +b + +
a b

299

Penase tube + +

Para-aminobenzoic acid (PABA) tube + +

Possible antibiotic type None Sulfonamide-type Penicillin-type Antibiotics except for penicillin and sulfonamide-type

at 3500 rpm for 5 min. The lower layer was concentrated under nitrogen gas. The residues were dissolved with 0.5 ml of 50% acetonitrile and 0.5 ml of HPLC mobile phase. The mixture was placed in an ultrasonic bath for 10 min and centrifuged at 14,000 rpm for 10 min. Supernatant was ltered through a 0.2 lm nylon lter. Twenty microlitres of solution were injected onto the HPLC. 2.10. HPLC analytical procedures The HPLC analysis of various antibiotics was carried out as shown in Table 4. The analytical methods were slightly changed from the Korea Food Code and published papers (Boudra & Morgavi, 2006; Jung, Chae, Kim, & Kim, 1993; Ramrez et al., 2003). For identifying antibiotics, standards were added into samples and analysed by exactly the same method. The increment of peak height of antibiotic was checked for conrming each antibiotic. 2.11. Standard curves of antibiotics and determination of limit of detection (LOD) and limit of quantication (LOQ) Stock solution (0.1 lg ml1 in methanol) of antibiotics was diluted to 0.5, 0.2, 0.1, 0.05 and 0.01 lg ml1 concentrations with HPLC mobile phase for making standard curves. All samples were prepared in triplicate. For determining the LOQ, 0.01 lg ml1 of each antibiotic was injected seven times and the standard deviation was calculated. LOD and LOQ were calculated based on the following equations (Harris, 2001): LOD (limit of detection) = 3 SD/slope, LOQ (limit of quantication) = 10 SD/slope. 2.12. Recovery test

Negative result. Positive result.

subtilis BGA spores were inoculated in Mueller Hinton agar. Kocuria varians ATCC 9341 was inoculated in medium test agar at pH 8.0. The spores of B. cereus ATCC 11778 were inoculated in medium test agar at pH 6.0. E. coli ATCC 11303 was inoculated in medium test agar at pH 8.0. The spores of B. stearothermophilus ATCC 10149 were inoculated in DST medium, and 0.5 lg/ml trimethoprim was then added to the agar to 1% (v/v). Paper discs (diameter 10 mm) were dipped in each milk sample. Four discs per sample were then placed on the surface of the media, at equal distances apart on each of the ve plates. The plates of B. subtilis BGA and B. cereus were incubated at 30 C for at least 18 h. The plates of K. varians and E. coli were incubated at 37 C for at least 24 and 18 h, respectively. The B. stearothermophilus plate was incubated at 55 C for 1215 h. The milk sample was considered positive when the inhibition zone was greater or equal to 2 mm on at least one of the ve plates. 2.7. Comparison of minimum detectable concentration in the two microbiological screening methods Sulfonamide and quinolone-free milk, conrmed by HPLC, was fortied with various amounts of the seven sulfonamide and quinolone antibiotics (0800 lg l1). The minimum detectable concentration of each antibiotic was determined according to the two microbial assays. In Table 3 the levels of fortied antibiotics for determining minimum detectable concentrations are shown.

For the recovery test, 0.1 lg ml1 of each antibiotic was added to a milk sample without antibiotics, and analysis was then carried out according to the procedure described above. Then, the recovery rate was calculated based on the following equation (Harris, 2001).

% recovery

Concentration of spiked sample Concentration of unspiked sample 100 Concentration of added antibiotic

2.8. Sample preparation for HPLC analysis of sulfonamide type antibiotics Each sample (1 g) was taken and homogenised for 2 min with 1 ml of 0.1% potassium phosphate solution. The homogenised sample was centrifuged at 4000 rpm for 15 min with 10 ml of acetonitrile. Supernatant was mixed with 10 ml of n-hexane and centrifuged at 3500 rpm for 5 min. The lower layer of solution was concentrated by nitrogen gas. The concentrates were dissolved with 0.5 ml of 50% acetonitrile and 0.5 ml of water. The mixture was placed in an ultrasonic bath for 10 min and centrifuged at 14,000 rpm for 10 min. Supernatant was passed through a 0.2 lm nylon lter. Fifty microlitres of solution were injected onto the HPLC. 2.9. Sample preparation for HPLC analysis of quinolone type antibiotics Each sample (1 g) was taken and homogenised for 10 min with 1 ml of 2.5% trichloroacetic acid. The homogenised sample was centrifuged at 5000 rpm for 20 min with 10 ml of acetonitrile. Supernatant was mixed with 10 ml of n-hexane and centrifuged

3. Results 3.1. Measurement of minimum detectable concentration by TTC and STAR microbial screening assays The minimum detectable concentrations (MDCs) of milk fortied with various concentration of sulfonamide and quinolone type antibiotics were measured in TTC and STAR screening assays. The MDCs of sulfonamide and quinolone type antibiotics by TTC test are shown in Table 3. In TTC assay, the MDCs of sulfamonomethoxine (SMM), sulfadimethoxine (SDM) and sulfaquinoxaline (SQX) were 25 lg l1, and those of sulfamerazine (SMR) and sulfamethazine (SMT) were 50 lg l1. However, enrooxacin (ERF) and ciprooxacin (CIP) in milk were not detected at levels up to 800 lg l1 by TTC assay. The MDCs for sulfonamide-type antibiotics in the STAR protocol were much higher than those of the TTC test. The MDC of SDM and SMM was 175 lg l1 and that of SQX and SMR was 200 lg l1. The MDC of SMT was 600 lg l1 on a B. stearothermophilus plate. However, the MDCs of ERF were 20 and 300 lg l1 on E. coli and B. subtilis plates, respectively. Those of CIP were 10 and 400 lg l1 on E.

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Table 3 The minimum detectable concentration of antibiotics measured by TTC and STAR test Antibiotics TTC test Fortied concentrations (lg l Sulfaquinoxaline Sulfadimethoxine Sulfamonomethoxine Sulfamethazine Sulfamerazine Enrooxacin Ciprooxacin
a b c d 1

STAR test ) Minimum detectable concentration (lg l1) 25 25 25 50 50 N.D.b N.D. Fortied concentrations (lg l1) 0, 0, 0, 0, 0, 0, 25, 50, 100, 150, 175, 200, 400, 800 25, 50, 100, 150, 175, 200, 400, 800 25, 50, 100, 150, 175, 200, 400, 800 50, 100, 200, 400, 500, 550, 600, 800 25, 50, 100, 150, 175, 200, 400, 800 5, 10, 15, 20, 50, 100, 200, 300, 400 Plate Bsta Bst Bst Bst Bst Ecc Bsd Ec Bs Minimum detectable concentration (lg l1) 200 175 175 600 200 20 300 10 400

0, 0, 0, 0, 0, 0,

10, 25, 50, 75, 10, 25, 50, 75, 10, 25, 50, 75, 10, 25, 50, 75, 10, 25, 50, 75, 5, 10, 30, 100,

100, 100, 100, 100, 100, 200,

200, 200, 200, 200, 200, 400,

300 300 300 300 300 800

0, 5, 10, 30, 100, 200, 400, 800

0, 5, 10, 15, 20, 50, 100, 200, 300, 400

Bacillus stearothermophilus ATCC 10149. Not detected. Escherichia coli ATCC 11303. Bacillus subtilis BGA.

Table 4 Analytical conditions for HPLC analysis of the antibiotics Antibiotic Column Sulfonamide-type 250 mm 4.6 mm C18 column 40 C Quinolone-type 250 mm 4.6 mm C18 column 40 C Fluorescence detector k ex. = 278 nm, k em. = 455 nm Water (including 0.4% triethylamine and 4 ml 85% phosphoric acid): methanol: acetonitrile (85:7:8) 1.5 ml/min

analysis no sample was identied to contain sulfonamide antibiotics. Three samples possibly contained quinolone antibiotics in the STAR assay. Among the three samples, sample No. 19 was identied to contain CIP (16.7 lg kg1) by HPLC. 3.3. Standard curves, recovery tests, LOD (limit of detection), and LOQ (limit of quantication) determination In Table 6, standard calibration equations, recovery rates, LOD (limit of detection) and LOQ (limit of quantication) of tested antibiotics are shown. All samples were prepared in triplicate. The regression coefcient (r2) of standard curves was between 0.9962 and 0.9998, which showed superior linearity. Recovery rates ranged from 72.4% to 101.3%. The LOD and LOQ of each antibiotic is shown in Table 6. The LODs of sulfonamide antibiotics were between 4.0 and 11.3 lg kg1. Their LOQs were from 10.3 to 37.8 lg kg1. The LOQs of CIP and ERF were 23.2 and 3.0 lg kg1, respectively. 3.4. Analysis of antibiotic residues in milk by HPLC In the TTC and STAR assays, 15 and 3 samples, respectively, possibly contained sulfonamide antibiotic residues among 269 samples. In this study, we carried out HPLC analysis for all 269 samples to determine the correlation between microbial assays and HPLC analysis. The level of antibiotics in samples analysed using HPLC with UV or uorescence detector was shown in Table 5. In HPLC analysis 4 samples were detected to contain SMR (3 samples) and CIP (1 sample). The concentration of SMR in cows milk (No. 120 sample) was 12.2 lg kg1, which was higher than the LOQ and LOD. The levels of SMR and CIP in the other 3 samples were higher than their LODs but lower than their LOQs. 4. Discussion and conclusion For the quick and accurate detection of sulfonamide residues in milk, the TTC assay showed more sensitive results than those of the STAR protocol. The minimum detectable concentration (MDC) of sulfonamide type 5 antibiotics in TTC assay was lower than 50 lg l1. The MDC in the STAR protocol for sulfonamide type 5 antibiotics was higher than 175 lg l1. The milk sample identied to contain a low concentration of sulfamerazine by HPLC analysis was screened not by the STAR protocol but the TTC test. When test organisms were prepared, the TTC test could be performed rapidly within 3 h while the STAR protocol needed 2 days. The TTC test is specically designed for sulfonamide type antibiotics.

Column temperature Detector UV detector k = 270 nm Mobile phase Acetonitrile: 0.1% potassium phosphate solution (16:84)

Flow rate

1.2 ml/min

coli and B. subtilis plates, respectively. The MDCs of sulfonamide and quinolone-type antibiotics by STAR protocol are shown in Table 3. 3.2. Analysis of antibiotic residues in commercial milk products TTC and STAR assays Milk products including cows milk (n = 239) and goats milk (n = 30) were obtained from different areas of the Republic of Korea and measured for antibiotics by TTC and STAR protocols. The number of samples possibly containing sulfonamide-type antibiotics was 15 of 269 species by the TTC assay. Among 15 samples which showed a positive response in the TTC assay, only two samples (samples No. 55 and 120) were identied to contain SMR by HPLC, as shown in Table 5. Though sample No. 121 showed a negative response in the TTC assay, it contained 4.4 lg kg1 of SMR by HPLC. In the STAR assay, only 3 samples were identied as possibly having sulfonamide antibiotics, including penicillin, cephalosporin, baquiloprim, thiamphenicol and trimethoprim. However, by HPLC
Table 5 The level of antibiotic residues in milk analysed by HPLC Sample No. Source Concentration of antibiotic residues (lg kg1) CIP: 16.7 4.6 (<LOQ) SMR: 9.1 2.1 (<LOQ) SMR: 12.2 2.8 SMR: 4.4 2.6 (<LOQ) N.D.a Regulation of Korea Food Code None None None None

19 55 120 121 118, 2054, 56 119, 122269

a

Cows milk Cows milk Cows milk Cows milk Cows milk and goats milk

N.D.: not detected.

H.-H. Chung et al. / Food Chemistry 113 (2009) 297301 Table 6 Parameters of calibration curves, recovery rates, limit of detection (LOD), and limit of quantication (LOQ) of antibiotics Antibiotics Standard calibration equation (y = ax + b) a Class of sulfonamide Sulfamerazine (SMR) Sulfamethazine (SMZ) Sulfamonomethoxine (SMM) Sulfadimethoxine (SDM) Sulfaquinoxaline (SQX) Class of quinolone Ciprooxacin Enrooxacin 7E + 06 7E + 06 8E + 06 5E + 06 7E + 06 1E + 08 4E + 08 b 70259 68275 7516.4 8517 43769 3E + 06 5E + 06 r
2

301

Recovery rates (%)

LOD (lg kg1)

LOQ (lg kg1)

0.9998 0.9998 0.9962 0.9995 0.9986 0.9989 0.9986

95.1% 97.9% 79.2% 85.2% 90.8% 101.3% 72.4%

4.0 7.0 6.0 4.9 11.3 6.5 1.1

10.3 22.9 19.9 16.5 37.8 23.2 3.0

In the case of the STAR protocol, the inhibitory zone of sulfonamide type antibiotics appeared on the same plate with other antibiotics, such as penicillin, cephalosporin, baquiloprim, thiamphenicol, and trimethoprim. For detecting sulfonamide-type antibiotics in commercial milk products using STAR protocol, selectivity and sensitivity were relatively lower than the TTC assay. However, the STAR protocol had the ability to detect a low level of quinolone-type antibiotics, such as enrooxacin and ciprooxacin, in milk. In previously reported articles, liquid chromatographic determination of oxytetracycline in milk was reported (Furusawa, 1999). In that paper, 20 milk samples were analysed, but oxytetracycline was not detected in the milk samples. There has been no paper reporting the simultaneous analysis of sulfonamide and quinolone-type antibiotics in milk products. In HPLC analysis, the concentration of sulfamerazine in sample No. 120 was 12.2 lg kg1. The maximum residue level (MRL) of sulfamerazine in milk products has not been established in the Republic of Korea. Therefore, these data indicate that the regulatory authorities should set and regulate the MRL of sulfamerazine in milk products. Based on this study, we were able to establish an analytical method for accurate and robust detection of residual antibiotics in milk. In particular, TTC test could detect sulfonamide-type antibiotics over MRLs of EU and Codex in milk, and STAR assay could detect quinolone-type antibiotics over EU MRL. In a future study, we will develop a new microbial assay to decrease false-positive readings based on the current assays. Acknowledgment This work was supported by a grant from the KFDA (Korea Food and Drug Administration) R&D Project (06042-Hangnaemo-118). References
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