Food Chemistry 113 (2009) 297–301

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Food Chemistry

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Analytical Methods

Analysis of sulfonamide and quinolone antibiotic residues in Korean milkusing microbial assays and high performance liquid chromatography

Hyun-Hee Chung a, Jung-Bin Lee b, Yun-Hee Chung a, Kwang-Geun Lee b,*

a Korea Consumer Agency, 300-4, Yumgok-dong, Sucho-gu, Seoul, Republic of Koreab 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

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

Keywords:Antibiotic residuesSulfonamideQuinoloneMilkMicrobial assaysHigh performance liquid chromatography(HPLC)

0308-8146/$ - see front matter � 2008 Elsevier Ltd. Adoi:10.1016/j.foodchem.2008.07.021

* Corresponding author. Tel.: +82 2 2260 3370; faxE-mail address: kwglee@dongguk.edu (K.-G. Lee).

a b s t r a c t

A new protocol to analyse sulfonamides and quinolones from cows’ and goats’ milk was developed. Sevenantibiotics, sulfamonomethoxine (SMM), sulfadimethoxine (SDM), sulfamethazine (SMZ), sulfamerazine(SMR), sulfaquinoxaline (SQX), enrofloxacin (ERF), and ciprofloxacin (CIP), were analysed by microbialassays and HPLC. In microbial assays, 21 of 269 samples were screened to have possible antibiotic resi-dues. In HPLC analysis 4 samples were detected to contain SMR (3 samples) and CIP (1 sample). The con-centration of SMR in the cows’ milk was 12.2 lg kg�1, which is higher than the LOQ (limit ofquantification) and LOD (limit of detection). The levels of SMR and CIP in the other 3 samples werebetween their LOD and LOQ. Based on this study, we were able to establish an analytical protocol foraccurate and robust detection of sulfonamide and quinolone-type antibiotics in milk using two assays.

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1. Introduction

Antibiotics are widely used in dairy farms for the treatment ofdiseases involving bacterial infections, such as mastitis (Knechtet al., 2004). Their use may produce residues in milk, and subse-quently induce allergic reactions in humans (Wang, Leung, & Lenz,2006). In addition, antibiotics give rise to an increase in the antibi-otic resistance of pathogenic bacteria, which may result in healthproblems (Choma, Grenda, Malinowska, & Suprynowics, 1999;Schenck & Callery, 1998). In many countries, governmental author-ities have established monitoring programmes to determine theantibiotic levels in food and set a maximum residual level (MRL)for these antibiotics (Ramírez et al., 2003). In Table 1, MRLs of sul-fonamide and quinolone-type antibiotics in milk are shown.

Synthetic antibiotics such as sulfonamide and quinolone typeshave a similar function to natural antibiotics (Kishida, 2007; Malin-tan & Mohd, 2006; Rubies Vaquerizo, Centrich, Compañó, Granados& Prat, 2007). Commonly used sulfonamide-type antibiotics are sul-famonomethoxine (SMM), sulfadimethoxine (SDM), sulfametha-zine (SMZ), sulfamerazine (SMR), and sulfaquinoxaline (SQX). Thequinolone-type antibiotics include ciprofloxacin (CIP) and enroflox-acin (ERF). They are all used in veterinary medicine to treat live-stock diseases, such as gastrointestinal and respiratory tractinfections (Hormazabal, Steffenak, & Yndestad, 1993). If antibiotic

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treatments are inappropriately applied to livestock without moni-toring safety recommendations, antibiotic residues can remain inthe animal tissues after slaughter (Zayas-Blanco, Garcia-Falcon, &Simal-Gandara, 2004). These residues may also result in a decreasein the quality of fermented milks (Yoon, Kim, Shin, & Beak, 2003).The presence of antibiotic residues in milk gives rise to publichealth concerns due to the development of drug resistance in intes-tinal bacteria populations (Zayas-Blanco et al., 2004).

Various analytical methods have been described to determineantibiotic residues in milk (Ramírez et al., 2003; Yoon et al.,2003; Zayas-Blanco et al., 2004). They include microbial assaysand instrumental analysis. Microbial assay can be used as a screen-ing technique and instrumental techniques such as high perfor-mance liquid chromatography (HPLC), thin layer chromatography(TLC), and electrophoresis, are confirmatory methods (Ramírez etal., 2003). Though methods of instrumental analysis such as HPLCproduce accurate data of the level of antibiotic residues, expensiveinstruments and skillful researchers are needed for this technique.Microbial assays are time- and labour-efficient because they in-volve prescreening of possible antibiotic residues. However, unde-sirable false-positive and false-negative results can occur if onlythe microbial assay is used. For detection of sulfonamide and quin-olone type antibiotics in milk, two microbial assays, STAR protocoland TTC (2,3,5-triphenyltetrazolium chloride reduction) test, werecompared in the present study. STAR protocol presented by Gaudinet al. (2004) is a plate culture method. TTC test, registered for Kor-ean poultry and livestock products, is a tube culture method (Korea

Table 1Maximum residual levels (MRLs) of sulfonamide and quinolenes in milk

Antibiotics EU (lg kg�1)a CODEX(lg kg�1)b

Republic of Korea(lg kg�1)c

SulfonamideSulfamonomethoxine 100 – The combined total residues of all substances within

the sulfonamide group should not exceed 100 lg kg�1– –

Sulfadimethoxine – 10Sulfamethazine 25 25Sulfamerazine – –Sulfaquinoxaline – –

QuinoloneEnrofloxacin 100 – Sum of enrofloxacin and ciprofloxacin – –Ciprofloxacin – –

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

298 H.-H. Chung et al. / Food Chemistry 113 (2009) 297–301

national veterinary research, 2006). Sulfonamides, inhibiting thesynthesis of folic acid compounds needed to grow microorganisms,compete with para-aminobenzoic acid (PABA) and synergisticallyact with trimethoprim (Stockstad & Jukes, 1987). The TTC test usesPABA acid and trimethoprim to detect sulfonamide residues.

In this study, we developed a protocol that combined microbialassays and HPLC for analysis of sulfonamide and quinolone-typeantibiotics in Korean milk products.

2. Materials and methods

2.1. Chemicals

Sulfamerazine (SMR), sulfamethazine (SMT), sulfamonome-thoxine (SMM), sulfaquinoxaline (SDM), sulfadimethoxine (SQX),ciprofloxacin (CIP), enrofloxacin (ERF), and trimethoprim werebought from Sigma–Aldrich corporation (Seoul, Republic of Korea).Potassium phosphate (KH2PO4), phosphoric acid, and sodium sul-fate were purchased from Dae Jung Co. (Seoul, Republic of Korea).HPLC-grade solvents such as acetonitrile, methanol, water, n-hex-ane, and ethyl acetate were purchased from J.T. Baker & Co. (Phil-lipsburg, NJ).

2.2. Culture media

Skim milk and diagnostic sensitivity test (D.S.T) agar werebought from Oxoid Limited (Cambridge, UK). Mueller Hinton agarat pH 7.4, medium test agar at pH 6.0 and medium test agar atpH 8.0 were purchased from Merck Korea Ltd. (Seoul, Republic ofKorea).

2.3. Microorganisms for the microbial assays

Streptococcus thermophilus ATCC 14485 was obtained from Kor-ea National Veterinary Research & Quarantine Service (Seoul,Republic of Korea) and Bacillus subtilis BGA was purchased fromMerck Korea Ltd. Kocuria rhizophila ATCC 9341, Bacillus cereus ATCC11778, Escherichia coli ATCC 11303, and Bacillus stearothermophilusATCC 10149 were obtained from Korean Culture Center of Microor-ganisms (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 Koreancities: Seoul, Suwon, Osan, Busan, Daegu, Gwangju, Iksan, Jeonju,Cheongju, Daejeon, Chuncheon, Gangneung, and Wonju. Purchasedsamples were stored at 4 �C after collection and tested by microbial

assay. For the HPLC analysis of antibiotics, the samples were keptat �45 �C.

2.5. Microbial assay I: 2,3,5-triphenyltetrazolium chloride reductiontest (TTC test)

For the detection of sulfonamide residues in milk the TTC testproposed by Cho et al. was used (Cho, Kim, Sohn, Jin, & Park,1993; Lee, Lee, Lee, Do, & Park, 1999). S. thermophilus ATCC14485 was incubated in the 10% skim milk medium at 37 �C for12 h. Yellow supernatant liquid in the incubated medium wastransferred to an equivalent volume of 10% skim milk medium.An 8 ml aliquot of milk known not to contain antibiotics was addedto 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 ml�1) was addedto the trimethoprim control tube and the sample tube. The control,trimethoprim control, and the sample tube were placed in a waterbath 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 for2 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 for30–60 min. If the pink colour of an incubated sample tube didnot reach that of the trimethoprim tube, the sample was judgedto be presumed positive.

The next procedure was performed for the presumed positivesamples only. Presumed positive sample (8 ml) was transferredto each of three tubes marked as ‘‘trimethoprim” (TMP), ‘‘penase”and ‘‘PABA”. One millilitre of trimethoprim solution (50 lg ml�1)was added to both the trimethoprim and penase tubes; 1 ml ofpara-aminobenzoic acid solution (500 lg ml�1) was added to thePABA tube. The TMP, penase and PABA tubes were placed in awater bath at 82 ± 2 �C for 2.5 min and then immediately dippedinto tap water to cool below 37 �C. One hundred microlitres of pen-icillinase solution (1000 IU ml�1) was added to the penase tube.One millilitre of prepared S. thermophilus inocula was added toeach 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 �Cfor 30–60 min. When the pink colour of the TMP tube reached tothat of the negative control tube, the reaction was considered tobe complete. The possible antibiotic type was identified presump-tively by combination of the results for each tube. Interpretationaccording 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, five test organisms were prepared accord-ing to the method previously published (Gaudin et al., 2004). The B.

Table 2Possible antibiotic type deduced from TTC test

Trimethoprim(TMP) tube

Penasetube

Para-aminobenzoicacid (PABA) tube

Possible antibiotic type

�a � � None+b + � Sulfonamide-type+ � + Penicillin-type+ + + Antibiotics except for penicillin

and sulfonamide-type

a Negative result.b Positive result.

H.-H. Chung et al. / Food Chemistry 113 (2009) 297–301 299

subtilis BGA spores were inoculated in Mueller Hinton agar. Kocuriavarians ATCC 9341 was inoculated in medium test agar at pH 8.0.The spores of B. cereus ATCC 11778 were inoculated in medium testagar at pH 6.0. E. coli ATCC 11303 was inoculated in medium testagar at pH 8.0. The spores of B. stearothermophilus ATCC 10149were inoculated in DST medium, and 0.5 lg/ml trimethoprimwas 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 med-ia, at equal distances apart on each of the five plates. The plates ofB. subtilis BGA and B. cereus were incubated at 30 �C for at least18 h. The plates of K. varians and E. coli were incubated at 37 �Cfor at least 24 and 18 h, respectively. The B. stearothermophilusplate was incubated at 55 �C for 12–15 h. The milk sample wasconsidered positive when the inhibition zone was greater or equalto 2 mm on at least one of the five plates.

2.7. Comparison of minimum detectable concentration in the twomicrobiological screening methods

Sulfonamide and quinolone-free milk, confirmed by HPLC, wasfortified with various amounts of the seven sulfonamide and quin-olone antibiotics (0–800 lg l�1). The minimum detectable concen-tration of each antibiotic was determined according to the twomicrobial assays. In Table 3 the levels of fortified antibiotics fordetermining minimum detectable concentrations are shown.

% recovery ¼ Concentration of spiked sample� Concentration of unspiked sample� 100Concentration of added antibiotic

2.8. Sample preparation for HPLC analysis of sulfonamide typeantibiotics

Each sample (1 g) was taken and homogenised for 2 min with1 ml of 0.1% potassium phosphate solution. The homogenised sam-ple was centrifuged at 4000 rpm for 15 min with 10 ml of acetoni-trile. Supernatant was mixed with 10 ml of n-hexane andcentrifuged at 3500 rpm for 5 min. The lower layer of solutionwas concentrated by nitrogen gas. The concentrates were dissolvedwith 0.5 ml of 50% acetonitrile and 0.5 ml of water. The mixturewas placed in an ultrasonic bath for 10 min and centrifuged at14,000 rpm for 10 min. Supernatant was passed through a 0.2 lmnylon filter. Fifty microlitres of solution were injected onto theHPLC.

2.9. Sample preparation for HPLC analysis of quinolone type antibiotics

Each sample (1 g) was taken and homogenised for 10 min with1 ml of 2.5% trichloroacetic acid. The homogenised sample wascentrifuged at 5000 rpm for 20 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 was concentrated undernitrogen gas. The residues were dissolved with 0.5 ml of 50% ace-tonitrile and 0.5 ml of HPLC mobile phase. The mixture was placedin an ultrasonic bath for 10 min and centrifuged at 14,000 rpm for10 min. Supernatant was filtered through a 0.2 lm nylon filter.Twenty microlitres of solution were injected onto the HPLC.

2.10. HPLC analytical procedures

The HPLC analysis of various antibiotics was carried out asshown in Table 4. The analytical methods were slightly changedfrom the Korea Food Code and published papers (Boudra & Morgavi,2006; Jung, Chae, Kim, & Kim, 1993; Ramírez et al., 2003). For iden-tifying antibiotics, standards were added into samples and analysedby exactly the same method. The increment of peak height of anti-biotic was checked for confirming each antibiotic.

2.11. Standard curves of antibiotics and determination of limit ofdetection (LOD) and limit of quantification (LOQ)

Stock solution (0.1 lg ml�1 in methanol) of antibiotics was di-luted to 0.5, 0.2, 0.1, 0.05 and 0.01 lg ml�1 concentrations withHPLC mobile phase for making standard curves. All samples wereprepared in triplicate. For determining the LOQ, 0.01 lg ml�1 ofeach antibiotic was injected seven times and the standard devia-tion was calculated. LOD and LOQ were calculated based on the fol-lowing equations (Harris, 2001):

LOD (limit of detection) = 3 � SD/slope,LOQ (limit of quantification) = 10 � SD/slope.

2.12. Recovery test

For the recovery test, 0.1 lg ml�1 of each antibiotic was addedto a milk sample without antibiotics, and analysis was then carriedout according to the procedure described above. Then, therecovery rate was calculated based on the following equation(Harris, 2001).

3. Results

3.1. Measurement of minimum detectable concentration by TTC andSTAR microbial screening assays

The minimum detectable concentrations (MDCs) of milk forti-fied with various concentration of sulfonamide and quinolone typeantibiotics were measured in TTC and STAR screening assays. TheMDCs of sulfonamide and quinolone type antibiotics by TTC testare shown in Table 3. In TTC assay, the MDCs of sulfamonomethox-ine (SMM), sulfadimethoxine (SDM) and sulfaquinoxaline (SQX)were 25 lg l�1, and those of sulfamerazine (SMR) and sulfametha-zine (SMT) were 50 lg l�1. However, enrofloxacin (ERF) and cipro-floxacin (CIP) in milk were not detected at levels up to 800 lg l�1

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 andSMM was 175 lg l�1 and that of SQX and SMR was 200 lg l�1. TheMDC of SMT was 600 lg l�1 on a B. stearothermophilus plate. How-ever, the MDCs of ERF were 20 and 300 lg l�1 on E. coli and B. sub-tilis plates, respectively. Those of CIP were 10 and 400 lg l�1 on E.

Table 4Analytical conditions for HPLC analysis of the antibiotics

Antibiotic Sulfonamide-type Quinolone-type

Column 250 mm � 4.6 mm C18

column250 mm � 4.6 mm C18 column

Columntemperature

40 �C 40 �C

Detector UV detector k = 270 nm Fluorescence detectork ex. = 278 nm, k em. = 455 nm

Mobile phase Acetonitrile: 0.1% potassiumphosphate solution (16:84)

Water (including 0.4%triethylamineand 4 ml 85% phosphoric acid):methanol: acetonitrile (85:7:8)

Flow rate 1.2 ml/min 1.5 ml/min

Table 3The minimum detectable concentration of antibiotics measured by TTC and STAR test

Antibiotics TTC test STAR test

Fortified concentrations (lg l�1) Minimum detectableconcentration (lg l�1)

Fortified concentrations(lg l�1)

Plate Minimum detectableconcentration (lg l�1)

Sulfaquinoxaline 0, 10, 25, 50, 75, 100, 200, 300 25 0, 25, 50, 100, 150, 175, 200, 400, 800 Bsta 200Sulfadimethoxine 0, 10, 25, 50, 75, 100, 200, 300 25 0, 25, 50, 100, 150, 175, 200, 400, 800 Bst 175Sulfamonomethoxine 0, 10, 25, 50, 75, 100, 200, 300 25 0, 25, 50, 100, 150, 175, 200, 400, 800 Bst 175Sulfamethazine 0, 10, 25, 50, 75, 100, 200, 300 50 0, 50, 100, 200, 400, 500, 550, 600, 800 Bst 600Sulfamerazine 0, 10, 25, 50, 75, 100, 200, 300 50 0, 25, 50, 100, 150, 175, 200, 400, 800 Bst 200Enrofloxacin 0, 5, 10, 30, 100, 200, 400, 800 N.D.b 0, 5, 10, 15, 20, 50, 100, 200, 300, 400 Ecc 20

Bsd 300Ciprofloxacin 0, 5, 10, 30, 100, 200, 400, 800 N.D. 0, 5, 10, 15, 20, 50, 100, 200, 300, 400 Ec 10

Bs 400

a Bacillus stearothermophilus ATCC 10149.b Not detected.c Escherichia coli ATCC 11303.d Bacillus subtilis BGA.

300 H.-H. Chung et al. / Food Chemistry 113 (2009) 297–301

coli and B. subtilis plates, respectively. The MDCs of sulfonamideand quinolone-type antibiotics by STAR protocol are shown in Ta-ble 3.

3.2. Analysis of antibiotic residues in commercial milk products TTCand STAR assays

Milk products including cows’ milk (n = 239) and goats’ milk(n = 30) were obtained from different areas of the Republic of Koreaand measured for antibiotics by TTC and STAR protocols. The num-ber of samples possibly containing sulfonamide-type antibioticswas 15 of 269 species by the TTC assay. Among 15 samples whichshowed a positive response in the TTC assay, only two samples(samples No. 55 and 120) were identified to contain SMR by HPLC,as shown in Table 5. Though sample No. 121 showed a negative re-sponse in the TTC assay, it contained 4.4 lg kg�1 of SMR by HPLC.

In the STAR assay, only 3 samples were identified as possiblyhaving sulfonamide antibiotics, including penicillin, cephalosporin,baquiloprim, thiamphenicol and trimethoprim. However, by HPLC

Table 5The level of antibiotic residues in milk analysed by HPLC

Sample No. Source Concentration ofantibiotic residues(lg kg�1)

Regulation ofKorea Food Code

19 Cows’ milk CIP: 16.7 ± 4.6 (<LOQ) None55 Cows’ milk SMR: 9.1 ± 2.1 (<LOQ) None120 Cows’ milk SMR: 12.2 ± 2.8 None121 Cows’ milk SMR: 4.4 ± 2.6 (<LOQ) None1–18, 20–54, 56–

119, 122–269Cows’ milkand goats’milk

N.D.a

a N.D.: not detected.

analysis no sample was identified to contain sulfonamide antibiot-ics. Three samples possibly contained quinolone antibiotics in theSTAR assay. Among the three samples, sample No. 19 was identi-fied to contain CIP (16.7 lg kg�1) by HPLC.

3.3. Standard curves, recovery tests, LOD (limit of detection), and LOQ(limit of quantification) determination

In Table 6, standard calibration equations, recovery rates, LOD(limit of detection) and LOQ (limit of quantification) of tested anti-biotics are shown. All samples were prepared in triplicate. Theregression coefficient (r2) of standard curves was between 0.9962and 0.9998, which showed superior linearity. Recovery rates ran-ged from 72.4% to 101.3%. The LOD and LOQ of each antibiotic isshown in Table 6. The LODs of sulfonamide antibiotics were be-tween 4.0 and 11.3 lg kg�1. Their LOQs were from 10.3 to37.8 lg kg�1. The LOQs of CIP and ERF were 23.2 and 3.0 lg kg�1,respectively.

3.4. Analysis of antibiotic residues in milk by HPLC

In the TTC and STAR assays, 15 and 3 samples, respectively, pos-sibly contained sulfonamide antibiotic residues among 269 sam-ples. In this study, we carried out HPLC analysis for all 269samples to determine the correlation between microbial assaysand HPLC analysis. The level of antibiotics in samples analysedusing HPLC with UV or fluorescence detector was shown in Table5. In HPLC analysis 4 samples were detected to contain SMR (3samples) and CIP (1 sample). The concentration of SMR in cows’milk (No. 120 sample) was 12.2 lg kg�1, which was higher thanthe LOQ and LOD. The levels of SMR and CIP in the other 3 sampleswere higher than their LODs but lower than their LOQs.

4. Discussion and conclusion

For the quick and accurate detection of sulfonamide residues inmilk, the TTC assay showed more sensitive results than those of theSTAR protocol. The minimum detectable concentration (MDC) ofsulfonamide type 5 antibiotics in TTC assay was lower than50 lg l�1. The MDC in the STAR protocol for sulfonamide type 5antibiotics was higher than 175 lg l�1.

The milk sample identified to contain a low concentration ofsulfamerazine by HPLC analysis was screened not by the STAR pro-tocol but the TTC test. When test organisms were prepared, the TTCtest could be performed rapidly within 3 h while the STAR protocolneeded 2 days. The TTC test is specifically designed for sulfon-amide type antibiotics.

Table 6Parameters of calibration curves, recovery rates, limit of detection (LOD), and limit of quantification (LOQ) of antibiotics

Antibiotics Standard calibration equation (y = ax + b) Recovery rates (%) LOD (lg kg�1) LOQ (lg kg�1)

a b r2

Class of sulfonamideSulfamerazine (SMR) 7E + 06 70259 0.9998 95.1% 4.0 10.3Sulfamethazine (SMZ) 7E + 06 �68275 0.9998 97.9% 7.0 22.9Sulfamonomethoxine (SMM) 8E + 06 �7516.4 0.9962 79.2% 6.0 19.9Sulfadimethoxine (SDM) 5E + 06 8517 0.9995 85.2% 4.9 16.5Sulfaquinoxaline (SQX) 7E + 06 �43769 0.9986 90.8% 11.3 37.8Class of quinoloneCiprofloxacin 1E + 08 �3E + 06 0.9989 101.3% 6.5 23.2Enrofloxacin 4E + 08 �5E + 06 0.9986 72.4% 1.1 3.0

H.-H. Chung et al. / Food Chemistry 113 (2009) 297–301 301

In the case of the STAR protocol, the inhibitory zone of sulfon-amide type antibiotics appeared on the same plate with other anti-biotics, such as penicillin, cephalosporin, baquiloprim,thiamphenicol, and trimethoprim. For detecting sulfonamide-typeantibiotics 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 ofquinolone-type antibiotics, such as enrofloxacin and ciprofloxacin,in milk.

In previously reported articles, liquid chromatographic determi-nation of oxytetracycline in milk was reported (Furusawa, 1999). Inthat paper, 20 milk samples were analysed, but oxytetracyclinewas not detected in the milk samples. There has been no paperreporting the simultaneous analysis of sulfonamide and quino-lone-type antibiotics in milk products.

In HPLC analysis, the concentration of sulfamerazine in sampleNo. 120 was 12.2 lg kg�1. The maximum residue level (MRL) ofsulfamerazine in milk products has not been established in theRepublic of Korea. Therefore, these data indicate that the regula-tory authorities should set and regulate the MRL of sulfamerazinein milk products. Based on this study, we were able to establish ananalytical method for accurate and robust detection of residualantibiotics in milk. In particular, TTC test could detect sulfon-amide-type antibiotics over MRLs of EU and Codex in milk, andSTAR assay could detect quinolone-type antibiotics over EU MRL.In a future study, we will develop a new microbial assay to de-crease false-positive readings based on the current assays.

Acknowledgment

This work was supported by a grant from the KFDA (Korea Foodand Drug Administration) R&D Project (06042-Hangnaemo-118).

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