Simple Continuous and Simultaneous Determination of Multiple Sulfonamide Residues

1067

CHIN-EN TSAI and FUSAO KONDO*

Department of Veterinary Public Health, Faculty of Agriculture, Miyazaki University, Kibanadai-Nishi, Gakuen, Miyazaki-shi 889-21, Japan Downloaded from http://meridian.allenpress.com/jfp/article-pdf/56/12/1067/1659673/0362-028x-56_12_1067.pdf by guest on 30 September 2021

(Received for publication September 14, 1992)

ABSTRACT trimethoprim (TMP) has been used as a test medium for sulfonamides. Sulfonamides and TMP inhibit the enzymes A new continuous separation method was developed for the of the tetrahydrofolate biosynthetic pathway in two sequen determination of nine different sulfonamides (sulfaguanidine, sulfamethazine, sulfapyridine, sulfadiazine, sulfathiazole, tial steps and have a synergistic effect which has been used sulfamethizole, sulfamethoxazole, sulfadimethoxine, and for therapeutic purposes for some years (7). For extraction, sulfaquinoxaline). Bioassay on minimum medium seeded with the cleanup method is complicated, involving Bacillus subtilis ATCC 6633 was carried out for detection. An deproteinization and removal of fatty acids from biological extract taken from an agar block of the clear inhibition zone on materials that are shown to be residual positive by bioassay. minimum medium produced by a mixture of sulfonamides was This has usually been done in accordance with each anti then subjected to high-performance liquid chromatography. bacterial agent tested, and much time is needed to obtain For identification, high-performance liquid chromatography the test sample for identification (3,8). As MM does not analyses were performed using two different columns and analyti contain any peptone, the cleanup method for extraction cal conditions. Using a u-Bondapak C column, the sulfonamides 18 would therefore be comparatively easy. were separated at room temperature using a mobile phase of methanol: 0.1 M KH2P04 (30:70, vol/vol) at a flow rate of 1.0 ml/ HPLC is becoming increasingly popular for the identi min. A variable wavelength detector set at 265 nm and recorder fication of chemical substances, and there are many reports set at 4 mm/min were used for the detection. The entire mixture on the identification of single sulfonamides (1-6,9,10,17) was resolved as eight peaks from 4.68 to 50.78 min. When an by HPLC analysis. Recently, however, more reports have Asahipak GS-320 column was employed, nine peaks were sepa appeared describing the simultaneous determination and rated with retention times ranging from 12.62 to 54.43 min using identification of multiple sulfonamides in animal tissues a mobile phase of acetonitrile: 1% acetic acid (25:75, vol/vol) at (8,11,16), animal feed (12,15), and food products (13) a flow rate of 2.0 ml/min. using HPLC. Generally, however, it has proved difficult to Correlation coefficients of standard curves for individual separate a large number of drugs in test samples by HPLC sulfonamides were linear (>0.99) with recoveries ranging from analysis. 25.2 ± 8.6% to 64.1 ± 8.6% for a concentration range of 1.0-25 lig/ml. Many analysts use a reverse-phase column for elution by the gel-permeation chromatography mode in sulfona Sulfonamides are widely used as antimicrobial agents mide determination by HPLC (1,4-6,12). It is difficult to and growth promoters in veterinary practice and animal clearly separate the early eluting drug peaks from protein production. The safety of these drugs for consumers has peaks originating from the biological samples themselves been questioned because of their apparent toxicity (9). in the reverse-phase column. Therefore, a sensitive and reliable method is needed for However, a new Asahipak GS-320 column has been monitoring of sulfonamide residues. developed recently by Asahi Kasei Co., Ltd., Tokyo, Japan. In general, the inspection of residual antimicrobial This column has characteristics suitable for rapid separa agents in meat is performed according to the following tion of relatively hydrophilic high-molecular-weight com steps: i) detection by microbiological assay, ii) extraction pounds, including most proteins, nucleosides, and glycans, (cleanup) from the residual-positive tissue, and iii) identifi by gel permeation. Low-molecular-weight hydrophobic com cation by high-performance liquid chromatography (HPLC). pounds are eluted slowly. Thus, gel-permeation chromatog In microbiological screening, Mueller-Hinton agar raphy can separate drugs and their metabolites, and with (MHA, Difco) and antibiotic medium No. 5 (Difco) have this column, it is possible to use incompletely deproteinized often been used as assay media. These media contain some biological samples. peptone for the growth of test organisms. Minimum me In the present study, a new separation method was dium (MM), which does not contain peptone, was devel tested for determination of nine different sulfonamides. oped by Spizizen (14) for biochemical research on the MM seeded with B. subtilis ATCC 6633 was used for DNA of Bacillus subtilis. Also, MHA containing detection, followed by extraction from the clear area of

JOURNAL OF FOOD PROTECTION, VOL. 56, DECEMBER 1993 1068 TSAI AND KONDO inhibition on the agar produced by mixed sulfonamides, Quantitation of sulfonamides was based on the linear rela and identification of the extract by HPLC. For the HPLC tionship between the peak heights (uV) in the chromatograms and analysis, we compared a reverse-phase |i-Bondapak C the known concentration (ug/ml) of the drugs. column and an Asahipak GS-320 column for simultaneous Preparation of agar taken from the inhibition zone on the agar determination of sulfonamides. plate Each concentration of the mixed sulfonamide solution, 1.0, MATERIALS AND METHODS 2.5, 5.0, 10.0, or 25.0 ug/ml, was used in the formation of an Chemicals used inhibition zone on MM without TMP. The agar surrounding the A total of nine sulfonamides were used in this study: paper disc on the MM plates was transferred into an injection sulfaguanidine (SGN), sulfamethazine (SMT), sulfapyridine (SPR), syringe using a small spoon or knife. The plunger of the syringe sulfadiazine (SDZ), sulfathiazole (STZ), sulfamethizole (SMI), was pushed by hand, and the agar was broken into pieces by sulfamethoxazole (SMX), sulfadimethoxine (SDM), and passage through the syringe needle. The broken agar was trans sulfaquinoxaline (SQ). All except STZ were purchased from ferred immediately to a centrifuge tube, then 2.0 ml of ethyl Sigma Chemical Co., St. Louis, MO. STZ was purchased from acetate (Wako Pure Chemical Industries, Ltd., Osaka, Japan) was

Tokyo Kasei Kogyo Co., Tokyo, Japan. Each sulfonamide, except added to the tube for extraction, followed by centrifugation at Downloaded from http://meridian.allenpress.com/jfp/article-pdf/56/12/1067/1659673/0362-028x-56_12_1067.pdf by guest on 30 September 2021 SDM, was first dissolved in N.N-dimethylformamide at 10% 1,000 x g for 5 min. The supernatant was evaporated to dryness wt/vol. SDM was dissolved in 28% ammonia to a final concentra in a 40°C water bath under nitrogen gas. The sediment was tion of 10% wt/vol. These stock solutions were further diluted to suspended in 0.08 ml of a mobile phase of acetonitrile: 1% acetic a concentration of 1,000 Ug/ml. SPR and SDZ were diluted in acid (25:75, vol/vol) and then centrifuged at 7,000 x g for 6 min. 0.05 M NaH2P04 buffer (pH 7.0). The other seven sulfonamides Fifty microliters of the supernatant was injected into the HPLC. A were diluted to 1,000 ug/ml with distilled water. These solutions schematic representation of the procedure for extraction from the are referred to as working solutions. The working solutions were agar within the clear inhibition zone for each antibacterial agent diluted in twofold serial dilutions from 100 to 0.05 ug/ml in water on MM is shown in Fig. 1. The extraction time was less than 30 for the bioassay or in the mobile phase for HPLC analysis. The min. mixture of nine sulfonamides was prepared by adding together 1 ml of each working solution. Recovery The recoveries from the agar were determined by HPLC Disc diffusion method analysis using a GS-320 column with six replicates. The recover Trimethoprim (TMP, Nakarai Tesque Co., Kyoto, Japan) was ies of each sulfonamide from the agar were determined by used for examining any synergistic effect in the sulfonamides comparing the peak heights obtained by chromatography with bioassay (11). those of pure drug standards. Eight milliliters of MM or MHA seeded with B. subtilis ATCC 6633 was poured into a disposable, sterilized petri dish (90 mm diameter) to a thickness of 2 mm. The media were compared with or without TMP (final concentration 0.05 (ig/ml). Hinimum nediura seeded with B. subtilis ATCC-6633 (spore) All drugs were tested at a concentration of 100 ug/ml and in Incubate'Yor 18 h, at 37°C twofold serial dilutions to 0.05 ug/ml in sterile distilled water. A • Paper disc filter paper disc (type 27, 10 mm diameter, Toyo Roshi Co., Ltd.) was moistened with 0.08 ml of the drug solution and put on the " Inhibition zone surface of the plate. The plates were incubated at 37°C for 18 h, and the diameter of the clear inhibition zone surrounding the paper disc was measured. The lowest concentration which com pletely inhibited growth of the test organism was recorded for Agar block • each antibacterial agent.

HPLC analysis

(i) Apparatus. The apparatus consisted of a Model 600E [Extraction method] multisolvent delivery system, a U6K injection system, a Model Syringe for breaking up agar 481 variable-wavelength detector (Waters Associates, Inc., Milford, MA), and a chromatopac C-R6A (Shimadzu Seisaku Co., Ltd., Kyoto, Japan) used as an integrator. The recorder was set at a chart speed of 4 mm/min. Add 2 il ethyl acetate to centrifuge tube (ii) HPLC column. Two analytical columns, a 100 mm x 5 mm ID, prepacked 10-um particle size, u.-Bondapak C column Mix with vortex mixer (Waters Associates) and a 500 mm x 6.0 mm ID Asahipak GS- i Centrifuge (1,000 x g, 5 Bin) 320 column (Asahi Kasei Co., Ltd.) were used for comparison. 1 (iii) Mobile phases. The following two mobile phases were Supernatant 1 used: methanol: 0.1 M KH2P04 (30:70, vol/vol) according to the Evaporate 1 same mobile phase as Smedley and Weber (13) for the u.- Dissolve with mobile phase Bondapak C column, and acetonitrile: 1% acetic acid (25:75, i vol/vol) for the Asahipak GS-320 column. Centrifuge (7,000 X g, 6 uiiO 1 (iv) Analytical conditions. Analytical conditions were as Supernatant for HPLC analysis follows: flow-rate, 1.0 ml/min for the u-Bondapak C18 column and 2 ml/min for the GS-320 column; temperature, ambient (~25°C); Figure 1. Schematic representation of procedure used for extrac detection, UV265 nm and 0.004-0.04 absorbance units, full scale tion of drugs from agar inside the inhibition zone on MM for (AUFS) for both columns. HPLC analysis.

JOURNAL OF FOOD PROTECTION, VOL. 56, DECEMBER 1993 DETERMINATION OF MULTIPLE SULFONAMIDE RESIDUES 1069 RESULTS fonamides are shown in Fig. 2. As can be seen, the GS-320 column eluted nine separate peaks, one for each sulfon Inhibition diameter and minimum detectable concentration amide. The peaks were quite symmetrical, without tailing, Comparative data for inhibition zone diameter and and each had a characteristic retention time. The elution minimum detectable concentration in MM and MHA me orders and retention times (min) of the sulfonamides were: dium for each of the nine sulfonamides are shown in Table SGN (12.62), SMT (15.38), SPR (16.15), SDZ (17.40), 1. Inhibition zone sizes were different for the nine sulfona STZ (19.88), SMI (22.47), SMX (28.67), SDM (33.84), and mides at any given concentration. Tests performed using SQ (54.43). MM generally showed a larger inhibition diameter and a lower detectable concentration for all sulfonamides com In contrast, only eight peaks appeared when the 9- pared with MHA. MM seeded with B. subtilis showed component mixture was analyzed on the ^i-Bondapak C)g better antibacterial activity at 25-0.78 |ig/ml than MHA column. On this column, the fastest eluting drug peak even without addition of TMP to the assay medium. appeared earlier (4.68 min) than on the GS-320 column. The elution orders and retention times (min) of the sulfon amides were: SGN (4.68), SDZ and STZ (same retention HPLC analysis of standard solutions of sulfonamides Downloaded from http://meridian.allenpress.com/jfp/article-pdf/56/12/1067/1659673/0362-028x-56_12_1067.pdf by guest on 30 September 2021 Chromatograms analyzed on both GS-320 and \i- time, 7.72), SPR (8.57), SMI (10.82), SMT (13.03), SMX Bondapak C columns using the mixture of the nine sul (16.12), SDM (43.69), and SQ (50.78).

TABLE 1. Comparison of inhibition zone size (mm) in minimum medium (MM) and Mueller-Hinton agar (MHA), with and without trimethoprim, of nine sulfonamides by disc method using B. subtilis.

Drugs Medium Trimethoprim Concentration (Ug/ml) (ug/ml) 100 50 25 12.5 6.25 3.12 1.56 0.78 0.39

MM + 0 47.6 42.1 40.7 36.9 31.7 25.3 21.4 17.1 _ MM + 0.05 59.8 56.6 51.3 47.0 42.2 38.3 28.6 20.5 13.4 SQ MHA + 0 22.7 21.3 18.4 17.1 12.7 - - - - MHA + 0.05 33.4 30.9 28.1 24.4 20.8 17.6 14.1 - -

MM + 0 46.2 41.6 38.5 34.6 30.1 26.5 21.7 17.2 - MM + 0.05 58.6 54.8 47.8 45.0 39.5 32.6 24.6 19.6 - SDM MHA + 0 22.8 19.7 17.6 15.1 12.3 - r - - MHA + 0.05 32.8 31.2 28.0 24.8 21.2 16.3 12.2 - -

MM + 0 51.9 47.2 42.0 36.2 32.8 23.1 17.0 14.0 - MM + 0.05 56.3 52.0 47.0 39.1 32.0 25.0 18.1 15.1 - STZ MHA + 0 19.0 17.1 12.8 12.0 - - - - - MHA + 0.05 34.9 32.0 29.6 26.2 23.5 17.2 13.0 - -

MM + 0 46.2 41.3 37.5 33.9 27.1 19.4 13.2 - - MM + 0.05 61.7 57.3 52.4 46.0 37.4 30.0 22.7 - - SMX MHA + 0 24.2 22.3 18.5 15.0 - - - - - MHA + 0.05 35.4 32.0 29.1 27.1 23.0 17.6 12.8 - -

MM + 0 44.8 35.4 31.0 25.2 19.3 16.0 - - - MM + 0.05 56.4 51.6 47.0 38.6 32.7 19.2 12.6 - - SDZ MHA + 0 22.4 20.8 16.0 13.8 - - - - - MHA + 0.05 33.5 31.4 27.5 24.0 19.6 - - - -

MM + 0 37.7 31.0 28.5 24.3 15.4 - - - - MM + 0.05 48.4 43.1 35.7 26.0 17.0 - - - - SMI MHA + 0 18.1 14.3 ------MHA + 0.05 26.2 23.6 19.6 13.7 - - - - -

MM + 0 35.3 26.7 21.4 17.5 12.2 - - - - MM + 0.05 47.4 40.8 32.2 23.0 12.0 - - - - SPR MHA + 0 21.7 18.7 14.5 ------MHA + 0.05 31.1 26.6 23.1 19.2 11.9 - - - -

MM + 0 36.3 30.2 19.8 16.0 - - - - - MM + 0.05 46.4 38.3 31.0 21.4 - - - - - SMT MHA + 0 19.1 14.7 12.6 ------MHA + 0.05 29.3 25.8 21.5 17.4 - - - - -

MM + 0 29.1 20.0 12.9 ------MM + 0.05 29.8 20.2 15.0 ------SGN MHA + 0 12.7 ------MHA + 0.05 20.8 17.6 12.0 ------

JOURNAL OF FOOD PROTFCTION, VOL. 56, DECEMBER 1993 1070 TSAI AND KONDO

Retention tine Cain)

Figure 3. HPLC chromatograms produced using the GS-320 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/56/12/1067/1659673/0362-028x-56_12_1067.pdf by guest on 30 September 2021 Retention tide (Bin) column and an extract from agar within the inhibition zone on MM formed by a mixture of nine standard sulfonamides (each at 10 \ig/ml), together with a blank of medium alone (lower). Analysis was done under the conditions described in the text, except for AUFS 0.008.

Figure 2. HPLC chromatograms of nine standard sulfonamides using GS-320 (upper) and \y-Bondapak C]s (lower) columns. Analysis was done at a concentration of 10 \ig/ml for each sulfonamide under the conditions described in the text except that the injection volume was 50 u/ for the GS-320 column and 25 \il for the [l-Bondapak C column.

The standard calibration curves were linear, and the Retention tine (uin) correlation coefficient for each of the nine drugs was more than 0.99. Linear response curves (|ig/ml versus peak Figure 4. HPLC chromatograms produced using the \i-Bondapak height) were obtained when the method was used to deter CJS column and an extract from agar within the inhibition zone on mine the concentrations of the nine sulfonamides. MM formed by a mixture of nine standard sulfonamides (each at 10 \lg/ml), together with a blank of medium alone (lower). Recovery for HPLC analysis of the extracted agar Analysis was done under the conditions described in the text, The extracted agar from the inhibition zone on MM except for AUFS 0.016. was used for drug identification on the two HPLC columns, as shown in Fig. 3 and 4. The GS-320 column perfectly and accurate, since many kinds of drugs are used in pigs, separated nine sulfonamide peaks. On the p.-Bondapak C]g column, the earliest eluting peak (SGN) was partially ob cattle, and broiler chickens which are slaughtered for hu scured by an unidentified peak originating from the agar man consumption every day in all countries. itself. Therefore, the GS-320 column was used for the MM was developed by Spizizen {14) for biochemical recovery study. The recovery results for the nine sulfon research on DNA in B. subtilis. It is known that only B. amides in the extracted agar using the GS-320 column are subtilis and Escherichia coli can grow on this medium. shown in Table 2. The detection limit for drugs in the Accordingly, a strict aseptic procedure does not need to be inhibition zone on the agar was 1 ^g/ml. The recoveries of used for MM, unlike the case for the routine assay medium the nine sulfonamides extracted from the agar (n = 6) MHA, when carrying out investigations of residual drugs. ranged from 25.2 ± 8.6% to 64.1 ± 8.6%. SGN and SMI Because MM does not contain any peptone, the clear showed quite low recovery in comparison with the other inhibition zone produced is larger than that on MHA, and drugs. extraction (deproteinization) is very easy to perform. The resolution achieved by the p.-Bondapak C18 and DISCUSSION GS-320 columns was evaluated and compared using nine different sulfonamides at several concentrations. The nine It is desirable that all tests for the detection, extraction, drugs were eluted as 9 separate peaks within about 55 min and identification of residual drugs should be simple, rapid, on the GS-320 column. However, only eight peaks ap-

JOURNAL OF FOOD PROTECTION. VOL. 56. DECEMBER 1993 DETERMINATION OF MULTIPLE SULFONAMIDE RESIDUES 1071

TABLE 2. The correlation coefficient of standard curves and recovery results of nine sulfonamides on the agar block extraction by Asahipak GS-320 column.

•"Concentration of each sulfonamide (|jg/rul)

Drugs "Corn coeff. 1.0 2.5 5 10 25 cMean ± SD

SGN 0.9945 ± 0.0030 24.0 ± 8.3 27.9+ 11.8 27.7 ± 9.3 25.0 ± 6.1 21.7 ± 4.8 25.2 + 8.6 SMT 0.9987 ± 0.0005 53.4 + 13.1 58.5 + 9.4 60.5 ± 15.0 71.9 + 9.8 69.0 + 4.5 63.1 ± 12.8 SPR 0.9988 ± 0.0004 49.8 + 12.2 62.5 ± 7.6 65.0 + 11.3 69.7 ± 8.7 67.8 ± 4.2 63.4 ±11.4 SDZ 0.9987 + 0.0005 52.9 + 1.2 65.0 ± 4.1 63.0+ 11.0 67.2 ± 6.6 61.0 ± 5.6 63.1 ± 8.0 STZ 0.9987 ± 0.0005 45.5 + 3.4 54.0+ 8.1 51.4 ± 9.5 56.3 ± 6.4 54.1 + 3.7 52.4 ± 7.6 SMI 0.9985 ± 0.0008 35.7 ± 8.9 31.1 ±9.5 32.0 + 12.1 30.0 ± 7.4 25.3 ± 5.6 30.6 + 9.6 SMX 0.9987 + 0.0005 48.5 ± 8.4 59.1 +5.3 62.4 + 12.0 64.0 + 10.6 70.8 + 4.8 61.5 ± 11.4 SDM 0.9987 ± 0.0005 54.5 ± 7.9 60.5 ± 3.3 65.1 ± 7.8 70.3 ± 8,5 68.1 ±2.4 64.1 ± 8.6

SQ 0.9977 ± 0.0011 39.3 ± 5.3 58.2 ± 7.4 62.1 +6.8 60.7 ± 13.1 60.0 ± 6.0 56.3 ± 11.6 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/56/12/1067/1659673/0362-028x-56_12_1067.pdf by guest on 30 September 2021 a Mean + SD, 6 standard curves, n 6. b Mean + SD, n = 6. c n = 30.

peared on the |i-Bondapak Clg column (two drugs, SDZ and and allows simultaneous analysis of multiple drugs. It STZ, having the same retention time). When the sulfon should prove useful for routine laboratory testing of re amides were extracted from the agar, the GS-320 column sidual sulfonamides in food. As only sulfonamides were was superior to the u-Bondapak C18 because peaks originat used in this study, the present procedure should be inves ing from the agar did not interfere with the early sulfon tigated further employing other antimicrobial agents. amide peaks. However, the earliest eluting drug peak (SGN) was obscured by the peaks from the agar when the u- REFERENCES Bondapak C column was employed. ]g Barry, C. P., and G. M. Maceachern. 1983. Reverse phase liquid As the elution order of certain drugs (SMT, SPR, chromatographic determination of sulfathiazole residues in honey. J. SDZ, STZ, SMI) differed between the two columns, the Assoc. Off. Anal. Chem. 66:4-7, use of both columns allowed more reliable identification Belliardo, F. 1981. Determination of sulfonamides in honey by high- pressure liquid chromatography. J. Agric. Res. 20:44-48. of sulfonamides with different retention times. SDM and Blanchflower, W. J., and D. A. Rice. 1988. Extraction of SQ were eluted later than the other sulfonamides on both sulfamethazine from feed samples. J. Assoc. Off. Anal. Chem. columns. Earlier elution of such late eluting drugs might 71:302-303. be achieved by the use of a gradient analytical HPLC Carignan G., and K. Carrier. 1991. Quantitation and confirmation of sulfamethazine residue in swine muscle and liver by LC and GC/MS. system. J. Assoc. Off. Anal. Chem. 74:479-482. If the diameter of the inhibition zone on MM was too Cox, B. L., and L. F. Krzeminski. 1982. High pressure liquid small to obtain an adequate amount of agar for extraction, chromatographic determination of sulfamethazine in pork tissue. J. it was possible to perform the extraction using more than Assoc. Off. Anal. Chem. 65:1311-1315. 6. Endoh, Y. S„ Yamaoka, and N. Sasaki. 1987. Liquid chromato two agar plates (2 paper discs). As shown in Table 2, the graphic determination of sulfamoyldapsone in swine tissues. J. Assoc. mean recovery for the nine sulfonamides was 44.8% at 1 Off. Anal. Chem. 70:1031-1032. lig/ml. The low recovery was due to incomplete drug Gudding, R. 1976. An improved bacteriological method for the diffusion in the paper disc. Good recovery was obtained detection of sulfonamide residues in food. Acta Vet. Scand. 17:458- 464. when agar was gathered from 5 and 10 separate plates Horii, S., C. Momma, K. Miyahara, T. Maruyama, and M. Matsumoto. bearing each sulfonamide at 1 lig/ml. The recovery com 1990. Liquid chromatographic determination of three sulfonamides pared with 1 lig/ml standard sulfonamide reached 152.6 in animal tissue and egg. J. Assoc. Off. Anal. Chem. 73:990-992. and 337.2% for 5 and 10 agar plates, respectively. Larocque L., G. Carignan, and S. Sved. 1990. Sulfamethazine (sulfadimidine) residues in Canadian consumer milk. J. Assoc. Off. A new continuous separation method was studied for Anal. Chem. 73:365-367, the determination of nine different sulfonamides. Bioassay 10 Long, A. R., L. C. Hsieh, M. S. Malbrough, C. R. Short, and S. A. using MM seeded with B. subtilis ATCC 6633 was used for Barker. 1990. Matrix solid phase dispersion isolation and liquid detection, and ethyl acetate was used for extraction from chromatographic determination of sulfadimethoxine in catfish {Ictalurus punctatus) muscle tissue. J. Assoc. Off. Anal. Chem. the agar obtained from the clear inhibition zone produced 73:868-871. on the plate by a mixture of sulfonamides. The extract was 11 Parks, O. W. 1982. Screening test for sulfamethazine and sulfathiazole then identified by HPLC. The detection limit was about 1 in swine liver. J. Assoc. Off. Anal. Chem. 65:632-634. lig/ml for each sulfonamide using this approach. However, 12, Smallidge, R. L., E. J. Kentzer, K. R. Stringham, E. H. Kim, C. Lehe, R. W. Stringham, and E. C. Mundell. 1988. Sulfamethazine as shown in Table 1, this was not the case for all sulfon and sulfathiazole determination at residue levels in swine feeds by amides by the bioassay method. Because the nine sulfon reverse-phase liquid chromatograph with post-column derivatization. amides show different antibacterial activities, it is possible J. Assoc. Off. Anal. Chem. 71:710-717. that certain drugs would be lost if the bioassay method was 13 Smedley, M. D., and J. D. Weber. 1990. Liquid chromatographic determination of multiple sulfonamide residues in bovine milk. J. used in the first step to screen samples suspected to contain Assoc. Off. Anal. Chem. 73:875-879, residuals. However, the present method is simple and rapid, 14. Spizizen, J. 1958. Transformation of biochemically deficient strains

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