1067 Journal of Food Protection, Vol. 56, No. 12, Pages 1067-1072 (December 1993) Copyright©, International Association of Milk, Food and Environmental Sanitarians Simple Continuous and Simultaneous Determination of Multiple Sulfonamide Residues 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.