Improved Method for the Determination of Residual Aminoglycoside Antibiotics in Animal Tissues by Electrophoresis and Bioautography

Home , Bekanamycin

FUSAO KONDO* and SACHIKO HAYASHI

Department of Veterinary Public Health, Faculty of Agriculture, Miyazaki University, I, l-chome, Kibanadai-nishi, Gakuen, Downloaded from http://meridian.allenpress.com/jfp/article-pdf/52/2/96/1655464/0362-028x-52_2_96.pdf by guest on 28 September 2021 Miyazaki-shi, Japan 889-21

(Received for publication July 19, 1988)

ABSTRACT bioautographic visualization of the separated components has been reported by several authors (1,2,3,4,7,9,11,12,15). The use of agar gel electrophoresis and bioautography for Most of these methods involve the application of antibi the identification of small amounts of seven aminoglycoside otic solutions of high potency. In order to obtain a more antibiotics (streptomycin, dihydrostreptomycin, kanamycin, sensitive method, the procedure described by Lightbown bekanamycin, gentamicin, fradiomycin and paromomycin) was and Rossi (4) was evaluated. After agar gel electrophoresis, studied. The procedure used for the electrophoretic separation of these components is described. The antibiotics were detected the gel was covered with a second layer of agar seeded with high sensitivity using Bacillus subtilis ATCC 6633 as a test with a test organism. However, they reported difficulty in organism, Tris buffer at pH 8.0, and Bacto-Agar as the support obtaining thin layers of uniform thickness due to larger gel ing medium for each of the components. The limits of detection tray (94 x 19 cm), which is a prerequisite for high repro obtained for the various antibiotics ranged between 0.078 and ducibility and sensitivity. Because of the long duration of 0.313 ug/ml and the standard deviation ranged from 0.05 and electrophoresis ( 1 to 3 h), growth of contaminants often 0.34. Recoveries from extracts of bovine kidney tissue contain occurred. Many previous reports on TLC and bioautogra ing each drug ranged from 59.0% to 90.2%. The sensitivity of phy have dealt with a limited number of different antibi this method can be adjusted through minor modifications, thus otics, but no studies have focused on antibiotics having a allowing it to be used for routine residual analysis of aminogly cosides in biological samples. similar chemical structure. The method described in this paper involves a 40-min period of electrophoresis and permits the identification of up to seven aminoglycoside antibiot Aminoglycoside antibiotics (AGs) show stronger anti ics. Better sensitivity was obtained than that described in bacterial activity against Gram-negative bacteria in com the literature. parison with Gram-positive microorganisms (5) and they are extensively used for the treatment and prevention of MATERIALS AND METHODS infections in livestock. However, this group of drugs are Antibiotics examined known to show high long-term residual concentrations in The aminoglycoside antibiotics studied and their source: the kidney in comparison with other antibiotics (6). For streptomycin (SM), and dihydrostreptomycin (DSM) (Meiji Seika this reason, a 30-d drug-clearance period for cows and Co., Tokyo, Japan), kanamycin (KM) and bekanamycin (BKM) pigs is stipulated in Japan. Accordingly, the inspection of (Takeda Chemical Industries, Osaka, Japan), gentamicin (GM) residual aminoglycosides in animal tissues must be inten (Sigma Chemical Co., St. Louis, MO), and fradiomycin (FM) sified. and paromomycin (PRM) (the National Veterinary Assay Labo There have been many reports on microbioassay (10), ratory, Tokyo, Japan). thin-layer chromatography (TLC), TLC/bioautography (7), Aqueous solutions of each drug were prepared at suitable and gas-liquid chromatography (GLC) (8,16,17) as the ana concentrations by dilution in electrolyte. lytical methods for AGs. In the TLC method it is difficult for AGs to be visualized on the TLC plate using standard Culture Media Heart infusion broth (HIB; Difco Laboratories, Detroit, MI) developing solvent. On the other hand, specimens for GLC was used for preculture of the test organism and Antibiotic Medium analysis must be initially converted chemically to volatile No. 1 (AMI; Difco) was used as the layer medium after addition substances. Measurement of AGs by enzyme immunoas of 1.5% Bacto Agar (Difco). say has also been reported (13,14), but this method is not yet in general use. Bacteria The separation of antibiotics by TLC followed by Bacillus subtillis ATCC 6633 was used as the test organ-

JOURNAL OF FOOD PROTECTION, VOL. 52, FEBRUARY 1989 DETERMINATION OF ANTIBIOTICS IN ANIMAL TISSUES 97

ism. The culture medium was inoculated with B. subtilis cells to water bath at 70 to 80°C. After centrifuging for 10 min at 900 achieve a concentration of 104 cfu/ml in the layer medium (AMI). G, the supernatant was placed in a rotary flask and evaporated The size of the gels poured was 60 x 25 x 0.2 cm. to dryness under reduced pressure at 70°C using a rotary evapo rator. After dissolving the residue with a small amount of phosphate Electrophoresis equipment buffer, the sample was passed through a filter paper, Toyo Roshi The DNA Sub Cell Electrophoresis System 170-4304 (Bio- Type No. 2, (Toyo Roshi Kaisya, Ltd., Tokyo). The filtrate was Rad Laboratories, Milano, Italy) with a Model Elepos PS-2515 further passed through a Sep-Pak silica cartridge (Waters Asso (Toyo Kagaku Sangyo Ltd., Tokyo) power pack capable of ciates, Inc., Milford, MA) and the cartridge washed with 5 ml delivering a maximum current of 200 mV or 2500 volts was of distilled water. Next, elution was performed with 4 ml of used for electrophoresis. The electrophoresis gel plate was connected 10% acetic acid, and the eluate was dried and condensed by to a "Neocool", model BP-51 aspirator (Yamato Instruments spraying with nitrogen gas. The sediment was finally dissolved Co., Tokyo, Japan) for cooling at 5°C. with 1 ml of distilled water and used for the electrophoresis.

Electrolytes RESULTS The electrolytes used were buffers containing (g/1): Tris, Downloaded from http://meridian.allenpress.com/jfp/article-pdf/52/2/96/1655464/0362-028x-52_2_96.pdf by guest on 28 September 2021 1.82, succinic acid, 0.98; pH 6.0; and Tris, 3.03; succinic acid, Effect of migration to supporting media and electrolytes 0.85; pH 8.0, prepared from analytical-grade reagents. Various The results for the migration distance and direction of phosphate buffers at 1/15 M were also prepared to give pH mobility of AGs, when Tris and phosphate buffer were values of 6.5, 7.0, 7.5 and 8.0 with 0.1 N phosphoric acid or 0.1 used as electrolytes, are shown in Tables 1 and 2, respec N sodium hydroxide, respectively. These electrolytes were also used as solvents for the supporting media. tively. AGs generally migrated further in Bacto Agar than in Agar Noble. All drugs moved in the direction of the Supporting media anode in Tris buffer at both pH values in the Bacto Agar. The supporting gels were 1% (w/v) Bacto Agar (Difco) and However, it differed depending on the antibiotics present 1% (w/v) Agar Noble (difco) prepared independently after dis in the Agar Noble (Table 1). GM did not move at all in solving in the two buffers used as electrolytes, according to the the Agar Noble and Tris buffer, pH 8.0. All drugs moved method of Smither and Vaughan (II). in the direction of the cathode, irrespective of pH, when After cooling to 50°C, the agar gels were poured onto the phosphate buffer was used as the electrolyte (Table 2). leveled gel plate to give a uniform gel depth of 2.0 mm. When B. subtilis was used as the test organism in phos phate buffer, no growth on several plates sometimes occurred. Electrophoresis procedure Accordingly, it was judged that phosphate buffer was not Ten microliters of each diluted drug solution was infused a good electrolyte. onto a spot on the gel plate, using the same buffer as that employed to prepare the plate. After setting, the gel layers were connected to the electrolyte in the buffer boxes by a layer of thick cotton Detection limits and migration distance of aminoglyco fabric. After closing the lid of the safety box, the electrophoresis sides was normally run for 40 min at a constant current of 190 mA, When analysis of the drugs was carried out using Tris which gave a starting potential of 800 volts. During the electro buffer and Bacto Agar, the detection limit of each drug phoresis, water at 5°C from the cooling aspirator was run under ranged from 0.313 to 5.0 |ig/ml at pH 6.0 and from 0.078 the plate bed to prevent the occurrence of so-called joule heat. to 0.313 Ug/ml at pH 8.0, respectively. The detection limit After electrophoresis, Antibiotic Medium No. 1 containing was smaller in the alkaline buffer at pH 8.0 (Table 3). The B. subtilis was layered 0.2 cm thick over the gel plate asepti- cally into a germ-free box and the plates were incubated over 4-times-average migration distance of the drugs ranged night at 37°C. Then, by precisely measuring the length of central from 21.8 to 109.1 mm under the analytical conditions of inhibition zone that appeared no growth from a center of each pH 8.0, Tris buffer and Bacto Agar. The standard devia drug spot, the migration distance of each drug was determined. tion among the seven drugs ranged from 0.05 to 0.34 (Table The direction of migration and also the forms of the inhibition 4). zone were observed. To assess reproducibility, the migration distances of each drug were measured 4 times under the best analytical conditions TABLE 1. Migration distance of aminoglycosides using Tris and the result expressed as the mean average migration dis Buffer as electrolyte. tances plus standard deviation. Bacto Agar Agar Noble Drug pH 6.0 pH 8.0 pH 6.0 pH 8.0 Drug recovery from bovine kidney DSM (+7'"74.8 "(+) 41.5 (-) 107.0 (-) 110.0 Each drug (100 ug/g) was added to 10 g of bovine kidney SM (+) 79.3 (+) 59.6 (-) 104.0 (-) 108.0 cut up in small pieces, 25 ml of 70% methanol was then added KM (+) 110.1 (+) 66.2 (+) 32.0 (-) 110.0 and the tissue was homogenized for 10 min using high-speed ho- AKM (+) 118.4 (+) 100.0 (+) 51.6 (-) 87.8 mogenizer, Ultra-Turrax T18 (Janke & Kunkel GmbH & Co., GM (+) 115.01 (+) 118.1 (+) 74.01 0 Switzerland). The homogenate was transferred to a centrifuge FRM (+) 81.71 (+) 120.0 (+) 13.21 (+) 22.8 tube and separation was performed at ambient temperature for PRM (+) 100.0 (+) 112.8 (+) 52.5 (-) 44.8 30 min at 900 G. The supernatant was transferred to another Migration distance: mm. centrifuge tube, centrifugation was again done and the superna (+): anode, (-): cathode. tant was collected. The supernatant was heated for 10 min in a t: tailing.

JOURNAL OF FOOD PROTECTION. VOL. 52, FEBRUARY 1989 98 KONDO AND HAYASHI

TABLE 2. Migration distance of aminoglycosides using phosphate buffer as electrolyte. Drugs pH 6.5 pH 7.0 pH 7.5 pH 8.0 DSM Bacto Agar '-) 104.8 '-) 92.6 '-) 72.4 -) 55.2 Agar Noble -) 109.2 -) 71.9 -) 52.8 -) 42.6 SM Bacto Agar -) 108.8 '-) 85.0 ;-) 72.0 -) 49.6 Agar Noble -) 106.3 -) 59.7 -) 50.0 -) 38.1 KM Bacto Agar ,-) 82.0 -) 72.0 ,-) 60.0 -) 50.2 Agar Noble -) 82.5 '-) 51.0 -) 37.6 -) 38.6 AKM Bacto Agar -) 52.1 -) 54.5 -) 46.2 -) 46.0 Agar Noble -) 55.1 -) 35.4 -) 30.2 -) 31.7 GM Bacto Agar -) 43.0 -) 70.0 -) 66.5 -) 61.0 Agar Noble -) 93.9 -) 73.5 -) 55.0 -) 49.0 FRM Bacto Agar -) 20.8 t -) 29.7 t -) 24.8 t -) 29.6 t Agar Noble -) 51.71 -) 25.0 t -) 14.8t -) 20.9 t PRM Bacto Agar -) 56.7 -) 65.6 -) 61.6 -) 45.4

Agar Noble -) 80.0 -) 50.4 -) 40.5 -) 36.1 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/52/2/96/1655464/0362-028x-52_2_96.pdf by guest on 28 September 2021 Migration distance: mm. (-): cathode. t: tailing

TABLE 3. Detection limits of seven aminoglycosides by Electro- Recovery of antibiotics from bovine kidney phoresislBioautographic Method using Tris Buffer as electrolyte Analyses were carried out on extracts from bovine and Bacto Agar as supporting medium. kidney tissues containing each drug using analytical con Drugs Detection limits (u.g/ml) ditions of pH 8.0, Tris buffer and Bacto Agar. pH 6.0 pH 8.0 DSM 0.625 0.156 Recoveries of the seven aminoglycosides from bovine SM 1.25 0.313 kidney ranged from 59.0 to 90.2% (Table 5). An extract KM 0.313 0.078 from bovine kidney containing antibiotics showed almost AKM 0.625 0.156 the same migration distance as standard solutions of each GM 0.313 0.078 aminoglycoside. FRM 5.00 0.156 PRM 1.25 0.313 DISCUSSION

Analytical conditions for AGs using electrophoresis and bioautography were examined. The migration distance of Ags was greatest under test conditions using Bacto Agar TABLE 4. Average migration distance of aminoglycosides using as the supporting medium with Tris buffer (pH 8.0). The Tris Buffer (pH 8.0) and Bacto Agar. migration distances for SM and SDM were similar and Drug Average S.D. they were difficult to separate. DSM 41.5 0.05 Smither and Vaughan (11) have carried out electro SM 59.5 0.19 phoresis using agarose as a supporting medium. They have KM 66.2 0.16 AKM 100.0 0.20 reported that in agarose electroendosmosis has been low, GM 118.1 0.28 the mobility of the molecule being dependent mainly on FRM 120.0 0.34 its net electrical charge, while in agar the higher electro- PRM 112.8 0.26 endosmotic activity caused an apprecable flow of water, Migration distance: mm. resulting in longer migration distances. Accordingly, we S.D.: Standard deviation. examined this using agar in place of agarose. Because the inhibitory circle zone was dispersed remarkably as a result of this, the separation and identification of each AG was impossible. Agarose shows strong absorption and separa tion zone effects at a low voltage and this is considered to TABLE 5. Recoveries of extracts from bovine kidney tissue have produced the remarkable dispersion. containing 100 [ig/g of each aminoglycoside. Drug Found (u.g/g) and Recovery (%) Because a Sep-Pak silica cartridge is used for prepa DSM 90.2 ration of the extract instead of a glass column, the opera SM 86.5 tion will be easy for many laboratories. The migration KM 80.5 distance for PRM and AKM was lower than the standards, AKM 59.0 but the others were similar to the standard solution of GM 76.8 antibiotic. Furthermore, reproducibility was very high. FRM 71.6 Therefore, it was judged that there was not obstruction to PRM 65.1 the separation of AGs. This form of analysis would un-

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