APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 1978, p. 517-520
0099-2240/78/0035-0517$02.00/0 Copyright © 1978 American Society for Microbiology
Vol. 35, No. 3
Printed in U.S.A.
Improved Microbiological Assay Procedures for Dihydrostreptomycin Residues in Milk and Dairy Productst JEAN M. INGLIS AND STANLEY E. KATZ* Department of Biochemistry and Microbiology, Cook College, Rutgers-The State University of New Jersey, New Brunswick, New Jersey 08A903 Received for publication 18 July 1977
Because dihydrostreptomycin can remain as a slowly removed antibotic residue in dairy animals and because of the need for more sensitive procedures with which to provide information concerning antibiotic residues in food products, procedures were developed for more sensitive assays of dihydrostreptomycin in milk and some representative dairy products. The cleanup procedures that aided these improvements were (i) precipitation of milk proteins by acidification and (ii) centrifugation to extract cheeses and to remove physical barriers to diffusion in the cylinder plate assay. In addition, a thin, single seeded assay layer was used to maximize diffusion. Levels of dihydrostreptomycin as low as 0.06 jig/ml in milk and 0.2 to 0.4 lug/g in cheeses were measurable; these levels were some fourfold more sensitive than those presently recommended by the Food and Drug Administration.
Residues of dihydrostreptomycin can appear in milk and other dairy products from treated mastitic cows when milk is not withheld from market for the recommended time. These residues can have adverse effects on human health, causing direct toxic effects ranging from sensitization to the drug to anaphylactic shock (3). In addition, the ingestion of antibiotic residues may result in the development of resistance among the microorganisms of the human intestinal tract (2). Dihydrostreptomycin residues present special problems, since this antibiotic can be sequestered in tissue for extended periods of time, 30 days or more. It also remains heat stable longer than many other antibiotics (3). The most complete compendium of reproducible procedures for detecting antibiotics in milk and dairy products is suggested by the Food and Drug Administration (FDA) (7). The sensitivity of the procedures for detecting dihydrostreptomycin residues (in milk) is approximately 0.2 ,ug/ml. Although the FDA-suggested procedures combine both reliability and a fair degree of sensitivity, more sensitive procedures are needed to provide information on antibiotic residues in food products. Improved sample preparation procedures shown useful in increasing the sensitivity of tetracycline assays in dairy products (4, 5) were applied to the assay for dihydrostreptomycin. t Journal paper of the New Jersey Agricultural Experiment Station, Rutgers-The State University of New Jersey, New Brunswick.
The procedures presented here for the assay of dihydrostreptomycin in milk and some representative dairy products incorporate cleanup methods that remove sensitivity-lowering materials which form physical barriers to diffusion in the cylinder plate assay. These cleanup techniques, such as precipitation of proteinaceous materials and centrifugation of solid samples, coupled with the use of single-layer assay plates, combine to increase sensitivity and recoveries. MATERIALS AND METHODS Media. Maintenance medium consisted of 6.0 g of peptic digest of meat, 4.0 g of pancreatic digest of casein, 3.0 g of yeast extract, 1.5 g of beef extract, 1.0 g of glucose, 15.0 g of agar, and 1,000 ml of deionized water. The pH of this medium should be 6.5 to 6.7 after sterilization. Antibiotic medium no. 1 (BBL or Difco) is satisfactory. Sporulation medium was maintenance medium containing 300 mg of magnesium sulfate per liter. Assay medium consisted of 1.5 g of beef extract, 3.0 g of yeast extract, 6.0 g of peptone, 15.0 g of agar, and 1,000 ml of deionized water. The pH of this medium should be 8.0 ± 0.1 after sterilization. Streptomycin assay agar with yeast extract (BBL) antibiotic medium no. 5 (Difco) are satisfactory. Test organism. A spore suspension of Bacillus subtilis (ATCC 6633) was prepared by growing the organism for 5 days at 37°C in a Roux bottle containing 300 ml of sporulation medium. The resultant growth was suspended in 50 ml of sterile physiological saline and centrifuged, and the supernatant was decanted.
The sediment was reconstituted in 70 ml of sterile physiological saline, and the suspension was heat treated for 30 min at 65°C. The spore suspension was diluted 1:1,000 with physiological saline, and 5 ml of 517
518
APPL. ENVIRON. MICRO1310L.
INGLIS AND KATZ
the diluted suspension was used to inoculate 1,000 ml of the assay medium. Preparation of the standard response curve. A master standard solution containing 1,000 ,ug of dihydrostreptomycin per ml was prepared. This solution can be stored at 5°C for 2 weeks. The concentrations used in the standard curve were 0.0625, 0.125, 0.25, 0.50, 1.00, and 2.00 ,ug/ml, with 0.50 ,ug/ml used as the reference concentration. The master standard solution and the standard curve were prepared in pH 8.0 phosphate buffer (16.73 g of dipotassium phosphate and 0.523 g of monopotassium phosphate per liter of deionized water). The standard response curve was used to determine the absolute recoveries of dihydrostreptomycin. The assay medium was cooled to 65°C and inoculated with the B. subtilis spores. Six milliliters of the inoculated assay medium was spread evenly into plastic petri dishes (15 by 100 mm). The plates were allowed to cool with the covers ajar to allow water of condensation to evaporate. Six cylinders were placed on each plate by using a Shaw dispenser. For each sample, three petri dishes were used. On each dish, three alternate cylinders were filled with the reference concentration, and the others were filled with the solution to be assayed. The plates were incubated at 300C for 18 to 24 h. After incubation, the cylinders were removed, the plates were washed gently, and the zones of inhibition were measured by using a Fisher-Lilly zone reader. The potency of the samples was then determined according to the wellestablished procedures of the Association of Official Analytical Chemists (1). Procedures for determinating dihydrostreptomycin residues in milk and dairy products. (i) Preparation of compensatory standard curve in milk. The concentrations used in the compensatory curve, 0.00, 0.0625, 0.125, 0.25, 0.50, 1.00, and 2.00 ug of dihydrostreptomycin per ml, with 0.50 ,ug/ml as the reference concentration, were prepared with milk as the diluent. Each concentration was acidified with 6 N phosphoric acid to a pH of 4.5 ± 0.1. After centrifugation at 6,000 rpm (5,900 x g) for 15 min at 20°C, the liquid phase of each sample was decanted. The pH of the supernatant was adjusted to 8.0 with 4 N sodium hydroxide. About 15 ml of the pH-adjusting supernatant was centrifuged at 2,000 rpm (1,000 x g), and the resulting clear supernatants were used as the assay solution. (ii) Assay for dihydrostreptomycin in milk. A 200-ml sample of milk was acidified with 6 N phosphoric acid to pH 4.5. The total liquid was decanted from the precipitated milk solids after centrifugation at 6,000 rpm (5,900 x g). The pH of the decanted liquid was adjusted to 8.0 with 4 N sodium hydroxide. The above procedure for preparing the compensatory standard curve was followed with the clear supernatant as the assay solution. The zones of inhibition were compared with the compensatory standard curve, and the concentration was calculated. Correction for volume changes resulting from pH adjustments did not change the observed levels. (iii) Assay for dihydrostreptomycin in nonfat dry milk. The dry milk was reconstituted according to the directions on the package and assayed according to the procedure for fresh milk.
(iv) Assay for dihydrostreptomycin in cottage cheese. A 100-g sample of cottage cheese was placed in a 250-ml plastic centrifuge bottle and centrifuged for 30 min at 7,500 rpm (9,000 x g) at 200C. The liquid extractant was decanted, and the pH was adjusted to 8.0 with 4 N sodium hydroxide. Approximately 15 ml of this solution was further clarified for 15 min at 2,000 rpm (1,000 x g). The clear supernatant was used as the assay solution. The zones of inhibition were compared against a standard curve prepared in pH 8.0 phosphate buffer. Corrections for recoveries from samples were made, using cottage cheese supplemented with known quantities of dihydrostreptomycin. (v) Assay for dihydrostreptomycin in cream cheese. A 50-g sample of cream cheese was blended with 100 ml of deionized water in a high-speed blender. The pH of the blended cheese was adjusted to 8.0 with 4 N sodium hydroxide. About 15 ml of the homogenate was centrifuged for 15 min at 2,000 rpm (1,000 x g). The assay solution was the middle layer, between the precipitated protein and the upper lipid layer. The zones of inhibition were compared against a standard curve prepared in pH 8.0 phosphate buffer. Corrections for recoveries from samples were made, using cream cheese supplemented with known quantities of
dihydrostreptomycin. (vi) Assay for dihydrostreptmycin in buttermilk. A 200-ml sample of buttermilk was placed in a centrifuge bottle and centrifuged at 6,000 rpm (5,900
x g) for 25 min at 20°C. The supernatant was decanted and placed in a beaker, and the pH was adjusted to 8.0 with 4 N sodium hydroxide. The previously described procedures for milk were then followed. Zones of inhibition were compared against a standard response curve. Corrections for recoveries from samples were made, using buttermilk supplemented with known amounts of dihydrostreptomycin.
RESULTS AND DISCUSSION The improved methodology resulted in almost a fourfold increase in sensitivity compared with the procedure suggested by the FDA (7) (Fig. 1). Actual recoveries from milk samples supplemented and assayed by both procedures are FDA - Smggeeted 2.0
1.00 g
E
0.5
e
f
/Improved
/
ffi
0.25 .
/
a i
.12
*
Zone Diameter (mm)
FIG. 1. Comparison of standard response curves for dihydrostreptomycin.
NEW ASSAY FOR DIHYDROSTREPTOMYCIN RESIDUES
VoL. 35, 1978
compared in Table 1. Although both procedures give quantitative recoveries as determined by their respective compensatory response curves, the difference in ability to measure low levels is striking. The improved procedure measured 0.0625 ug/ml, versus 0.25 ,ug/ml for the FDAsuggested method. The improved procedure can detect 0.05 ,lg/ml, but cannot measure any zone size at this level. The organism density around the cylinders containing 0.05 ,ug/ml is thinned significantly without the formation of an actual zone. This phenomenon also was noted in the assay of chlortetracycline in eggs (6). Student's t test for paired samples showed a significant difference at the 99% confidence limits between the recoveries found by the two methods. The calculated t of 3.195 exceeds the critical t value of 2.845 for the 99% confidence limits for 20 df (8). The importance of improved cleanup procedures is illustrated by comparing recoveries and detection of dihydrostreptomycin in nonfat dry milk (Table 2). These data indicate the importance of removing physical barriers to diffusion. In the improved procedure, the milk is reconsti-
tuted and assayed as fresh milk, whereas the FDA-suggested method uses 1 + 3 extractiondilution of the dry powder. The milk solids prevent diffusion of the antibiotic, hence the poor sensitivity. Dihydrostreptomycin could not be detected in buttermilk at any of supplementation levels by the FDA-suggested procedure, whereas the improved procedure could detect 0.125 jg/ml (Table 3). Recoveries for the new method averaged 53.7% and levels of 0.25 ,ug/ml could be measured when compared with a pH 8.0 phosphate buffer response curve. A significant amount of liquid was separated from the milk solids after highspeed centrifugation of buttermilk. Because of the acidic nature of the buttermilk, pH 4.0 to 4.5, the acidification step was unnecessary. Adjustment of the liquid portion to pH 8.0 and further centrifugation yielded a clear assay solution. Cottage cheese could also be directly clarified by high-speed centrifugation, which after pH adjustment and reclarification yielded a clear assay solution. Because solids were removed, the TABLE 2. Comparison of recoveries of dihydrostreptomycin from nonfat dry milk
TABLE 1. Comparisons of recoveries of dihydrostreptomycin from milk
Dihydrostreptomycin found
Dihydrostreptomycin found
Dihydrostreptomycin added
(pg/mi)
Improved % Recovg/ml ,i,nl ery
b NDa 0.00 DC 0.05 0.06 0.0625 96.0 0.12 120.0 0.10 0.125 0.12 96.0 0.11 88.0 0.125 0.14 107.6 0.13 0.12 92.3 0.13 0.15 0.13 86.7 0.17 0.18 105.9 0.15 88.2 0.17 0.19 76.0 0.25 0.29 116.0 0.25 0.28 112.0 0.25 0.27 108.0 0.25 102.5 0.41 0.40 0.50 0.48 96.0 0.50 0.62 124.0 0.50 0.44 88.0 0.50 0.80 105.0 0.80 0.94 94.0 1.00 1.20 120.0 1.00 1.08 108.0 1.00 0.84 84.0 1.00 a Not detectable. b -, None recovered. c Detectable but not measurable.
FDA-suggested gm g/i
519
% Recovery
ND ND ND ND ND ND ND ND ND ND ND 0.24 0.24 0.23 0.27
_
96.0 96.0 92.0 108.0
0.58 0.50 0.48 0.54
106.0 100.0 96.0 108.0
1.00 0.84 0.95 0.91
100.0 84.0 95.0 91.0
-
-
Dihydrostreptomycin added (Ug10 (jLg/10 g))
0.00 6.25 10.00 12.5 20.0 25.0 30.0 50.0 a
b
Improved
FDA-suggested
yg/1Ogg% Recovery ery
iLIog% g/l g Recover
tg/1
__b
NDa ND
-
12.5
100.0
26.0
104.0
54.0
108.0
ery
ND
-
ND
-
ND
-
ND ND
-
-
ND, Not detectable. None recovered. TABLE 3. Comparison of recoveries of dihydrostreptomycin in buttermilk Dihydrostreptomycin found
Dihydrostreptomycin added
(pLg/ril)
Improved
jA/1% Recovery
ND a 0.00 _b ND 0.0625 Dc 0.125 56.0 0.14 0.25 54.0 0.27 0.50 51.0 0.51 1.00 a Not detectable. b_, None recovered. c Detectable but not measurable.
FDA-suggested AI
ND ND ND ND ND ND
% Recovery -
-
520
INGLIS AND KATZ
APPL. ENVIRON. MICROBIOL.
improved method measured a level of 0.20 ,ug/g and recoveries ranged between 55.0 and 86.0% (average, 73.7%). The FDA-suggested procedure could not detect residues in the range of 0.2 to 1.0 ,ug/g (Table 4). Although the direct separation of the aqueous TABLE 4. Comparison of recoveries of dihydrostreptomycin from cottage cheese Dihydrostreptomycin found Dihydrostreptomycin added
(JLg/g)
L/g yg/g ND a ND 0.16 0.22 0.28 0.41 0.46 0.58 0.65 0.74 0.86
0.00 0.10 0.20 0.40 0.40 0.60 0.60 0.80 0.80 1.00 1.00
FDA-suggested
Improved % Recovery
_b -
80.0 55.0 70.0 68.3 76.7 72.5 81.2 74.0 86.0
igg
Lg/g ND ND ND ND ND ND ND ND ND ND ND
% Recovery -
-
Not detectable. b-, None recovered.
a
TABLE 5. Comparison of recoveries of dihydrostreptomycin in cream cheese Dihydrostreptomycin found
(jig/g)
Improved igg
yg,g
ND a 0.0 ND 0.3 0.43 0.4 0.78 0.8 1.05 1.2 1.29 1.6 1.56 2.0 a, b See Table 4.
% Recovery
_b 108.0 99.8 87.5 80.6 78.1
FDS-suggested Agg
/g/g ND ND ND ND ND 0.60 0.85
ACKNOWLEDGMENT These investigations were supported by the Food and Drug Administration under contract no. 223-74-7179.
LITERATURE CITED 1. Association of Official Analytical Chemists. 1970. Official methods of analysis, 11th ed. Association of Official Analytical Chemists, Washington, D.C. 2. Food and Drug Administration. 1972. Report of the FDA Task Force on the Use of Antibiotics in Animal Feeds-Human Health Hazard. Fed. Reg. Rep. 72-6008. 3. Huber, W. G. 1971. The impact of antibiotic drugs and their residues. Adv. Vet. Sci. Comp. 15:102-132. 4. Katz, S. E., and C. A. Fassbender. 1970. Sensitive microbial assay procedure for chlortetracycline in milk and related products. J. Assoc. Off. Anal. Chem.
53:968-972.
Dihydrostreptomycin added
phase was unsuccessful when applied to cream cheese, the assay of "spiked" cream cheese by the improved procedure showed significant improvements in recoveries and in the limit of detectability over the FDA-suggested method, a factor of 4 (Table 5). Student's t test for paired samples showed a significant difference between recoveries obtained by the two methods. The calculated t value of 7.376 exceeds the critical t value of 4.604 for 4 df at the 99% confidence limits. In general, the greater sensitivities and higher recoveries obtained with the improved procedures were the result of smaller dilutions, the removal of physical barriers to diffusion, and the thin, single assay layer.
% Recovery -
-
40.6 42.5
5. Katz, S. E., and C. A. Fassbender. 1972. Improved procedures for the determination of oxytetracycline in milk, milk products; chicken muscle, liver and eggs. Bull. Environ. Contam. Toxicol. 7:229-236. 6. Katz, S. E., C. A. Fassbender, and J. J. Dowling. 1972. Chlortetracycline residues in eggs from hens on chlortetracycline-supplemented diets. J. Assoc. Off. Anal. Chem. 55:128-133. 7. Kramer, J., G. G. Carter, B. Arret, J. Wilner, W. W. Wright, and A. Kirshbaum. 1968. Antibiotic residues in milk, dairy products and animal tissues: methods, reports and protocol. Food and Drug Administration, Washington, D.C. 8. Steele, R. G. D., and J. H. Torrie. 1960. Principles and procedures of statistics. McGraw-Hill, New York.
0099-2240/78/0035-0517$02.00/0 Copyright © 1978 American Society for Microbiology
Vol. 35, No. 3
Printed in U.S.A.
Improved Microbiological Assay Procedures for Dihydrostreptomycin Residues in Milk and Dairy Productst JEAN M. INGLIS AND STANLEY E. KATZ* Department of Biochemistry and Microbiology, Cook College, Rutgers-The State University of New Jersey, New Brunswick, New Jersey 08A903 Received for publication 18 July 1977
Because dihydrostreptomycin can remain as a slowly removed antibotic residue in dairy animals and because of the need for more sensitive procedures with which to provide information concerning antibiotic residues in food products, procedures were developed for more sensitive assays of dihydrostreptomycin in milk and some representative dairy products. The cleanup procedures that aided these improvements were (i) precipitation of milk proteins by acidification and (ii) centrifugation to extract cheeses and to remove physical barriers to diffusion in the cylinder plate assay. In addition, a thin, single seeded assay layer was used to maximize diffusion. Levels of dihydrostreptomycin as low as 0.06 jig/ml in milk and 0.2 to 0.4 lug/g in cheeses were measurable; these levels were some fourfold more sensitive than those presently recommended by the Food and Drug Administration.
Residues of dihydrostreptomycin can appear in milk and other dairy products from treated mastitic cows when milk is not withheld from market for the recommended time. These residues can have adverse effects on human health, causing direct toxic effects ranging from sensitization to the drug to anaphylactic shock (3). In addition, the ingestion of antibiotic residues may result in the development of resistance among the microorganisms of the human intestinal tract (2). Dihydrostreptomycin residues present special problems, since this antibiotic can be sequestered in tissue for extended periods of time, 30 days or more. It also remains heat stable longer than many other antibiotics (3). The most complete compendium of reproducible procedures for detecting antibiotics in milk and dairy products is suggested by the Food and Drug Administration (FDA) (7). The sensitivity of the procedures for detecting dihydrostreptomycin residues (in milk) is approximately 0.2 ,ug/ml. Although the FDA-suggested procedures combine both reliability and a fair degree of sensitivity, more sensitive procedures are needed to provide information on antibiotic residues in food products. Improved sample preparation procedures shown useful in increasing the sensitivity of tetracycline assays in dairy products (4, 5) were applied to the assay for dihydrostreptomycin. t Journal paper of the New Jersey Agricultural Experiment Station, Rutgers-The State University of New Jersey, New Brunswick.
The procedures presented here for the assay of dihydrostreptomycin in milk and some representative dairy products incorporate cleanup methods that remove sensitivity-lowering materials which form physical barriers to diffusion in the cylinder plate assay. These cleanup techniques, such as precipitation of proteinaceous materials and centrifugation of solid samples, coupled with the use of single-layer assay plates, combine to increase sensitivity and recoveries. MATERIALS AND METHODS Media. Maintenance medium consisted of 6.0 g of peptic digest of meat, 4.0 g of pancreatic digest of casein, 3.0 g of yeast extract, 1.5 g of beef extract, 1.0 g of glucose, 15.0 g of agar, and 1,000 ml of deionized water. The pH of this medium should be 6.5 to 6.7 after sterilization. Antibiotic medium no. 1 (BBL or Difco) is satisfactory. Sporulation medium was maintenance medium containing 300 mg of magnesium sulfate per liter. Assay medium consisted of 1.5 g of beef extract, 3.0 g of yeast extract, 6.0 g of peptone, 15.0 g of agar, and 1,000 ml of deionized water. The pH of this medium should be 8.0 ± 0.1 after sterilization. Streptomycin assay agar with yeast extract (BBL) antibiotic medium no. 5 (Difco) are satisfactory. Test organism. A spore suspension of Bacillus subtilis (ATCC 6633) was prepared by growing the organism for 5 days at 37°C in a Roux bottle containing 300 ml of sporulation medium. The resultant growth was suspended in 50 ml of sterile physiological saline and centrifuged, and the supernatant was decanted.
The sediment was reconstituted in 70 ml of sterile physiological saline, and the suspension was heat treated for 30 min at 65°C. The spore suspension was diluted 1:1,000 with physiological saline, and 5 ml of 517
518
APPL. ENVIRON. MICRO1310L.
INGLIS AND KATZ
the diluted suspension was used to inoculate 1,000 ml of the assay medium. Preparation of the standard response curve. A master standard solution containing 1,000 ,ug of dihydrostreptomycin per ml was prepared. This solution can be stored at 5°C for 2 weeks. The concentrations used in the standard curve were 0.0625, 0.125, 0.25, 0.50, 1.00, and 2.00 ,ug/ml, with 0.50 ,ug/ml used as the reference concentration. The master standard solution and the standard curve were prepared in pH 8.0 phosphate buffer (16.73 g of dipotassium phosphate and 0.523 g of monopotassium phosphate per liter of deionized water). The standard response curve was used to determine the absolute recoveries of dihydrostreptomycin. The assay medium was cooled to 65°C and inoculated with the B. subtilis spores. Six milliliters of the inoculated assay medium was spread evenly into plastic petri dishes (15 by 100 mm). The plates were allowed to cool with the covers ajar to allow water of condensation to evaporate. Six cylinders were placed on each plate by using a Shaw dispenser. For each sample, three petri dishes were used. On each dish, three alternate cylinders were filled with the reference concentration, and the others were filled with the solution to be assayed. The plates were incubated at 300C for 18 to 24 h. After incubation, the cylinders were removed, the plates were washed gently, and the zones of inhibition were measured by using a Fisher-Lilly zone reader. The potency of the samples was then determined according to the wellestablished procedures of the Association of Official Analytical Chemists (1). Procedures for determinating dihydrostreptomycin residues in milk and dairy products. (i) Preparation of compensatory standard curve in milk. The concentrations used in the compensatory curve, 0.00, 0.0625, 0.125, 0.25, 0.50, 1.00, and 2.00 ug of dihydrostreptomycin per ml, with 0.50 ,ug/ml as the reference concentration, were prepared with milk as the diluent. Each concentration was acidified with 6 N phosphoric acid to a pH of 4.5 ± 0.1. After centrifugation at 6,000 rpm (5,900 x g) for 15 min at 20°C, the liquid phase of each sample was decanted. The pH of the supernatant was adjusted to 8.0 with 4 N sodium hydroxide. About 15 ml of the pH-adjusting supernatant was centrifuged at 2,000 rpm (1,000 x g), and the resulting clear supernatants were used as the assay solution. (ii) Assay for dihydrostreptomycin in milk. A 200-ml sample of milk was acidified with 6 N phosphoric acid to pH 4.5. The total liquid was decanted from the precipitated milk solids after centrifugation at 6,000 rpm (5,900 x g). The pH of the decanted liquid was adjusted to 8.0 with 4 N sodium hydroxide. The above procedure for preparing the compensatory standard curve was followed with the clear supernatant as the assay solution. The zones of inhibition were compared with the compensatory standard curve, and the concentration was calculated. Correction for volume changes resulting from pH adjustments did not change the observed levels. (iii) Assay for dihydrostreptomycin in nonfat dry milk. The dry milk was reconstituted according to the directions on the package and assayed according to the procedure for fresh milk.
(iv) Assay for dihydrostreptomycin in cottage cheese. A 100-g sample of cottage cheese was placed in a 250-ml plastic centrifuge bottle and centrifuged for 30 min at 7,500 rpm (9,000 x g) at 200C. The liquid extractant was decanted, and the pH was adjusted to 8.0 with 4 N sodium hydroxide. Approximately 15 ml of this solution was further clarified for 15 min at 2,000 rpm (1,000 x g). The clear supernatant was used as the assay solution. The zones of inhibition were compared against a standard curve prepared in pH 8.0 phosphate buffer. Corrections for recoveries from samples were made, using cottage cheese supplemented with known quantities of dihydrostreptomycin. (v) Assay for dihydrostreptomycin in cream cheese. A 50-g sample of cream cheese was blended with 100 ml of deionized water in a high-speed blender. The pH of the blended cheese was adjusted to 8.0 with 4 N sodium hydroxide. About 15 ml of the homogenate was centrifuged for 15 min at 2,000 rpm (1,000 x g). The assay solution was the middle layer, between the precipitated protein and the upper lipid layer. The zones of inhibition were compared against a standard curve prepared in pH 8.0 phosphate buffer. Corrections for recoveries from samples were made, using cream cheese supplemented with known quantities of
dihydrostreptomycin. (vi) Assay for dihydrostreptmycin in buttermilk. A 200-ml sample of buttermilk was placed in a centrifuge bottle and centrifuged at 6,000 rpm (5,900
x g) for 25 min at 20°C. The supernatant was decanted and placed in a beaker, and the pH was adjusted to 8.0 with 4 N sodium hydroxide. The previously described procedures for milk were then followed. Zones of inhibition were compared against a standard response curve. Corrections for recoveries from samples were made, using buttermilk supplemented with known amounts of dihydrostreptomycin.
RESULTS AND DISCUSSION The improved methodology resulted in almost a fourfold increase in sensitivity compared with the procedure suggested by the FDA (7) (Fig. 1). Actual recoveries from milk samples supplemented and assayed by both procedures are FDA - Smggeeted 2.0
1.00 g
E
0.5
e
f
/Improved
/
ffi
0.25 .
/
a i
.12
*
Zone Diameter (mm)
FIG. 1. Comparison of standard response curves for dihydrostreptomycin.
NEW ASSAY FOR DIHYDROSTREPTOMYCIN RESIDUES
VoL. 35, 1978
compared in Table 1. Although both procedures give quantitative recoveries as determined by their respective compensatory response curves, the difference in ability to measure low levels is striking. The improved procedure measured 0.0625 ug/ml, versus 0.25 ,ug/ml for the FDAsuggested method. The improved procedure can detect 0.05 ,lg/ml, but cannot measure any zone size at this level. The organism density around the cylinders containing 0.05 ,ug/ml is thinned significantly without the formation of an actual zone. This phenomenon also was noted in the assay of chlortetracycline in eggs (6). Student's t test for paired samples showed a significant difference at the 99% confidence limits between the recoveries found by the two methods. The calculated t of 3.195 exceeds the critical t value of 2.845 for the 99% confidence limits for 20 df (8). The importance of improved cleanup procedures is illustrated by comparing recoveries and detection of dihydrostreptomycin in nonfat dry milk (Table 2). These data indicate the importance of removing physical barriers to diffusion. In the improved procedure, the milk is reconsti-
tuted and assayed as fresh milk, whereas the FDA-suggested method uses 1 + 3 extractiondilution of the dry powder. The milk solids prevent diffusion of the antibiotic, hence the poor sensitivity. Dihydrostreptomycin could not be detected in buttermilk at any of supplementation levels by the FDA-suggested procedure, whereas the improved procedure could detect 0.125 jg/ml (Table 3). Recoveries for the new method averaged 53.7% and levels of 0.25 ,ug/ml could be measured when compared with a pH 8.0 phosphate buffer response curve. A significant amount of liquid was separated from the milk solids after highspeed centrifugation of buttermilk. Because of the acidic nature of the buttermilk, pH 4.0 to 4.5, the acidification step was unnecessary. Adjustment of the liquid portion to pH 8.0 and further centrifugation yielded a clear assay solution. Cottage cheese could also be directly clarified by high-speed centrifugation, which after pH adjustment and reclarification yielded a clear assay solution. Because solids were removed, the TABLE 2. Comparison of recoveries of dihydrostreptomycin from nonfat dry milk
TABLE 1. Comparisons of recoveries of dihydrostreptomycin from milk
Dihydrostreptomycin found
Dihydrostreptomycin found
Dihydrostreptomycin added
(pg/mi)
Improved % Recovg/ml ,i,nl ery
b NDa 0.00 DC 0.05 0.06 0.0625 96.0 0.12 120.0 0.10 0.125 0.12 96.0 0.11 88.0 0.125 0.14 107.6 0.13 0.12 92.3 0.13 0.15 0.13 86.7 0.17 0.18 105.9 0.15 88.2 0.17 0.19 76.0 0.25 0.29 116.0 0.25 0.28 112.0 0.25 0.27 108.0 0.25 102.5 0.41 0.40 0.50 0.48 96.0 0.50 0.62 124.0 0.50 0.44 88.0 0.50 0.80 105.0 0.80 0.94 94.0 1.00 1.20 120.0 1.00 1.08 108.0 1.00 0.84 84.0 1.00 a Not detectable. b -, None recovered. c Detectable but not measurable.
FDA-suggested gm g/i
519
% Recovery
ND ND ND ND ND ND ND ND ND ND ND 0.24 0.24 0.23 0.27
_
96.0 96.0 92.0 108.0
0.58 0.50 0.48 0.54
106.0 100.0 96.0 108.0
1.00 0.84 0.95 0.91
100.0 84.0 95.0 91.0
-
-
Dihydrostreptomycin added (Ug10 (jLg/10 g))
0.00 6.25 10.00 12.5 20.0 25.0 30.0 50.0 a
b
Improved
FDA-suggested
yg/1Ogg% Recovery ery
iLIog% g/l g Recover
tg/1
__b
NDa ND
-
12.5
100.0
26.0
104.0
54.0
108.0
ery
ND
-
ND
-
ND
-
ND ND
-
-
ND, Not detectable. None recovered. TABLE 3. Comparison of recoveries of dihydrostreptomycin in buttermilk Dihydrostreptomycin found
Dihydrostreptomycin added
(pLg/ril)
Improved
jA/1% Recovery
ND a 0.00 _b ND 0.0625 Dc 0.125 56.0 0.14 0.25 54.0 0.27 0.50 51.0 0.51 1.00 a Not detectable. b_, None recovered. c Detectable but not measurable.
FDA-suggested AI
ND ND ND ND ND ND
% Recovery -
-
520
INGLIS AND KATZ
APPL. ENVIRON. MICROBIOL.
improved method measured a level of 0.20 ,ug/g and recoveries ranged between 55.0 and 86.0% (average, 73.7%). The FDA-suggested procedure could not detect residues in the range of 0.2 to 1.0 ,ug/g (Table 4). Although the direct separation of the aqueous TABLE 4. Comparison of recoveries of dihydrostreptomycin from cottage cheese Dihydrostreptomycin found Dihydrostreptomycin added
(JLg/g)
L/g yg/g ND a ND 0.16 0.22 0.28 0.41 0.46 0.58 0.65 0.74 0.86
0.00 0.10 0.20 0.40 0.40 0.60 0.60 0.80 0.80 1.00 1.00
FDA-suggested
Improved % Recovery
_b -
80.0 55.0 70.0 68.3 76.7 72.5 81.2 74.0 86.0
igg
Lg/g ND ND ND ND ND ND ND ND ND ND ND
% Recovery -
-
Not detectable. b-, None recovered.
a
TABLE 5. Comparison of recoveries of dihydrostreptomycin in cream cheese Dihydrostreptomycin found
(jig/g)
Improved igg
yg,g
ND a 0.0 ND 0.3 0.43 0.4 0.78 0.8 1.05 1.2 1.29 1.6 1.56 2.0 a, b See Table 4.
% Recovery
_b 108.0 99.8 87.5 80.6 78.1
FDS-suggested Agg
/g/g ND ND ND ND ND 0.60 0.85
ACKNOWLEDGMENT These investigations were supported by the Food and Drug Administration under contract no. 223-74-7179.
LITERATURE CITED 1. Association of Official Analytical Chemists. 1970. Official methods of analysis, 11th ed. Association of Official Analytical Chemists, Washington, D.C. 2. Food and Drug Administration. 1972. Report of the FDA Task Force on the Use of Antibiotics in Animal Feeds-Human Health Hazard. Fed. Reg. Rep. 72-6008. 3. Huber, W. G. 1971. The impact of antibiotic drugs and their residues. Adv. Vet. Sci. Comp. 15:102-132. 4. Katz, S. E., and C. A. Fassbender. 1970. Sensitive microbial assay procedure for chlortetracycline in milk and related products. J. Assoc. Off. Anal. Chem.
53:968-972.
Dihydrostreptomycin added
phase was unsuccessful when applied to cream cheese, the assay of "spiked" cream cheese by the improved procedure showed significant improvements in recoveries and in the limit of detectability over the FDA-suggested method, a factor of 4 (Table 5). Student's t test for paired samples showed a significant difference between recoveries obtained by the two methods. The calculated t value of 7.376 exceeds the critical t value of 4.604 for 4 df at the 99% confidence limits. In general, the greater sensitivities and higher recoveries obtained with the improved procedures were the result of smaller dilutions, the removal of physical barriers to diffusion, and the thin, single assay layer.
% Recovery -
-
40.6 42.5
5. Katz, S. E., and C. A. Fassbender. 1972. Improved procedures for the determination of oxytetracycline in milk, milk products; chicken muscle, liver and eggs. Bull. Environ. Contam. Toxicol. 7:229-236. 6. Katz, S. E., C. A. Fassbender, and J. J. Dowling. 1972. Chlortetracycline residues in eggs from hens on chlortetracycline-supplemented diets. J. Assoc. Off. Anal. Chem. 55:128-133. 7. Kramer, J., G. G. Carter, B. Arret, J. Wilner, W. W. Wright, and A. Kirshbaum. 1968. Antibiotic residues in milk, dairy products and animal tissues: methods, reports and protocol. Food and Drug Administration, Washington, D.C. 8. Steele, R. G. D., and J. H. Torrie. 1960. Principles and procedures of statistics. McGraw-Hill, New York.
