Anal. Chem. 1983, 55, 1974-1977
1974
Potentiometric Microbiological Assay of Gentamicin, Streptomycin, and Neomycin with a Carbon Dioxide Gas-Sensing Electrode D. L. Simpson' and R. K. Kobos*2 Department of Chemistry, Virginia Commonwealth University, Richmond, Virginia 23284
A potentiometric method that uses a carbon dloxlde gas sensor has been developed for the microbiological assay of antlblotics. The method Is based upon the antlblotlc lnhlbltion of carbon dloxlde production of a suspenslon of Eschsrlchla CON, which is measured with the potentlometrlc gas sensor after a 2-h Incubation perlod. Linear log dose-response curves were obtained for the antlbiotlcs gentamlcln, streptomycln, and neomycln in the ranges of 0.017-3.33, 0.33-16.67, and 0.17-3.33 pg/mL, respectively. The method has been shown to be useful for pharmaceutical label clalm assays. The feaslbillty of applying the method to the determlnatlon of serum gentamlcln has also been studled.
Traditional microbiological assays for antibiotics are based upon the inhibition of growth of a sensitive microorganism (I). These assays provide a measure of the biological activity of the antibiotic, whereas nonmicrobiological methods measure concentration. The traditional microbiological assays, however, require long incubation times, i.e., 18-24 h, and a relatively high expertise in handling the microorganisms and the data interpretation. Respirometric assays for antibiotics, which are based upon the inhibition of respiration of microorganisms, have been developed (2-7). These assays are faster than the traditional methods, generally requiring 1-4 h. The basic method (2-7) uses a spectrophotometer to determine the carbon dioxide produced by the microorganism after an incubation period of 30-60 min. The carbon dioxide is separated from the incubation mixture by acidification and absorption into an alkaline solution containing phenolphthalein as the colorimetric indicator. The separation step is necessary because the turbidity and color of the incubation mixture will interfere with the spectrophotometric measurement. Oxygen uptake by microorganisms during respiration is also inhibited by antibiotics. Nystatin has been determined with the yeast cells Saccharomyces cereuisiae immobilized in a membrane which was placed over an oxygen-sensing electrode (8). A major problem with the use of immobilized microorganisms to determine antimicrobial agents is the need to replace the microbial membrane after each determination, since the effect of the agent is generally irreversible. Thus, the response of the sensor must be assumed to be unchanged by the membrane replacement. In a recent publication (9) we have demonstrated that tetracycline hydrochloride can be determined with suspensions of Escherichia coli ATCC no. 9637 and a potentiometric carbon dioxide gas sensor (IO, 11). The method uses the principle of inhibition of carbon dioxide production of the bacterial cells by the antibiotic. This potentiometric microOn sabbatical leave from Philip Morris, Inc. 2Present address: E. I. du Pont de Nemours and Co., Experimental Station, Photo Products Department, Wilmington, DE 19898.
biological assay is fast, simple, inexpensive, and reproducible. The log dose-response curve for the antibiotic, following a 2 h incubation at 37 "C in nutrient broth-tetracycline hydrochloride mixtures, was linear in the potency range of 33-167 pg/mL. Good agreement with the label claim was obtained in the assay of a pharmaceutical preparation of tetracycline hydrochloride capsules by using the 2 x 2 parallel line bioassay method (12). The purpose of the study reported herein was to extend the recently developed potentiometric method to the aminoglycoside class of antibiotics represented by gentamicin, streptomycin, and neomycin. Since the range of sensitivity of the gentamicin assay was found to be appropriate, a feasibility study for the determination of serum gentamicin was done.
EXPERIMENTAL SECTION Apparatus. An HNU Model 10-22-00or an Orion Model 95-02 carbon dioxide sensor was used with a Corning Model 130 pH/mV meter to measure the carbon dioxide generated by the bacterial cells. The HNU sensor was used for the optimization studies and the pharmaceutical preparation bioassays, whereas the Orion model was used for the serum gentamicin determinations. Measurements were made in a sealed cell thermostated at 25.0 k 0.5 "C. Prior to the electrochemical measurement of carbon dioxide, the bacterial cell suspensions were incubated with nutrient broth-antibiotic mixtures in a Lab-Line Orbit shaker bath at a selected temperature controlled to within k1 "C. Bacterial cell suspensions were standardized and measured by turbidimetry by using a Beckman Model DB-G spectrophotometer with 1-cm silica cells. Variable volume Finnpipettes, in the ranges of 5-50, 200-1000, and 1000-5000 rL, were used for all solution preparations. Gilmont 0.2- and 2.0-mL microburets were used for the electrode calibrations. Disposable 10-mL syringes were used for transferring incubated bacterial suspensions to the measurement cell. Reagents. All chemicals used were of reagent grade. Solutions were prepared with distilled-deionized water. Reference grade gentamicin sulfate (50 mg/mL), streptomycin sulfate (740 rg/mg), and neomycin sulfate (681 pg/mg) were obtained from ICN Life Sciences. The pharmaceutical preparations used for the bioassays were obtained from the Medical College of Virginia Commonwealth University. Schering Garamycin Pediatric Dose (10 mg/mL) and Pfizer Streptomycin Sulfate powder dose (1g) forms were assayed for gentamicin and streptomycin, respectively. A sample of plasma at a gentamicin potency of 4.0 gg/mL was supplied as a blind sample by Berry Kline of the Department of Pharmacy and Pharmaceutics at Virginia Commonwealth University. The serum used in the optimization studies was freeze-dried control serum and diluent, type 1-A, normal obtained from Sigma Chemical Co. Difco nutrient broth was obtained from General Scientific. The bacterium Escherichia coli no. 9637 was obtained from the American Type Culture Collection, Rockville, MD. Procedures. The bacterial cell suspension was prepared and stored as previously described (9). For the optimization studies, solutions of the antibiotics in the 0.05-500 gg/mL potency range were prepared daily and stored in an ice-filled Dewar flask. The stock nutrient solution was also prepared daily at the appropriate
0003-2700/83/0355-1974$01.50/00 1983 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 55, NO. 12, OCTOBER 1983
I
24!
-1
Flgure 1. log dose-response curves for gentamicln at pH 7.0 (0)and pH 7.8 (A):2 h incubation, :37 "C,stock cell suspension concentration
x 10' cells/mL, I.L
0.1 M.
concentration in phosphate buffer and stored in ice. The log dose-response curves for gentamicin and streptomycin were optimized in a study of the effects of incubation parameters, i.e. pH, temperature, cell concentration, and ionic strength. One milliliter each of stock E. coli suspension, stock nutrient broth, and standard antibiotic solution were mixed in a 15-mL centrifuge tube, which was than stoppered to prevent loss of carbon dioxide during incubation. The procedure used in these optimization studies has been previously described (9). The method was tested by assaying pharmaceutical preparations of gentamicin sulfate and streptomycin sulfate under the conditions found to be optimal for each antibiotic. A 2 X 2 parallel line bioassay (9,12)was idone by using two dose levels each for standard and sample solutions. The dose levels were chosen to be within the linear range of the optimized log dose-response curves, Le., 2 and 40 yg/mL for streptomycin and 0.1 and 5.0 yg/mL for gentamicin. The standard and sample solutions were each run in duplicate at each dose level in a single bioassay. Three bioassays using freshly prepared solutions of standard and sample were done for each antibiotic. A single dose of the pharmaceutical preparations of either streptomycin sulfate or gentamicin sulfate was used for each assay. The serum gentamicin determinations were not done by using the 2 X 2 parallel line bioassay procedure, because this method requires an a priori estimate of the sample potency. Since such estimates are generally not available for serum samples, the calibration curve method was used. Solutions of gentamicin mixed with pH 7.8 phosphate buffer and blank control serum were prepared by serial dilution and used to determine the log doseresponse curve. T h e semm carbon dioxide present as bicarbonate was removed before the serial dilutions by acidification of the initial high potency solution to pH 5.0 followed by stirring in air for 30 min. The pH was then adjusted to 7.8 with known volumes of 1.0 M NaOH before the serial dilutions. The amount of serum in the highest potency sample was adjusted to yield a range of serum concentrations upon dilution to the lowest potency which would approximate that of the range expected by dilution of a real serum sample by a factor of 1:25 or 150. In the gentamicin serum assay, standard samples were prepared at potencies of 0.046,0.230,and 0.919 Fg/mL, using pH 7.8 phosphate buffer and the same blank plasma used in the preparation of the unknown gentamicin sample. The same degassing procedure previously described was used on the highest potency standard solution prior to the serial dilution. The three standard solutions were assayed once each. The unknown plasma sample was diluted by a factor of 1:5 with pH 7.8 phosphate buffer, degassed, pH adjusted t,o pH 7.8, and diluted 1:26 with pH 7.8 phosphate buffer. The unknown was assayed in replicates of 4. In order to determine the effect of the blank plasma, a second experiment was done in which the standard solutions were prepared with the plasma diluted by a factor of 1 : l O relative to that of the first experiment. Three standard, degassed solutions were prepared at potencies of 0.046,0.232,and 0.929 pg/mL and were assayed once each. The unknown plasma sample was degassed, diluted by a factor of 1510, and assayed in triplicate.
2
1
0
log concn,pg/ m I
of 4.5
1975
log concn,yg/ml
Flgure 2. log dose-response curves for gentamicin at incubation temperatures of 30 "C (0)and 37 "C (A):2 h incubation, stock cell concentration of 4.5 X 10' cellslml, pH 7.8, p = 0.1 M.
24
2
"%5 n
0" E
0
t - - - - - C
1
.
'T
- ... ....
A
12,
l
-8
81 ~
-1
0
1
2
log concn,,vg/ m l
Flgure 3. log dose-response curves for gentamicin at ionic strengths of 0.1 (A)and 0.53 (0)M: 2 h incubation, 37 "C, stock cell con-
centration of 4.5 X 10' cells/mL, pH 7.8. RESULTS AND DISCUSSION The optimization of incubation conditions for the bioassay of tetracycline hydrochloride (9),streptomycin, and gentamicin has shown that pH, temperature, and time are the most important parameters. Reduction of the p H from 7.8 to 7.0 or temperature from 37 "C to 30 "C decreases both the total carbon dioxide produced and the slope of the linear region of the log dose-response curves. Figure 1 shows the effect of the incubation p H on the log dose-response curve for gentamicin. The effect of temperature on the log dose-response curve for gentamicin is shown in Figure 2. The optimium values of incubation p H and temperature for both streptomycin and gentamicin were p H 7.8 and 37 "C. The log dose-response curves for streptomycin and gentamicin were not significantly affected by changes in the bacterial cell concentration of the stock suspension in the range 2.2 X los to 4.5 X lo8 cells/mL. The use of higher cell concentrations results in decreased sensitivity, whereas low cell concentrations produce insufficient amounts of carbon dioxide. Heller et al. (13)have shown that the uptake of gentamicin by E. coli is diminished in incubation media of high ionic strength, i.e. I.L > 0.2 M. Experiments with the E . coli-gentamicin system under study were done a t ionic strengths of 0.05,0.10, and 0.53 M. The results obtained a t ionic strengths of 0.10 and 0.53 M are compared in Figure 3, which shows that the lower limit of detection is raised from 0.05 pg/mL a t 0.1 M to about 2.0 bg/mL a t 0.53 M. At an ionic strength of 0.53 M, the upper limit also appears to increase. However, the sensitivity, which is proportional to the slope in the linear region, does not appear to change significantly in the 2.0-10.0 pg/mL potency range. The linear range of the log dose-re-
1976
ANALYTICAL CHEMISTRY, VOL. 55, NO. 12, OCTOBER 1983
Table I. Results of the Bioassay of Gentamicin in Schering Garamycin Pediatric Dose, 10 mg/mL sample reference solution pooled reference solution pooled reference solution pooled reference solution pooled a
run no.
slope, M x 104/decade
potency, mg/mL
1 -4.52 1 -4.33 1 -4.42
11.21
2 2
11.74
-6.59 -5.63 2 -6.11 3 -6.84 3 -6.74 3 -6.79 av -5.98 av -5.57 av -5.77
9.68
* * i-
Standard deviation.
Table 11. Analysis of Variance for the Bioassay of Gentamicin in Schering Garamycin Pediatric Dose (10 mg/mL, 2 mL)
% claim
112.1
114.8 96.8
1.27a 1 . 2 1 10.8 i 1 . 3 b 107.9 c 11.6b 1.22 95% confidence limits.
sponse curve a t an ionic strength of 0.05 M was 0.05-2.0 pg/mL with no change in sensitivity. An ionic strength of 0.1 M was chosen as optimum because it produced the widest linear range, i.e., 0.05-10.0 pg/mL. Furthermore, the buffer capacity of the 0.05 M ionic strength solution was low, which limited its usefulness for bioassays. The optimum incubation conditions chosen for the bioassays of streptomycin and gentamicin were pH 7.8, T = 37 "C, time of 120 min, stock E. coli cell concentration of 4.5 X lo8 cells/mL, and a stock nutrient solution of 2.4 g/100 mL nutrient broth. These were the same as the optimum incubation conditions found for the tetracycline hydrochloride system (9). Under these optimum conditions the linear ranges of the log dose-response curves for streptomycin and gentamicin stock solutions were 1-50 and 0.05-10 pg/mL, respectively. On the basis of the dilution used in the assays, Le., 1:3, the actual linear ranges were 0.33-16.67 and 0.017-3.33 pg/mL for streptomycin and gentamicin, respectively. The log dose-response curve for neomycin was determined at the same optimum conditions used for gentamicin and streptomycin. The neomycin curve is linear in the stock solution potency range of 0.5-10.0 pg/mL. This corresponds to an actual range of 0.17-3.33 pg/mL on the basis of the dilution used in the assay. The results of the bioassay of a pharmaceutical preparation of gentamicin are shown in Table I. The results are in good agreement with the manufacturer's claim. A bioassay was also performed on a pharmaceutical preparation of streptomycin. The potency determined was 99.4 f 1.8% of the manufacturer's claim, with 95% confidence limits. These results are well within the uniformity of content for individual capsules as defined by U.S.P. (14). Although the standard uniformity of content assay using 30 capsules was not done, the good agreement obtained demonstrates the feasibility of the potentiometric method for pharmaceutical label claim assays. The validity of the bioassays of streptomycin and gentamicin was determined by an analysis of variance (ANOVA). The results for the gentamicin bioassay are shown in Table 11. Similar results were obtained for the streptomycin bioassay. These results indicate that both assays were valid. The regression terms are highly significant and the deviations from parallelism are insignificant. The preliminary studies reported herein for serum gentamicin were done with two different types of blood samples. One was a commercially available freeze-dried serum, which was used for the determination of the log doseresponse curve and optimization studies. Pooled human plasma was used in the assay of the unknown gentamicin sample. Both types of samples required removal of background carbon dioxide before analysis. After degassing and dilution of the samples
sum of degrees of squares, freedom M ZX lo8
source of variance regression parallelism between doses error within doses
variance, Mz x l o 8
3
577.42 0.72 578.32
577.42 0.72 192.77
20
57.53
2.88
1 1
regression 577.42 - 200.49 >> Fo.05(l, 20) = 4.35 error 2.88 Regression is significant. parallelism 0.72 - -= 0.25 < Fo.05(l, 20) = 4.35 error 2.88 Deviation from parallelism is insignificant. by 150-15 in pH 7.8 phosphate buffer, no significant increase in background carbon dioxide relative to the buffer was found. A new gas-dialysis procedure described by Fraticelli and Meyerhoff (15) could provide a more efficient method for carbon dioxide removal. The two types of blood samples studied had different effects on the slope of the gentamicin log dose-response curve and the amount of carbon dioxide produced. The gentamicin prepared with the commercial freeze-dried serum resulted in about 15-20% less carbon dioxide and ca. 20% lower slope relative to gentamicin in phosphate buffer. The linear range obtained with the freeze-dried preparation was only 0.05-1.0 pg/mL, which was significantly reduced from the 0.05-10.0 pg/mL range obtained in phosphate buffer. The system with plasma gentamicin standard solutions produced about the same amount of carbon dioxide as was obtained with the phosphate buffer solutions. However, the slope of the gentamicin log dose-response curve was ca. 100% greater than that obtained with the buffer solutions. The effect of the plasma on the linear range is not known, since solutions were prepared only in the 0.05-1.0 pg/mL range. The reasons for the different effects obtained with the two types of blood samples are not known and will require further study. The results of the plasma gentamicin assays are promising. The average potencies at 1:25 and 1 5 0 plasma dilutions were 3.26 f 0.19 and 3.30 f 0.30 pg/mL, respectively. The good agreement between the averages at these two dilution levels suggests that there were no significant effects from dilution of the plasma in the range used. Therefore, the data obtained a t the two plasma dilution levels were combined and the average potency was determined to be 3.28 f 0.20 pg/mL for the five determinations. The average of the combined data has a -18% error relative to the 4.00pg/mL potency at which the plasma gentamicin sample was prepared. Although more research is required to optimize this method for serum gentamicin determinations, the preliminary results obtained demonstrate the feasibility of the approach.
ACKNOWLEDGMENT We thank Berry Kline, Department of Pharmacy and Pharmaceutics, for his many suggestions and for providing the serum gentamicin samples.
LITERATURE CITED (1) Kavanagh, F. I n "Methods in Enzymology"; Hash, J. H., Ed.; Academic Press: New York, 1975; Volume 43, pp 55-69. (2) Dewart. R.;Nandts, F.; Lhoest, W. Ann. N.Y. Acad. Scl. 1965, 730, 686. (3) Shaw, W. H.; Duncombe, R. E. Ann. N.Y.Acad. Sci. 1965, 130. 647.
Anal. Chem. 1983, 55, 1977-1979 (4) Shaw, W. H. C.; Duncombe, R. E. Analyst (London) 1963, 8 8 , 694. (5) Greely, V. J.; Holl, W. W.; Michaels, T. P.; Sinotte, L. P. Ann. N . Y . Acad. Scl. 1985, 130,657. (6) Haney, T. A.; Gerke, J. R.; Madlgan, M. E.; Pagano, J. F. Ann. N . Y . Acad. Scl. 1962, 93, 627. (7) Kubin, H. Chem. Abstr. 1980, 9 3 , 210363s. (8) Karube, I . : Matsunaga, T.; Suzuki, S. Anal. Chim. Acfa 1979, 109, 39. (9) Simpson, D. L.; Kobos, R. K. Anal. Lett. 1982, 75, 1345. (10) Stow, R. W. ; Baer, R. F.; Randall, B. F. Arch, p h p . Med. Rehab//. 1957, 38, 646. (11) Severinghaus, J. W.; Bradley, A. F. J . Appl. Physlol. 1958, 13,515. (12) Goldstein, A. "Biostatistics: An Intrductory Text"; Macmlllan: N~~ York, 1964; Chapter 4.
1077
(13) He!ler, A. H.; Spector, R.; Aalyson, M. J . Anliblot. 1980, 33, 604. (14) United States Pharmacopela; United States Pharmacopeia1 Convention, Inc.; Rockvllie, MD, 1980, 20th Rev.; p 956. (15) Fraticelli, Y. M.; Meyerhoff, M. E. Anal. Chem. 1983, 55, 359.
RECEIVED for review April 12, 1983. Accepted July 11, 1983. We acknowledge the financial support of Philip Morris, Inc., and the National Science Foundation (Grant No. CHE8215809). Presented in part a t the Ninth Annual Meeting of the Federation Of Analytical Chemistry and Spectroscopic Societies, Philadelphia, PA, Sept 20, 1982.
Extraction of Palladium Isotopes in Irradiated Actinides Arthur L. Erikson,* Robert L. Tromp, Robert A. Nielsen, Myra D. Anderson, Terry C. Chapman, and W. A. Emel Exxon Nuclear Idaho Company, P.O. Box 2800, Idaho Falls, Idaho 83401
A method Is described for the carrier-free separatlon of microgram quantltles of fission product palladlum from dissolver solutlons of Irradiated actlnldes. The separatlon Is effected by extractlng the dlniethylglyoxlme (DMG) complex wlth chloroform. This extractlon Is followed by the destruction of the DMG complex wlth nltrlc and perchlorlc aclds. Analysis of the sample Is then performed by surface lonlzatlon mass spectrometry using a modlfled slllca gel technique. Radloactive tracers were used to follow the palladlum and the most likely mass Interferences, ruthenium, sllver, and cadmlum, through the procedure. A palladlum yield of 50-60% and decontamlnatlon factors for ruthenlum of >4 X lo4, for cadmlum of >lo4 and for silver of >lo5 were obtained at the mlcrogram level. Analysis of several allquots of a dlssolver solution of Irradiated saePudemonstrated that the achleved purtflcatlon was sufflclent for mass spectrometric analysis of palladlum at the mlcrogram level.
Fission product P d is of particular interest in the analysis of irradiated nuclear fuels because of the position of its isotopes on the valley side of the light mass peak of the fission yield curve. Fission yields in this portion of the fission yield curve vary more with fissioning species than do those in other portions of the curve. The fission product P d isotopes with masses, 105, 106, 107, 108, and 110 span the area of the greatest change in yield with changing fissioning mass. Therefore, the relative fission yields of the P d isotopes are important to studies of the nuclear fission process. The analysis of Ptl by mass spectrometry requires the separation of P d from the sample matrix and from materials that interfere with the ionization process and from elements with isotopes of the same masses as the P d isotopes, such as ruthenium, cadmium, and silver. Carrier separation of Pd by precipitation of the palladium dimethyglyoxime (DMG) complex is the classical procedure for separation of P d from irradiated actinides. Young (1) demonstrated that the Pd(DMG) complex could be extracted by chloroform. Micrograms of Pd were successfully separated from fire assay silver beads for subsequent assay by dithizone titration. Fraser, Beamish, and McBryde ( 2 )quantitatively
extracted up to 1 mg of P d from a lead and nickel matrix. Alexander, Schindewolf, and Coryell (3) used the DMG extraction to separate short-lived Pd isotopes formed by the bombardment of U by 15-MeVdeuterons. They also reported stripping Cd and Ag isotopes formed by decay of the Pd in the organic phase using 0.015 M nitric acid. This suggested that cadmium and silver are not extracted into chloroform using DMG. Therefore, a Pd-DMG extraction was investigated as a mass spectrometry preparation procedure. To our knowledge a P d separation procedure a t microgram levels suitable for mass spectrometric analysis has not previously been reported.
EXPERIMENTAL SECTION Apparatus. Phase separations were performed with BioRad 11 mL Econo-Columns with molded plastic frits at the bottom of the columns. When the inside of the column was rinsed with chloroform, the plastic frit acted as a phase separator. All glassware was leached with hot aqua regia for 2 h and rinsed once with distilled water and twice with quartz distilled water. Reagents. Demineralized and laboratory distilled water, reagent grade hydrochloric acid, and reagent grade nitric acid were redistilled in a quartz apparatus. Analytical reagent grade chloroform (Mallinckrodt),analyzed reagent grade dimethylglyoxime (J. T. Baker) and double distilled reagent grade 70% perchloric acid (G. Frederick Smith) were used without further purification. The dimethylglyoximewas used as a 1%solution in ethanol. The carrier-free Io3Pdtracer (0.243 mCi/mL and 1.0 pg of Pd/mL) was obtained from Amersham Corp.; the llOmAg (11.5 mCi/mL and 1.0 mg of Ag/mL) and carrier-free lo9Cd (5 mCi/mL and 13.9 wg of Cd/mL) were obtained from New England Nuclear. The 'OBRuwas obtained by separating a few micrograms of Ru from a fission product solution. Procedure. The sample in a conical bottom Pyrex tube was dried with a jet of heated, filtered air. The sample was then boiled down twice with 5-mL portions of aqua regia rich in hydrochloric acid until the final volume was about 2 mL. The sample was then diluted to 12 mL with 0.1 M HC1. This resulted in a final HCl molarity of 0.5-1.0 M, the nitric acid having been removed during the digestion. Two chloroform extractions of the Pd-DMG complex were performed as follows. Three milliliters of DMG solution was added and the sample was allowed to stand for 10 min. Five milliliters of chloroform was added and the sample was extracted by vortex mixing for 2 min. The phases were separated in an Econo-Column, and the organic phase was collected in a clean 40-mL tube. The
0003-2700/83/0355-1977$01.50/00 1983 American Chemlcal Society
