Turbidimetric Bioassay for Carbenicillin

Transcription

1 ANTMWICGOBAL AGENTS AND CHEMOTHEAPY, Mar. 1973, p Vol. 3, No. 3 Copyright 1973 American Society for Microbiology Printed in U.S.A. Turbidimetric Bioassay for Carbenicillin JOSEPH P. STANKEWICH AND RONALD P. UPTON Analytical Research and Methods Development, Quality Control, Pfizer Inc., Groton, Connecticut 634 Received for publication 18 December 1972 A quantitative turbidimetric bioassay has been developed for carbenicillin with the use of a strain of Escherichia coli. The assay is relatively specific for carbenicillin and is not affected by 1% (wt/wt) benzylpenicillin. It has been demonstrated to be more precise than the plate diffusion assay. Since it is readily automated, it is also faster and less expensive. The influence of ph, time, and extent of growth on the assay has been evaluated. Carbenicillin (a-carboxybenzyl penicillin) is a new semisynthetic penicillin active against both gram-negative and gram-positive bacteria. In particular, carbenicillin shows activity against Pseudomonas aeruginosa, strains of Proteus, and Escherichia coli. The in vitro activity of carbenicillin has been described by a number of authors (3, 4, 9). Carbenicillin can exist in many salt forms, including monosodium carbenicillin (MSC) and disodium carbenicillin (DSC). MSC monohydrate is an intermediate in the production of DSC. Both result in carbenicillin when dissolved in ph 7. buffer solution. The standard unit of carbenicillin is defined as carbenicillin free acid (CFA) where pure CFA contains 1, jg of CFA/mg. Carbenicillin samples can contain up to 5% benzylpenicillin (Addendum 1969 to the British Pharmacopeia 1968). An antibiotic must conform to rigid biological and chemical specifications as an assurance of its quality, and an important part of antibiotic testing is the determination of biopotency. A number of biological methods for assaying carbenicillin have been reported. Jones and Palmer (5) described an automated microbiological assay for carbenicillin using E. coli. This procedure requires an AutoAnalyzer and is relatively complex. Burnett and Sutherland (1) described a procedure for the bioassay of carbenicillin in body fluids. Their method, which is a plate diffusion assay with P. aeruginosa (NTTC 149) as the test organism, is basically similar to that described in the Code of Federal Regulations (2). Turbidimetric bioassays are more advantageous than plate bioassays (6). This report describes the development, specificity, precision, and accuracy of a turbidimetric bioassay for carbenicillin with a Pfizer strain of E. coli (51A266) as the test organism. A comparison of potencies obtained by the plate diffusion assay and the turbidimetric assay on samples of MSC and DSC is included. MATERIALS AND METHODS Carbenicillin. The samples of carbenicillin used in this investigation were the monosodium and disodium salts of carbenicillin (Pfizer Co., Inc.). Their respective structures are shown in Fig. 1. Solutions were made daily in sterile.1 M phosphate buffer (ph 7.). This buffer was chosen as most desirable from a study on the effect of ph and buffers on reconstituted stability. The MSC reference sample which was used as the standard for assaying both MSC and DSC was stored at -2 C. Test organism. A culture of E. coli (51A266), which was found to be more sensitive to carbenicillin than to benzylpenicillin, was generously supplied by Arthur R. English, Medical Research Laboratories, Pfizer Co., Inc., Groton, Conn. Instrumentation. An Elanco Autoturb was used throughout the investigation. The Autoturb is an automated system for photometric microbiological analyses. An extensive description of the Autoturb has been given by Kuzel and Kavanagh (7). Standard curve response. The relationship of growth to various concentrations (.5 to.9 jsg) of CFA per milliliter of test broth was investigated. The effects of time, relative amount of growth, and ph of the test broth on the concentration-response curve were evaluated. Basic turbidimetric assay procedure. The assay inoculum was prepared from a fresh slant of E. coli on standard nutrient agar (BBL) incubated at 37 C for 18 to 24 h. The resulting growth was washed into a Roux bottle containing seed agar (BBL) by use of 5 ml of sterile USP saline. The Roux bottle was incubated at 37 C for 18 to 24 h, and the resulting growth was washed from the bottle with about 5 ml of sterile saline into a sterile flask. This cell suspension was diluted with sterile USP saline to give a 25% light transmission 364

2 VOL. 3, 1973 TURBIDIMETRIC BIOASSAY FOR CARBENICILLIN 365 MONOSODIUM <HH5yCH3 HN%CH3 H CARBENICILLIN /H :a~~~~qn OQOONa DISODIUM CARBENICILLIN FIG. 1. Structures of monosodium carbenicillin and disodium carbenicillin. The assignment of the proton to the a-carboxyl group on monosod.ium carbenicillin has not been unambiguously proven. at a wavelength of 58 nm with the use of a 1.27-cm cuvette. This standardized cell suspension can be stored under refrigeration (5 C) for a maximum of 1 week. The inoculum that was used for the assays was 7. ml of the standardized cell suspension per liter of chilled (5 C) antibiotic assay broth (BBL). A carrousel of 2 test tubes was filled with distilled water, standard, and sample solutions. The standard curve solutions consisted of 6., 7., and 8. pg of carbenicillin/ml diluted in.1 M phosphate buffer (ph 7.). The samples were diluted to approximately 7.,g of carbenicillin/ ml. The distilled water tubes were included as a reference of growth in the absence of antibiotic. Four samples of approximately.1 ml of solution were automatically removed from each test tube in the carrousel, and each was diluted with approximately 9.9 ml of chilled inoculated antibiotic assay broth, resulting in a 1:1 dilution of antibiotic. The final concentration of antibiotic was.6,.7, and.8 pg of carbenicillin/ml of assay broth. The racks, consisting of 8 tubes, were then incubated p at 37 C.5 C for about 3 h and 15 min. After incubation, bacterial growth was stopped by placing the rack in a water bath at 85 to 9 C for 2 to 3 mi. The racks were then cooled to ambient temperature and read at 53 nm on an Autoturb Reader Module equipped with a model 33 Turner spectrophotometer with a 1.-cm flow cell. The reader was equipped with both a Thomas print tape and a paper tape punch. The paper tapes were loaded via a teletype tape reader into a time-share computer for analysis. Analysis of samples. Samples of MSC and DSC were assayed by the turbidimetric method and the results were compared with those obtained from the plate diffusion assay. The plate diffusion assay, was basically that described in the Code of Federal Regulations (2) with P. aeruginosa (NCTC 149) as the test organism. A single turbidimetric assay consisted of one sample weight which was manually diluted with.1 M phosphate buffer (ph 7.) to approximately 7. ;&g/ml and placed in five carrousel tubes. Four samples were removed from each tube, thereby producing a total of 2 replicates. A single plate diffusion assay consisted of one sample weight diluted to 7. Ag/ml. Three penicylinders on each of 36 petri plates were filled with the sample solution, producing a total of 18 replicates. RESULTS Standard curve response. The relationship of growth to various concentrations of CFA per ml of test broth was investigated. Figure 2 depicts a representative concentration-response standard curve. The response is plotted as absorbance versus final concentration of CFA per milliliter of test broth. The concentration range of.55 to.85,ug of CFA per ml generates a reasonably linear response. Analysis of these points by linear regression showed no significant departure from linearity (deviation from linearity <.2%). Concentrations of.6,.7, and.8 E 5 Lr( F-- 'I:.4 wllj z <.3 o <.2.1 jf a.9 FIG. 2. Typical concentration-response curve of Escherichia coli to a range of carbenicillin concentrations. The absorbance of the reference growth in the absence of antibiotic was.48.

3 366 STANKEWICH AND UPTON Mg of CFA/ml of test broth were used as standard curve points for subsequent analyses. Influence of time and relative amount of growth. Series of concentration-response tests were conducted and were terminated at various time intervals. Figure 3 depicts the family of curves obtained. Note that the slope increases with time or the extent of growth. The concentration range of.6 to.8 Mug of CFA/ml of test broth remains linear throughout the time period indicated. Conducting the assay over a longer period results in a steeper slope and a decrease in linearity. Good assay results were obtained when the assay was incubated until the reference growth reached an absorbance of about.5. Generally, an incubation time of 3 h and 15 min was required. However, as illustrated in Fig. 3, some flexibility due to day-to-day variability can be tolerated. Influence of ph. Smith and Finland (9) reported on the in vitro activity of carbenicillin and found that lowering the ph increased the activity against P. aeruginosa (NCTC 149). Possible effects of ph in the carbenicillin turbidimetric assay were evaluated by running standard curves in antibiotic assay broth at ph 7.5, 7., 6.5, and 6.. The family of curves obtained is illustrated in Fig. 4. Note that the curves are very similar for ro L E.5 O H- 4 zo cr n 2.1 FIG. 3. Effect of various incubation times on the concentration-response curve for carbenicillin. The absorbances of the reference growth with increasing time were.35,.4,.48, and.6. The absorbance values at.85 and.9 jg of carbenicillin/ml for the various times were similar. Symbols for various incubation times: () 3 hr, (A) 3 hr 6 min, () 3 hr 15 min, (A) 3 hr 4 min..6 F E o.5 r OA LIC Lii z <.3 m (I) 4- <.2 u ph 6 v. I. ph 7. ph 7.5 ANTIMICROB. AG. CHEMOTHER. ph 6.5 'C \ FIG. 4. Effect of varying the ph of the antibiotic assay on the concentration-response curve for carbenicillin. The absorbances of the reference growth are depicted as zero carbenicillin concentration. the test broths at ph 6.5, 7., and 7.5, and that they remain linear. The small changes noted in slope are within experimental variability and, as previously shown, are influenced by extent of growth. At ph 6., the activity of carbenicillin drops off considerably; this is indicated by more extensive growth and a decrease in the slope of the curve. The assay is relatively insensitive to small ph fluctuations in the range from 6.5 to 7.5. Influence of benzylpenicillin. The effect of benzylpenicillin on the assay is important because carbenicillin can contain up to 5% benzylpenicillin. Its effect under assay conditions was measured by adding 11. mg of Pfizer benzylpenicillin standard (1,662 units/mg) to 114. mg of MSC standard (877,ug/mg). The mixture was then assayed in duplicate. The two potencies obtained, each of which is a total of 2 replicates, were 886 and 877 Mg/mg. The average of 881 Mg/mg indicates that 1% (wt/wt) benzylpenicillin does not interfere with the assay. Precision of the turbidimetric assay. Two lots of MSC were tested to determine assay precision. Five weights of each of the two MSC lots were tested daily for 5 days by the plate diffusion assay (2) and the proposed turbidimetric assay. An evaluation of the relative standard deviation to determine precision is presented in Table 1. The average relative standard deviation for the turbidimetric assay was 1.1% compared with 3.8% for the plate assay.

4 VOL. 3, 1973 TURBIDIMETRIC BIOASSAY FOR CARBENICILLIN 367 TABLE 1. Precision of turbidimetric and plate assays on two lots of monosodium carbenicillino Assay Lot Lot A CFA (%) SD N CFA (%) SD N Plate diffusion Turbidimetric CFA = carbenicillin free acid; SD = percent relative standard deviation; N = number of assays. TABLE 2. Comparison of carbenicillin free acid (CFA) determinations by turbidimetric assay and composition material balance Determination Percent CFA Lot Lot A Turbidimetric assay Composition material balance (CMB) Percent deviation from CMB Accuracy of the turbidimetric assay. To document the accuracy of the turbidimetric assay, we established the potencies of the above two carbenicillin lots by the composition material balance technique (1). Composition material balance is achieved by account for the total composition of a bulk sample. This technique involves measuring all known components and impurities of carbenicillin other than CFA. The percent CFA is determined by subtracting the sum of the abundance of nonactive components and impurities from 1%. The measurement of each nonactive component and impurity was an average of three determinations. The results in Table 2 show that the average difference between percent CFA determined by the turbidinmetric assay and the composition material balance was -.7%. Correlation between the turbidimetric and plate bioassays. Eleven samples of MSC and eight samples of DSC were analyzed by both the plate diffusion and turbidimetric bioassays. As shown in Tables 3 and 4, all assay results are in close agreement for both MSC and DSC, respectively. The average variation is approximately 2% in either case. A statistical evaluation of these data, by use of the two-sample t test (8) and the average relative standard deviations from Table 1, showed that the mean average potencies obtained by both assays are not statistically different at a level of significance of.25. TABLE 3. Comparative bioassay results on monosodium carbenicillin samples Sample Avg Carbenicillin free acid (%) Turbidimetric assay 85.3 (4)a 85.2 (4) 85.3 (3) 87.8 (4) 85.9 (3) 87.6 (2) 86.5 (2) 85.4 (3) 85.4 (4) 83.9 (4) 86.2 (4) a Number of assays. Plate assay 82.1 (2) (2) 86.6 (2) 84.7 (2) 85.3 (3) 84.4 (2) 84.7 (2) 83. (3) 85.2 (3) 81. (3) 84.6 (3) Deviation of turbidimetric assay from plate asay (%) TABLE 4. Comparative bioassay results on diisodium carbenicillin samples Sample Avg Carbenicillin free acid (%) Turbidimetric assay 86.2 (4)a 86.1 (4) 84.2 (6) 84. (6) 83.8 (6) 84.6 (4) 84.1 (5) 84.8 (5) a Number of assays. Plate asay 84.4 (2) (2) 82.9 (2) 83.4 (2) 82.6 (2) 83.2 (2) 82.3 (2) 82.7 (2) Deviation of turbidimetric assay from plate asay (%) DISCUSSION A quantitative turbidimetric bioassay has been developed for carbenicillin, with a strain of E. coli as the test organism. The advantages of a

5 368 STANKEWICH AND UPTON turbidimetric bioassay are that it is readily automated, less expensive, faster, and more precise than a plate assay. Results from a turbidimetric assay can be obtained on the same day, whereas the plate assay requires two days. The quality of the plate diffusion assay for carbenicillin is not optimal. The test organism (P. aerunosa) is aerobic and surface growth predominates. The definition of the zones of inhibition is complicated by a halo which appears at the periphery of the zones. The test organism for the plate assay is not suitable for a turbidimetric assay because it is aerobic and forms a pellicle. It is difficult to obtain reproducible photometric readings because growth in liquid culture is not uniform. Benzylpenicillin is more active than carbenicillin against the usual test organisms for penicillin, such as Staphylococcus aureus. The test organism for carbenicillin should be sensitive to carbenicilin but relatively insensitive to benzylpenicillin because of its presence in carbenicillin. The turbidimetric assay for carbenicillin with E. coli is relatively specific for carbenicillin. The presence of 1% (wt/wt) benzylpenicillin has been demonstrated not to interfere with the assay. The turbidimetric assay for carbenicillin produces a linear response when absorbance is plotted against concentration (Fig. 2). A linear relationship between concentration and response increases the accuracy of interpolation of data and facilitates useful calculations of line slope and intercept. Reasonable changes in time, relative amount of growth, and ph do not adversely affect the linearity and reproducibility of the standard curve. The turbidimetric assay has been shown to be more precise than the plate assay. The average relative standard deviation for the turbidimetric assay was 1.1% as compared with 3.8% for the plate assay. The accuracy of the turbidimetric assay has also been documented. The average ANTIMICROB. AG. CHEMOTHER. difference between CFA determined by the turbidimetric assay and the composition material balance was -.7%. The MSC and DSC sample results obtained by the turbidimetric assay correlate well with the plate bioassay. On the average, the two assays agree within about 2%. The average of the means for the turbidimetric and plate assays for both MSC and DSC has been shown not to be statistically different at a level of significance of.25. The turbidimetric assay for carbenicillin has been demonstrated to have decided advantages over the presently used plate diffusion assay. ACKNOWLEDGMENTS We thank the Pfizer Quality Control Laboratories, Groton, Conn., for their assistance in providing some of the data in this publication. Special thanks go to F. C. Hartl for the many carefully performed assays which represent the bulk of the data. LITERATURE CITED 1. Burnett, J., and R. Sutherland Procedures for the assay of carbenicillin in body fluids. Appl. Microbiol. 19: Code of Federal Regulations , CFR U.S. Government Printing Office, Washington, D.C. 3. English, A. R Laboratory studies with carbenicillin. Antimicrob. Ag. Chemother. 1968, p Isenberg, H. D., and M. Siegel In vitro action of carbenicillin against bacteria isolated from clinical material. Appl. Microbiol. 18: Jones, A., and G. Palmer Automated microbiological assay. Analyst (London) 95: Kavanagh, F Analytical microbiology. Academic Press Inc., New York. 7. Kuzel, N. R., and F. W. Kavanagh Automated system for analytical microbiology. II. Construction of system and evaluation of antibiotics and vitamins. J. Pharm. Sci. 6: Miller, I., and J. E. Freund Probability and statistics for engineers. Prentice-Hall, Inc., Englewood Cliffs, N.J. 9. Smith, C. B., and M. Finland Carbenicillin: activity in vitro and absorption and excretion in normal young men. Appl. Microbiol. 16: Wagner, R. L Development of quality control methodology for new drugs. Bull. Parenteral Drug Ass. 26:76-86.