Table 11. Concentrations of Nitrite Found in Air Particulate Samples'' Weight % Location as NO; BYU Campus 0-0.04 New York City 0-0.14 St. Louis 0-0.04 Fly ash from an oil fired power plant 0.02 Plume from an oil fired power plant 0-0.02 a Unpublished data, this laboratory. determination. Even though nitrite is known to react with sulfite, Fe(III), and amines, the reactions are apparently slow in the dilute, acidic solution used in this study. Cu(I1) was tested for interference because it is often found in environmental samples. Interferences that may occur during the extraction of environmental samples would be difficult t o detect. In the solid particulate state, there may be reducing or oxidizing agents, or amines for example that may react with the nitrite when it is dissolved in the 0.1 M HCl, 2.5 mM FeC13solution. For this reason, the nitrite found in a sample by the anaiysis procedure given here should probably be regarded as a lower limit of the nitrite t h a t was present in the solid. The limit of detection is determined by the error encountered in setting the bath and hence the titrant temperature equal to the reaction vessel temperature. The titrant temperature, with respect to a temperature measurement in the Dewar, can only be known to A 60 WV(-0.002 "C). This means t h a t the sulfamic acid may be 60 pV hotter or cooler than the reaction vessel temperature when it is injected and it will cause a change in temperature even though there may be no reaction. Therefore the limit of detectability is -3 nmol
per 2 mL of solution, Le., A (0.15 mL)(0.002 "C)(1 cal/ mL-"C)/(107 cal/mmol). This conclusion was verified by the blank runs where t h e standard deviation of an observation was 1.7 nmol.
ACKNOWLEDGMENT Thanks are due to David H. Jones for performing some of the DIE experiments.
LITERATURE ClITED P. N. Masee and P. F. Swann, B r . Meed. Bull., 25, 240 (1969) and references therein. B. S. Garg, Y L. Mehta, and M. Katyal, Taalanta, 23,71 (1976). B. F. Raer ana M. G. Mellon, Ind. Eng. C!hem.,Anal. Ed., 18, 96 (1946). J. M. Pappenhagen and M. G. Mellon, Anal. Chern., 25, 341 (1953). B. J. Meehan, S. A . Tariq, and R. J. Magee, Microchem., J.. 21, 302 (1976) K . Toei and T. Kiyose, Anal. C h m Acta3 88, 125 (1977). S. W. Boese, V. S. Archer, and J. W. 0'1-aughiin, Anal. Chem., 49, 479 (1977). J. T. Stock and R. G . Bjork, Microchem. J . , V I , 219 (1962). J. T. Stock and R . G . Bjork, Talanfa. 11, 315 (1964). P. W. West and F. Ordoveza, Anal. Chem., 34, 1324 (1962). R . C. Brasted, A y l . Cnem., 24, 11 11 (1952). G. A. Vaughan, Thermometric and Elnthalpimatric Titrimevy" Van NostraandReinholdCo., London, 1973, pp 1 6 1 7 ; J. Barthel, "Thermometric Titrations", Jonn Wiley & Sons, New York, 1975, pp 66-69. L. D. Hansen, L. Whiting, D. J. Eatough, T. E. Jensen, and R. M. Izatt, Anal. Chem., 48, 634 (1976). D.6.Wagman, W. H. Evans, V. B. Parker, I. Halow, S. M. Bailey, and R. H. Schurnm, U.S. AM/. Bur. Stand. Tech. a t e , 270-3, US. Department of Commerce, 1968. A. K. Covington, J. V. Dobson, and Lord WynneJones, Trans. Faraday SOC..61. 2057 (1965) L. D. Hansen, T.'E. Jensen, S . Mayne, C). J. Eatouyh, R. M. Izatt, and J. J. Christensen, J . Chem. Therrnodyn., I,919 (1975).
RECEIVEU for review April 22, 1977. Accepted July 5 , 1977. This work was supported in part by Contract No. E(11-1)-2988 from the U S . Energy Research and Development Administration.
Bioassay of Antifungal Antibiotics by Flow Microcalorimetry Anthony E. Beezer,' Babur 2. Chowdhry, Roger
D. Newell,'
and H. J. Valentine Tyrrell
Chemistry Department, Chelsea College, University of London, Manresa Road, London
This bioassay system relies upon the inhibition of the microcalorimetricaliy observed respiration of Saccharomyces cerevisiae In buffered glucose. The application of the technique to other antimycotics, which have differing modes of action to the polyenes, 5-fluorocytosine and clotrimazole is described. The observed thermograms reveal some of the differences in tho modes of action of the antibiotics. A discussion is given of the assay of formulations of nystatin and of the investigation of the effects of drugs in combination. The features of the assay are: the use of inocula stored in liquid nitrogen; improved reproduclbility (f3 % compared with f5-10% derived from the classlcai agar plate diffusion technique); improved sensitivity; speed of assay (1 h compared to 16 h); simplicity of experimentation and capability of automation.
Calorimetric methods of analysis have been extensively reviewed (1,2). In the main, however, these applications have Present address, Department of Oral Medicine and Pathology, Guy's Hospital Medical School, London SE1 9RT.
SW3 6LX
been restricted to inorganic chemical systems. I t is only recently that extension of the principles to organic systems (3)and biochemical systems ( 4 ) has become a t all appreciable. These systems are chemically reasonably well defined. Rather poorly defined from a physicochemical point of view are biological systems with more or less intact life functions. Each metabolic event taking place is associat,edwith thermai effects and the smailest fluctuations in metabolism will be shown in the heat output rate, if the instrument sensitivity is sufficient. In this, principally microbiological field it is perhaps the analytical applications of microcalorimetry which have the most potential for the immediate future, particularly for those areas where present analytical methods are time consuming, laborious, and of relatively poor accuracy and precision. There have been relatively few reports of the effects of metabolic modifiers on the calorimetric behavior of microorganisms. Of those where flow microcalorimetry was used, three are qualitative (5-7) and the remaining report describes the bioassay of nystatin bulk material (8). The bioassay technique involved tne use of liquid nitrogen stored inocula of' Saccharomyces cerevisiae (9). T h e developed method proved superior in speed, reproducibility, and sensitivity to the classical agar plate diffusion technique (10). However, to be truly a potential substitute for the established procedures, the ANALYTICAL CHEMISTRY, VOL. 49, NO. 12, OCTOBER 1977
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Table I. Results of the Assay of Raw Antibiotics‘ Anti biotic Pimnricin Amphotericin-B Candicidin Lucensomycin
Time of assay for 6.5 x s o h , min see text
Concn range studied, M 1-7 x 1 x i w 6 - 7 x 10-5
Ma
83
4.2-8.3 X 69 see text (a) < 5 x 35 ( b ) 5 X 10-b-lO-’ 0.2-6.5 X 10-o 25 Nystatin 1 x 10-6-1 x lo-’ 60 Filipin see text Clotrimazole 3 X 10-’-5.2 X 10.‘ N o effect within range studied 5-Fl~oroc ytosine~ Reproducibility of control 12.5%;reproducibility of drug assay 23.0%. Lowest concentration quoted is lowest determinable concentration. 5FC is converted by susceptible fungi t o 5-fluorouracil which is highly toxic; it blocks methylation of desoxy(ribonuc1eic)uridylic acid and thymidilate synthesis. It is, therefore, anticipated that 5FC would only show significant effects on growing yeast cultures. This conclusion has been confirmed and will be reported in a subsequent paper. microcalorimetric technique must be capable of dealing with a wide range of antimycotics and with drug formulations in some simple fashion. It must also be capable of investigating possible “potentiation” of one antibiotic by the presence of another. T h e simplicity requires that the method should preferably not require preliminary separation of the active ingredients from excipients in the formulations. Calorimetry being a general and nonspecific tool registers the thermal consequences of all participating reactions whether of analytical interest or not. Thus the proof of specificity is required. Furthermore the insistence of national authorities (11)that many antibiotic assays be performed on viable organisms rather than by purely chemical procedures has led to little investigation of alternative methodologies for antibiotic assay. The reports that exist on the thermometric analysis of drugs have dealt with pure sulfonamides (12, 13) and with formulations of chlorpromazine hydrochloride and aminophylline (14). In the latter investigation, aqueous titration replaced nonaqueous extraction procedures, thus introducing greater simplicity. A modified flow microcalorimetric system of antibiotic sensitivity testing has been developed (15) but cannot be used as a quantitative analytical procedure. A possible calorimetric assay procedure has been proposed (16) for penicillin G and tetracycline hydrochloride. The technique involved observation of the alteration in heat output rate during the exponential phase of growth in treated and untreated samples of Streptococcus faecalis grown in rich medium. Wadso (17) has recently described the effects of tetracyclines upon Escherichia coli. T h e present paper reports the extension of a microcalorimetric assay procedure established for nystatin to other polyene antibiotics whose modes of action have been discussed (18). In addition other antimycotics with different modes of action, 5-fluorocytosine (19) and clotrimazole (20) have been investigated. Formulations of nystatin have likewise been investigated. One of the tested formulations contains another antibiotic, tetracycline hydrochloride. Previous attempts (21) t o measure any synergistic effects of the tetracycline hydrochloride on the measured potency of nystatin concluded that the effect was small and that variation in the yeast inoculum precluded estimation of the magnitude of the effect. We report here measurement of this potentiating effect.
EXPERIMENTAL Methods. The method of preparation, storage, recovery, and assay of liquid nitrogen stored inocula of Saccharomycescereuisiae (NCYC 239) are as previously described (9). The flow microcalorimeter (LKB type 107001-1, LKB Produkter AB, S-161,25 Bromma 1, Sweden), its design (22), and operation have been described (9). 1782
ANALYTICAL CHEMISTRY, VOL. 49, NO. 12, OCTOBER 1977
time
+
Figure 1. Control thermogram and thermogram showing interaction of nystatin, candicidin, amphotericin-6, filipin, and iucensomycin (at high concentration)
Materials. Nystatin and Amphotercin-B (E. R. Squibb and Sons Ltd. Merseyside, U.K.), Pimaricin (Gist Brocades N.V., Holland), Filipin (Upjohn, Kalamazoo, Mich.), Candicidin (Pharmax, Crayford, U.K.), Lucensomycin (Farmitalia, Milan, Italy), 5-fluorocytosine (5FC; Hoffmann-La Roche Inc., Nutley, N.J.) and clotrimazole 1-(o-chloro-a,a-diphenylbenzyl)imidazole (Bayer U.K. Ltd., Hayward’s Heath, U.K.) were all stored, as recommended, under nitrogen at 250 K until required. All antibiotics were subjected to UV spectroscopic investigation on receipt. The values of urnax recorded were in all cases in accord with the literature (23). In addition clotrimazole and 5FC were subjected t o mass spectrometric investigation; previous results (24,251 were confirmed. Formulated products containing nystatin (Mysteclin Tablets and Mycostatin Oral Suspension, placebos of these products, individual components of the tablets, and tetracycline hydrochloride) were generously provided by E. R. Squibb and Sons Ltd. Solutions of antibiotics and dacebos were first made UD in DMF (except for 5FC which is soluble in water) and then difuted with buffer to give the final concentration of drug required, such that the concentration of DMF in the assay was 0.3% (except for clotrimazole where it was 0.1%). All solutions were freshly made and stored in the dark during use. The operation of the microcalorimeter and the design of the assay experiment have been described previously (9).The calorimeLer was operated at 303 K in a room maintained at 298 K.
RESULTS AND DISCUSSION The results of the assay of the raw antibiotics are described in Table I. The general forms of the derived thermograms are as shown in Figure 1. The form is essentially (i.e., return of the thermogram to the baseline) the same as that described previously (8). The reason for the decrease in heat output rate with time is the inhibition of the respiration of Saccharomyces cereuisiae. However, in this case the microcalorimeter was operated a t 303 K and not 298 K as previously
Table 11. Potency Order of Drugs Microcalorimetric potency ranking (concn) 1 / 2 MIC” Equimolar solns 1 2 2 1
Minimum inhibitory concn (wg mL-’ ) b 0.03 0.09-0.5
Antibiotic Assumed MW Candicidin 1200 Amphotericin B 924 Nystatin 0.8-3.1 926 5 5 Filipin 5.0 654 3 4 Pimaricin 0.9-1 5.0 665.7 4 3 Lucensomycin 707.7 between 4 and 3 a l / 2 MIC concn taken was half the lowest value in the quoted range. Note the wide variations i n MIC’s. This is characteristic of interlaboratory results f o r the assay of polyenes; see, for example, the WHO report ( ‘ 2 7 )establishing the international standard for nystatin. Table 111. Comparison of Results Complexed material 1 2
Formu!ations Mysteclin Tablets Nvstan Oral SusDension
Potency (agar plate diffusion: unit mL-I) 1174 2348 250 000 uitablet + 10% overage 100 000 u m L - ’ + 10% overage
(8). This modest change in temperature has led to a surprisingly different correlation between the response and the applied dose. The assay procedure reported earlier for nystatin raw material alone operated a t 298 K gave a linear relationship between a response parameter and the logarithm of the applied dose, the “normal” bioassay relationship. At 303 K, however, the relationship between dose and response found for the polyenes was linear. Moreover the time response recorded (as described under the Experimental section) is that for a complete kill (Le., return of the thermogram to the baseline) of the added yeast inoculum. The thermogram is believed to result solely from the heat changes associated with the respiration of the yeast. Thus the return of the thermogram to the baseline implies that all yeast cells added have ceased respiring under the influence of the added antibiotic. The form of the thermograms as a function of temperature and comparison with those reported previously suggest that the kinetics and hence the mechanism of the interaction process is exceedingly complex, as might be expected. I t is apparent that the thermograms recorded for the assay of pimaricin, clotrimazole, and lucensomycin (at less than 5 x lo4 M concentrations) are quite different in form from those observed for the other antimycotics. They do, in fact, more resemble the effect of an inhibitor upon an enzyme (26) than they do the effects of the other antibiotics (Figure 2 ) . T h e inhibitory effects of these antibiotics are much more rapid than those of the other antibiotics studied since they react within 3 min (the flow time from the reaction vessel to the calorimetric cell) of their introduction to the yeast inoculum. Only in the case of lucensomycin can a thermogram form similar to that obtained for the other polyenes be achieved by variation in concentration of the antibiotic. I t is interesting to note the borderline position of lucensomycin between the two extremes of the observed calorimetric responses. Lucensomycin differs from pimaricin only in the alkyl side chain as depicted in Figure 3. T h a t this simple change has produced such a marked difference in response is striking. The reasons for this differing “mode of action” as a function of concentration in lucensomycin is currently under investigation. The other polyenes, however, have a slower interaction, the kinetics of which, though as yet unexplained in detail, are faithfully recorded by the calorimeter.
Microcalorimetric assay __ Unit, mL-’ Std dev, % 1110
1719 2 2 7 256
1 4 3 084
Difference, %
5.5 26.7
3.0 2.7
-
3. 1. 3.2
+30.1
-
- 9.1
=l
t
I
time
+
Figure 2. Control thermogram and thermograms showing interaction
of pimaricin. clotrimazole, and lucensomycin (at low concentration)
OH
”2
Figure 3. R = CH3-, pimaricin; R = CH3(CH2)3-,lucensornycin
That 5FC does not interact with respiring yeast cells is as expected since this antimycotic interferes with DNA synthesis and would, therefore, only demonstrate its effects in growth medium. I t is interesting to note that the microcalorirneter allows some measure of the relative rates of the reactions. At equimolar concentrations, the drugs appear to have the potency order described in Table 11. The potency judgment used here is that of “time to kill”; the most potent drug being that one which, a t equimolar concentration, inhibits the respiration of the standardized liquid nitrogen stored inoculum in the shortest time. T h e order for equimolar concentrations of the antibiotics is different from that obtained when half the minimum inhibitory concentrations of the drugs were applied (Table 11). The “normal” ranking order is that of MIC’s as that published previously (27). This differs substantially from the other two ANALYTICAL CHEMISTRY, VOL. 49, NO. 12, OCTOBER 1977
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ranking orders-particularly with respect to nystatin. There is, therefore, a problem here of the definition of potency. T h e results for the formulations of nystatin and nystatin complexed with polyvinylpyrrolidone (28) are described in Table 111. T h e results obtained are compared in Table I11 with those obtained from agar plate diffusion experiments. T h e agreement obtained is within the sum of the individual standard deviations only for complexed material 1 and the Mysteclin tablets. T h e placebo materials, both individually and in combination (up to 2 g was added), had no observable effect on the respiration of the S. cereuisiae. The differences between the other two results, for the nystatin oral suspension in particular, are rather difficult to explain except to note that the agar plate diffusion technique depends upon inhibition of growth whereas the microcalorimetric assay depends upon the inhibition of respiration in S. cereuisiae. Moreover, the microcalorimetric assay technique employs a homogeneous aqueous suspension containing only 0.3% DMF whereas the agar plate diffusion technique depends, obviously, upon diffusion of the antibiotic through the gel from a solution containing ca. 10% DMF. Therein may lie an explanation. Tetracycline hydrochloride which is present in Mysteclin tablets shows only a small synergistic effect when studied in combination with bulk nystatin raw material alone. The magnitude of its “potentiating” effect lies only just outside the error limits attached to the experiment. I t is perhaps in the quantitative determination of synergistic effects that the microcalorimeter has one of its more obvious applications. Tetracycline hydrochloride plus nystatin is not an usual combination of drugs in dosage form. More common perhaps are combinations of antifungal drugs and, for example, the polymixins, rifampycin etc. T h e use here of standardized inocula of S. cerevisiae recovered from storage in liquid nitrogen for periods up t o 4 years (29) has been essential for this comparative study of a group of antimycotics and of possible synergistic effects. I t has also been useful in showing differences in the modes of action of the investigated drugs. Some aspects of the microcalorimetric investigation of modes of action of antimycotics will be discussed in a subsequent paper.
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ANALYTICAL CHEMISTRY, VOL. 49, NO. 12, OCTOBER 1977
The extension of the application of microcalorimetry t o antibacterial drugs is now under active investigation.
LITERATURE CITED (1) A. E. Beezer in “MTP International Reviews of Science”, physical Chemkty Series 1, T. S. West, Ed., Voi. 13, Butterworths, London, 1973. (2) H. J. V. Tyrreil and A. E. Beezer, “Thermometric Titrimetry“, Chapman and Hall, London, 1968. (3) E.J. Greenhow “Cataly?ic Thermometric Titrimetry”. Chem. Rev.. in press (4) C. Spink and 1. Wadso, in “Methods of Biochemical Analysis”, D. Glick. Ed., Vol. 23A, Wiiey-Interscience, New York, N.Y., 1976. (5) H. Prat, Rev. Can. Biol., 12, 19 (1953). (6) E. Catvet and H. Prat, in “Recent Progress in Microcalorimetry“, banslat& by H. A. Skinner, Ed., Pergamon Press, Oxford, 1963. (7) S. Deiin, P. Monk, and 1. Wadso. Sci. Tools, 16. 12 (1969). (8) A. E. Beezer, R. D. Newell, and H. J. V. Tyrrell, Anal. Chem., 49, 34 (1977). (91 A. E. Beezer, R. D. Newell, and H. J. V. Tyrrell, J . Appl. Bacteriol., 41, 197 (1976). (10) F. Kavanagh, Ed., “Analytical Microbiology”, Academic Press, New ‘fork, N.Y., 1963. (1 1) D. W. Hughes and W. L. Wilson, Can. J . Pharm. Sci., 8 (3), 67 (1973). (12) J. Jordan, R. A. Henry, and J. C. Wasilewski, Microchem. J . , 10, 260 ( 1966). (13) H. Schafer and E. Wilde. fresenius 2. Anal. Chem., 130, 396 (1949). (14) A. B. De Leo and M. J. Stern, J . Pharm. Sci.,53, 933 (1964); 5 4 , 91 1 (1965); 55, 173 (1966). (15) J. S. Binford, L. F. Binford, and P. Adler, Am. J . Ciin. Pathol., 59, 86 (1973). (16) T. E. Jensen, L. D. Hansen. R . D. Saaers. and R. M.Izatt. Thermochfm. Acta, 17, 65 (1976) (17) P A Mardh, T Ripa, K-E Andersson. and I Wadso, Anbmcrob Agents Chemother., I O , 604 (1976). (18) J. M.T. Hamilton-Miller. Adv. Appl. Microbiol., 17, 109 (1974). (19) W. H. Beggs, G. A. Sarosi, and N. M. Steeie, Antimicrob. Agents Chemother. 9, 863 (1976). (20) R. J. Hoit and R. L. Newman, J . Clin. Pafhoi., 25, 1089 (1972). (21) T. C. Forster, personal communication. (22) P. Monk and I.Wadso. Acta Chem. Scand., 22, 1842 (1969). (23) B. Z.Chowdhry, M.Sc. Dissertation, University of London, 1975. (24) C. S. Good, Bayer Ltd. Sussex, U.K., personal communication. (25) From P. G. Philpott, Roche Products, Hertfordshire, England, personal communication, (26) A. E. Beezer and C. D. Stubbs, Talanfa, 20,27 (1973). (27) J. W. Lightbrown, M. Kogurt, and K. Uemura. Bull. W . H . O . ,29, 87 (1963). (28) M. B. Dexter, J . Pharm. Pharmacol., 27, 58 (1975). (29) R . D. Newell, Ph.D. Thesis, University of London, 1975.
RECEIVED for review April 29, 1977. Accepted June 29, 1977. One of us (R.D.N.) thanks E. R. Squibb and Sons Ltd. and the Governors of Chelsea College for the award of a scholarship. B.Z.C. thanks the SRC for the award of a research studentship.
