A Cylinder-Plate Method for the Quantitative Determination of Diffusion of Antibacterial Drugs from Ointments*

A Cylinder-Plate Method for the Quantitative Determination of Diffusion of Antibacterial Drugs from Ointments* By ELMER M. PLEIN and JOY BICKMORE PLEIN Plotting the squares of the diameters of the inhibition zones produced by varying concentrations of antibacterial drugs against the logarithms of the concentrations resulted in straight line curves for mercury bichloride, chlortetracycline hydrochloride, oleandomycin phosphate, salicylic acid, and sodium sulfacetamide. A cylinder-piate merhod for quantitative determination of diffusion of antibacterial drugs from ointments by reference to the standard curves is presented. The results of simple cylinder-plate-inhibition zone diffusion tests in six different ointment bases are compared with the quantitative data obtained in testing diffusion from the six ointments by the cylinder-plate standard curve method.

HE MOST WlDELY USED method Of Ointment Tbase evaluation is by means of the agar plate or agar cup plate procedures or modifications of these methods. Ruehle and Brewer (1) in Circular 198 of the U. S. Department of Agriculture described these two procedures for determining the diffusion of antibacterial drugs from ointments, and although there have been numerous modifications of their methods, the procedures have not changed essentially. In the assay of a number of differentantibiotics, penicylinders are placed on agar plates which have been seeded with a microorganism susceptible to the antibiotic. The penicylinders are then filled with solutions containing varying concentrations of the antibiotic and the resulting inhibition zones are plotted against the logarithms of the concentrations in constructing a standard curve. The potency of unknown antibiotic solutions is then determined by reference to the standard curve (2, 3). Since this logarithmic concentration-inhibition zone relationship is true for antibiotic solutions, it was felt that this function or other mathematical relationships might also be valid for other antibacterial drugs. This study was undertaken in order to determine whether the inhibition zones produced by varying concentrations of several antibacterial drugs were functions of the drug concentrations, to construct standard curves from the data if this relationship were found, and to use these standard curves in obtaining quantitative data on diffusion of antibacterial drugs from ointments.

* Received May 3, 1957, from the College of Pharmacy, University of Washington, Seattle. Presented to the Scientific Section, A. PH. A , , New York meeting May 1957. The iilicone oils were supplied by Dow Corning Corp., the chlortetracycline hydrochloride by Lederle Laboratories, Division of American Cyanamid Co., and the oleandomycin phosphate by Pfizer Laboratories, Division of Charles Pfizer and Co.

t

STANDARD CURVE

-

MERCURY BlCHLORlDE

2.0

0

Fig. 1.-Standard

curve for mercury bichloride.

EXPERIMENTAL

Ammoniated mercury, chlortetracycline hydrochloride, oleandomycin phosphate, salicylic acid, and sodium sulfacetamide were selected for this study. Micrococcus pyogenes var. aureus (Staph. aureus), A. T. C. C. #6538,was used as the test organism for all five drugs and in addition, B. cereus, A. T. C. C. #9634, was used in the chlortetracycline study. The Staph. uureus was maintained and tested on nutrient agar prepared by rehydrating Difco Nutrient Agar 0001-01. The organism was grown for testing in nutrient broth containing 5.0 Gm. of peptone and 3.0 Gm. of beef extract per 1000 ml. of distilled water. The usual bacteriological media inhibit sulfonamide action (4, 5 ) . Therefore, for sodium sulfacetamide standard curve and ointments the medium of Straws, et al. (4), was used to

716

December 1957

SCIENTIFIC EDITION

I-

717

7.0

STANDARD CURVE- CHLORTETRACYCLINE HCI

STANDARD CURVE- SALICYLIC ACID

6.C

'oo"4

5.c

.F

- 4.c E

z

3.c

z

e

G c W

E 2.c d J

I.!

I .c

0

400 600 BOO 1000 1200 1400 SQUARE OF DIAMETER I N H I B I T I O N ZONE. m m 2

,

200

Fig. 2.-Standard

curves for chlortetracycline hydrochloride.

-

50

I

I

I

150

100

SOUARE O F D I A M E T E R

Fig. 4.-Standard

250

200

I N H I B I T I O N ZONE,

3 0

mrn'

Curve for Salicylic Acid in 20% Alcohol.

F

STANDARD CURVE -0LEANDOMYCIN PHOS.

to

0.lL

I

I

200

'

400

I

600

SQUARE OF DIAMETER

Fig. 3.-Standard

curve for oleandomycin phosphate.

grow the Staph. aureus and a solid medium for the agar plates was prepared by the addition of 15.0 Gm. of Baeto-agar (Difco) per liter of gel to the Strauss, et al., formula. The B. cereus was maintained on rehydrated Difco penassay seed agar 0263-02 and a spore suspension

Fig. 5.-Standard

'

I

800

'

1000

1

'

1200

INHIBITION ZONE. mm2

Curve for Sodium Sulfacetamide.

of the organism was prepared as described by Grove and Randall (2). Preparation of Test Solutions.-Sterile technic and sterile diluent were used in preparing the solutions. The antibacterial concentrations used may be seen by referring to the points in Figs. 1 to 5.

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Distilled water was used as the diluent for oleandomycin phosphate and for sodium sulfacetamide solutions. Phosphate buffer p H 4.5 (2) was used as the diluent for chlortetracycline solutions and alcohol 20% v/v was used as the diluent for salicylic acid. An aqueous solution of mercury bichloride was used in determining a standard curve for mercury, and diffusion of ammoniated mercury ointments was determined by reference to this curve (see Discussion). Preparation of Plates.-Staph. aureus organisms were transferred into appropriate nutrient broth for a t least five consecutive daily transfers before use. Ten milliliters of the appropriate melted nutrient agar was poured into 20 x 100 mm. petri dishes equipped with porcelain covers. The agar layer was allowed t o cool and harden, and then a 10-ml. quantity of nutrient agar seeded with 0.1 ml. of a twenty-four-hour culture of Staph. aureus was poured over the first agar layer. For the testing of chlortetracycline solutions and ointments with B. cereus, the plates were prepared as described by Grove and Randall (2) for chlortetracycline solutions. Difco penassay medium was used for these determinations, and 0.1 ml. of the spore suspension of B. cereus was mixed with the second layer. Standard Curve Determinations.-When the seeded layer had hardened, porcelain penicylinders (Fischer Scientific) were placed on the agar surface. With all drugs, except chlortetracycline, six cylinders per plate were placed on the agar at 60" intervals with the center of the cylinder a t about a 3.5-cm. radius. A seventh cylinder was placed in the center of the plates which were used for low concentration solutions in order t o determine that the diluent itself was not bacteriostatic. A solution midpoint in the range of concentrations of those being tested was selected as the reference solution and was placed in three alternating cylinders on each plate. The other three cylinders were filled with a test solution of another potency, and the seventh cylinder in the center of the plate was filled with the solution diluent. Three plates were prepared for each concentration. The inhibition zones obtained in testing chlortetracycline ointments were large, and therefore, only four cylinders were placed on each plate used in chlortetracycline determinations. For the standard curve, alternate cylinders were filled with the reference solution and with another test solution, and four plates were prepared for each concentration. The plates were incubated at 34' for seventeen hours (B. ceyeus at 30") and the diameters of the zones of inhibition were read by placing the plate over an A 0 Spencer colony counter. These inhibition zones were corrected (2, 3) by referring t o the reference solution, and a standard curve was constructed by plotting the squares of the diameters of the inhibition zones (on the arithmetic scale) against the logarithms of the concentrations of the antibacterial drug (on the logarithmic scale) on semilogarithmic paper. The standard curve was drawn in such a way that the curve invariably went through the point representing the reference solution. Diffusion of Drugs from Ointments.-Six ointment bases: simple petrolatum ointment, simple silicone ointment, petrolatum absorption base, Eiljcone absorption base, petrolatum emulsion base,

ASSOCIATION

Vol. XLVI, NO. 12

and silicone emulsion base were prepared (6). Each of the medicinal agents was incorporated into each of these six ointment bases in the following concentrations: ammoniated mercury 595, chlortetracycline hydrochloride 3 %, oleandomycin phosphate 3%, salicylic acid 5y0, and sodium sulfacetamide 10%. Thus, 30 ointments were tested. The plates were prepared just as those for the standard curves. Three cylinders were placed a t 120" intervals on each plate and these cylinders were filled with the same reference solution which was used as the reference solution for the standard curve. For each plate three cylinders were filled with the test ointment and these cylinders were placed on the plates between the cylinders containing the reference solution. Three plates were prepared for each of the six ointment bases in testing the diffusion of a particular medicinal agent. Two cylinders containing a chlortetracycline ointment and two cylinders containing chlortetracycline reference solution were used on each plate for testing chlortetracycline ointments, and four plates were prepared for each ointment. The plates were incubated and the inhibition zones read as with the standard curve determinations. The mean of the inhibition zones of the 54 reference solution determinations was calculated and the mean of the three reference solution determinations for each plate was also calculated. The mean inhibition zone for the three ointments on each plate was corrected t o what it would have been had the mean inhibition zone of the reference solution on that plate been equal to the mean inhibition zone of the 54 determinations of the reference solution. The mean inhibition zone of the reference solution for all the ointment determinations usually varied slightly from the mean of the reference solutions in the standard curve determination. Therefore, a line was drawn through the point representing the reference solution of the ointment determinations and parallel to the standard curve. The concentrations of active medicinal agent which had diffused from the ointments could then be read directly from the standard curve (corrected for this particular group of data) by squaring the corrected diameters of the ointment inhibition zones and reading across the graph to the concentration. Standard curves for chlortetracycline were conducted with both Sfaph. aureus and B. cereus, and since the inhibition zones were more distinct in the B. cereus determination, this organism was used for the chlortetracycline ointment tests.

RESULTS The results of the standard curve determinations are shown graphically in Figs. 1-5. As is seen in these figures, a good straight line curve is obtained when the logarithms of the concentrations of the antibacterial drugs are plotted against the squares of the diameters of the zones of inhibition. This relationship was found t o hold true whether concentrations extending over only one cycle were needed for assay of ointments (salicylic acid ointments), or whether a concentration range of five cycles was needed (oleandomycin ointments). The results of quantitative determination of the diffusion of the five drugs (in rng./ml. obtained from standard curves) from each of the six ointment

719

SCIENTIFIC EDITION

December 1957 AMMONIATED MERCURY

CHLORTETRACYCLINE HCI I. 2. 3. 4. 5. 6.

20

E E

z - 15

: 0

10

SIMPLE PETROLATUM OINTMENT SIMPLE SILICONE OINTMENT PETROLATUM ABSORPTION BASE SILICONE ABSORPTION BASE PETROLATUM EMULSION BASE SILICONE EMULSION BASE

N

5

I 2 3 4 5 6

I 2 3 4 5 6

I 2 3 4 5 6

OLEANDOMYCIN PHOSPHATE

1-

1

DIFFUSION EOUIVALEN

IO.O\

.

n

I 2 3 4 5 6

SODIUM SULFACETAMIDE

SALICYLIC ACID

Ir.

8

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E

f6

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i

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0

w 24

0 N

I

I 2 3 4 5 6

-

15

-

I 2 3 4 5 6

EQUlVALENl

45 \

g 10-

€ 2

N 0

2

20

I

g30

5k 1 2 3 4 5 6

15

I 2 3 4 5 6

Fig. 6.-Results shown by the quantitative cylinder plate method for determination of drug diffusion from ointments compared with results shown by inhibition zone measurement alone. “Diffusion equivalent” refers to mg./ml. on the standard curve. Inhibition zones reported are from edge of cylinder t o edge of zone of inhibition.

bases is shown in Fig. 6 (right half of each section). For purpose of comparison the uncorrected inhibition zones (in mm. from edge of cylinder t o edge of inhibition zone) from the same data are also shown in Fig. 6 (left half of each section). The importance of the determination of logarithmic concentration-inhibition zone relationships in evaluation of diffusion of drugs from topical vehicles is apparent by comparing the inhibition zones produced by these 30 ointments with the actual quantity of medicinal agent diffused from the ointments. It may be seen that with salicylic acid and ammoniated mercury the inhibition zones as determined by the cylinderplate method gave an indication of the relative diffusion from the six ointment bases. However, with ammoniated mercury, the differences in inhibition zones were not proportional to the differences in ammoniated mercury actually diffused. With the other three medicinal agents, the results obtained by the cylinder-plate method and the actual determination of diffusion by reference to a standard curve are not even comparable. For example, the inhibition zone of oleandomycin in silicone emulsion base was only slightly greater than twice the zone produced by the drug in simple petrolatum ointment, but the amount of oleandomycin diffused from silicone emulsion base was 345 times the quantity diffused from the simple petrolatum ointment. Also, the inhibition zones produced by the different chlortetracycline ointments varied only from 11.6 mm. t o 14.8 mm., whereas the quantity of chlortetracycline diffused from these ointments varied from 17.5 to 600 rncg./rnl. Other comparisons of

the data of this investigation emphasize that only by use of a standard curve will inhibition zones show a valid evaluation of drug diffusion.

DISCUSSION The usual method for the assay of antibiotics (2, 3) employs the diameters of the zones of inhibition instead of the squares of the diameters. This procedure yields good results since the range of the concentrations is relatively small. In this study, when the diameters of the zones of inhibition were plotted against the logarithms of the concentrations, a good straight line curve did not result if the concentrations were extended through a wide range. With salicylic acid i t was necessary t o extend the concentrations through only one cycle in order to produce inhibition zones both greater than and less than those produced by the salicylic acid ointments, and a fairly straight line was obtained when the diameters of the inhibition zones were plotted against the logarithms of the salicylic acid concentrations. With the other four drugs, whose concentrations extended over three to five logarithmic cycles, the standard curves were decidedly curved when the data were plotted in this manner (using diameters rather than diameters squared). It should be pointed out that use of a reference solution on each plate is critically necessary for accurate determinations since certain important factors, principally temperature variables, cannot be absolutely controlled. The importance of the reference solution to the accuracy of agar plate methods

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is evidenced by examination of the data for oleandomycin diffusion from petrolatum emulsion base and from silicone emulsion base. While there was no difference in the uncorrected inhibition zones produced by the two ointments, after correction the inhibition zone of oleandomycin in silicone eiuulsion base was found to be 0.75 mm. greater than that of the drug in petrolatum emulsion base. Since these zones were large (about 31 mm.), this small difference (0.75 mm.) accounted for a difference in antibiotic diffusion of 3.1 uig./ml., or an increase of about 50T0 (compare standard curve with the data in Fig. 6). In order to introduce practical problems involving the diffusion of insoluble or relatively insoluble drugs from ointments, ammoniated mercury and salicylic acid were included in this study. Inasmuch as ammoniated mercury is insoluble in water and no satisfactory agent was found to solubilize the drug, mercury bichloride was selected for the determination of the standard curve, and concentrations were calculated on a basis of mercury content. It was felt that this liberty in using mercury bichloride t o evaluate diffusion of ammoniated mercury was justified because it is the mercury ion which is responsible for the activity of ammoniated mercury. Salicylic acid is not sufficiently soluble in water t o produce solutions of the strength necessary to construct a standard curve which will extend over the range of the inhibition zones produced by the salicylic acid ointments. Twenty per cent alcohol v/v was selected as the solvent for this drug after it was determined that Staph. azireus developed colonies beneath solutions containing alcohol in 40% v/v concentrations or less. Since it was thought that 20% alcohol might potentiate the activity of salicylic acid, inhibition zones produced by an aqueous solution of salicylic acid, 2 mg./ml., were compared

statistically with the zones produced by the acid in 20% alcohol. The alcoholic solutions produced slightly larger zones than did the aqueous solutions; however, the difference was not significant (P = 0.054). Preliminary determinations with mercury bichloride and ammoniated mercury were conducted with stainless steel penicylinders. However, the results on subsequent determinations became erratic, and these unusual inhibition zones were found to be due to mercury which had been adsorbed on the cylinders. Usual cleaning processes did not decontaminate the cylinders, and porcelain cylinders were used thcreafter. CONCLUSIONS Standard curves are neither too difficult nor too time consuming for use in routine ointment diffusion tests. T h e fallacy i n comparing diffusion from ointments o n a basis of inhibition zones alone has been pointed out, and it is recommended that the standard curve method be adopted to test t h e diffusion of antibacterial drugs from ointment bases. REFERENCES (1) Ruehle, G. L. A., and Brewer, C. M. “Circular No. 198,” United States Department of Agriculiure (December iaxij ----,. (2) Grove! Donald C., and Randall, W;,A.. “Assay hfethods of Antibiotics, A Laboratory Manual, Medical Encyclopedia, Inc , New York (1955). (3) “B. M y v i d e s Cup Plate Method for Determination of Chlortetracycline, T e t r a c y c h e and Oxytetracycline Concentrations in Serum and Body Fluids,” Copy, Department of Health Washington 25 D. C. (4) Striuss. E., Dingle: J. H., and Finland, M., J. Immunology. 42, 331(1941). 43, (5) Naimark, G. M.,and White, L., THISJOURNAL, 7 ( 1954). ( 6 ) Plein. J. B., and Plein, E. M . . THIS JOURNAL, 46. 705 (1957).

Paper Chromatographic Evaluation of Rauwolfia Species* By B. P. KORZUN, A. F. ST. ANDRE,t and P. R. ULSHAFER A method for the assay of Rauwolfia root material by paper chromatography is presented. A number of Rauwolfia species are evaluated for reserpine content and for the distribution of other alkaloids. HE INTEREST AROUSED b y the unique bioTlogical activity of reserpine, a n alkaloid first isolated from the Indian R. serpentina ( l ) , ~~

* Received April 23, 1057, from the Research Department, CIBA Pharmaceutical Products Inc., Summit, N. J . t Deceased October 24, 1955.

has lead to a n extensive investigation of the alkaloid content of other Rauwolfia species. Previous investigators have used partition chromatography o n paper (2-4), and we have found this procedure, with the extensions and modifications outlined in this paper, to be of invaluable aid in the examination of t h e alkaloids present in the many root samples available to us. B y a visual comparison of the chromatograms of root extracts with those produced by known concentra-