Chemical Assay of Crystalline Penicillins DOROTHY J. HISCOX Laboratory of Hygiene, Department of National Health and Welfare, Ottawa, Canada
A method for the chemical assay of crystalline penicillins uses potassium ferricyanide as an oxidizing agent and ceric sulfate as a volumetric standard. The method is rapid and sensitive and may be applied to procaine penicillins.
D
URING investigations undertaken in this laboratory into chemical methods of analyzing penicillins for total penicillin activity, the use of potassium ferricyanide and ceric sulfate was initiated because it seemed that such a method would possess several advantages: rapidity, sensitivity, and the use of only one standard solution which is stable and may be used for long periods without restandardization. The sensitivity of the method is proved by the fact that 1 mg. of penicillin requires 5.25 ml. of standard ceric sulfate for its titration, while by the iodometric method ( 1 ) the same weight requires only 2.52 ml. of standard sodium thiosulfate. REAGENTS REQUIRED
Potassium ferricyanide Solution A, 15 grams per liter. Solution B, 15 grams of potassium ferrocyanide and 40 grams of sodium hydroxide per liter. Sulfuric acid, 10 N solution (approximate). Ceric sulfate, 0.01 N solution. Setopaline, 0.1%solution. PROCEDURE
To a 1- to 5-ml. aliquot containing 0.75 to 1.25 mg. of penicillin in a 25 X 200 mm. test tube add 5 ml. of potassium ferricyanide Solution A. To another similar aliquot add 5 ml. of Solution B (not more than one week old). Heat both aliquots in boiling water for 15 minutes. Cool in running water, add 5 ml. of 10 N sulfuric acid, and titrate with 0.01 iV ceric sulfate, usingsetopaline as indicator. Run blank determinations on the ferricyanide solutions daily and subtract these from the penicillin titrations t o determine the net titrations. Net titration B minus net titration A measures the amount of penicillin present in the aliquot. Boiling time is not critical and may be extended to 20 minutes. Titrations need not be carried out immediately. Standing for 1 hour has not affected the titration of samples. For reference a regression line was established using the above rocedure. As most crystalline penicillins are largely penicillin crystalline sodium penicillin G in amounts varying from 0.10 to 2.25 mg. was used as standard. Titrations varied from 0.40 to 12.25 ml. Both water and a 1% phosphate buffer a t pH 6.0 were used as solvents for penicillin. Using 113 points determined over a period of 2 months, the following equation was established
8,
Y
= 0.0489
+
Y = 0.0054 0.145615X,the correlation coefficient was +0.9966 and the standard error was +0.05 mg. Later, determinations were made with a sample of procaine. These points fell as close to the line as most of those used in its determination; so the line was not changed. Taking the molecular weight of penicillin G as 334 and procaine as 236, the proportion by weight of penicillin to procaine in 1 mg. of procaine penicillin must be 0.586 t o 0.414. The penicillin results used to determine the regression equation m r e modified by multiplying the titrations by 0.586; the titrations of the procaine regression were multiplied by 0.414. New lines using these values were established. In Figure 1 are plotted the original and the corrected lines. I t is readily seen that, for values from 0.35 to 0.60 mg. of penicillin or procaine, there is little differ,
+ 0.180996X
where X represents milliliters of ceric sulfate used and Y milligrams of penicillin. The correlation coefficient of this equation was +0.9975 and the standard error of prediction *0.04 mg.
/
Results of the analysis of a number of samples of penicillin by the ferricyanide method are compared with results by the iodometric method and bioassay in Table I. Aliquots containing 0.75 to 1.25 mg. of penicillin were used for the ferricyanide method and aliquots containing 2.5 to 3.0 mg. for the iodometric method. Results by the ferricyanide method approximate those of the bioassay more closely than do results of the iodometric method.
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APPLICATION TO PROCAINE PENICILLINS
When the ferricyanide method was used to determine the potency of procaine penicillins, results were approximately double those of the bioassay. To determine the validity of halving the results, a procaine regression line was established using the method outlined above with amounts of procaine hydrochloride varying from 0.25 to 2.5 mg. The factor 0.8663 was used to determine the procaine content. The equation in this case was
9
10
il
TITRATION, ML. OF CERIC SULFATE
Figure 1. Regression Lines for Ferricyanide Determination of Penicillin and Procaine A . Penicillin B . Procaine
C. Penicillin titrations X 0.586 D . Procaine titrations X 0.414
V O L U M E 21, NO. 6, J U N E 1 9 4 9
659
Table 11. Assay of Procaine Penicillin Sample 43,165 43,331 43,298 43,300 43,321 43,322 43,323 43,329 43,163 43,1P,4
(Results in units per milligram) Bioassay Iodometric 99 3 444 548 1060 942 1015 948 1000 970 1030 970 1040 942 1035 942 1077 522 942 410 839 Mean 1003 764
Ferricyanide 902 872 877 1081 1017 904 964 1028 1053 959 966
ence between the corrected lines. Thus, by the ferricyanide method, it is permissible to determine procaine penicillin as penicillin and divide the results by 2 if an aliquot containing approsimately 1 mg. of procaine penicillin is used. Results of the analysis of procaine penicillin by this method are compared in Table I1 nith results by the bioassay and iodometric methods. The mean difference between the bioassay and ferricyanide methods is 37 units, and between the bioassay arid iodometric methods it is 239 units. I t is obvious that the ferricyanide method can be used for the analysis of procaine penicillins only while they retain their full potency. If a loss in potency is suspected, the compound should be analyzed before and after the addition of penicillinase. The penicillinase prepared in this laboratory ( 2 ) does not interfere with the determination nor affect procaine. Although penicillinase destroys completely the biological activity of penicillin, it does not destroy all its chemical activity as measured by the
ferricyanide method. The loss in activity varies with the conditions under which the penicillinase acts but is relatively constant under given conditions. Once this loss has been determined for the given conditions, the difference in the ferricyanide assay before and after the action of penicillinase will give a good estimate of the potency. CONCLUSIOhS
The ferricyanide method possesses several advantages. I t is rapid; determinations require less than 30 minutes. I t is sensitive, using less than 1 mg. of penicillin per determination. I t is simple and needs only a minimum of standard equipment. The number of standard reagents necessary is reduced to one which is stable. The chief disadvantage of the method is that it may be applied only to crystalline penicillin. Many penicillin preparations contain substances that interfere with the procedure. 4CKIOWLEDGMENTS
Gifts of crystalline sodium penicillin G were received from Merck & Company of Montreal, and Ayerst, McKenna and Harrison of Montreal. Bioassays were carried out under the direction of Ruth Thomas. Frances Connell made the iodometric determinations. LITERATURE CITED
(1) Alicino, J. F., IXD. EXG.CHEM.,ANAL.ED.,18, 619 (1946). (2) Morgan, J. F., and Campbell, M. E., J . Biol. Chem., 169, 465
(1947).
RECEIVED J u l y 16. 1948.
Penicillin in Broths and Finished Products Chemical Method f o r Estimation of Types KIYOSHI HIGUCHI
AND
W. I€. PETERSON
University os Wisconsin, Madison, Wis.
T
HE multiplicity of penicillin types has made necessary a method that permits estimation of the individual penicillins present in mixtures. Various techniques have been developed for this purpose. Applications have been made of ultraviolet and infrared spectroscopy ( 1 , 10, 11), adsorption chromatograms (B),parition procedures involving paper strip (8, 9, as),as well as packed column chromatography ( 7 , 18) and countercurrent distribution systems ( 4 , 1 5 ) . Other methods, such as the chemical precipitation of penicillin G as the A7-ethylpiperidine derivative ( 1 4 , PO) and niicrobiological differential assays, have been useful (12, 19). Each method has certain limitations, such as a requirement of rather pure sample, applicability to only a single penicillin, or lack of clear-cut distinction of types. I n the development of the present method advantage was taken of the fact that the various penicillins are characterized by the so-called “ f l groups,” which are in the form of carboxylic acids joined by amide linkages to the penicillin molecule (3). The acids were liberated by boiling in 5 S alkali and estracted into benzene. The individual R acids were separated and identified by the use of partition chromatograms of the type described by Peterson and Johnson (17). The procedure has been used in the determination of penicillins G, I
