ANALYTICAL CHEMISTRY
1490 I t s sensitivity permits the determination of quantities down to 0.5 mg. with a low limit of 10 micrograms per milliliter, when a sample aliquot of 50 ml. is used. The method shows good precision and its accuracy compares favorably with conventional procedures. ACKNOWLEDGMENT
The authors are indebted to E. G. E. Shafer and Norbert Steiger for their helpful suggestions, and to James Brydon for his assistance. J. T. Woods, Calco Chemical Division, American Cyanamid Co., kindly provided a number of diazonium salts, while technical 4methoxy-2-nitroaniline was furnished by .\ntara Chemicals, Division of General Dyestuff Corp. LITERATURE CITED (1) Bessey, 0. A,, J . Bid. Chem., 126, 771 (1938).
(2) Gyorgy, Paul, “Vitamin Nethods,” Vols. I and 11, New York, Academic Press, 1950. (3) Lange, N. A., and Forker, G. %I., “Handbook of Chemistry,” 6th ed., Appendix, p. 27, Sandusky, Ohio, Handbook Publishers, 1946. ( 4 ) “Pharmacopeia of the United States,” p. 686, XIV rev., 1960. (5) Ponting, J. D., IND. ENG.CHEX, ANAL.ED., 15, 389 (1943). (6) Robinson, W. B., and Stots, E., J . Bid. Chem., 160, 217 ( I W 6 ) . (7) Roe, J. H., and Kuether. C . A , , Ibid., 147, 399 (1943). (8) Roe, J. H., and Kuether, (’, .\., Scicnce, 95, 77 11942). (9) Rubin, S.H., Jahns, F. W., a n d Haue~rifeind,J. C., Fruii P r o d ucts J., 24,327 (1945). (10) Scudi, J. V., and Ratish, B . D , , 1 x 0 . EKG.CHEM.,AN.\I.. En., 10,420 (1938). (11) Ibid., 11, 98 (1939). (12) Stewart, A. P., Jr., and Sharp, P. F.,Ihi‘d., 17, 373 (1946) (13) Weidenhagen, R., and Wegner, H., Ber., 72, 2010 (1939). RECEIVED for review February 17, 1953. .iccepted July 25, 1953. l’resented at the hleeting-in-hliniature of the North .Jersey Section, . 4 ~ l E R I c A x CHEMICAL SOCIETY, January 26. 1953.
Determination of Penicillin G in Penicillin 0 J. L. JOHNSON, W. A. STRUCK, E. J. SCOTT, AND J. E. STAFFORD The Upjohn Co., Kalamazoo, Mich.
This investigation was undertaken to devise a method for determining the benzylpenicillin (penicillin G) cantent of allylmercaptomethylpenicillin (penicillin 0). Penicillin 0 is useful in the treatment of patients who are sensitive to penicillin G; therefore, an accurate determination of small amounts (< 1%) of penicillin G that occur in penicillin 0 is important. Oxidation of penicillin G with alkaline permanganate yields benzoic acid which is determined spectrophotometrically. Interfering oxidation products, which derive predominantly from penicillin 0, are removed by simple extractions. Studies of known added increments of penicillin G have shown a recovery of 86 i 4%. The method is directly applicable to potassium penicillin 0, and a preliminary extraction of the organic base makes it applicable to the procaine or 2-chloroprocaine salt. The method has been found to be the most generally applicable and sensitive for determining small amounts of penicillin G in penicillin mixtures.
A
LLYLMERCAPTOMETHYLPENICILLIN (3), penicillin 0, has therapeutic activity comparable to benzylpenicillin, penicillin G, but elicits allergic manifestations in a much lower percentage of patients (1). In producing penicillin 0, the formation of small amounts of penicillin G has not been prevented because of the penicillin G precursors inherent in the fermentation medium and in the metabolic products of the mold by which it is produced. For these reasons, a sensitive method for measuring the penicillin G content is essential.
R-CONH-CH-CH”
I
OH
S
t
\
CHa $CHs
C-N+H-COOH Penicillin
Penicillin 0, R = CHF=CH-CH*-S-CH~Penicillin G, R =
O--CH~-
The determination of penicillin G as a major constituent has received considerable attention (2, 6, 8, 9, 13-15, 18). The techniques represented by there methods are gravimetry and infrared and ultraviolet spectrophotometry. These approaches lack the sensitivity necessary for the determination of penicillin G in mixtures in which penirillin 0 predominates. As a minor constituent, penicillin G has been measured by a colorimetric method which is an adaptation (4, 5. 10, 16) of the Kapeller-Adler reaction for phenylalanine (11). I n the mixtures under consideration here, this method was not easily made reproducible, especially in the hands of different technicians. Del Vecchio and Argenziano ( 7 ) used permanganate oxidation and nitration to measure penicillin G colorimetrically. Their oxidation was carried out under acid conditions which undoubtedly incurred a serious loss of benzoic acid through oxidation. This has also been observed by Leman and Montaigne (1.2). Philpotts, Thain, and Twigg (17) reported the use of permanganate oxidation and a spectrophotometric measurement of benzoic acid based on its absorption maxima a t 273 and 281 mp. T o achieve the sensitivity required for determining the small amounts of penicillin G in penicillin 0 (0.1 to 0.5%) and to avoid the interferences that penicillin 0 introduces, the basic method of Philpotts, Thain, and Twigg has been modified extensively. The benzoic acid is extracted from the ultraviolet absorbing materials resulting from oxidation of penicillin 0 and determined by means of its absorption maximum a t 224 mp (Figure 1). The use of this maximum gives a fifteenfold greater sensitivity than the maxima in the 270- to 280-mr region. REAGENTS
All chemicals are reagent grade Potassium hydroxide, 2.5% w./v. solution Potassium permanganate, crystals and 4.5% n-./v. solution Oxalic acid dihydrate, 10% w./v. solution Sulfuric acid, 18 N Sodium chloride, finely powdered crystals Chloroform Ammonium hydroxide, 0.1 N and concentrated PROCEDURES
Potassium Penicillin 0. A 300-mg. sample of potassium penicillin 0 is weighed into a200-ml. round-bottomedflaskfitted with a reflux condenser. One milliliter of 2.5% potassium hydroxide
V O L U M E 25, NO. 10, O C T O B E R 1 9 5 3 solution, 50 ml. of 4.5Oh potassium permanganate solition, and several glass beads or platinum tetrahedra are added, and the mi\ture is heated under reflux for 1 hour. The condenser is rinsed with a small amount of water and removed. After cooling the solution to room temperature with t a p water, 25 ml. of the 10% oxalic acid solution are added. When the initial action has subsided, 15 ml. of 18 N sulfuric acid are added in portions with continual agitation and cooling, so that the temperature of the solution does not rise above 25’ C. a t any time during the acidification. The reduction may be hastened by using a stirring rod flxttened on one end to crush any lumps of manganese diovitlr
1491 The solution is cooled and 2.5 grams of potassium permanganate are added slowly and the mixture is heated under reflux for 1 hour. With one exception, the use of 30 ml. of the 10% oxalic acid solution instead of 25 ml. for the reduction of t h e manganese dioxide, the procedure from this point on is identical n-ith the procedure for potassiuni penicillin 0. CALCUL4TIONS
The percentage of peiiirillin G in a sample is calculated according t o the equations below. Potassium penicillin G. %
=
.3.05 X X X 50 X 100 -- ___
Procaine penicillin G , %
=
4.82 X X X 50 X 100 x t . of sample, mg.
2-Chloroprocaine penicilliii G,
x =
R
x
-
kB.w - k 8 . m
wt. of sample, mg.
x x x 50 x 100 yo = 5.10 wt. of sample, mg.
= concentration of benzoic acid
in
sample solution, mg./ml.
whcre
Ax
~ B , x
R = ‘210 220
230 240 250 260 270 280 290 300
absorbance of sample solution a t the specified wave length = absorbance index of U.8.1’. benzoic acid a t the specified wave length
=
absorbance of blank at 2 2 3 m p (measured against 0.1 V . absorbance of blank at 224 mp ammonium hydroxide)
WAVE LENGTH IN MILLIMICRONS
Figure 1. Ultraviolet Absorption Spectrum of Benzoic Acid in 0.1 N Ammonium Hydroxide Car, recording spectrophotometer
Khen a clear, colorless solution is obtained, it is transierred quantitatively t o a 250-ml. separatory funnel. The common proprietary stopcock lubricants should not be used because they give ultraviolet absorption which interferes with the subsequent determination of benzoic acid. Glass plugs lubricated with white petrolatum or unlubricated Teflon plugs are satisfactory. Thirty grams of sodium chloride are dissolved in the reaction misture, and it is extracted successively with three portions of chloroform-30, 20, and 10 ml., respectively-which are filtered through a glass wool pledget in the funnel stem and collected in a second separatory funnel. Exactly 50 ml. of 0.1 S ammonium hydroxide are added to the combined chloroform extracts, and the funnel is shaken vigorously for 5 minutes. The chloroform layer is discarded, and the ammonium hydroxide solution is filtered through glass ~ o o placed l in the funnel stem into a glassstoppered container. The absorbances of the ammoniacal solution are measured a t 220 and 224 mp using a Cary recording spectrophotometer or a Beckman llodel DU instrument. These measurements are referred t o a blank prepared by equilibrating 0.1 iV ammonium hydroxide with chloroform as in the final extraction of the sample. Both the sample and blank solutions should be kept in contact with a few drops of chloroform in the containers until transferred to the cuvette. Even this precaution does not assure identical chloroform concentrations in the solutions. I n recognition of this, the system is treated as a two-component mixture in calculating the penicillin G content of the sample. Procaine and 2-Chloroprocaine Penicillin 0. -4500-mg. sample of procaine penicillin 0 monohydrate or 2-chloroprocainepenicillin 0 monohydrate is weighed into a 250-ml. separatory funnel. This sample size is approximately equivalent t o 300 mg. of the potassium snlt. The material is dissolved in 50 ml. of chloroform, and the solution is extracted with 50 ml. of ammonium hydroxide (1part of concentrated ammonium hydroxi le and 4 parts of water). The chloroform layer is discarded and the ammoniacal solution is filtered through a pledget of glass wool in the funnel stem and collected in a 250-ml. round-bottomed flask. The funnel is rinsed with 20 ml. of water which is added to the contents of the flask, Three milliliters of 2.5% potassium hydroside solution and several glass beads are added to the solution, and i t is boiled vigorously until the volume is reduced by about 20 ml. Very active boiling is essential in order to remove dissolved chloroform and other volatile components that would consume large and variable amounts of permanganate during the oxidation.
DISCUSSION A h l ) RESULTS
The important advantages of thc, method are its sensitivity and reproducibility. h typical 300-mg. sample of potassium penicillin 0 containing 0.4% of potassium penicillin G, when carried through the procedure, gives 0.4 mg. of benzoic acid in the final 50 ml. of ammoniacal solution. The absorbance of this solution at
Table I. Major Component
Reproducibility of Results
Sample
Potassium penicillin 0
1
2
3 4 5
6 7
Procaine penicillin 0 monohydrate
2-Chloroprocaine penicillin 0 monohydrate
8 9 10 11
12 13 14 15 16
17
Lab. A Lab. B Lab. C Potassium Penicillin G Found, % 0.44 0.52 0.44 0.80 0.47 0.46 0.48 0.40 0.44 0.42 0.39 0.40 0.43 0.21 0.24 0.19 0.21 0.21 0.21 0.22 0.23 0.67 0.76 0.71 0.76 0.30 0.30 0.28 0.32 0.46 0.48 0.42 0.58 0.60 Procaine Penicillin G Monohydrate Found, % 0.40 0.45 0.98 1.08 0.57 0.59 0.74 0 73 2-Chloroprocaine Penicillin G Monohydrate Found, % 1.13 1.07 1.53 1.64 0.90 0.87 0.38 0.34 0.36 0 40 0.50 0.45 0.46 0.48 0.44 0.21 0 20
ANALYTICAL CHEMISTRY
1492 224 mp is about 0.5. This order of sensitivity is superior to that of any method previously described for determining penicillin G as a minor component. The reproducibility of the method was established by technicians working independently in separate laboratories. Table I lists the results obtained for samples of potassium, procaine, and 2-chloroprocaine penicillin 0. The use of ammonium hydroxide as the base for extracting benzoic acid from the chloroform contributes greatly to the reproducibility of the method. Stronger bases such as sodium or potassium hydroxide absorb carbon dioxide from the air, and the resulting carbonates contribute variable absorptions a t 220 and 224 mp. The accuracy and validity of the method were established by studying the recoveries obtained (Table 11) when known increments of potassium penicillin G were added to a base sample of potassium penicillin 0.
Recovery, % = 100
x
total potassium penicillin G found, potassium penicillin G originally present, % potassium penicillin G added, 7 0
loss must occur during the oxidation of the penicillins and/or the reduction of excess permanganate and manganese dioxide. When the oxidation mixture was acidified before adding oxalic acid, a serious and variable destruction of benzoic acid resulted from the action of the acidified excess permanganate. In the procedure, this loss is avoided by adding the oxalic acid before the sulfuric acid.
Table 111. Recovery of 2-Chloroprocaine Penicillin 0 2-Chloroprocaine Penicillin G Added, % Xone
0.42 0.67 0.52
2-Chloroprocaine Penicillin G Found, % 0 21
n
Recovery. %
22
0.53
0 54 0 74 0 64
76 78 81
The r f f e c t s of oxidation time and of permanganate concentration on the recovery have been examined by adding benzoic acid to potassium penicillin 0. Recoveries of 95 to 100% were obtained by shortening the oxidation time to 40 minutes and reducing the amount of permanganate to 42 ml. However, the recovery dropped back to the usual 86% when penicillin G was added to penicillin 0 and oxidized under these same conditions. This suggested that the greater part of the loss occurs during the formation of the benzoic acid rather than as a result of its destruction. Accordingly, an adaptation of the procedure was applied to phenylacetamide and phenylacetic acid. The recoveries in these experiments were also in the 86% range. I t has been concluded, therefore, that the more important factor in reducing recoveries is the mode of attack of alkaline permanganate on the methylene group of the phenylacetic acid moiety, ACKNOWLEDG.RZEFT
00
1
,
-0.1
, 0
,
0.1
,
,
0.2 03
,
,
,
,
,
0.4
0.5
0.6
07
08
I
as
Y, POTASSIUM PENICILLIN G ADDED
Figure 2.
The authors wish to express appreciation to 0. R. Woods, J. H. Ford, C. L. Graham, G. c‘. Prescott, and W. Bradbury for their cooperation, suggestions, and stimulating interest in this
Recovery of Added Potassium Penicillin G LITERATURE CITED
Figure 2 shows the results of this study plotted with per cent found aa abscissa and per cent added as ordinate. This curve, of slope 0.86, showed that the operating recovery is 86 f 4%. Results may be corrected to absolute on this basis, but in absolute units the error is small. Similar data on the recovery of known increments of 2-chloroprocaine penicillin 0 are recorded in Table 111; in this case the operating recovery is 78 i 4%. Attempts have been made to achieve quantitative recoveries of penicillin G as benzoic acid. When benzoic acid was added to the oxidation mixture just prior to extraction it was repeatedly recovered completely. It was then apparent that the 10 to 15%
Table 11. Recovery of Added Potassium Penicillin G Potassium Penicillin G Added, % None 0.21 0.35 0.40 0.47 0.73 None 0.34 0.51 0.69
Potassium Penicillin G Found, % ’ Lab. A 0.21 0.39 0.51 0.55 0.61 0.85 Lab. B 0.24 0.54 0.67 0.83
Recovery, %
86 86 85 85 88
88 84 86
(1) .4dair, C. V., Woodin, W. G.. Bunn, P. A, and Canarili, L., Am. J . Med., 11, 188 (1951). (2) Barnes, R. B., Gore, R. C., Williams, E. F., Linsley, S. G., and Peterson, E. IT.,AKAL.CHEM.,19, 620 (1947). (3) Behrens, 0. K., Cerse, J., Edwards, J. P., Garrison, L., Jones,
R. G., Soper, Q. F., Van Abeele, F. R., and Whitehead, C. W., J . B i d . Chem., 175, 793 (1948). (4) Boxer, G. E., and Everett, P. hI., ANAL.CHEM.,21, 670 (1949) (5) Canback, T., Farm. Revy., 46, 97 (1947). (6) Colon, A. A., Herpick, G. E., Neuss, J. D., and Frediani, H. A , ,
J . Am. P h a r m . Assoc., Sci. Ed., 38, 138 (1949). (7) Del Vecchio, G., and Argenaiano, R., Boll. soc. ital. biol. sper., 22, 1190 (1946). (81 Gottlieb. K. R.. D a n s k Tidsskr. Farm.. 26. 1 (1952). (9j Grenfell,~T. C.,’Means,J. A., and Brok,’E. $., J . Bid. Chem., 170, 527 (1947). (10) Hiscox, D. J., ANAL.CHEM.,22, 723 (1950). (11) Kapeller-Adler, R., Biochem. Z.,252, 185 (1932). (12) Leman, A., and Montaigne, M. T., Compt. rend., 231, 412 (1950). (13) Levy, G. B., Fergus, D., and Caldas, J. M., ANAL.CHEM.,21, 664 (1949). (14) Levy, G. B., Shaw, D., Parkinson, E. S., and Fergus, D., Ibid., 20, 1159 (1948). (15) Mader, W. J., and Buck, R. R., Ibid,, 20, 284 (1948). (16) Page, J. E., and Robinson, F. A , , A’ature, 158, 910 (1946). (17) Philpotts, A. R., Thain, W., and Twigg, G. H., Ibid., 159, 839 (1947). (18) Sheehan, J. C., Mader, W. J., and Cram, D. J., J . Am. Chem. Soc., 68, 2407 (1946). R E C ~ I V Efor D review March 11, 1953. .4ccepted July 30. 1953.
