Infrared Assay of Procaine Salt of Benzylpenicillin N. H. COY, C. W. SABO, A N D B . T. KEELER E. R . Squibb and Sons, N e w B r i m s w i c k , N . J . An infrared method of assay of the procaine salt of benzylpenicillin is based on the measurement of the depth of the /3-lactam band of penicillin at 5.6 p and is performed with fixed cells using chloroform solutions. The method has been applied to a variety of preparations containing procaine penicillin. Comparison of results indicates good agreement with the biological assay.
T
Fourteen plant samples of the procaine salt of penicillin were measured as indicated above. Table I1 lists the results of these tests along with the biological values and calculated conversion factors. The test was further applied to miscellaneous samples of the procaine salt of benzylpenicillin, including aqueous suspensions and also mixtures in oil gelled with aluminum stearate. In the case of the aqueous suspension, the water was first removed by
HE infrared spectra of the sodium salt of benzylpenicillin and of its degradation products have been investigated by several workers in studies on the structure of the penicillin molecule (3). Barnes et al. (1) have published these spectra along with those of the crystalline sodium salts of other types of penicillin and have developed a method for the assay of the crystalline sodium salt of benzvlpenicillin making use of the characteristic band a t 14.2 p. I t was found in these investigations that all the different types of penicillin possessed a characteristic band a t 5.6 p which was not present in any degradation product of penicillin. This band v a s attributed to the presence in the penicillin molecule of the P-lactam carbonyl group. The introduction of the procaine salt of benzylpenicillin has led to an attempt to assay this salt by infrared methods. The fact that chloroform was transparent to infrared rays in the region 5.0 to 5.2 p (4, coupled with the fact that the procaine salt of benzylpenicillin was sufficiently soluble in chloroform to give a concentration adequate to produce a strong absorption band a t 5.6 p, indicated a possible method of assay of this salt. The procaine moiety of the penicillin salt does not interfere with the strength of the 5.6 p band.
Table 11.
1 2 3 4 5 6 7 8 9 10 12 13 14
Optical Density 0.300
0.155 0.180 0.240 0.315
1010
1032 929 936 988 977 985
Sample Procaine salt of benaylpenicillin with aluminum stearate Procaine salts of benzylpenicillin with peanut oil containing 2% aluminum stearate 1
1. 2.
2 3 4 5 6 7 8
Aqueous suspensions of procaine salts of benzylpenicillin Sample 1 Initial potency 2 - 5 O C. 1-month storage 37O C. 1-month storage Sample 2 Initial potency 2-5O C. 3-month storage 37O C. 3-month storage Sample 3 Initial potency 2-5" C. 1-month storage 2 4 O C. 1-month storage 37' C. 1-month storage
3.
Reproducibility of Measurement of a Single Sample
0,393 0.198 0,234 0.312 0.410
923 958 1000 989 992 989 997 1009 994 999 983 982 998 989
963 978 999 985 998 991 1003 1010 1013 964 960 985 988 987
Conversion Factor 1270 1290 1300
1300
1330 1290 1300 1310 1320 1270 1260 1300 1280 1280 conversion factor = 1290 * 2%
Table 111. Comparison of Values Obtained by Infrared Method with Biological Assays of Various Samples and Preparations
T o test the validity of Beer's law, concentrations ranging from 0.2 to 1.0% were measured. There is a straight-line relationship beh-een optical density and concentration over a range of concentrations from 0.2 to 0.8%. Results shoiying the reproducibility of measurements of any one sample are given in Table I. Each density reading is an average of two aliquots of each concentration. For accuracy the concentrations tested "ere such as to give density readings in the range 0.15 to 0.40.
Concentration, %
1003 999 997 981 1005 992 1009
Average
The instrument used was a Perkin-Elmer infrared spectrometer hfodel 12B. .4fixed cell holder as supplied by the Perkin-Elmer Corporation was fitted with sodium chloride plates and polyethylene spacers and gaskets. The cell was assembled as suggested by Carol et al. ( 2 ) ; the cell thickness was approximately 0.5 mm. The sample solution was prepared by weighing accurately a 20to 40-mg. sample of the procaine salt of benzylpenicillin and dissolving it in chloroform, using a IO-ml. volumetric flask. The cell was then filled n i t h chloroform, the wave length drum of the instrument n a s set a t the position of maximum absorption of the 5.6 p band, and the slit waq found for full-scale deflection, usingthe glass shutter for the density reading of infinity. The gain of the amplifier !vas kept constant. The chloroform in the cell TT as then replaced bv an aliquot of the procaine penicillin solution in chloroform and the density value read directly from the chart.
Optical Density Concn., %
Biological Test, Units/Ma. Lab, Lab, I1 Av. I
0.76 0.76 0.77 0.78 0.75 0.77 0.77 0.77 0.77 0.76 0.76 0.76 0.77 0.77
11
EXPERIWENTAL PROCEDURE AND RESULTS
Table I.
Derivation of Conversion Factor
Infrared Test Optical Densit$]
1
0.77 0.78 0.77 0.77 0.77
a
669
k X 1290 (conversion factor)
Infrared Testa, p/m.
Biological Test,
880
870
p/ml. 320,000 320,000 314,000 329,000 333,000 341,000 340,000 331,000
332,000 318,000 322,000 334,000 313,000 351.000 332,000 338,000
c/m.
Difference, 70
+1
c/mL -4
+1 -2
-1
+6 -3
?;
308,000 321,000 240,000
218,000
+ 10
303,000 321,000 93,000
127,000 306,000
297,000 297,000 286,000
291,000 286,000
-27
A N A L Y T I C A L CHEMISTRY
670
spreading the sample on a watch glass and drying in a vacuum desiccator. For samples containing oil, those compounds which interfered with the infrared absorption a t 5.6 p were removed by treating the sample with petroleum ether and centrifuging. The solvent was decanted and the residue taken up in chloroform and tested. The results of such tests are listed in Table 111. DISCUSSION OF RESULTS
Table I1 shows that the value of the physical constant, k , is the same, within the error of the method, for all the samples tested. This does not imply a lack of sensitivity on the part of the infrared test as, theoretically, the potency of all the samples listed should be the same. The density readings obtained with any cell vary with the thickness of the liquid in the cell; hence the values of k depend on the cell used and should be established whenever a cell is assembled. Table I11 establishes the fact that a variety of samples of the procaine salt of benzylpenicillin, includ-
ing certain samples that have broken down on storage, can be tested with accuracy by the infrared method. ACKNOWLEDGMENT
The authors are indebted to R. Blue, Quality Control Division, E. R. Squibb & Sons, for the biological data. The interest and helpful suggestions of R. B. McCormack, Biochemical Development Division, E. R. Squibb & Sons, are gratefully acknowledged. LITERATURE CITED
(1) Barnes, R. B., Gore, R. C., Williams, E. F., Linsley, S. G., and , Petersen, E. M., ANAL.CHEX.,19, 620-7 (1947). (2) Carol, J., Molitor, J. C., and Haenni, E. O., J . Am. Pharm. Assoc.,
37, 173-9
(1948).
(3)
Editorial Board, Monograph on Chemistry of Penicillin, Science,
(4)
Torkington, P., and Thompson, H. W., Trans. Faraday SOC.,41,
105, 653-9 (1947). 184-6 (1945).
RECEIVED October 19,1948.
Colorimetric Determination of Benzylpenicillin Colorimet ric Determination of Total Penicillins GEORGE E. BOXER AND PATRICIA M . EVERETT Merck & Co., Znc., Rahway, N . J.
A colorimetric method for the determination of benzylpenicillin in samples of any purity and in fermentation liquors, based on the determination of the phenylacetyl side chain by the Kapeller-Adler reaction, is described. Separation from interfering substances is obtained by solvent extraction of penicillin and by the use of blanks obtained by alkali inactivation. The results are reproducible to +3%.
V
ARIOUS methods for the chemical assay of benzylpenicillin have been described in the literature. (The terms benzylpenicillin and penicillin G are used synonymously throughout this paper, as both terms have been employed in the literature cited.) The rather weak absorption of the phenylacetyl side chain at 263 mp has been used by Philpotts, Thain, and Twigg (13) and by Grenfell, Means, and Brown (6) to determine penicillin G in penicillin preparations. Sheehan, Mader, and Cram (14) and Mader and Buck (11) devised a gravimetric procedure which is based on the fact that the 1-ethylpiperidine salt of benzylpenicillin is considerably less soluble in amyl acetate and acetone than the corresponding salts of the other penicillins. Colorimetric procedures for the determina$ion of the phenylacetyl group of penicillin G, proposed by Page and Robinson (Id) and by Del Vecchio and Argenziano ( 4 ) , are adaptations of the Kapeller-Adler (9) reaction for phenylalanine. It is stated that all these methods are satisfactory if applied t o penicillin G preparations of 50% or higher purity and, in general, best results can be expected with nearly pure preparations. The colorimetric procedures are specific far any phenyl monocarboxylic acid derivatives and do not distinguish the biologically active penicillin G from its biologically inactive degradation product, benzylpenicilloic acid, which will be present t o some extent in most isolation intermediates. Particular dBioulties must be expected with the assay of fermentation
Analytical recovery of penicillin G added to fermentation liquors was 97 * 5%. A hydroxylamine method for the colorimetric determination of total penicillin in samples of any purity and in fermentation liquors is described. Simplification of procedure and improved stability of the final color complex have been obtained, Analytical recovery of penicillin added to fermentation liquors was 97 * 5%.
liquors which contain the various derivatives of phenylacetic acid used for the stimulation of penicillin G formation (9). The method for the assay of benzylpenicillin described in this paper is based on analytical separation of the active penicillin from the above-mentioned interfering materials, followed by the colorimetric determination of the phenylacetyl side chain of penicillin G by the Kapeller-Adler reaction (9). Most of the substances used to stimulate the production of penicillin G in fermentation liquors are either basic or neutral phenylacetyl derivatives ( 2 ) . If the penicillins are extracted from the broth a t low p H into an organic solvent and then reextracted into a neutral aqueous phase, they will be effectively separated from the precursor material. It is apparent that intermediates in the purification following the solvent extraction of penicillin will present different and simpler analytical problems than broth itself. Two types of phenylacetyl derivatives other than benzylpenicillin are encountered with the various intermediates of penicillin purification: (a) benzylpenicilloic acid, and ( b ) various further degradation products of penicillin G and any other unrelated phenyl monocarboxylic acid derivatives. The active penicillins are readily and quantitatively separated from the penicilloic acids by extraction with chloroform at p H 2, inasmuch penicilloic acid-is exas none of the dicarboxylic acid-i.e., tracted by chloroform. This separation also affords a simple
