Determination of Penicillin G Using C3Isotope as a Tracer JOHN T. CRAIG, J. B. TINDALL,
AND
MURRAY SENKUS, Commercial Solvents Corp., Terre Haute, Ind.
A precise method was developed for the determination of penicillin G in relatively impure samples of penicillin broth and crystalline samples. Labeled potassium benzylpenicillinate was enriched with respect to Cla at the carbon attached to the benzyl group. The enrichment in analytical samples was determined by hydrolysis of the penicillin to phenylacetic acid, followed by decarboxylation of the acid to carbon dioxide. The C'S content of the gas sample
T
HREE chemical and physical methods have been developed for the determination of penicillin G in penicillin samples.
I n the gravimetric method, the material is isolated quantitativelj a i the X-ethylpiperidinium salt ( 1 0 ) . I n one spectroscopic mrthod the penicillin G content is obtained by measuring it8 ultraviolet absorption in solution ( 4 ) ,while in another method it ir obtained by measuring th(, infrared absorption of a cri-stalline wmple ( 2 ) These three procedure\ are the most precise methods that are :rvnilable for the purpose at hand. Honever, their utility is limited, inasmuch aq they are confined to relatively pure samples. I n the evaluation of a recovery process, they can be used only for thr analysis of final products, and they have proved to be indisjwnuable for this purpose ever since industry started to produce ri 1 qtalline penicillin. Obviously, differentiation betwen penicillink in each of the preceding steps is highly desirable, particularljI\ lien attempts arc made to separate the penicillins during the rrcovery and to control the production of penicillin G in the broth through the use of precuisoiq or through thc selection of molds. Several microbiological methods have been described for thr clifferentiation between penicillins ( 3 , 6, 1 1 ) in impure samples. In this laboratory the Thorn and Johnson method ( 1 1 ) has proved to be rspeciall> useful, inasmuch as it is particularly iuited to the determination of penicillin G in samples ranging in purity from that found in broth to that in crystalline samples. The precision of this method is * 10%. The method described herein is an isotope dilution method for tlio determination of penicillin G, using C13as a tracer. RittenIwrg and Foster (9) have developed an isotope dilution method u ith application to the determination of amino acids through the use of 5 ' 5 and fatty acids using deuterium. Labeled penicillin 13 as prepared by using as a precursor in the frrmentation phenylacetamide containing excess CI3 at the amide group. The method has been applied to the determination of pcnicillin G in broth as well as in commercial crystalline samples. EXPERIMENTAL
Microbiological Preparation of Labeled Benzylpenicillinic Acid. Eastman Kodak potassium cyanide containing 16 atom % C13 was converted to phenylacetonitrile according to the method of Adams ( I ) . The nitrile was then hydrolyzed to phenylacetamide as described by Purgotti ( 7 ) . To a medium suitable for the production of penicillin G was added 0.04% by weight of the amide. The mixture was sterilized and inocculated with Penicillium chrysogenum Wisconsin strain Q-176. The fermentation was a]lowed to proceed e t 25' C. in 2-liter Erlenmeyer flasks agitated on reciprocal shakers. The broth was harvested after 5 days. To each 3-liter portion of the C13 filtered broth were added 2 grams of pure potassium benzylpenicillinate in order to reduce the V13/C1zratio a t the amide carbon to about 4/100, because it was
was determined in a mass spectrometer. The method of analysis should prove to be of great value in developing and improving process for the isolation of penicillin G. The analysis of a broth is of special significance in this connection, inasmuch as it reveals the amount of penicillin G per unit volume. A biological assay of broth can be obtained, but these data cannot be used to calculate the penicillin G content.
calculated that thik \I ould be a suital)le ratio for the 1al)eled standard. Isolation of Crystalline Labeled Potassium Benzylpenicillinate. Crystalline potassium benzylpenicillinate was isolated from the broth by way of crystalline benzylpenicillinic acid diisopropyl etherate, which has been reported by Trenner (I!?), with some modification. Any method for the isolation of crystalline benzylpenicillin from broth could be used a t this point. The etherate was converted to the potassium salt by dissolving in butyl acetate, then adding dropwise with good agitation 0.5% aqueous potassium hydroxide until the pH of the aqueous phase had reached 7.5. The aqueous layer was separated and dried from the frozen state. Crystallization of the solid product from n-butyl alcohol according to the method of Kintersteiner ( I S ) gave 1.8 grams of crystalline potassium benzylpenicillinate, the labeled standard, Several %liter portions of broth were processed to obtain a large quantity of labeled potassium benzylpenicillinate, followed by subsequent recrystallization of the composite material. Isotope Concentration of Labeled Standard. A mixture consisting of about 1 gram of the labeled potassium benzylpenicillinate and 50 ml. of 4 A- sulfuric acid was allowed to reflux for 2 hours. The solution was extracted with ethyl ether. The cxtract was distilled to remove the ethyl ether. To the residue were added 15 ml. of warm petroleum ether to dissolve the crude phenylacetic acid. The solution was filtered and the filtrate was caooled to permit the phenylacetic acid to recrystallize. Filtration of the mixture yielded about 0.25 gram of phenylacetic acid per gram of potassium benzylpenicillinate. The melting point was checked to assure purity of the acid, The 0.25 gram of r e c r y ~ t a h e dphenylacetic acid was then decarboxylated with quinoline and copper chromite according to the method of Huggett (6). The carbon dioxide which was evolved was entrained in nitrogen and the mixture was bubbled through excess barium hydroxide. The barium carbonate which formed was isolated under nitrogen. A sample of carbon dioxide for the mass spectrometer was then isolated from the carbonate according to the method of Rittenberg- (8). The C13/C12 ratio was founa to be 0.0466. Preparation of Primary Standard. Potassium benzylpenicilh a t e was used as the Drimarv standard in the oresent analytical method. I-se was again made of the isopropql etherate for the preparation of potassium benzylpenicillinate of the desired purity. Commercial benzylpenicillin was converted to the etherate as described by Trenner (12). The crude crystals were recrystallized several times according to Trenner's method. The pure etherate was then converted to the potassium salt and recrystallized as before. Purity of Labeled Standard. The potassium benzylpenicillinate content of the labeled standard was determined by the dilution of 0.25 gram of the material with 1.00 gram of uure potassium benzylpenicillinate, the primary standaFd. A caibon dioxide sample was isolated from the primary standard, the labeled standard, as well as from a mixture of the two according to the method described previously. The C13/C12ratios of these samples were obtained in the mass spectrometer. ANALYSIS OF PENICILLIN SAMPLES
Analysis of Broth Samples. I n the analysis of broth, 0.25 gram of labeled potassium benzylpenicillinate was added to 3
332
V O L U M E 23, NO. 2, F E B R U A R Y 1 9 5 1 ~
~
_
Table I.
Sample
SO. 59 83 104 107
,945
547 393 43 1 44OC 440D
Labeled Standard Added,
Bio-
G./L Broth 0.083 0,090
assay,
0.083 0.083 0.083 0.083 0.042
482 458 540
p/-111.
550 4;78
0.093 0.087
,500 44.3
458 192
0,042
208
_-
_
~_
333
_ ~ _
~
~
___ ~
Penicillin G in Broth Samples
+ 1)
+ 1)
xherc, X is the weight, of potassium penicillin G in the unknown sample, R is t’he C13/C12 ratio of the labeled standard. C is the C13/C1*ratio of ordinarl0.265 pmicillin G, D is the C13/C12ratio of the misturc of 0.235 0.270 thr tn-o, and E is the weight of labeled standard :tdded. In onc analysis, a broth sample contained 00 .. 21 39 27 0 . 2 i 0 gram of penicillin :is potapsiuni henzylpeni0.184 0.195 cillinate per liter. 0,276 0.106 The penicillin C: contents of t e n samples of broth 0.123 were determined (Table I ) . Penicillin G in Crystalline Samples. The peni~cillin G contents of seven crystalline samples w r t ~ determined (Table 11). Some of these samples \ v c ~ r (analyzed ~ by the ultraviolet method (4)as well as by the N-ethdpiperidinium method (10). Thesc data are included i n the, table for comparison.
Penicillin G as Potassium Benaylpenioillinate, G./L. Broth Electrostat. Electromag.
c 1 3 0 2 :c1202
(C1?0? = 100) Hectrostat. Electromag.
-
1 . 9 1 (1.13)a 2.02(1.10) 1.96(1.11) 2.01 (1.11) 2.08(1.12) 2,19(1,15) 2.16(1.15) 1.91(1.15) 2 Og(1.15) 2.01 (1.15)
A- = E ( R - D ) (C (D - C ) (B
1.96(1.16)“ 2.09(1.16) 2.02(1.16) 2.07(1.16) 2.17(1.17) 2.26(1.20) 2.22(1.20) 1.98(1.19) 2.16(1.20) 2.06(1.20)
0.270 0.238 0.270 0.236 0,207
0.186 0.194 0.283 0.107 0.125
Valiics in parenthese- are C’302:C1?02 ratios of ordinary CO?
liters of the sample. The mixture was filtered. Crystalline potassium benzylpenivillinate was isolated froni the filtrate as previously described. The crystalline sample way hydrolyzed to phenylacetic acid. Thc acid vias purified, then decarboxylated to obtain the carbon dioxide sample for assay as described above. Analysis of Commercial Crystalline Samples. In the analysis of crystalline penicillin, 0.6 gram of a sample \vas mixed with 0.2 gram of labeled potassium benzylpenicillinate. The misture was then analyzed according to the procedure described in the experimental section. CALCULATIOK OF RESULTS
Purity of the Labeled Standard. The procedure for the ibolation of carbon dioxidc samples from the primary standard, the labeled standard, as well as from a mixture of the two is described above. The weight of potassium benzylpenicillinate in 0.2500 gram of Iabelrd standard r a s determincd by solving the following equation:
n-here X is the weight of p o t a s h i n penicillin G in a known weight of laheled standard, :I is weight of primary standard added, R is the C13/CI2 ratio of the labeled standard, C is the C 1 3 / C 1ratio 2 of the ordinary penicillin G, and 11 is the C13/C12 ratio of the mixture of the two. I t was found that 0.2500 gram of labeled standard contained 0.229 gram of potassium benzylpenicillinate or 91.6% by \\.eight.
DISCUSSION
The penicillin G valurs that were calculated from the electromagnetic readings were about 1% l o n w than the values calculated from the electostatic readings. I n view of the better agreement among the electromagnetic readings on normal carbon dioxide, it appears likely that these values are more accurate. I n the preparation of penicillin for clinical use, it is necessary to separate penicillin G from the other penicillins during the recovery from the broth. The present method of analysis should prove to be of great value in developing a process for the isolation of penicillin G. The analysis of a broth is of special significance in this connection, inasmuch as it reveals the amount of penicillin G per unit volume. I t is recognized that the biological assay of the broth can he obtained more readily, but these data cannot be used to calculate the penicillin G content with the same degree of accuracy. ACKNOWLEDGMENT
The authors are indebted to R. J. Hickey for the niicrobiological preparation of labeled penicillin broth. The esaminations of the carbon dioside samples in the mass spectrometer by James Seerman are also gratefully acknon-ledged. LITERATURE CITED
Table 11.
(1) Adanis, It.. wild Thal, .-I.F., Ory. Sunthcses, Collective Vol. I, 101
Penicillin G Crystalline Samples
Potassium Benzylpenicillinate, % (cl20z = 100) by Weight Electro- ElectroUltraElectrostat. Electromag. stat. mag. N.E.P.= violet c13o2:c12o?
Code So.
2.03(1.15) 1 . 9 0 (1.15) 1 . 8 6 (1.15) 1,87(1,15) 1.97 (1.15) 1.79(1,15) l.gl(1.15) I‘
2.08(1.20) 1 , 9 7(1.20) 1.92(1.20) 1.93(1.20) 2.03 (1.20) 1.86(1.20) 1.97(1.19)
70 87 93 91 77 101 85
71 85 93 91 77 100
84
67 79
..
... 87 ...
79 93 88 68
...
78
100
..
.V-Ethyl piperidinium.
Potassium Benzylpenicillinate in Broth Samples. The amount of “penicillin G” in broth samples was determined by dilution of a given amount of sample with a given weight of the labeled standard. The dilution of penicillin in broth with the labeled standard and the isolation of potassium benzylpenicillinate therefrom, as well as the preparation of a carbon dioxide sample fiom the crystalline product, are described above. The per cent by weight of potassium benzylpenicillinate in the Ilroth samples was calculated by substituting the knon-n values i n the following formula:
(1932). (2) Barnes, It. B., Gore, 11. C.. TVilliarns, E. P..Linsley, S . G., and Peterson, E. hl.,.\NAI.. HEM.. 19, 620 (1947). (3) Craig, L. C., J . B i d . Chcni.. 155, 519 (1944). (4) Grenfell, T. C.. Means, .J. A,. and I3town, E. T., Ihid., 170, 527 (1947). (5) Higuchi, K., and Petcrson, 15’. H.. Ais.~r.. HEX., 19, 68 (1947). (6) Huggett, C., Arnold. R. T., and Taylor, T. I., J . .Am. Chem. S O C . . 64, 3043 (1942). (7) Purgotti, A., Gam. c h i m . itul., 20, 173 (1890). (8) Rittenberg, D., “Preparation and Measurement of Isotopic Tracers,” p. 41, Ann Arbor, hfich., Edwards Brothers, 1947. (9) Rittenberg. D., and Foster, C.: L., J . B i d . Chem., 133, 737 (1940). (10) Sheehan, J. C., hlader, 1V. J.,and Cram, D. ,J., J . A m . Chem. S O C . , 68, 2407 (1946). (11) Thorn. J. A , and Johnson, &I.J., . ~ N L . CHEY.,20, 614 (1948). (12) Trenner, K. R., and Buhs, R. P., J . A7n. Chem. S O C . , 70, 2897 (1948). (13) Wintersteiner, O., “Chemistry of Penicillin,” p. 89, Princeton, N. J.,Princeton University Press, 1948. RECEIVEDApril 24, 1950. Presented before the Division of Analytical Chemistry a t the 116th Meeting of the AMERICAKCHEMICALSOCIETY, Atlantic City, S . J.
