Determination of Phenylacetic Acid and Pheny lacetamides in Samples from Penicillin Fermentations S. C. PAN and DAVID PERLMAN Squib6 lnstitute for M e d i c a l Research, New Brunswick,
A modification of the Kapeller-Adler procedure for the determination of phenylalanine has been used for the determination of phenylacetic acid. The procedural changes include carrying out the nitration a t 40" to 44" C. instead of 96" to 98" C., thus eliminating the formation of all chromogenic nitro derivatives except the compound which gives a stable violet color on reduction in a n ammoniacal solution. Use of this as well as other minor modifications has led to the development of a n analytical method with precision of the order of &3%. Phenylacetic acid can be quantitatively separated from benzylpenicillin or its degradation products by extraction from acidified aqueous solutions with toluene. The extracted phenylacetic acid is then determined by the modified Kapeller-Adler reaction. Modifications of the developed procedure have been proposed which make possible the determination of phenylacetic acid in samples containing as little as 0.01 mg. per milliliter. Phenylacetamides and its neutral derivatives--e.g., N-methyl- or N-(2-hydroxyethyl)phenylacetamides-can be separated from phenylacetic acid and benzylpenicillin or its degradation products by extraction with chloroform from a n alkaline solution. The extracted phenylacetamides can be hydrolyzed into phenylacetic acid, which is then determined by the modified Kapeller-Adler reaction. The phenylacetic acid when mixed with these neutral phenylacetamides can also be separately determined after removing the phenylacetamides by chloroform extraction of a sample which has been made alkaline.
N. J. the two nitro groups are ortho to each other. Beer, Dicken8, and Salmony ( d ) have shown that the cherry-red complex formed from benzoic acid is due to the 2,5-dinitrobenzoic acid-Le., the two nitro groups are para to each other. Many investigators have attempted to utilize this color reaction for the quantitative determination of benzoic acid (16, 10), phenylalanine ( 5 , 6 ) , and the phenylacetyl group of benzylpenicillin ( 7 , 10). Beer et al. (2) have shown that these chromogenic nitro compounds are produced only in small quantities during nitration of these aromatic acids, the major product being the 2,4-dinitro derivative which remains colorless after reduction. Their studies suggest that this reaction cannot he easily adapted for the quantitative determination of these acids. Hiscox (10) concurs with this conclusion while pointing out the difficulties in obtaining reproducible results. Because other specific methods for determining phenylacetic acid are not available, the llohler or Kapeller-Adler reaction has been re-examined in an effort to develop a procedure which would permit a fairly accurate determination of phenylacetic acid. EXPERIMENTAL
Modifications on the Procedure of the Kapeller-Adler Reaction. In the conventional Kapeller-Adler reaction (6, 7 , 12, 16) a sample of phenylalanine solution is first evaporated to dryness and treated with 1 to 2 ml. of a nitrating reagent (10 or 20 grams of potassium nitrate in 100 ml. of concentrated sulfuric acid) in a boiling water bath (96' to 98" C.) for 20 to 30 minutes. To the nitration mixture, 0.5 to 1.0 ml. of a concentrated hydroxylamine solution is added, followed by neutralization with excess concentrated ammonium hydroxide. Hiscox (10) commented on such a procedure: "Considerable error arises from the heat evolved and the spitting that occurs when concentrated ammonium hydroxide is added to concentrated sulfuric acid." Preliminary dilution ( 6 ) or partial neutralization accompanied by cooling ( 7 ) has been suggested to avoid this excessive heating. In the present study, this difficulty was removed by employing a smaller amount (0.25 ml.) of the nitrating reagent, which wa5
A
COMMON practice has been to add phenylacetic acid, phenylacetamide, or derivatives of these compounds to penicillin fermentations to serve as precursors for the biosynthesis of benzylpenicillin (5,8, 18). ilccurate and reliable analytical methods have been needed to follow the metabolism of these precursors during the fermentations. Ishima and Tanno (11) have proposed a method for the determination of phenylacetic acid in which the acid is extracted from the fermentation samples with benzene and then determined gravimetrically as the ammonium salt. This appears to be the only method for the determination of phenylacetic acid reported in the literature and no method has so far been proposed for the determination of phenylacetamides. In the present study, attempts have been made to develop analytical methods for the determination of phenylacetic acid and phenylacetamides by making use of the Kapeller-Adler reaction for phenylalanine.
LOG
+Q
0.600 0.m 0.400
0.m 0.200
ADAPTATION OF THE KAPELLER-ADLER REACTION TO THE DETERMINATION O F PHENYLACETIC ACID
0.100 '
1
In Mohler's method for the determination of benzoic acid (16) and a similar procedure proposed by Kapeller-Bdler for the determination of phenylalanine (IZ), nitration of these aromatic acids followed by reduction-e.g., with hydroxylamine in an ammoniacal solution-results in the formation of colored compounds. Block and Bolling ( 5 , 6 ) believe, in agreement with the experimental results of Meisenheimer (14) and Xfeisenheimer and Patzig ( 1 5 ) ,that the violet color formed from phenylalanine is that of the aci-form of the reduced 3,4-dinitro derivative-Le.,
420
440
480
460
500
520
540
560
560
600
620,
WAVE LENGTH m,u
Figure 1. Absorption Spectra of Colored Solutions from Conventional Kapeller-Adler Reaction of Phenylacetic Acid, Phenylalanine, and Benzoic Acid 0.0368 millimole (equivalent to 0.5 mg. of phenylacetic acid) of each compound was used. Nitration was carried out at 96' to 98' for 30 minutes. Nitrated products are reduced with hydroxylaminein an. ammoniacal solution
1432
V O L U M E 26, NO. 9, S E P T E M B E R 1 9 5 4
1433
later neutralized with 6.25 ml.
of 1 t o 3 diluted ammonium hydroxide ( 4 . 7 N ) . Here a small amount of already diluted sul-
Table I.
Effect of Nitration Temperature on Results of Kapeller-Adler Reaction
Nitratedb a t 40-44O C., Nitratedb a t 96-98' C., 30 Min. 15 Min. % Trans7?TransCompounds Tested6 mittancec Color mittancec Color Phenylacetic acid (CsHaCHnCOOH) 33.75,40 Dull violet 50.75,52 Bright violet Benzoic acid (CaHsCOOH) 57.75,55 5 Cherry red 98.5, 99.5 DL-Phenylalanine (CsHsCHzCHNHzCOOH) 38.25,38 Violet 43.75,45.5 Brigit'biolet DL-hfandelic acid (CeHsCHOHCOOH) 35.75,37 Dull violet 75,75.75 Violet cinnamic acid (C~H~CH=CHCOOH) 50.5, 43 5 Red-brown 95.75,94 Salicylic acid ( c ~ I , ( O H ) C O O H ) 98,98.75 Yellow 100 ... 0 0.0147millimole (equivalent t o 0.2mg. of phenylacetic acid) of each compound was used. b T h e nitrating reagent contained 20 grama of potassium nitrate and 100 ml. of concentrated sulfuric acid: 0.25ml. of the reagent was used. After nitration, 0.5 ml. of 15% hydroxylamine hydrochloride and 6.25 ml. 1 t o 3 ammonium hydroxide (4.7N)were added. 0 Values are duplicate readings in an Evelyn photoelectric colorimeter with a 540-mr filter.
furic acid was neutralized with diluted ammonium hydroxide; €!xcesSive heating and spitting were entirely avoided. A second modification for facilitating the operation of the test consisted of shortening the time required for evaporating the samples to dryness. The test tubes containing the Samsolutions were immersed in a 50" to 70" C. water bath and a gentle current of air, filtered through a column of cotton, was passed over the liquid surface. By employing a few glass manifolds, a number of samples can be conveniently dried a t the same time.
With the procedure rendered convenient by these minor modifications, detailed studies were made on the effect of nitration conditions upon the color development. When tested under the conditions of heating 20 t o 30 minutes a t 96" to 98" C., benzoic acid produced a cherry-red color, phenylalanine a violet color, and phenylacetic acid a color varying from violet to reddish brown. As a result of this variation, the per cent transmittance of the A colored complex from phenylacetic acid, as determined in an Evelyn photoelectric colorimeter with a 540-mp filter, was hardly reproducible. The absorption spectra of these three colored B solutions, as determined with a BeckC man Model DU spectrophotometer, are presented in Figure 1. Examination of these curves as well as the data assembled by Beer et al. ( 2 ) suggested that the spectrum obtained with the phenylacetic acid was probably a com0 bination of those obtained with benzoic acid and phenylalanine-Le., a mixture of 2,5- and 3,4-dinitro derivatives. The production of Aeveral nitro deE rivatives when phenylacetic acid was nitrated a t 96' to 98" C. was also SOLVENT FRONT demonstrated by filter paper chromatography. A 3-mg. sample of sodium Figure 2. Paper phenylacetate was nitrated with 1 ml. Chr o m a t o g r a m of Nitrated Phenof the nitrating reagent a t 96" to 98" C. ylacetic Acid for 30 minutes. After cooling, 3 ml. of Sample 1, products of water were added and the solution was nitration a t 96' to extracted with 10 ml. of chloroform. 98' C. Sample 2, producp of The chloroform extract was evaponitration a t 40 t o 440 c. rated to dryness and the residue Solvent system, bufdissolved in 0.5 ml. of ethyl alcohol. A fered phenol Deteeting reagents, strip of Whatman No. 1filter paper was stannous chloride and Ehrlich reagent spotted with this solution and the chroA . Pink matogram was developed for 18 hours B . Pink C. Ochre with a buffered phenol system (4)-120 D . Yellow (deep purple on grams of phenol plus 10 ml. of a solution standing) containing 6.3% sodium citrate and E . Yellow-brown 3.7% potassium dihydrogen phosDhate-and the mixture was completely . liquefied a t 50" to 60' C. Descending technique was employed and the spots were detected by spraying first with 1% stannous chloride in 1N hydrochloric acid, followed by Ehrlich reagent ( p dimethylaminobenzaldehyde) ( 1 ) . A diagrammatic sketch of the results obtained is presented in Figure 2. Sample 1in this sketch
.
I
.
LW+
0.500 0.400
0.300
0.200
O.'OO
tI .
420
1
440
460
480
500
520 540 WAVE LENGTH
580
580
600
J
620
Figure 3. Absorption Spectra of Colored Solutions from Modified Kapeller-Adler Reaction of Phenylacetic Acid and Phenylalanine 0.0368 millimole (equivalent t o 0.5 m g . phenylacetic acid) of each compound was used. Nitration was carried o u t a t 40' t o 4.4' C. for 15 minutes. Nitrated products were reduced with hydroxylamine i n an ammoniacal solution
represents the chromatogram of products from nitrating phenylacetic acid a t 96" to 98' C. Fivecolored spots aere noted on this chromatogram. The colors of the spots differed from each other; the spots labeled B and C were not well separated but were of different hue. Spot E was partially masked by a colored band coincident with the solvent front. Because these spots appeared on the chromatogram only after spraying with both reagents, they must represent nitro derivatives of benzene. When a sample of twice-recrystallized phenylacetamide (melting point 155" C.) was hydrolyzed and the phenylacetic acid in the hydrolyzate was extracted with toluene and tested with the same chromatographic method, an identical chromatogram as described in Figure 2 was obtained, showing that these spots were not due to the impurities in the phenylacetic acid sample. These results, therefore, led to the conclusion that the nitration of phenylacetic acid a t 96" to 98' C. resulted in the formation of a t least five different nitro derivatives. The possible variation in the proportion of these chromogenic compounds produced and the differences in their color development with time and in the stabilities of the colors would naturally account for the nonreproducibility of the transmittance readings. After a few futile attempts to produce only one chromogen, it was found that nitration a t lower temperatures resulted in the formation of only a violet chromogen in the Kapeller-Adler reaction. Further study showed that treatment with nitrating reagent for 15 minutes a t 40" to 44' C. gave the best result. Paper chromatographic study of the nitro derivatives of phenylacetic acid thus obtained showed that only one chromogen was present as indicated in Figure 2 (sample 2). The absorption spectra of this violet-colored complex obtained from phenylacetic acid and that from phenylalanine by the modified procedure of KapellerAdler reaction are shown in Figure 3. The shape of the curves apparently supports the chromatographic result that only one
ANALYTICAL CHEMISTRY
1434
Table 11.
Effect of Water Content of Nitrating Reagent" on Color Development
Water Content of Transmittanceb, 3' % Nitrating Reagent, M1./100 M1. Phenylacetic itcidc DL-Mandelic acid e 0 51.25, 50 68 5, 71 51.75. 50.5 5 10 50.75, 50.5 93, 96.5 96.25, 97.25 99.75, 100.25 20 a The nitrating reagent and the procedure as described in footnotes of Table I were used. Kitration was carried out a t 40° t o 44O C. for 15 minutes. b Values are duplicate readings in a n Evelyn photoelectric colorimeter with a 540-m@filter. C 0.0147 millimole (equivalent t o 0.2 mg. of phenylacetic acid) of each compound was used.
chromogen was present. No chromogenic compound was produced when benzoic acid was nitrated a t 40" to 44" C. A study on the effect of the length of color development time upon the color intensity is summarized in Figure 4. The color intensity reached its maximum 55 minutes after addition of ammonium hydroxide and the maximum intensity remained practically unchanged for a 45-minute period following.
2
t 20-
5 z
a I-
10-
ap
0'
Ib
20
io
i o 50 60
io
So 90 i o i o
I i O I30
MINUTES
'
Figure 4. Stability of the Color from Modified Kapeller-Adler Reaction of Phenylacetic Acid 0.4 mg. of phenylacetic acid was used i n this test. The t i m e a t which a m m o n i u m hydroxide was added was taken as zero t i m e
Table I presents a comparison of color intensities obtained when six related aromatic acids were tested with the original (nitration a t 96' to 98" C.) and the modified (nitration at 40" to 44' C.) Kapeller-Adler reaction. Kitration a t 40" to 44' C. resulted not only in a better reproducibility but also in the elimination of chromogen formation from benzoic and cinnamic acids. Further study showed that the color formation from nL-mandelic acid could be reduced when a small amount of water was added to the nitrating reagent, as indicated in Table 11. Addition of as much as 10 ml. of water per 100 ml. of reagent did not affect the formation of the colored complex from phenylacetic acid. These modifications therefore made the method more specific for phenylacetic acid. Proposed Procedure for the Determination of Phenylacetic Acid. On the basis of the results discussed in the preceding section, the following procedure was finally adopted. The procedure for constructing a calibration curve is given as an example. Duplicate aliquots of a phenylacetic acid solution, representing 0.00, 0.05, 0.10, 0.20, 0.30, 0.40, and 0.50 mg. of phenylacetic acid are pipetted into absorption tubes for use in the Evelyn photoelectric colorimeter. The aliquots are made alkaline with 0.1 ml. of 0.1N sodium hydroxide and dried a t 50" to 70" C. under a gentle current of air (see above). To each of the tubes, 0.25 ml. of a nitrating reagent (20 grams of potassium nitrate, 100 ml. of concentrated sulfuric acid, and 10 ml. of water) is added and the
tubes are kept in a 40' to 44" C. water bath for 15 minutes. At the end of that period, 0.5 ml. of hydroxylamine hydrochloride (15% in water) is added, followed by 6.25 ml. of 1 to 3 diluted ammonium hydroxide (4.7-V). The contents are well mixed after addition of each reagent and the tubes are allowed to stand a t room temperature (22" to 28" C.) for 60 minutes. The per cent transmittance is then read in an Evelyn photoelectric colorimeter equipped with a 540-mp filter, with the tube containing only the reagents set a t 100%. The reading should be completed within 30 minutes. The transmittance readings showed that the colored complex followed Beer's law throughout the entire range tested. A number of repeated tests showed that the day-to-day variation as well as that between duplicates hardly exceeded i1% transmittance or approximately =k3% in the results of the analysis. (Mathematically, a difference of 1% transmittance means a difference of 2.5 to 4% in absorbance within the range of 15 to 70% transmittance.)
DETERMINATION OF PHENYLACETIC ACID IN S-IMPLES FROM PENICILLIN FERMENTATIOYS The second problem in the present study has been to apply the modified Kapeller-Adler reaction (as described in the preceding section) to the determination of phenylacetic acid in samples from penicillin fermentations. Experimentation on such a problem is reported here. Benzylpenicillin and its degradation products, including benzylpenillic acid and benzylpenicilloic acid can all be regarded as derivatives of phenylacetic acid and will naturally also produce colored compounds in the Kapeller-A4dlerreaction. A separation of phenylacetic acid from these compounds is thus a prerequisite for a satisfactory procedure. Phenylacetic acid may be extracted from aqueous solutions by nonpolar solvents, including benzene and toluene (19),while penicillin has been reported to be relatively insoluble in these solvent's ( 2 1 ) . -4s benzylpenillic and benzylpenicilloic acids are dibasic, they should be less soluble in these nonpolar solvents than benzylpenicillin. Higuchi and Peterson (9) made use of these principles for separating the "R-group acids" from alkali-treated penicillins. Quantitative separation of phenylacetic acid from benzylpenicillin and its degradation products by means of extraction with a suitable solvent is obviously feasible. EXPERIMENTAL
Test Solutions and Reagents. Phenylacetic acid obtainedfronn Eastman Kodak Co. was used without special purification. The, criteria used for examining the purity of this material are men-tioned in other parts of this communication. An aqueous solheion containing 2.5 mg. per milliliter was prepared as the sbndard' solution. Pure potassium benzylpenicillinate (kindly supplied, by the Division of Chemical Development of this instibute; it contained 1590 units per mg.) and nbphenylalaninie. ("erck), were also used in the present study. The solvents, toluene and, chloroform, the anhydrous sodium sulfate, and other chemicals, were all of C.P. grade and they were used without special purification. Extraction Procedure. The extraction procedure developed,, as illustrated by the procedure for constructing a calibration curve, is as follows: A 1.0-nil. aliquot of the standard phenylacetic acid solutioii, is placed in each of two 5 X 5/8 inch test tubes. It is acidified' with 0.1 ml. of 10N sulfuric acid, and 0.25 to 0.3 gram of anhydrous sodium sulfate (use of a calibrated spoon simplifies the, operation) and 5 ml. of toluene are added. The mixture is shaken vigorously for 30 seconds with the thumb as a stopper. Test t,ubes or other containers equipped with ground-glass stoppers can also be used for this purpose but the use of rubber stoppers or corks must be avoided. The mixture is poured after shaking. into a test tube in case other containers are used. The tubes are loosely corked and centrifuged for 5 minutes to separate the solvent layers. Aliquots of the toluene layer, representing 0.0,O. I, 0.2, 0.3, 0.4, and 0.5 mg. of phenylacetic acid are transferred into duplicate absorption tubes for use in the Evelyn photoelectric colorimeter, together with 0.1 ml. of 0.1N sodium hydroxide. The procedure described in the first part of this communication< is then applied. I
V O L U M E 2 6 , NO. 9, S E P T E M B E R 1 9 5 4 _.-___.____
1435
_ _ _ . . _ _ _ _ ~ ~ ~
A n a l y s i s of F e r m e n t a t i o n Samples. A 1-ml. sample of a cell-free filtrate (obtained by Vol. Sodium Init. Ratio Sulfate in Concn. in Extraction centrifuging the fermentation Solvent to A s . Phase, Aq. Soln.. Efficienoyb, sample) is acidified with 0.1 ml. Compounds Solvent Aq. Soln. G./100 M I . Mg./Ml. % Phenylacetic acid Toluene 1 to 1 30 4.0 93.5 90.0 of ION sulfuric acid Testing Toluene 2 to 1 30 4.0 91.5 95.0 with a minute drop of thymol Toluene 5 to 1 30 4.0 95.0 98.0 Chloroform 1 to 1 30 4.0 92.5 95.0 blue indicator on a spot plate Chloroform 2 to 1 30 4.0 90.0 98.0 should give a definite pink color: Chloroform 5 to 1 30 4.0 96.0 103.0 Toluene 5 to 1 20 4.0 97.5 . . . more acid should be added if a Toluene 5 to 1 10 4.0 98.0 . . . Toluene 5 to 1 0 4.0 92.5 yellow or blue color results. The Toluene 5 to 1 30 4.0 95.5 100:s Toluene 5 to 1 30 1.0 90.0 93.3 extraction and color development Toluene 5 to 1 30 0.2 87.5 . . . procedures as described abovc Penicillin Toluene 5 to 1 30 3.0 0.0 . . . Chloroform 5 to 1 30 3.0 ca. 36d ... are then followed. An aliquot of Phenylalanine Toluene 5 to 1 30 4.86 0.0 ... the toluene extract containing Chloroform 5 to 1 30 4.85 0.0 . . . 0.04 to 0.6 mg. of phenylacetic a T h e aqueous phase was made strongly acid I N with sulfuric acid. T h e extraction efficiency denotes the perceitage of a compound under test extracted b y shaking once with t h e acid should be used in the color solvent. Values given were from duplicate determinations. development procedure. e Potassium benzylpenicillinate was used a n d its acid inactivation products were actually being tested. J Determined after hydrolysis (see below). Table IV illustrates that re__-coveries of phenylacetic acid added to fermentation samples X straight line should result when the percentage transmittance when analyzed wit,h the procedure recommended above were entirely satisfactory. Results of analyses of fermentation samples values are plotted against the amounts of phenylacetic acid used on a semilogarithmic scale. For routine analysis, two phenylbefore and after additions of benzylpenicillin indicated that the presence of benzylpenicillin did not affect the determination of acetic acid levels, namely, 0.2 and 0.4 mg.-are sufficient for constructing the standard curve. phenylacet,ic acid. When samples of unfermented cornsteep This procedure was developed from a series of experiments medium or fermented medium containing no added phenylacetic testing t'he effec,ts of different variables-e.g., sodium sulfate acid were analyzed, a slight yellow color often appeared, equivaconcentration, initial phenylacetic acid concentration, etc.-upon lent in absorbance at 540 mp to 0.00 to 0.02 mg. of phenylacetic the extraction efficiency. The term "extraction efficiency" acid per milliliter (Table IV). used in this report denotes the percentage of phenylacetic acid Determination of Small Amounts of Phenylacetic Acid in Fer(or any other compound under test) extracted after shaking once mentation Samples. The smallest quantity of phenylacetic with a solvent. I t is another way of expressing the distribution acid that can be determined with a reasonable degree of accuracy coefficient. The data thus obtained are summarized in Table 111. by the described method is 0.05 mg. per 1-ml. sample. Since it Phenylacetic acid can be extracted efficiently from its aqueous has often been desirable to have a method that could determine solution by either chloroform or tolume. Rlaximum extraction as little as 0.01 mg. per milliliter, attempts have been made to rfficiency was ensured when the aqueous phase cont,ained 10 grams or more of anhydrous sodium sulfate per 100 ml., and 5 volumes of toluene per volume of aqueous phase were used. The Table IV. R~~~~~~~ of Phenylacetic Acid -$dded to presence of sodium sulfate apparently shifted the distribution Fermentation Samples equilibrium in favor of the toluene, probably as a result of the BenzylTable 111. Extraction"of Phenylacetic Acid, Benzylpenicillin, and Phenylalanine from Acidified Aqueous Solutions by Toluene and Chloroform
~~
~~
Phenylacetic
penicillin
Phenylacetic
salting-out effect. A separate experiment (data not given) Acid Used, Added, Acid Found, Recovery, showed that shaking for 10 seconds was the minimum shaking Medium Mg./Ml. Ng./Ml. Mg./Ml. % time needed to obtain the maximum extraction efficiency. L-nfermented" 0.0 .. 0.01b ... 0.40 0.388 97.0 As has been pointed out in the first part of this communication, 0.40 0.5 0.403 100.8 1.20 1.20 100.0 the analytical method for phenylacetic acid has a precision of 1.20 0.5 1.165 97.2 4 ~ 3 %and the variations between duplicate determinations as 2.50 .. 2.42 98.0 .. 0.02b 0.0 Ferrnentedc given in Table 111 would be expected. By allowing such pos0.40 .. 0.412 103:0 sible variations, it can be safely concluded that a t an initial 1.50 .. 1.42 95.0 phenylacetic acid concentration of 4 mg. per milliliter, over T h e unfermented medium contained 2.5% cornsteep solids, 3% lactose, 95% recovery of the acid in the toluene layer can be obtained. ~ ~ e ~ ~ ~ ~ ~ n c ~ ~a slight , ~ xyellou, ~ b color, i t e d C T h e same medium was fermented in shaken flasks for 72 hours with At lower initial concentrations, the recoveries are somewhat lower. Penicillium chrysogenum strain Wis. 49-133. No phenylacetic acid was Data presented in Table 111 added during the fermentation. also show that benzylpenicillin, or rather benzylpenicilloic and Table V. Removal of Interfering Substances for Determining Small Amounts of Phenylacetic Acid in Fermentation Samples benzylpenillic acids formed from acid inactivation of penicillin. o-HydroxyPhenylacetic Acid Found Phenylphenylacetic acetic J%-ithoutRe-extraction and After Re-extraction and wrrenot extracted by toluene unOxidation Acid" Oxidation Acid tlrr these conditions. When chloAdded, Recovery, Recovery. Added, Sample y'M1. -,/Ml. y/mI. % Color ?/mi. 7% Color 1 oform was uscd, approximateljWater 20.0 0.0 18.0 90.0 Violet 16.0 80.0 Violet 36% of the benzylpenicillin and Water 0.0 330 10 .O . .. Yellow 2.0 ... Colorless Fermented medium 0.0 0.0 14.0 ... Yellow 4.0~ Colorless its degradation products was esFermented medium 0.0 330 19.2 Yellow 3 ,o* : : Colorless Fermented medium 30 0 0 0 33.0 16::O Brown 17.8 89.0 Violet tracted. Chloroform is, thereFermented medium 10.0 330 21.0 250 Brown 12.0 120 Violet fore, not a suitable solvent for Fermented medium 20,O 330 30.0 I50 Brown 21.0 105 Violet Fermented mediumC 40 0 330 41.3 103 Brown39.0 97.5 Violet s e p a r a t i n g phenylacetic acid violet ;This material was kindly supplied hy t h e Division of Chemical Development of this institute. from benzylpenicillin. Seither Very close t o 100% transmittance. solvent extracted phenylalanine Same fermentation samples as described in footnotes of Table 11. from its aqueous solutions.
ANALYTICAL CHEMISTRY
1436
It seems possible to apply the alkaline potassium ferricyanide Concn. oxidation directly to samples Apparent Degree of Hydrolysisd, % during Sodium Hydrolysis Hydroxide c , After heating for from fermentations, thus elimiCompounds Tested Millimoles/Ml. h' 30 min. 90 min. 180 min. nating one of the extraction steps 95.0,98.0 ... ... Phenylacetamide 0,0173 1.0 in the procedure for small (CaHaCHzCONHz) amounts of phenylacetic acid. N - (2-hydroxyethyl)0.0173 1.0 70.0 95.0 ... Phenylacetamide 0.0173 2.5 95.0 95.0 However, since the fermentation (CsHICHzCONHCHiCHzOH) 0.0173 5.0 95.0 15.0,98.0 1OO:O samples often contain large 0.0047 Benzylpenicillin 1.0 27.2 60.3 . . . (potassium benzyl 0.0047 2.5 64.0 89.5 ... amounts of reducing sugars, penicillinate) 0.0047 5.0 91.0 96.5,92.5 98.0 penicillin, and other substances N-methyl phenylacetamide' 0.0156 1.0 ... 84.0 ... (CsHsCHzCONHCH:) 0.0156 2.5 ... 87.5 ... which will also consume the fer0.0156 5.0 86.2 84.5 84.0 ricyanide, the amount of ferricy0.0173 2.5 98.0 ... Phenaceturic acid fCoHaCHzCONHCHzCOOH) 0.0173 5.0 iOi:5 95.0 ... anide to be used must be excesPhenylacetic acid 0.0173 5.0 96.2 ... sively high, and the oxidation be(control) ( 2 . 3 5 mg./ml.) comes far less effective than by The hydrolysis was carried out in a boiling water bath with the test tubes loosely corked *e Refers to the concentration after addition of sodium hydroxide. the procedure recommended Refers to that in the whole hydrolyzate. here. Repeated use of these recd Calculated, from the phenylacetic acid determined in the hydrolyzate. e The material was probably not pure (see text). ommended procedures in routine analyses showed them to be entirely satisfactory. use, a larger aliquot of fermentation sample for analysis. In so The procedures described here are applicable only to samples doing, however, the nonspecific yellow color mentioned became from penicillin fermentations to which phenylacetic acid alone so deep as to invalidate the analytical result. is added as precursor. If phenylacetamide or other derivatives are also used as precursor, these procedures cannot be used t o One of the compounds responsible for this yellow color was found to be o-hydroxyphenylacetic acid, which has been shown to determine the free acid present. The method described below must be consulted. occur in penicillin fermentations (13,17). (An analytical method for the determination of o-hydroxyphenylacetic acid will be reDETERMINATION O F NEUTRAL PHENYLACETported elsewhere.) As this compound is phenolic in nature, AMIDES AND PHENYLACETIC ACID IN SAMPLES oxidation by potassium ferricyanide will convert it into products FROM PENICILLIN FERMENTATIONS insoluble in toluene. After some experimentation, the following procedure was found to be satisfactory: Besides the use of phenylacetic acid as the precursor for the biosynthesis of benzylpenicillin, many of its derivaPlace a 5-ml. sample of a cell-free filtrate containing 0.01 to tives have also been used for this purpose ( 3 ,8,f8). I t has, there0.12 mg. of phenylacetic acid per milliliter in a 6 X a/, inch test tube. Acidify the sample with 0.3 ml. of 10N sulfuric acid (pink fore, been considered desirable to extend the analytical method to thymol blue). Add 1.5 grams of anhydrous sodium sulfate and for phenylacetic acid further to the determination of other pre18.0 ml. of toluene from a buret. Shake the mixture vigorously cursors. One group of these precursors consists of phenylacetfor 30 seconds and centrifuge for 5 minutes with the tubes loosely amide and its neutral derivatives-e.g., N-methyl- and N-( 2corked. Transfer 15 ml. of the toluene extract to another 6 X I / , inch test tube. Add 1.0 ml. of 0.1M sodium carbonate and hydroxyethy1)-phenylacetrtmides. This part of the report repeat shaking and centrifuging. Remove most of the toluene deals with the development of a method for determining this layer with the aid of suction, care being taken not to remove any group of compounds. Because free phenylacetic acid can also aqueous layer. Add 0.1 ml. of a 10% solution of potassium ferribe formed from enzymatic hydrolysis of these phenylacetamides cyanide and heat the tubes in a boiling water bath for 10 minutes. While maintaining the water bath a t 70" to 80" C., pass a during penicillin fermentations, a method is also proposed for gentle current of air over the liquid surface for 5 minutes for determining phenylacetic acid in the presence of these neutral complete removal of the residual toluene as judged from the disderivatives. appearance of the toluene smell. Make up the aqueous residue to approximately 1 ml. with water. With this 1 ml. as the sample EXPERIMENTAL to be analyzed, follow the extraction procedure as described above. From the 5 ml. of toluene extract use a 4-ml. aliquot for drying Test Solutions and Reagents. The standard phenylacetic acid and color development. solution, potassium benzylpenicillinate, and the solvents, chloroData collected in experiments in which this modified method form and toluene, used were the same as those already described. was applied are summarized in Table V. Recoveries of phenylPhenylacetamide (Benzol Products), N-methyl-phenylacetacetic acid from the fermented medium or samples containing amide (kindly supplied by the Division of Chemical Development added o-hydroxyphenylacetic acid varied from 103 to 250% when of this institute, melting point 56" C.), N-(2-hydroxyethyl)no re-extraction and oxidation were used, while recoveries varying phenylacetamide (Givaudan Delawanna), and phenaceturic acid from 89 to 120% were obtained when these modifications were (Eastman Kodak) were used without purification. As has been mentioned previously in this report, twice-recrystallized phenyl'q;i$kodified method has also been shown to be applicable to the acetamide n'as used to check the purity of the phenylacetic acid determination of phenylacetic acid in samples of crystalline postandard by the procedure described below. Aqueous stock tassium benzylpenicillin. solutions of these compounds of convenient concentrations, usually 0.03M, were prepared for use in the followingexperiments. Hydrolysis of Phenylacetamide and Its Derivatives. PrelimCONCLUSIONS AND DISCUSSION inary tests showed that the direct application of the modified Although the method developed involves &3% or slightly Kapeller-Adler reaction to the phenylacetamides failed to produce equivalent color intensities as their phenylacetic acid contents. larger errors and is not to be recommended for highly accurate For quantitative determination of these phenylacetamides, it determination of phenylacetic acid, it is certainly reliable enough was considered essential to hydrolyze them into phenylacetic acid for studying problems relating to the metabolism of phenylaceand determine the latter in the hydrolyzate. To find the conditic acid during penicillin fermentations. The procedure is simple tions required for complete hydrolysis, aliquots of stock solutions were mixed with sodium hydroxide or hydrochloric acid a t difand convenient, allowing the handling of a large number of samples ferent concentrations and heated in a boiling water bath for a t the same time. When supplemented with the procedure for devarying periods. The hydrolyzates were then analyzed for termining small amounts of phenylacetic acid, the method can cerphenylacetic acid by the procedure already described in this tainly cover the entire desirable range of phenylacetic acid conreport and the apparent degree of hydrolysis was calculated. Results of alkaline hydrolysis are summarized in Table VI; those centrations which might occur during penicillin fermentations. Table VI.
Alkaline Hydrolysis" of Phenylacetamide and Its Derivatives
',
V O L U M E 2 6 , NO. 9, S E P T E M B E R 1 9 5 4
1437
Table VII. Extraction of Neutral Phenylacetamides, Benzylpenicillin, Phenaceturic Acid, and Phenylacetic Acid from Alkaline Solutions" Compounds Phenylacetamide
Init Concn , J l g /M1
N-methylphenylacetamide N-(2-hydroxyethyl)- phenylacetamide
4.0 4.0 4.0
4.0 4.0
Solvent Chloroform Chloroform Toluene Chloroform Chloroform
Volume Ratio Solvent t o Aa Soln 3 to 1 10 t o 1 8 to 1 5 to 1 10 to 1
Sodium Sulfate in Aq Phase, G / l o 0 RI1
Extractionb Efficiency,
30 30 30 20
90.2,95.0 70.0
20
93
88.5
81.2c 82.1C
5.2 5.2
Toluene 6 to 1 30 41.8 Chloroform 3 to 1 85.0 10 Chloroform 3 to 1 20 65.0 Chloroform 3 to 1 30 81.5 5.2 C hlorof orm 5 to 1 30 85.5 5.2 C hlorof arm 10 to 1 30 87.5,91,5 1 . 0 4 Chloroform 10 t o 1 25 84 5 0 . 2 1 Chloroform 10 to 1 25 82.8 Benzylpenicillind 3 . 0 Chloroform 5 to 1 30 0.0 Phenaceturic acid 5 . 8 Chloroform 5 to 1 30 0.0 Phenylaceticacid 4 . 0 Chloroform 5 to 1 30 0.0 a T h e aqueous phase was made 0.1.V with sodium hydroxide in each case. b The extraction efficiency denotes the per cent of the compound under test extracted after shaking once with the solvent. e Direct hydrolysis of .V-methyl phenylacetamide gave a maximum recovery of 85 to 87% (Table VI). These extraction efficiencies are therefore equivalent to 90 t o 95%. d Benzylpenicilloic acid was actually being dealt with here. From an acidified solution, chloroform extracts approximately 20% of the penicilloic acid. 5.2 5,2
tube. Make the sample alkaline with 0.1 ml. of 1N sodium hydroxide (blue to thymol blue) and add 0.3 gram of anhydrous sodium sulfate. Pipet in 10 ml. of chloroform and shake the test tubes vigorously for 30 seconds. Loosely cork the tubes and centiifuge them for 5 minutes. Remove the upper aqueous layer with the aid of suction and transfer 8.0 ml. of the chloroform layer into another 5 X 6 / * inch test tube. Evaporate the chloroform extract to dryness a t 30" to 35' C. under a gentle current of air. (A relatively low temperature was chosen for avoiding any possible loss through sublimation.) Dissolve the residue in 1 ml. of 5N sodium hydroxide. Heat the tubes in a boiling water bath for 60 minutes with the tubes loosely corked. Carefully neutralize the hydrolyzate by adding, dropwise, 0.2 ml. of concentrated sulfuric acid, while keeping the test tube immersed in cold water. The resulting solution should be acid toward thymol blue. Determine the phenylacetic acid content of the acidified hydrolyzate with the procedure described in the second part of this communication. No sodium sulfate needs to be added. A correction factor of 92YGrecovery must be applied to the calculation to account for the efficiency of the extraction by chloroform. Procedure for Determination of Phenylacetic Acid in Presence of Neutral Phenylacetamides. Further studies on differential extraction showed that it was not feasible to separate both phenylacetic acid and the neutral phenylacetamides from benzylpenicillin. Chloroform extracts from an acidified solution, not only phenylacetic acid, neutral phenylacetamides, and phenaceturic acid but also a part of benzylpenicillin or its degradation products from both acid or alkali inactivation. On the other hand, extraction with toluene-a nonpolar solvent-from an acidified solution failed to effect complete extraction of the phenylacetamides (Table VII). The procedure proposed here appears to be the only feasible method to determine phenylacetic acid in the presence of neutral phenylacetamides. A 2-ml. sample is made alkaline with 0.2 ml. of 1 N sodium hydroxide and 0.6 gram of anhydrous sodium sulfate is added. The mixture is extracted with 10 ml. of chloroform in the same manner as described for determining phenylacetamides. The chloroform layer is removed with the aid of suction, care being taken not to remove any aqueous layer. The extraction and the removal of chloroform are repeated twice. The aqueous layer is then acidified with 0.2 ml. of 10N sulfuric acid and extracted with 10 ml. of toluene, and the phenylacetic acid in the toluene extract is determined according to the procedure already described.
with acid hydrolysis were not as satisfactory and are not presented. Different derivatives of phenylacetamide were hydrolyzed a t different rates. To ensure complete hydrolysis of all the compounds tested, heating in 5N sodium hydroxide a t 96" to 98' C. (boiling tyater bath) for 60 minutes was required. The low recovery in the case of N-methyl-phenylacetamide was explained by the probably low purity of the compound used, since longer heating failed to increase the apparent degree of hydrolysis any further. Extraction of Phenylacetamide and Its Neutral Derivatives. I n order to separate the neutral phenylacetamides from acidic substances-e.g., phenylacetic acid, benzylpenicillin, and its degradation products-extraction from alkaline solutions was tested. The procedure followed was similar to that of extracting RESULTS phenylacetic acid by toluene (for details of the procedure, see a later section). Aliquots of the extracts were dried under a current Results on testing the recoveries of phenylacetamides, repreof air, the residue was hydrolyzed in 5 N sodium hydroxide, and sented by X-(2-hydroxyethyl)- phenylacetamide and phenylthe phenylacetic acid in the hydrolyzates was determined. From the quantity of phenylacetic acid determined, the extraction acetic acid added to unfermented and fermented cornsteep media, efficiencieswere calculated. Data showing the effects of different as determined by the procedures described, are summarized in factors affecting the extraction efficiency are presented in Table Table VIII. Fairly satisfactory results were obtained in every VII. Over 90Y0 (average being estimated a t 92%) of the three case. Of course, errors introduced through hydrolysis and chloroneutral phenylacetamides could be extracted from their aqueous solutions when one volume of the aqueous phase, which was form extraction enhanced the over-all error to more than f3%. saturated with sodium sulfate (30 grams per 100 ml.) was exAs the data show, however, an accuracy within 10% of the known tracted with 10 volumes of chloroform. The low extraction effivalues has been obtained in practically all cases. ciency of K-methyl-phenylacetamide was again explained by its probably low purity. Toluene was a far poorer solvent for exDISCUSSION AND CONCLUSIONS tracting these compounds than chloroform. The extraction efficiencies a t lower initial concentrations of these compounds were I t can be seen from the data collected in the present study that somewhat lower, data with K-(2-hydroxyethyl)- phenylacetamide the procedures developed involve certain inevitable errors and being given as an example. Table VI1 also presents data showing that benzylpenicillin and phenaceturic and phenylacetic acids are not to be recommended for uses where high accuracy is emwere entirelv not extracted from their alkalink solutions by chloroform. These compounds, therefore, do not interfere with the deTable VIII. Analysis of Mixtures of N-(2-Hydroxyethy1)- Phenylacetamide and termination of neutral phenyl Phenylacetic Acid in Samples of Fermentation Media acetamides by chloroform extrac.V-(Z-hydroxy- Phenylethyl)- Phenylacetic N-(Z-hydroxyethyl)Phenylacetic Acid tion. acetamide Acid Phenylacetamide Found Found Recommended Procedure for Determining Neutral Phenylacetamides. On the basis of these foregoing studies, the following p r o c e d u r e h a s b e e n recommended for determining neutral p hen y l a c e t a m i d e s in samples from penicillin fermentations. Place a 1-ml. sample of cellfree filtrate a in 5 X 5/8-inch test
Samplea Unfermented cornsteep medium
Added, Mg:./%fl. 0 0.25
Added, >fg./M1.
Mg./ml
*
Recovery,
%
Recovery,
%
hlg./ml.
0 0 0
1.04 Water 0.16 0.96 0.141 88.2 0,845 88.0 Unfermented corn1.66 1.28 1.48 89.5 1.18 91.2 steep medium 0.16 0.96 0.142 89.0 0.89 92.5 Fermented corn0.926 0.09 0.810 87.6 0.100 111 steep medium 0.129 0.09 0.128 99.2 0.094 105 T h e cornsteep medium contained 2.5% cornsteep ?olids, 3% lactose, and 1% calcium carbonate. The fermented medium refers to the same medium fermented In shaken flasks by P. chrysogenum stram Wis. 49-133 for 72 hours. No phenylacetyl precursors were added during the fermentation. A correction for the efficiency of chloroform extraction (92%) has been applied in the calculation. ~~
'
ANALYTICAL CHEMISTRY phasized. They are, however, reliable enough for studying problems on the metabolism of phenylacetamides during penicillin fermentations. Repeated use of the procedures in this Iaboratory has shown that they are entirely satisfactory. Differences in the rates of hydrolysis and utilization of different derivatives of phenylacetamide during penicillin fermentations as judged from the analytical results have been clearly demonstrated. Efficiency of chloroform extraction is lower than 90% when the concentrations of phenylacetamides in the samples are low. Of course, the efficiency can always be improved by repeated extraction. However, in so doing, the procedure will be necessarily long and involved. On the other hand, slightly lower recovery a t a low concentration exerts very little effect on the significance of the result. Repeated extraction is therefore not recommended. The method developed has failed to include the determination of phenaceturic acid or other acidic phenylacetyl derivatives, since they cannot be conveniently differentiated from benzylpenicillin. I n the absence of these acidic phenylacetyl derivatives, it seems possible to determine benzylpenicillin by difference. Since, however, a determination by difference always tends to exaggerate the experimental error which is already relatively large in the present method, the results will naturally become of little significance.
Beer, C. T., Dickens, F., and Salmony, D., Biochem. J . , 49, 700 (1951).
Behrens, 0. K., in “The Chemistry of Penicillin,” ed. by Clarke, H. T., Johnson, J. R., and Robinson, R., Chap. 19, Princeton, N. J., Princeton University Press, 1949. Berry, H. K., Sutton, H. E., Cain, L., and Berry, J. S., Unir. Texas Publ. No. 5109, 21-55 (1951). Block, R. J., and Bolling, D., J . Bid. Chem., 129, 1 (1939). Block, R. J., and Bolling, D., “The Amino Acid Composition of Proteins and Foods, Analytical hlethods and Results,” p. 107-9, Springfield, Ill., Charles C Thomas, 1946. Boxer, G. E., and Everett, P. >I., - 4 s ~CHEM., ~ . 21, 670 (1949). Florey, H . W., et al., ”Antibiotics,” Vol. 11, Chap. 18 and 30, London, Oxford University Press, 1949. Higuchi, K., and Peterson, W. H., Ax.4~.CHEM., 21, 669 (1949). Hiscox, D. J., “The Chemical Estimation of the Potency of Antibiotics,” p. 12, Ottawa, Laboratory of Hygiene, Dept. of National Health and Welfare, 1951. Ishima, M., and Tanno, T., J . Antibiotics ( J a p a n ) , 44, 169 (1951).
Kapeller-Adler, R., Biochem. Z., 252, 185 (1932). King, N. K., and Hambly, A4.N., Roy. Australian Chem. Inst. J . & Proc., 17, 403 (1950). Meisenheimer, J., Ber., 36, 4174 (1903). Meisenheimer, J., and Patzig, E., Ibid., 39, 2526 (1906). Mohler, M. E., Bull. SOC.Chim., Paris, 3, 414 (1890). Nishikida, T., J . Antibiotics (Japan),4, 299 (1951). Perlman, D., Botan. Rev., 16, 449 (1950). Seidell, A., “Solubilities of Organic Compounds,” Vol. 11,p. 583, New York, D. Van Nostrand Co., 1941. Waelsch, H., and Klepetar, G., Z. physiol. Chem., 236, 9 2
LITERATURE CITED
(1) Balston, J. N., and Talbot, B. E., ”-4Guide to Filter Paper and Cellulose Powder Chromatography,” p. 76, London, H. Reeve Angel, 1952.
(1935).
Whitmore, F. C., et al., Ind. Eng. Chem., 38, 942 (1946). RECEIVED for review November 7, 1953.
Accepted June 10, 1954
Simultaneous Determination of Penicillin and Penicilloic Acid In Fermentation Samples by a Colorimetric Method S. C . PAN Squibb Institute for M e d i c a l Research, N e w Brunswick,
Penicilloic acid, produced by treating penicillin with alkali, reduces arsenomolybdic acid directly at room temperature (22” to 28” C.) in the presence of traces of mercuric chloride. The absorbance of the molybdenum blue produced, as measured with a 660-mp filter, is proportional to the penicilloic acid concentration used. When penicilloic acid is heated in a boiling water bath in an acid solution (0.lN sulfuric acid), 98 to 99% of its reducing power is destroyed in 5 minutes. Penicillin can be extracted to the extent of 93%by a single extraction with methyl isobutyl ketone at pH 5.5 or lower when the aqueous phase is 80y0 saturated with ammonium sulfate. Penicilloic acid, which remains almost completely (97%)in the aqueous phase at pII 5.5, can also be extracted efficiently (93%) when the pH of the aqueous phase is lowered to below 3.0. Traces of interfering impurities can be determined separately as a blank after destroying the penicilloic acid by heating in an acid solution. Based on these principles, a method has been developed whereby these two compounds can be determined simultaneously in samples from penicillin fermentations.
P
EXICILLIN decomposes in aqueous solutions into penicilloic acid by the action of either an alkali or the bacterial enzyme penicillinase ( 5 , 6 ) . Paper chromatographic evidence ( 1 1 ) has shown that penicilloic acid is invariably produced during the later stages of a penicillin fermentation, possibly as a result of
N. J. such a decomposition. For studying problems related to the formation of penicilloic acid during penicillin fermentations, it is desirable to develop a method whereby these two compounds can be determined simultaneously. The first attempt in the present study consisted of a search for a convenient method for the determination of penicilloic acid. The iodometric titration ( 1 ) or the alkaline reduction of ferricyanide (8, 9) can obviously serve this purpose. However, the titration method is as a rule not as sensitive and simple as many colorimetric procedures while the reduction of ferricyanide is not considered specific enough for samples from penicillin fermentations. Attempts were niade to determine penilloaldehyde Tvhich could be readily produced from penicilloic acid by treatment Kith mercuric chloride in an acid solution ( 5 , 6 ) . It was found during such experiments that in the presence of traces of mercuric chloride, penicilloic acid could directly reduce arsenomolybdic acid at room temperature. Subsequent study on this reaction showed that the intensity of the molybdenum blue produced formed a quantitative basis for determining penicilloic acid. An account of this study is presented in this report. As a second effort in the present study a procedure for a diffeientia1 extraction of penicillin and penicilloic acid from their aqueous solutions was developed. It has long been known that penicillin can be extracted from its aqueous solution a t p H 6.4 with 1-butanol if the aqueous phase is partly saturated with ammonium sulfate ( 3 ) . Henstock made use of this extraction procedure for developing his hydroxamic acid method ( 7 ) . Using the same principle, P6nau et al. ( I S ) also obtained a satisfactory extraction of penicillin a t p R 3.6. I n view of the difference in the acid
