Colorimetric Determination of dydroxyphenylacetic Acid in Samples from Penicillin Fermentations S. C. PAN The Squib6 Institute for M e d i c a l Research, New Brunnvick,
Estimation of the amount of o-hldroxj phenylacetic acid in penicillin samples is necessary in the purification of penicillin. >lost of the known methods for determining phenols cannot be used for this purpose in the analjsis of penicillin fermentations, as it is difficult to differentiate the acid from nonphenolic interfering substances contained in the fermentation medium. i colorimetric method based on Stoughton’s nitrosation method for phenols was developed in which the interfering substances produced a color intensity so low- that it affected the anal3tical result negligibly. The procedure described is simpler and more sensitive than Stoughton’s original method and can determine as little as 3 y of o-hSdroxyphenylacetic acid in a penicillin sample.
T
N . J.
c~oncentrstionof 1 mg. per ml. This stock solution war further diluted t o 40 y per ml. with water. SODILW SITRITE SOLTTIOS. 1 grams in 100 ml. of water. hmx -~CET.ITE. Amvl ac,etate from Eastman Organic Chemicals Diviqion \vas u”sed without furt,her purification. Calibration Curve. After a few preliminary tests, the following procedure was developed for completing the color reaction. Transfer by means of pipets duplicate aliquots of standard ohydroxvphenylacetic acid solution, representing 0, 4, 8. 16, 24, 32, and 40 y t.o absorption tuheP for the Evelyn photoelectric colorimeter. Make up the volume to 1.0 ml. with water in each case. Add 1.0 ml. of 214’ sulfuric acid, followed by 0.1 ml. of the sodium nitrite solution. Keep the tubes loosely corked and heat in a boiling water bath for 10 minutes. Cool, and add 5 ml. of 1 to 3 diluted ammonium hydroxide solution (approximately 5AV). Read t,he per cent transmittance in the Evelyn colorimet’er equipped with a 420-mp filt,er. Set the tube containing only t.he reagents a t 100% t,ransmitt,ance. A plot’ of per cent transmitt,ance against the amount of ohydroxyphenylacetic acid w e d is shown in Figure 1. The yelloncolor follon-s Beer’s law u p t,o a concentration of 30 y per 7 ml. of t’he reaction mixture. There is a deviation from the straight line at o-hytiroxyphenylacetic acid concentrations higher than 30 y per i mi. of reaction mixture.. but the error introduced t’hrough thip deviation amounts to less t,han 5 % up to 40 y of o-hydroxyphenylacetic acid.
HE occurrence of c-hydroxyphenyiacetic acid in t,he mother
liquors left after the crystallization of potassium penicillin has been reported by King and Hambly ( 4 ) and by Sishikida ( 7 ) . It is apparently a product of phenylacetic acid metabolism during penicillin fermentations, because these tlvo compounds are closely related in structure and phenylacetic acid is widely used as a precursor for the biosynthesis of penicillin G (1, 9). Since the occurrence of hy hydroxy phenylacetic acid creates a purification problem in penicillin manufacture, a method for its estimation in samples of fermentation media or crude penicillin has been urgently needed. o-Hydroxyphenylacetic acid is phenolic in nature and can apparently he determined by a number of known methods (6). Preliminary teats revealed, however, that most of these methods cannot be adapted to the analysis of samples from penicillin fermentations, inasmuch as there is no simple ~ a ofydifferentiating o-hydiosvphenylacetic acaid from nonphenolic interfering substances contained in the fermentation medium. -4fter some experimentation, a method based on the same principle as that 0f the method proposed by Stoughton ( I I ) for phenols was developed and was found to be satisfactory. The color intensity developed from the interfering substances in a penicillin fermentation medium was so low that it affected the analytical result to an almost negligible extent. Stoughton’s original method (11) involves the conversion of phenols in an acet,ic acid solution into p-nitrosophenol (quinone monoxime), which exhibits a bright yellow color when the solution is neutralized with excess alcoholic ammonia. Different modifications have been propo-ed ( 5 , 10>1 2 ) and the method has been applied to the analyses of a number of different materials, including petroleum product’s ( 5 $ 12) and phenolic resins (2). As it is known that the nitrosation of phenols can he easily carried out in dilute aqueous acid solutions ( S ) , attempts were made t o modify the method by carrying out the reactions entirely in an aqueous medium. As a result of such a modification, it was shown that not only can t.he procedure be simplified, hut the method can be made somewhat more Pensitive. As little as 3 y of o-hydroxgphenylacetic acid can be accurately determined b y the procedure described.
101 0
’
4
’
8
I6
24
0-HYDROXYPHENYLACETIC
32
40
ACID ,PG.
Figure 1. Calibration Curve for Determination of o-Hgdroxyphenylacetic Acid Per cent transmittance determined with a 420-rnr filter
Each step in the foregoing procedure was studied in some detail. For maximum color production, 10 minutes’ heating in a boiling water bath was shown to he necessary. When the reaction was allowed to proceed a t room temperature, a reaction time of 60 minutes produced a far lower color intensity than was obtained after heating. The color intensity increased with increase in sulfuric acid concentration, reaching a plateau at 1N. Sodium nitrite concentrations lower than that specified lowered the color intensity slightly while higher concentrations tended t o destroy the colored complex. Other alkalies could be used in place of ammonium hydroxide, but the calibration curves obtained were not as satisfactory. I n agreement mith Stoughton’s result (11), the color developed was exceedingly stable. Identical transmittance readings were obtained before and after the colored solution was allowed to stand overnight a t room temperature.
EXPERIMENTAL
Test Solutions and Reagents. STAXDARD 0-HYDROXYPHENYL- Specificity of the Color Reaction. Phenylacetic acid and peniACETIC ACID SoLcrrIoN. The arid war dissolved in water t o a cillin G, both nonphenolic in nature, were found to yield ab-
65
66
ANALYTICAL CHEMISTRY
solutely no color in this test. This is, of course, of great importance for applying the method t.o the analyses of samples from penicillin fermentations, as phenylacet,ic acid and penicillin G are invariably present in t,hese samples. I n agreement with earlier report's ( 6 ) 11), different phenolic compounds produced different coloi intensities. Of about a dozen phenolic substances tested, however, none was found to produce as high a color intensit>- as +hydroxyphenylacetic acid. The color intensity produced by phenol itself amounted to 66% and that by p-hydrosypheri\-lacetic acid, the side chain acid of penicillin X, amounted to 25% of that produced by o-hydroxyphenylacetic acid on an equal molar basis. Phenolic substances which would interfere with the nnalysis are apparently present to only a negligible extent in 3 cornsteep medium either before or after fermentation by penicilliii-produring fungi (see beloiv). -
Table I.
-~ -
Extractiona of o-Hydroxy-phenylaceticAcid and Interfering Substances by Amyl Acetate Init. o-Hydroxyphenylacetic Acid Concn y 1\11
o-Hydroxyphenylacetic Acid Found, r/iri 42 216 448
EY-
traction h Efficiency,
Samples 5% FVater in 84.0 Ratel 2.50 86.2 Water io0 89.0 Unfermented cornsteeli medium I C 14-18 . . Unfermented cornsteep medium I I d 27-36 Fermented cornsteep mediume 17.0 . . a A I-ml. sample was mired with 1 i n l . of I S H,SOr and extracted nit11 8 nil. of amyl acetate. 6 Extraction efficiency denotw perceritazc of o-hydroxyphenylacetic acid rstracted after shaking once with solvent. C The medium contained 2% cornsteep qolids. 2% lactose, and 1% CaCOa d The medium contained 3 . 3 4 , rornstrep solids. 3.5% lactose, and 1";.
-
- A
LaLUa. e Cornsteep
for 96 hours. mentation. ~~~
~
medium I1 fermented xvith P . c h r y s o y e n u m strain Wis. 49-133 No phenylacetic acid was added as precursor during the fer~
~
~-
adapted for use in the Evelvn photoelectric colorimeter, add 0.1 ml. of 0.1'V sodium hydroxide Polution. and evaporate the whole contents to dryness in a 50" t o 70" ('. water bath under a gentle current of air. Dissolve the dried residue in 1 ml. of water and then follow the procedure as described in the section on the calibration curve. Iiistead of obtaining coniplete estract>ionof o-hydroxyphenylacetic acid by repeated shaking with solvent, a procedure considered too long and involved, the distribution coefficient was carefully determined. For convenience, the percentage of o-hydroxyphenylacetic acid extracted after shaking once with the solvent was used to express the distribution coefficient, and is termed extraction efficiency in this report. D a t a collected in determining the extraction efficiency for pure o-hydroxyphenylacetic acid in an aqueous solution and iii estimating the amount of interfering substances which were extracted from samples of penicillin fermentation media are summarized in Table I. With 2.5 volumes of amyl acetate per volume of aqueous solution, the extraction efficiency varied somewhat wit,h the initial concentration of the o-hydrosyphenj-lacet.ic acid. However, within the range of 50 to 500 y per nil., the estraction efficiency varied between 84 and 89%. An average value of 86% could obviously be used as a correct,ion factor and the errors introduced would be within &3%. Data summarized in Table I also showed that when an unfermented cornsteep liquor medium was estracted and tested in the same way, the color intcnsitj- produced depended upon the amount of cornsteep liquor used in the medium. However, even with 3.5 grams of cornsteep solids per 100 ml. of medium, a color intensity equivalent to less than 35 y of o-hydrosyphenylacetic acid per ml. ivas observed. When such a medium was fernientod with Penicillium chrysogenum (\Tis. strain So. 4913:0, for 4 days and tested again, the color intensity was even lower ( l i y per ml.). Therefore, when analyzed with this method, substances in a cornsteep medium which behaved like o-hydrosyphenylacetic acid amounted to an almost negligible value either before or after penicillin fermentation. This is again an essential requirement' in order to make the method applicable to the analysis of fermentation samples. The extraction efficiency could be increased to 90% or higher by saturating the aqueous phase with anhydrous sodium sulfate and using a larger volume of amyl acetate. However, the color intensity from interfering ,substances was also greatly increased by such a treatment. This disadvantage overbalanced any gain in the estraction efficiency. Recoveries. Recovery of o-liydroxyphenylacetic acid added to samples of unfermented and ferment,ed cornsteep media was studied with the extract'ion procedure described. The results are summarized in Table 11. The recovery values, calculated on t,he assumption that the extract,ion efficiency was 86y0in ever!-
Extraction of o-Hydroxyphenylacetic Acid. I n order to separate the o-hydroxyphenvlacetic acid from other ingredienk of a cornsteep liquor medium, estrwtion by a suitable solvent was apparently a feasible means. Preliminary t,ests showed that n number of solvents could be used for this purpose. Among all the solvents tested, amyl acetate \Tap rhosen because it. extr;tctetl the least, amount of interfering sul)stances contained in an unfermented cornsteep medium. The procedure finally adoptetl for extracting the o-hytlrosyphenylacetic acid from samples of fermentation media is as follows: Transfer by means of a pipet, n 1-nil. sample of cell-free filtrate into a 5 X 6 / * inch t,est tube. .ldd 1 ml. of 1N sulfuric acid and 5 ml. of amyl acetat.e. Shake the tubes vinorouslr for 80 s e c o n d s , usyng t h u m b a s stopper. [The same general Table 11. Recovery of o-HydroK?.phenvlacetic .tcid .\cltled to I'enicillirt Fermentation extractlion procedure as used >redia in similar extraction method. Apparent o-Ilyd, OX).o-Tiydroxydeveloped in this laboratory ( 8 ; plirnylaretic o-IIydrouyo-IIyrli~ossphenylacetic was f o l l o w e d . ] Other roriAcid in phenylacerir. plienylar.rtic Bcid t,ainers-e.g., volumetric fla~lii Saml)lrh .kcid Added .kcid Found. Rrcoveredc, Rccoi-eri. 1 , Samplea y , N l r/\ll. ,y/AIl. y/\Il. "; equipped with ground glass Unfermented cornsteep 2 0 . 9 100 117 4 9 6 . 5 90.: stoppers-can also be used, hut medium I 20 9 400 421.0 400.1 100 0 the use of rubber st,oppers or Unfermented cornsteep 31.4 ion 131.6 ion 2 100.2 corks must be avoided. Pour medium I1 137.1 31.4 400 406. n 101 3 t,he contents into 5 X 5,'s inch test tubes aft,er shaking, in caw Cornsteep medium I 16.3 100 109 4 93.1 03 1 fermented 72 hours 16.3 400 412 i 396.2 99.1 o t h e r c o n t a i n e r s a r e used. Centrifuge the tubes for 5 miriCornsteep medium I1 19.7 100 113.0 93.3 93 3 fermented 120 hours 19.7 400 418.7 399 0 90.8 utes to secure a satisfactoriSame samples a s described in footnotes of Table I. separation of the two layers. b A correction factor for extraction efficienry, 86%. has been applied in calculation t o obtain these values Pipet an aliquot of the ami1 Values obtained b y subtracting column 1 from column 3 . acetate extract containing 3 to d Values obtained here by x 100 column 2 30 y of o-hydroxyphenylacetic acid into an absorpt'ion tube C
V O L U M E 2 7 , NO. 1, J A N U A R Y 1 9 5 5 case, ranged from 93.1 to 101.5yo. This was considered satisfactory. K h e n the apparent o-hydrosyphenylaretic acid in the s:tniples (colriinn 1, Table 11),which represents the interfering substaiicea, is not subtracted from the o-li~drosyphenylacetic acid fourid (coluiun 3 , Table II), as is true in the analysis of unknown cnmples, the analytical results obtained are necessarily higher t l i u i i the t,rue o-hydroxyphenylacetic acid content. The effect of tliese unavoidable errors naturally deptlnds up011the o-hydroq-phr~i~-lacetic acid content. \\Iieii this value is 400 y per ml. or higher, the error ititrodured is less than 5% and can be cons i d r t d negligible for most prnctical purposes. As has been mentioned, when the Parnples are tested with other known tnrtliods for phenols-e.g., the use of Folin's phenol reagent (6'--tlie values for the int,etfet%igsubstances are so high :I< to inv:ilidate the significance of the analytical results. Analysis of Potassium Penicillin Samples. Since crystallirie potassium benzyl penirilliti is prepared by amyl acetate estractioii of fermentation broth, the extraction procedure as described Cor fermentation samples rail obviously be omitted. Direct applic-ation of the procedure ax described in the section on the ca1iI)ratiori curve to samples of pot:issium penicillin has been shiiwii t,o he entirely satisfncioi,!-.
67 other phenols, Stoughton's original method or other modificat,ions ( 5 , I O ) niight be prtderred. The present method has been developed on the basis th:i t the iiiterferirig substances contained in samples from ferrnentations of cornsteep medium hy penicillin-producing fungi amount t o an almost negligible value. This, of course, might not, be true with samples of an ent,irely different nature-e.g., blood, urine, etc.-it is not known whether the present method can be applietl to thew sninples. .ACKXOU LED(: I 1 5 Y T
The satnple of o-hydrosvpheii).lacetic acid was kindll- supplied by Helmuth Cords, Iliviaion of Chemical Developmelit,, Squibb Institute. I t was isolated from 3 sample of crude potawium periirillin and tiirlted :It 1 1 T " C. (uncorrected). LITERATI-RE CITEU
Florey. 11. It7,,rat d., "Anti1,ioti Vol. 11. Chap. 18, Oxford University I'WW, 1-0 Haslain, .J.. )17ietteiii. and Newlanil-;.G . , A n a l ~ s t 78, , 340 (19631). Hodgsorr. I f . Ti.. : ~ i i d ) l o o r e , F. FI., .I. ('herif. S o r . . 123, 2499 (1923). King, S . K.. uiid IIaiiihly, .\. S . , R o J ~.41~slralian . Chrm. I n s t , J . d. Proc.. 17, 408 (1950). I.ykkeii, I.., arid Treueder., 1:. S.. arid Z a l i i i . IT., 1st). E s a .
DISCI-SSIOS
(:HEM,, . % S X l , , 1':U.. 18, 109 (194ti). l l i i t o n , I
