Hydroxylamine Method of Determining Penicillins J.\RED H. FORD, Research Laboratories, The Upjohn Company, Kalamazoo, *Vich. effect of interfering substances. The method is based upon the fact that penicillin reacts rapidly with hydroxylamine to give a hydroxamic acid, which forms a purple complex with ferric ion, and this can he determined colorimetrically.
In a search for a rapid and precise method for assaying penicillins in fermentation liquors as well ab finished salts, the hydroxylamine method of Staab, Ragan, and Binkley was modified to include the use of penicillinase-inactivated blanks to eliminate the
Phosphate Buffer. Sterile 2.5 millimolar pH 7 phosphati buffer was prepared by placing the solution in a bottle equipped with two-holed rubber stopper which held a siphon and an airinlet tube attached to a glass tube packed with cotton. Thr entire apparatus was autoclaved for 15 minutes a t 15 pound.* per square inch (1.1kg. per sq. cm.) steam pressure. Acetate Buffer. One volume of 0.1 -11acetic acid was mixed with 4 volumes of 0.1 M sodium acetate. Ferric Chloride. A 10% solution of ferric chloride hexahydrate in 0.1 N hydrochloric acid was used.
A
RAPID and precise method for assaying penicillins in fermentation liquors as well as in finished salts was desired for determining yields in processing. The hydroxylamine method of Staab, Ragan, and Binkley (IS) appeared the most promising of those described in the literature. I t was found to give good results on finished salts that were 50 to 1007, pure, but the values .obtained on fermentation liquors were much higher than those indicated by the bioassay data. This difficulty was overcome by modifying the method to include the use of penicillinase-inactivated blanks. Since only a brief abstract of the original method has been published, the details are given in this paper. The method is based upon the fact that penicillin (3) (I) reacts rapidly with hydroxylamine to give a hydroxamic acid (11)which forms a our& comdex with ferric ion that can be determined colorimetrically (IS). It is very similar to thr mrthod of rip-
. .
S RCOXHCH-CH
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t
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C(CH3)Z
I
+ NHzOH +
CO-N-CHCOOKa
S
I
7‘ (CH3)
RCOSHFH--$Xd
2
I
c=o AH-~H-COON~ I
NHOH
I1
S
RCOSHCH--CH
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C(CH3)z
I
COOH kH---&HCOOSs
I11 mann and Tuttle ( 7 ) for the determination of acyl phosphates. This reaction is not specific for penicillin, since many esters, anhydrides, and amides also react with hydroxylamine to produce hydroxamic acids. The hydroxylamine can also react with aldehydes or ketones to give oximes that form colored complexes with ferric ion ( l a ) . However, the effect of these interfering substances can be eliminated by the use of a blank which differs from the test solution only in the fact that the penicillin has been hydrolyzed to penicilloic acid (111)by means of the enzyme penicillinase (4). The action of this enzyme is believed to be specific for penicillin and thr resulting penicilloic acid does not interfere
PROCEDURE
Samples of solid penicillin salts were dissolved in sterile 2.5 millimolar phosphate buffer and diluted to a concentration about 0.25 to 2.0 millimolar. For benzylpenicillin (penicillin G) a 1.0 millimolar solution is equivalent to 594 Oxford units per ml Fermentation liquors, if not previously clarified, were filtered through pa’ er. To a 5-ml. portion of the test sample was added one drop ofpenicillinase solution and the mixture was allowed t o stand a t room temperature for 4 hours. It was found by Fred Hanson of these laboratories that incubation a t 3 8 O for one hour was equally effective. Two milliliters each of the test solution and the acetate buffer were pipetted into a macrotube for the Evelyn photoelectric colorimeter. Two milliliters each of the penicillinase-inactivated test solution and acetate buffer were placed in a second colorimeter tube. (The colorimeter tubes should give identical readings with distilled water. The Evelyn “selected” tubes were found to be well matched but the Klett “calibrated” tube? varied as much as 10 scale divisions. From 90 of these “calibrated’’ tubes the author obtained a group of 12 that matched. The interval timer was started and 2 ml. of the hydroxylamine solution were pipetted into each tube. When the timer read exactly 5 minutes, 2 ml. of the hydrochloric acid and then 2 ml. of the ferric chloride solution were pipetted into each tube. In all cases the reagents were added to the sample tubes first. When the timer read about 9.5 minutes the blank tube was placed in the instrument (540 filter) and the zero point adjustment was made At exactly 10 minutes the reading on the sample was taken. With the Klett-Summerson instrument (Klett 54 filter) thtprocedure was the same, except that 1-ml. volumes were used. The above timing schedule makes it convenient to run test qamples in groups of five. When this is done it is advisable t o use volumetric pipets only for measurement of the sample and serological or “blow” pipets for measuring the reagents, as the! deliver more rapidly. STABILITY OF COLOR
The use of a strict timing schedule, especially for the interval between addition of the ferric chloride and reading the colorimeter, was made necessary by thr unstable nature of the color The change of color intensity with timc, using a 1millimolar penicillin solution, is shown in Table I. Table I.
REAGENTS
Penicillinase Solution. The contents of a 100,000-unit vial of Schenley Penicillinase A were dissolved in 25 ml. of distilled water and placed in a dropping bottle. This solution was found to be stable for periods as long as 3 months when stored in a refrigerator. Hydroxylamine Solution. A 4.0 M solution of hydroxylamine hydrochloride (70 grams of reagent grade hydroxylamine hydrochloride in 250 ml.) was mixed with an equal volume of 3.0 M sodium hydroxide. The resulting mixture (pH 6.4) has a limited stability and should not be used more than a few hours after it has been pre ared. The hydroxylamine hydrochloride solution may be storex for several weeks a t room temperature.
Change of Color Intensity with Time
Time after Addition of Ferric Chloride, Minutes 2 4 6
10
Relative Intensity of Color 100 78 69 5%
~~
Several attempts were made to stabilize the color by derreasinp the acidity. Citric acid held the iron in solution but changed the color c6mpletely. Oxalic acid, sulfuric acid, and the hydribchlorides of glycine, pyridine, and nicotinic acid gave satisfactor! colors but did not improvr thr strthilitp.
1004
DECEMBER 1947 Table iI.
1005
Effect of Phosphate Ion
Phosphate Concentration, Mole per Liter 0.0 0.0025 0.25
Relative Intensit3 of Color 100 99 58
Table 111. Effects of Interfering Su,bstances on Color Produced by 1.0 m M . Sodium Benzylpenicillin Relative Intensity of Color When Blanks Contained: Hydroxylamine, Penicillinaseferric chloride, and inactivated Interfering Substance hydrochloric acid test solution Kone 100 100 111 10% acetone (vol.) 85 101 Amyl acetatea, saturated solution 108 98 Acetamide, 100 mg./ml. 438 a Technical grade (Pentasol, Sharples).
Kith the Klett-Summerson instrument this range was from about 2 to 4 millimolar. The results obtained with benzylpenicillin (G) a t 2 5 O are given in Table IT (averages of 6 determinations). Using the Evelyn colorimeter, penicillins F, K, and X Rere found to obey Beer's law over about the same concentration range but the colors produced were slightly less intense than with G The relative intensities of color, measured in the range where Beer's law is applicahle, are listed in Table V.
Table IV.
Evelyn L" I,/ m M 0.0268 0.214 0.0788 0.202 0.100 0.200 0.148 0,197 0.198 0.198 0.297 0.198 0.396 0.198 0.780 0.195 1.48 0.188 2-log of galyanometrr reading. galvanometer reading (logarithmic scale). ~
U l ~ L
0.126 0,375 0,500 0,750 1 .oo 1.50
2.00 4.00 8.00 INTERFERING SUBSTANCES
Results with Benzylpenicillin
a b
L G
= =
.
Gb
Klett G/mXl
..
3'4 42 59 75 108 142 279
91 84
79
75
'
72 71 70
..
Effect of Phosphates. Lipmann and Tuttle reported that in their acyl phosphate determinations the color intensity of the iron-hydroxaniic acid complex was depressed by certain anions, Khile the differenwh in rolor intensity on a molar basis are including fluoride and phosphate and to a lesser extent oxalate rather large, they do not detract seriously from the usefulness of and sulfate. In the present investigation the effect of phosphates the method. For example, a mixture of 60% G, 15% F, and 286;; has been studied. The others have been neglected, as it seemed K would give a molar intensity of 97 as compared with 100 for unlikely that they would be present in amounts sufficient to cause pure G. On a weight basis the differences, with the exception of trouble. penicillin K, are less pronounced. The pronounced effect of phosphate ion can be seen from the The two aromatic penicillins G and X have higher molar illresults given in Table 11. A 1.0 millimolar solution of sodium tcnsities than do the aliphatic penicillins F and K. In only one benzylpenicillin was tested in the presence of p H 7 phosphate ( 5 )of the other published chemical methods ( I , $ , 6,8,9,11) has 8 buffer. direct comparison of results on the various penicillins been r e In the present investigation 2.5 millimolar phosphate buffer corded. was used to dissolve all solid penicillin salts. An average of ten Impure Mixed Penicillins. h n accurate comparison betweeil determinations indicated that it caused a 1% depression in the chemical and biological penicillin determinations is imposeiblr color intensity, but this is probably within the experimental error. because of the wide variation in biological potency (see Table V) Other Interfering Substances. Since the reaction with However, a series of comparisons indicated that the methods hydroxylamine to produce hyLiroxamic acids has been reported (6') agreed a,? well as could be cTxpertt'd (see Table VI). The bioto take place with amides, anhydrides, and esters, the effects of assays were carried out in three control lahoratories within The amyl acetate and acetamide as interfering subatances were studied briefly. Furthermore, as oximes have been reported to give a color test with ferTable V. Relative Color Intensities ric chloride ( l a ) ,acetone was also studied. .1 1.0 Penicillin G X F h' millimolar solution of sodium benzylpenicillin was K (see Formula I) ChHsCH2p.HOCeHsCH1-CACHXH=CHCH?-7l-CiHis tested by two methods in the presence of the interbIolecuiar weight 364 fering substances. First, the comparisons were Relative of sodium oolor salt in3nf3 372 334 made using a blank containing only hydroxylamine, tensity, molar 92 basis 100 99 93 ferric chloride, and hydrochloric acid in order to deRelative color intensity, weight termine the increase in color intensity caused by the 90 basis 100 45 99 reaction products of hydroxylamine and the interBiological activity, Oxford units per 230U feringsubstances. In the second series the samples mg. 1667 900 1550 were read against penicillinase-inactivated test solutions which contained the interfering substances. Table 1'1. Impure Mixed Penicillins The results are summarized in Table 111. Bioassay I n all cases the increase in Color intensity caused tJy the interNO. Colorimetric hssay Lab, 1 Lab. 2 fering substances was corrected by using a penicillinase-inactiA. Crude Calcium Salt6 vated portion of the test solution as the blank. The loa- result obtained in the presence of 10% acetone mas probably due to the fact that the amount of hydroxylamine that reacted with the acetone was great enough to decrease suhstantiallg the amount available for reaction with the penicillin.
K-4,20 K-4721 K-4724 K-4725 26-AJF-2' X1-.4 JF-20
RESULTS OBTAINED
Pure Penicillins. The relationship between color intensity and concentration was studied on four different penicillins, using sohtions that were prepared from analytically pure sodium salts. (-sing the Evelyn instrument, the color was found to be directly proportional to the concentration over the range of 0.4 t o 4.0 millimolar (240 to 2400 Oxford units per ml. for prnirillin G ) .
K-47 19 K-4719s K-4720 K-4721 K-4723 K-4722 K-4725 a
1080,1080 1080,1090 1200,1220 1210, 1230 970,1010 1160 1640,1620 16.50. 1640
1080 1200 1350 1290 970 1020 1640 1716
B. Fermentation Liquors 430,460 480 320.330 360 470, 490 480 380,420 420 530, 550 610 470,460 430 480, 420 420
Crystalline sodiuni salts.
1240 1230 1330 1450 1180 1280 1780 1.560 420 300 520 380 520 540 440
1170 1130 1240 1590 1140 1380
.~~ ...
.. ..
V O L U M E 19. NO. 1 2
1006 Upjohn Company, using an adaptation of the hollow-cup agarplate method (IO). ACKNOWLEDGMENTS
The author is indebted t o Fred Hanson and Mrs. Gayle Hinckley for the bioassays; t o Schenley Laboratories, Inc., for the penicillinase; and to the following people for the samples of pure crystalline penicillin sodium salts: Roberta Harris for penicillins F and G, D. W. RlacCorquodale of Abbott Laboratories for penicillin K, and A. F. Langlykke of the Xorthern Regional Research Laboratories for penicillin X. LITERATURE CITED
(1) Alicino, IND.ENO.CHEM., ANAL.ED.,18, 619 (1946). (2) Allinson, Proc. SOC. Exptl. Biol. Med., 60, 293 (1945).
Committee on Medical Research, O.S.R.D., Sn'ence, 102, 627 (1945). Foster, Zbid., 101, 205 (1945). Herriott, J . Biol. C h a . , 164, 725 (1946). Karrer, "Organic Chemistry," 2nd English ed., p. 212, New York, Elsevier Publishing Co., 1946. Lipmann and Tuttle, J. Biol. Chem., 159, 21 (1945). Mundell, Fischbach, and Eble, J . Am. Pharm. Assoc., 35, 37'3 (1946). Murtaugh and Levy, J. Am. Chem.SOC.,67, 1042 (1945). Savage and VanderBrook. J . Bad., 52,386 (1946). Scudi, J . Biol. Chem., 164, 183 (1946). Shriner and Fuson, "Identification of Organic Compounds," 2nd ed., p. 68, New York, John Wiley & Sons, 1940. Staab, Ragan, and Binkley, Abstracts of 109th Meeting, AM CHEM.SOC., p. 3B (April 1946). RECEIVEDRIay 15, 1947
Determination of the Alcoholic Hydroxyl Group in Organic Compounds Phthalic Anhydride Method PHILIP J. ELVING' A N D BENJ. WARSHOWSKYz,Publicker Industries, Znc., Philadelphia, Pa. Phthalization with phthalic anhydride'in hot pyridine solution can be readily applied to the determination of alcohols or of the alcoholic hydroxyl content of complex mixtures of the type obtained in vapor-phase catalytic reactions involving alcohols. Aqueous alcoholic solutions can be analyzed. Aldehydes and other compounds found in such condensates do not interfere. Phenolic hydroxyl groups do not react. Amines react quantitatively or to excess. Polyhydroxy compounds with primary and secondary hydroxyl groups give satisfactory results; tertiary hydroxyl groups do not give satisfactory results. Volatile alcohols can be analyzed without difficulty.
I
N THE course of a comprehensive investigation of methods for the quantitative determination of the hydroxyl group in organic compounds, the reaction of phthalic anhydride with various hydroxyl-containing compounds was studied, since it seemed to offer certain advantages over the acetic anhydride and other methods usually used for the estimation of this functional group. A simple method for the determination of the alcoholic hydroxyl group content of organic compounds, based on estcrification with phthalic anhydride in pyridine solution, was developcd and tested. It waa found that the method can be used for the determination of the alcohol content of dilute aqueous solutions, and that such substances as ketones, saturated and unsaturated aldehydea, acids, esters, and phenols do not intcrfere. This method has been successfully applied t o monohydric aliphatic alcohols from C, to Ca. various polyhydric compounds, and complex liquid condensates obtained from organic reactions. The principal advantages of the method described in this paper are its specificity for alcoholic hydroxyl groups and its applicability to aqueous solutions. Its principal limitations are its inapplicability to tertiary hydroxyl compounds due to the dchydration of the latter, and the apparent excessive reaction of certain amines as discussed below. The extensive literature up to 1037 on the dctcrmination of the hydroxyl group is reviewed by Meyer (10). The method most frequently used for the determination of hydroxyl groups, which was first proposed in 1901 by Verley and Rolsing (ZS), is esterification by acetic anhydride in pvridine Jolution. More reccnt modifications by this method are dcscribed by Oyg, Porter, and Rillits ( I d ) and Christeusen arid his coPresent address. Purdue University, Lafayette, Ind. tpresent addreas, Camp Detrick, Frederick, RId.
I
workers (IS). While this method has been successfully applied t o the determination of most primary alcohols and many secondary alcohols, it is not readily applied to volatile compounds; aldehydes, especially those of relatively low molecular weight, interfere by reacting with the acetic anhydride; phenols react partiall? or completcly, interfering with the direct determination of the alcoholic hydroxyl content of the sample; and the presence of more than a small amount of water in the sample renders the determination inexact or impossible (26). The use of an acetyl chloride-pyridine complex suspended in toluene aa the esterifying agent is described by Smith and Bryant ( 2 1 ) ,who extensively investigated the applicability of the method to various types of hydroxyl-containing compounds. This method seems to offer some advantage over the use of acetic anhvdride-eg., the interference of aldchyde is lens, although the presence of aldehydes has an unfavorable effect on the cnd point. Smith and Bryant give the average precision of the accbtyl chloride method as '0.2%; the absolute accuracy of the two methods is said to be comparable and, based on the data given, about 1% relative. Christensen, Pennington, and Dimick ( 2 ) obtained satisfactory results using acetyl chloride without a solvent. Bryant, Rlitchell, and Smith (I) have described a procedure based on the determination by the Karl Fischer method of the water produced on csterification of the hydroxyl group with acetic acid i n the presence of boron trifluoride as catalyst. While the method can be applied to dilute aqueous solutions, phenols react incomplctely, and carbonyl and somc other types of compounds interfere by rearting with the reagent or the catalyst,. Other recently suggested methods for the determination of hydroxyl groups involve reaction with hydrogen iodide ( 1 1 ) , with acid chlorides of the higher fatty acids (15), or with triphenylmethyl chloride ( 1 7 ) .
