1486
ANALYTICAL CHEMISTRY
No trouble was caused by precipitation of such ions as iron and aluminum a t p H values as high as 7.5, providing tartaric acid was present. If large quantities of ions which form insoluble chlorides are present, it is necessary to remove the insoluble compounds by filtration. SUMMARY AND CONCLUSIONS
A rapid, accurate method for determining copper spectrophotometrically is based upon the colored complex formed by 2,2'-biquinoline and copper( I). This complex can be extracted by n-amyl alcohol from a weakly acid solution. The procedure is suitable for samples containing between 0.001 and 10.0% copper, but material containing larger amounts of copper can also be successfully analyzed. The technique required is simple and results are reproducible under routine conditions. After sample dissolution, a trained analyst can complete a single determinntion in less than 15 minutes.
LITERATURE CITED
Bendix, G. H., and Grabenstetter, Doris, IND. ENG. CHEM., ANAL.ED., 15, 649-52 (1943). Breckenridge, J. G., Lewis, R. W. +J., and Quick, L. A,, Can. J . Research, B17, 258 (1939). Gillis, J., Hoste, J., and Fernandez-Caldas,E., Anales edufo?. u. fisol vegetal (Madrid), 9, 585-91 (1950). Greenleaf, C . A., J. Assoc. Ofic.Agr. Chemists, 25, 385 (1942). Hoste, J., A n a l . Chim. Acta., 4, 23-37 (1950). Hoste, J., Heiremans, A., and Gillis, J., Mikrochemie cer. Mikrochim. Acta., 36/37,349-61 (1951). Hoste, J., Research (London),1, 713-5 (1948). Sandell, E. B., "Colorimetric Determination of Traces of Metals," Vol. 111, p. 305, N e w York, Interscience Publishers, 1950. Smith, G. F., and McCiirdy, W.H., Jr., AXAL.CHEM.,24, 371-3 (1952). RECEIVEDfor review February 12, 1953. Accepted July 2, 1953
Determination of Ascorbic Acid by a New Colorimetric Reaction MORTON SCHMALL, CHARLES W. PIFER,
AND ERNEST G. WOLLISH Products Control Laboratory, Hoffmann-La Roche, Inc., Nutley, N. J .
Most methods for the colorimetric determination of ascorbic acid are based on the reduction of 2,6-dichlorobenzenone indophenol or the wupling of dehydroaswrbic acid with 2,4-dinitrophenylhydrazine, both of which are subject to interferences. A new assay method for ascorbic acid involves the reaction with diazotized 4-methoxy-2-nitroaniline in acid medium, followed by development of a blue color in alkaline solution. This color, with a maximum absorbancy at 570 mM, is compared with standards in a suitable photoelectric colorimeter. Because of the sensitivity of the reaction it is possible to determine very small quantities of ascorbic acid. The simplicity of the procedure permits rapid analysis, suitable for routine control. The method is highly specific for the determination of ascorbic acid in the presence of dehydroascorbic acid and all other vitamins normally found in pharmaceutical preparations. It has also been applied to various fruit j~iicesand processed foods.
A
GREAT number of methods have been proposed for the de-
termination of ascorbic acid. The majority of these are based on the oxidatian of ascorbic acid with 2,6-dichlorobenzenone indophenol ( I , 2 ) or iodine, both of which suffer from lack of specificity. A different approach was used by Roe and Kuether 17,8),who oxidized ascorbic acid t o its dehydro form and coupled the latter with 2,4dinitrophenylhydrazine to produce a red color, which is measured photometrically. Another method (18)is based upon the oxidation of ascorbic acid and interfering compounds with cucumber juice and the selective reduction of dehydroascorbic acid with Staphylococcus albus or Escherichia coli. Scudi and Ratish (10, 11) reacted ascorbic acid with a known quantity of diazotized sulfanilamide and determined the excess of the reagent. These authors assumed a reduction of the diazonium salt by ascorbic acid. Weidenhagen and Wegner (IS) treated ascorbic acid with toluene-diazonium sulfate and advanced a mechanism, inr-olving a reduction of the diazonium salt, folloQ-eclby cleavage, rearrangement, and coupling of the reaction product. The 6nnl compound is described as almost colorless. I n order to apply the reaction of ascorbic acid with diazonium salts to the development of a photometric method, it was considered necessary t o find an intensely colored reaction product. Various diazonium salts tested were found t o result in only yellowish-colored compounds, in both acid and alkaline medium,
which color appeared t o be insufficiently distinct from the sample blank such as can be expected from a multitude of preparations. However, when diazotized 2-nitroaniline reacted n-ith ascorbic acid, a yellow color resulted in acid solution, which turned purplish in alkaline medium. When diazotized 4methoxy-2-nitroaniline was used, a vivid blue color was obtained under the same conditions. I t was found that the blue color of the latter compound was more desirable for analytical purposes, as its E:?!. value was considerably greater and the blank due to the reagents was much loll-er than with the 2-nitroaniline reagent. Therefore, diazotized 4-methoxy-2-nitroaniline was adopted as reagent for a photometric method. Owing to the enediol character of ascorbic acid, the reaction was found t o be highly specific for this vitamin in presence of all other knon-n vitamins. The actual reaction mechanism has not yet been fully elucidated. The blue color formrd in alkaline solution is reversible upon acidification. The absorption spectrum of the blue color with a maximum a t 570 mp is shown in Figure 1. Plotting concentration of ascorbic acid against galvanometer readings obtained with a Klett-Summerson photoelectric colorimeter] a graph resulted, as shown in Figure 2. For concentrations between 0.5 and 2.0 mg. of ascorbic acid per 200 ml. of final solution, a straight line was obtained, which, however, did not pass through the point of origin. Therefore] the
V O L U M E 25, NO. 10, O C T O B E R 1 9 5 3 formula for a straight-line function ( 3 )was applied in calculating the results (see calculation). .
1487 Table 1.
Sequence of Addition of, and Amounts of Reagents and Sample (Ml.)
REAGEhTS
Amino Reagent. Technical 4-methoxy-2-nitroaniline (Antara Chemicals) was recrystallized froni ethyl alcohol and dried a t 60" C., melting point 125' C. (corrected). Five hundred milligrams of the pure compound were dissolved in 125 ml. of glacial acetic acid and diluted to 250 ml. with sulfuric acid 10% w./v. This reagent is stable a t room temperature for a t least 2 months. It is also available in pure form as No. 2094 from Eastnian Organic Chemicals or Fisher Scientific Co. Nitrite Reagent. A 0.2% solution of sodium nitrite in water was prepared. Alcohols. Ethyl alcohol or isopropyl :tlcohol ; 2B denatured alcohol may sometimes cause a turbidity. Sodium Hydroxide, 10% w./v. Oxalic Acid Reagent, 0.5y0 oxalic acid in water. Ascorbic Acid Standard. USP ascorbic acid reference standard or its equivalent, dissolved in sufficient 0.5% aqueous oxalic acid so as to result in a final concentration of 0.5 mg. of ascorbic acid per milliliter (prepared fresh daily)
Sample Blank, (If Required)
...
Amino reanent reagent Nitrite reagent Alcohol Oxalic acid reagent Sample Standard Sodium hydroxide.
2 75 5
5
...
10%
25
Water
Reagent Blank 2 2 75
Sample Soh 2 2 75
5
...
...
...
25
25.
...
Standard A 2 2 75
3
5
T o make 200 ml.
In
Standard B 2 2 75 2
...
,.
2
3
25
25
all flasks
In
W
f 0 a
W K
APPARATb S
.I suitable spectrophotometer or photoelectric colorimeter with :I filter having a mavimuni trmiamittance a t 570 mp may be UWti.
200' W W I-
I
0
z PROCEDURE
9
Sample Solution. Two milliliters of amino reagent are pipetted into a 200-mI. volumetric flask, followed by 2 ml. of nitrite reagent. The solution is swirled to the disappearance of the orange color of the amino reagent, 75 mi. of alcohol are added, and the contents of the flask are mixed. A 5 m l . aliquot of the sample or sample extract, diluted to contain approximately 0.5 to 2 mg. of ascorbic acid in a 0.5y0 solution of aqueous oxalic mid is added; the solution is mixed and rendered alkaline by the :iddition of 25 ml. of 10% sodium hydroxide. The volume is brought to the mark with water, and the contents again are well mixed. The blue color developed may be read after 1 minute without necessity of accurate timing, as it reaches its peak within less than a minute. This color is usually quite stable, as in the case of multivitamin preparations. However, when certain samples are assayed, such as fruit juices and chocolate powders, the color may begin to fade within 10 minutes. Standard Solutions. From the ascorbic acid standard, two
0
0
z a
m
j
100.
0.5
Figure 2.
I I I.o I .5 MOM ASCORBIC A C I D
I 2 .o
Concentration us. Galvanometer Readings
standard dilutions are prepared: solution A containing 1 mg. of ascorbic acid and solution B containing 1.5 mg. of ascorbic acid. Both solutions are treated exactly in the same manner as the sample solution. Reagent Blank. The reagent blank is prepared in the same manner as the sample solution, but with omission of the sample. Sample B i d . In case of colored sample solutions a sample blank is prepared, containing the same amount of sample aliquot and all reagents with the exception of the amino reagent. The addition of the reagents is illustrated in Table I. Colorimetry. The photometer, a t a wave length of 570 mp, is set a t zero absorbancy with the reagent blank in all cases where a saniple blank is not necessary. In case of colored sample solutions, the instrument is set a t zero absorbancy with water, and the sum of sample- and reagent-blank readings is subtracted from the reading of the sample solution, while only the reading of the reagent blank is subtracted from the readings of the standard solution.
K
0
CALCULATION
In m Q.
0.10
ao 5
1
= milligrams of ascorbic acid in sample aliquot
where
A B C D E
4 00
500
600 MILLIMICRONS
Figure 1. Absorption Curve C.
5 micrograms per ml.
E: FA. 830
700
= = = = =
milligrams of ascorbic acid in Standard h milligrams of ascorbic acid in Standard B reading (absorbancy) of Standard ii reading (absorbancy) of Standard B reading (absorbancy) of sample
.Is an alternative a standard graph may be prepared by plotting a t least two points obtained with standard solutions. (Range 0.5 to 2.0 mg. of ascorbic acid.) The concentration of the sample solution, which should be within the range of the standards, can be interpolated from such a graph. While the graph was found
ANALYTICAL CHEMISTRY
1488 to be reproducible, it is advisable to run standards with the sample when high precision is desired, especially when a filter photometer is used. DISCUSSION
Excess of Reagents. The quantity of amino reagent used should not exceed 3 ml. of a 0.2% solution. Large amounts will cause a decrease in color intensity. In accordance with the observation of Scudi and Ratish (IO), no interference was found due to an excess of nitrite reagent used in quantities up to 8 ml. of a 0.2% solution. The presence of one of the alcohols mentioned is necessary for coloi development andcolor stability a t the ascorbic acidconcentrations suggested. In the presence of larger quantities of water, the reaction time prior to the addition of sodium hydroxide is increased. In such cases it is advisable to let the solution stand for up to 45 minutes before adding the 25 ml. of sodium hydroxide. When quantities of sample solutions between 5 and 25 ml. are used, about 5-minutes standing time should be allowed for each additional 5 ml. of sample prior to the addition of the sodium hydroxide solution. If because of a very low level of ascorbic acid in the sample solution, an aliquot larger than 25 ml., but not exceeding 50 ml., must be employed, the quantity of alcohol used should be increased to 100 ml. Oxalic acid has been reported an efficient stabilizer of ascorbic acid solutions ( 5 ) . It has been used in this procedure as diluent or extractant in preference t o metaphosphoric acid, since the solubility of the sodium oxalate formed in the final alcoholic solution is greater than that of sodium metaphosphate. In cases where large aliquots of 0.5% oxalic acid extractant containing the ascorbic acid must be employed, the resulting final solution may sh-w a turbidity. Filtration or centrifuging of the solution is necessary in such event prior to colorimetry. A s an alternative, a 0.2% oxalic acid diluent for extractant may be used, as proposed by Ponting ( 5 ) . A total of a t least 25 mg. of oxalate should be present per 200 ml. of final solution in order to ensure good stability of the blue color. For best results, the concentration of sample and standard should be within the range of 0.5 to 2.0 mg. of ascorbic acid. Preferably, the concentration of the sample should lie within the two levels of the standards.
Table 11. Compounds Which Do Not Interfere B Complex Vitamins Thiamine Riboflavin Pyridoxine Pantothenate Folic acid Niacin Xiacinamide
Vitamins A, D , and E Dehydroascorbic acid 2,3-Diketogulonic acid Pantoyl lactone Phenol Glycerol Propylene glycol Tweens
ence. Likewise, when 570 mg. of stannous chloride dihydrate or 100 mg. of sodium sulfite were added to 100 mg. of ascorbic acid, the vitamin was fully recovered. The previously mentioned reducing substances may be present without interference in the following ratio:
$2; Sn
+
3 parts to 1 part of ascorbic acid 2 parts to 1 part of ascorbic acid 3 parts to 1 part of ascorblc acid
+ +
A s can be seen from Table 11, vitamins normally found in multivitamin preparations did not cause interference even when present in a concentration twice that of ascorbic acid. Seither did oxidation and decomposition products interfere, such as dehydroascorbic acid and 2,3-diketogulonic acid, when tested in twice the quantity of ascorbic acid. Pyruvic acid, however, resulted in a red color. Pantoyl lactone, a possible cleavage product from pantothenates, as well as phenols, were found to be noninterfering, when tested in the same ratio of 2 parts to 1 part of ascorbic acid. Solutions containing ascorbic acid together with large quantities of polyhydroxy alcohols and Tweens could be suc~er;~fully assayrd.
.
Table 111. Sample Blank 5
Sample ml Standard ml. Oxalic a d d reagent, ml. .' Acetate buffer, uH 4.0 ml. 5 Formaldehyde, 40%, ml. 2.5 Water, ml.
Sequence of Reagents Reagent Blank
Sample Soh.
Standard A
Standard
2 3
2
5
5
2':5
2.5
B
0
.. 5 3
..
2.5
.. 3
.. 2.5
3 '
,.
Mix, allow flasks to stand for 10 minutes, then add to each a mixed solution of the following, which may be prepared during this time interval. Amino reagent, ml. 2 2 2 2 2 2 2 2 2 2 Nitrite reagent, ml. 75 75 75 75 75 Alcohol, ml.
-4fter mixing the contents of the flasks, add 25 ml. of 10% NaOH t o each one and bring t o the mark with water. If not clear, centrifuge or filter the final solutions before taking colorimetric readings.
Reductones and Reductic Acid. In certain sugar-containing processed foods reductones or reductic acid may be encountered, the presence of which will cause erroneously high results. This interference couId be fairly satisfactorily eliminated by applying the formaldehyde modification, as outlined by Robinson and Stotz ( 6 ) .
All solutions were prepared in 200-ml. volumetric flasks, aa exemplified in Table 111, where a 5-ml. aliquot of the sample solution was used. If a sample of other size seems preferable, a volume of acetate buffer, pH 4.0, equal to that of the sample and half that volume of formaldehyde is required-e. g., 10 ml. of sample, 10 ml. of buffer, 5 ml. of formaldehyde. Buffer, pH 4.0. Dissolve 250 grams of sodium acetate trihydrate in sufficient water to make 500 ml. and bring to 1000-ml. volume with glacial acetic acid.
INTERFERENCES
MODIFICATIONS OF TECHNIQUE
Reducing agents, such as ferrous and stannous ions as well as sulfur dioxide, did not interfere, when present in quantities normally encountered. For example, when 750 mg. of ferrous sulfate heptahydrate was mixed with 100 mg. of ascorbic acid (experiment I), and an aliquot equivalent to 1 mg. of ascorbic acid was assayed, quantitative recovery was obtained. If, however, 1 gram of ferrous sulfate heptahydrate was mixed with 100 mg. of ascorbic acid (experiment 11), the absorbancy of the final solution using a 1-mg. ascorbic acid aliquot was decreased in comparison with a standard of similar concentration. When a sample aliquot of such a solution containing only about 0.5 mg. of ascorbic acid was utilized (experiment 111),the absorbancy found was equal t o that of the standard. Although the ratio of ferrous iron t o ascorbic acid was the same in experiments I1 and 111, the greater amount of ferrous iron in experiment I1 caused interfer-
Alcoholic Oxalic Acid Extraction. When an attempt was made to determine the ascorbic acid content of soluble coffee where it is added as an antioxidant, poor results were obtained. However, quantitative recoveries could be achieved by using the following extraction technique applicable to solids. The sample was well mixed with 50 ml. of a solution of 25 mg. of oxalic acid in ethyl alcohol, the extract was filtered into a 200-ml. volumetric flask containing the amino and nitrite reagents, and the assay was carried out in the usual manner. Immiscible Solvent Extraction. Another convenient technique utilizes the fact that the yellow colored compound is soluble in certain organic solvents, such as benzene. When interference was encountered due to highly colored samples, the basic procedure was followed up to and including the addition of the sample, but carried out in 250-ml. sepa-
V O L U M E 25, NO. 10, O C T O B E R 1 9 5 3
1469
ratore. About 25 nil. of benzene mas added a t that point and sufficient water t o effect formation of two distinct layers. Upon separation the bottom layer was again extracted with two further 25-ml. portions of benzene and all benzene extracts w i e combined in another 250-ml. separator. Twenty-five millilitei s of sodium hydroxide 10% were added, the contents were \+ell shaken, and the aqueous layer was transferred to a 200-ml. volumetric flask. The benzene layer was washed with several portions of water, which were added to the flask and the volume was brought to the mark with water. Standards, run simultsneously, were treated in exactly the same manner as the sample. Iodine Blank. .4n alternative means of preparing the sample blank consists in oxidation of the ascorbic acid in the sample with a 0.01 A; solution of iodine to the appearance of a faint yellow color due to unconsumed iodine. In such a case the amino rcagent should be added to the sample blank. A slight excess of iodine will not cause erroneouqly high readings of the blank. Determination of Total Vitamin C. Since dehydroascorbic acid is considered a physiologically active vitamin, total vitamin C, including both ascorbic acid and dehydroascorbic acid, is often determined in food products. ;Iscorbic acid can be determined as described above, while total vitamin C requires a reduction with hydrogen sulfide, a \ outlined by Bessey ( 1 ) or Rubin et al. (9). The same quantitie. of buffer volution should be added t o the standard as used i n the *ample.
Table VI.
Processed Foods % Recovery
Ascorbic hcid Added, Mg. 5 mg./g. 1 mg./50 ml.
Soluble coffee Beer Grape juice Peaches Apples Figs
99.6 101.7 105.0 101 .o
0 . 4 mg./ml.
1.0mg./lO
g. g.
0 . 6 nig./lO
g.
1.0mg./lO
94.0 97.0
Found, Mg./Ml. Roche method D I method 0.1 0.1
Canned tomatoes
again compared against the 2,6-dichlorobenzenone indophenol method (4). Both results agreed closely. Processed Foods. Other foods and beverages, to which known quantities of ascorbic acid had been added, were assayed (Table TI). I n case of the soluble coffee, the alcoholic oxalic acid extraction was used, while for canned apples, peaches, and figs the formaldehyde modification as outlined under reductones was applied. Biological Fluids. The method holds good promise for the determination of ascorbic acid levels in biological fluids. \\-ark on this subject is in progress. ACCURACY 4ND PRECISION
RESb LTS
Pharmaceutical Preparations. The basic method as drwrihed was applied to the determination of ascorbic acid in various pharmaceutical preparations (Table IV). Good recoveries were 01)tained with tablets and solutions, while in the case of gelatin capsules, the results showed excellent agreement with the U.S. E'. 2,Odichlorobenzenone indophenol method ( 4 ) .
Table I\-.
Aisthe method described is not based directly on the reducing power of ascorbic acid, as with most conventional procedures, it is less subject to interferences. X comparison of results obtained by ESP titration with 2,6-dichlorobenzenone indophenol ( 4 ) against the Roche method is shown in Table VII. The precision obtainable is evident from the same table. Multivitamin tablets and a multivitamin solution were assayed a t four different levels by the method described, resulting in a maximum deviation of 0.8% from the mean.
Jlultivitamin Preparations Roche Method. Vo Rerovery
Tablet mass containing 30 nig. ascorbic acid/g. Glus B complex vitamins. ferrum reductum, fire, Afn, and Fe +
Table VII.
50 mg./g.)
99.0
Tablet rnas8. containing 40 uig. ascorbic acid/gm. plus B complex vitamins, methionine, choline, and Titamins A, D , E Solution, containing 85 nig. asrorbic acid/ml. Ius B roinplex vitamins. vitamins and D, Tween, and propylene glycol
Multivitamin tablets Assay 1
99.3
3
4
1
Mean
100.0 Found Roche method D I method
Gelatin capsules, containing asrorbic acid, vitamins A, D, and E, plus B comolex vitamins Cbocoiate powder, containing ascorbic acid, vitamins A. D, and B complex vitamins
Multivitamin solution Assay 1
118 mg./20
117 mg./20 g.
g.
% Deviation from Mean (Roche Method)
47.5
-0.6
47.8
0.0 fO.8 0.0
48.2 47.8 47.8
Found, Mg./M1.
2
86..5 inz./cav. - .
47.2 46.9 47.5 47.2 47.2 99.6 99.2
..
3
86.0 mn./cav. - .
dccuracy and Precision Found, Mg./Tablet DI Roche method method
+
..
4 XIean
99.4
98.3 98.9 98.7 98.0 98.5
-0.2
4-0.4 f0.2
-0.5
CONCLUSIONS
Natural Juices. When the ascorbic acid content of natural juices was determined, as reported in Table T', the results were
Table T'.
Sample Frozen orange juice Canned grapefruit juice Frozen lemonade Canned tomato juice
Natural Juices
Dilution 1 part plus 3 parta
water
1 part plus 4 parts water
Ascorbic Acid Found, iVg./MI. Roche DI method method
0.44
0.43
0.35 0.048
0.33 0.048
0.13
0.14
The proposed method offers several advantages over heretofore used procedures. 1. The method has a high degree of specificity due to the enediol grouping of ascorbic acid. Other methods are based on the reducing power of ascorbic acid or-i.e., the dinitrophenylhydrazine method-required oxidation to dehydroascorbic acid prior to colorimetry. With the proposed method, 2,3-diketogulonic acid does not produce a color and reducible substances do not interfere when present in quantities usually encountered in multivitamin preparations. The procedure is very rapid and simple, suitable for routine control. It can be carried out directly, requiring extractions only in unusual cases.
ANALYTICAL CHEMISTRY
1490 I t s sensitivity permits the determination of quantities down to 0.5 mg. with a low limit of 10 micrograms per milliliter, when a sample aliquot of 50 ml. is used. The method shows good precision and its accuracy compares favorably with conventional procedures. ACKNOWLEDGMENT
The authors are indebted to E. G. E. Shafer and Norbert Steiger for their helpful suggestions, and to James Brydon for his assistance. J. T. Woods, Calco Chemical Division, American Cyanamid Co., kindly provided a number of diazonium salts, while technical 4methoxy-2-nitroaniline was furnished by .\ntara Chemicals, Division of General Dyestuff Corp. LITERATURE CITED (1) Bessey, 0. A,, J . Bid. Chem., 126, 771 (1938).
(2) Gyorgy, Paul, “Vitamin Nethods,” Vols. I and 11, New York, Academic Press, 1950. (3) Lange, N. A., and Forker, G. %I., “Handbook of Chemistry,” 6th ed., Appendix, p. 27, Sandusky, Ohio, Handbook Publishers, 1946. ( 4 ) “Pharmacopeia of the United States,” p. 686, XIV rev., 1960. (5) Ponting, J. D., IND. ENG.CHEX, ANAL.ED., 15, 389 (1943). (6) Robinson, W. B., and Stots, E., J . Bid. Chem., 160, 217 ( I W 6 ) . (7) Roe, J. H., and Kuether. C . A , , Ibid., 147, 399 (1943). (8) Roe, J. H., and Kuether, (’, .\., Scicnce, 95, 77 11942). (9) Rubin, S.H., Jahns, F. W., a n d Haue~rifeind,J. C., Fruii P r o d ucts J., 2 4 , 3 2 7 (1945). (10) Scudi, J. V., and Ratish, B . D , , 1 x 0 . EKG.CHEM.,AN.\I.. En., 10,420 (1938). (11) Ibid., 11, 98 (1939). (12) Stewart, A. P., Jr., and Sharp, P. F.,Ihi‘d., 17, 373 (1946) (13) Weidenhagen, R., and Wegner, H., Ber., 72, 2010 (1939). RECEIVED for review February 17, 1953. .iccepted July 25, 1953. l’resented at the hleeting-in-hliniature of the North .Jersey Section, . 4 ~ l E R I c A x CHEMICAL SOCIETY, January 26. 1953.
Determination of Penicillin G in Penicillin 0 J. L. JOHNSON, W. A. STRUCK, E. J. SCOTT, AND J. E. STAFFORD The Upjohn Co., Kalamazoo, Mich.
This investigation was undertaken to devise a method for determining the benzylpenicillin (penicillin G) cantent of allylmercaptomethylpenicillin (penicillin 0). Penicillin 0 is useful in the treatment of patients who are sensitive to penicillin G; therefore, an accurate determination of small amounts (< 1%) of penicillin G that occur in penicillin 0 is important. Oxidation of penicillin G with alkaline permanganate yields benzoic acid which is determined spectrophotometrically. Interfering oxidation products, which derive predominantly from penicillin 0, are removed by simple extractions. Studies of known added increments of penicillin G have shown a recovery of 86 i 4%. The method is directly applicable to potassium penicillin 0, and a preliminary extraction of the organic base makes it applicable to the procaine or 2-chloroprocaine salt. The method has been found to be the most generally applicable and sensitive for determining small amounts of penicillin G in penicillin mixtures.
A
LLYLMERCAPTOMETHYLPENICILLIN (3), penicillin 0, has therapeutic activity comparable to benzylpenicillin, penicillin G, but elicits allergic manifestations in a much lower percentage of patients (1). In producing penicillin 0, the formation of small amounts of penicillin G has not been prevented because of the penicillin G precursors inherent in the fermentation medium and in the metabolic products of the mold by which it is produced. For these reasons, a sensitive method for measuring the penicillin G content is essential.
R-CONH-CH-CH”
I
OH
S
t
\
CHa $CHs
C-N+H-COOH Penicillin
Penicillin 0, R = CHF=CH-CH*-S-CH~Penicillin G, R =
O--CH~-
The determination of penicillin G as a major constituent has received considerable attention (2, 6, 8, 9, 13-15, 18). The techniques represented by there methods are gravimetry and infrared and ultraviolet spectrophotometry. These approaches lack the sensitivity necessary for the determination of penicillin G in mixtures in which penirillin 0 predominates. As a minor constituent, penicillin G has been measured by a colorimetric method which is an adaptation (4, 5. 10, 16) of the Kapeller-Adler reaction for phenylalanine (11). I n the mixtures under consideration here, this method was not easily made reproducible, especially in the hands of different technicians. Del Vecchio and Argenziano ( 7 ) used permanganate oxidation and nitration to measure penicillin G colorimetrically. Their oxidation was carried out under acid conditions which undoubtedly incurred a serious loss of benzoic acid through oxidation. This has also been observed by Leman and Montaigne (1.2). Philpotts, Thain, and Twigg (17) reported the use of permanganate oxidation and a spectrophotometric measurement of benzoic acid based on its absorption maxima a t 273 and 281 mp. T o achieve the sensitivity required for determining the small amounts of penicillin G in penicillin 0 (0.1 to 0.5%) and to avoid the interferences that penicillin 0 introduces, the basic method of Philpotts, Thain, and Twigg has been modified extensively. The benzoic acid is extracted from the ultraviolet absorbing materials resulting from oxidation of penicillin 0 and determined by means of its absorption maximum a t 224 mp (Figure 1). The use of this maximum gives a fifteenfold greater sensitivity than the maxima in the 270- to 280-mr region. REAGENTS
All chemicals are reagent grade Potassium hydroxide, 2.5% w./v. solution Potassium permanganate, crystals and 4.5% n-./v. solution Oxalic acid dihydrate, 10% w./v. solution Sulfuric acid, 18 N Sodium chloride, finely powdered crystals Chloroform Ammonium hydroxide, 0.1 N and concentrated PROCEDURES
Potassium Penicillin 0. A 300-mg. sample of potassium penicillin 0 is weighed into a200-ml. round-bottomedflaskfitted with a reflux condenser. One milliliter of 2.5% potassium hydroxide
