Determination of Penicillin G A Spectrophotometric Method GABOR B. LEVY, DENMAN SHAW’, ELDON S. PARKINSON, AND DAVID FERGUS Schenley Laboratories, Inc., Lawrenceburg, Ind. A spectrophotometric technique has been developed for the determination of benzylpenicillin in penicillin G preparations. The method is particularly suited for routine use, but is subject to the inherent limitations of this type of analysis-e.g., interference of extraneous benzyl groups. Readings are taken at only two wave lengths corresponding to the maximum and minimum of a characteristic absorption band. Routine analyses by this technique yielded results comparable to the gravimetric method generally used.
P
ENICILLIN G or benzylpenicillin is distinguished from other penicillins by the presence of the benzyl group in the side chain ( 2 ) . For the determination of the penicillin G content of mixtures of penicillins a physical characteristic due to the benzyl group-e.g., the light absorption in the ultraviolet regioncan be utilized. A spectrophotometric procedure based on this principle has been used by the Northern Regional Research Laboratories of the U. S. Department of Agriculture ( 7 ) and recently other methods of this nature have been published. One such method developed by Grenfell, Means, and Brown ( 5 ) is based on the measurement of the optical density at 263 and 280 mp, Another, reported by Colon and Frediani ( I ) ,utilizes in a somewhat similar 1 Present address, National Advisory Corninittee for Aeronautics, Cleveland, Ohio.
fashion readings taken a t 267, 280, and 320 mp. Philpotts, Thain, and Twigg (8) developed a spectroscopic procedure by which photographs of the ultraviolet spectrum of the samples are taken and matched to spectrograms of standards. This last method is distinguished from the previous ones by the fact that the intensity of the individual bands is the basis of the analysis while the intensity of the background is ignored. In this fashion potential interference by ultraviolet-absorbing materials other than benzyl groups is minimized or eliminated. Similarly, the method presented here is based on the evaluation of the intensity of a single absorption band, above the background absorption. However, a spectrophotometer is used, and thus the method is simpler and better suited for routine analyses. For this reason it is described in detail. PRINCIPLES
P O T A S S I U M PENICILLIN G
@ @
I 9600
2650
66XG
-
3 4 % NON-C
45%G
-
SSXNON-G
@
I NON-G P E N I C I L L I N S
I
9700
WAVE LENGTH, 8,
Figure 1. Absorption Spectra of Mixtures of Penicillin
The inherent limitation of ultraviolet methods of benzylpenicillin determination is the presence of other benzyl groups. In addition, however, most methods are sensitive to some degree to ultraviolet absorption exhibited by compounds that contain no benzyl group. This ultraviolet absorption may be termed “background,” superimposed upon which is the benzene band spectrum. To separate the latter from the former, graphical methods can be used-e.g. the base-line technique (IO),application of which is shown in Figure 1. Curves 1 and 4 illustrate how the magnitude, E , may be used to evaluate the quantity of benzyl groups present-Le., benzylpenicillin. The general or background ultraviolet absorption may shift the curves upward or in an irregular fashion; nevertheless, the specific band due to benzylpenicillin is present,, superimposed, and can be evaluated. In this form the proposed method is applicable to unknown mixtures and is limited only by the presence of benzyl groups not due to benzyl penicillin. This technique is well suited for research work. Honever, for routine analysis of penicillin G preparations, the qualitative composition of which is known, a simplified technique has been developed. This TTas done on the basis of accumulated experience which showed that commercial penicillin G preparations usually do not contain penicillin X or inactivated penicillin G, but besides benzylpenicillin, only small amounts of penicillins F, K, and dihydro F and some inert pigment. Gndcr these conditions an average value for the angular displacement of the spectrum due to impurities-i.e., the slope of the “background” absorption-together with the measurement of the height of a characteristic band (maximum and minimum) suffices, and affords a rapid screening method for determining the benzyl content of penicillin G preparations. The method requires somewhat greater care in operation than most spectrophotometric procedures but the advantage gained is increased specificity or accuracy. 1159
ANALYTICAL CHEMISTRY
1160 1.0
Table 11. Dependence of AE on Temperature 0.9
Temperature of Cell Compartment, O C .
AE (Mean)
8 17 19 21 25
0.073 0.067 0,062 0.062 0.057
0.8
.J
The origin of this straight-line calibration curve is a t AE = 0, which is an unusual case, occurring when the impurities admixed with penicillin show no ultraviolet absorption. I n practice, materials of the general spectral characteristics shown in curve 5 of Figure 1 are encountered. I n this case the origin of the calibration curve falls to negative AE values, corresponding to the slope of the base line or background. I n the experiment illustrated in Figure 1, the value of AE a t 0% penicillin G content is -0.0176, while for other similar samples values ranging between -0.01 and -0.02 were obtained. Using the value -0.0176, a straight-line calibration curve can be used, as shown in Figure 4. There is a slight variation in the slope of the base line and correspondingly in the intercept of the calibration curve with the Y axis and for this reason it is recommended that this point be firmly established for the type of salt to be analyzed and occasionally rechecked. This point corresponds then to the average or normal impurities. Holvever, the importance of exactly locating this value diminishes Ivith increasing penicillin G content. In refining the method a comparatively large temperature effect was found, as shoxn in Table 11. Consequently, constant temperature should be maintained in the spectrophotometer cell compartment. In addition, daily standards of known penicillin G content may be included with the samples. This seems advisable, particularly in vieiv of the recent findings reported by Ewing and Parsons ( 3 ) . .Is an additional refinement, index lines and magnifiers are used as recommended by Gibson and Balcom (4). By taking these precautions, a precision of the order of 1 to 2% can be achieved, as shown in Table 111.
0.6
2
0.5
0.4
0.3
0.9
0.1
9600
9500
WAVE LENGTH,
Figure 2.
Table I.
9700
800
A.
Absorption Spectrum of Penicillin G
Dependence of AE on Ethyl Alcohol Content
% Ethanol by Volume 94.3 92.4 87.3 71.0 48.9 0
AEo’ % 1 cm. 0.062 0.062 0.056 0.048 0.032 0.018
Reagents. Ethyl alcohol (spectroscopic grade), 95%. Standard penicillin salt of known penicillin G content. Apparatus. Beekman spectrophotometer model DV, equipped Kith ultraviolet light source and four 1-cm. quartz cuvett’es. Research Procedure. Prepare accurately an approximately 0.003 M solution of the standard penicillin salt in 95% ethanol. Determine the spectrogram of this salt a t constant predetermined temperature using 0.50 mm. slit width. Using tke ethanol blank take readings at. every 2.5 A. from 2600 to 2680 A. Calculate the values found as 0.1 yo solution of pure benzylpenicillin and plot the results a3 shown in curve 1 of Figure 1 . Dissolve the un-
EXPERIMENTAL
The ultraviolet light absorption of penicillin G due to the benzyl group appears in bands around 2525, 2585, 2645, and 26!5 .&., as can be seen in Figure 2. Because the 2525 and 2685 A. bands are rather weak, the choice was made between the two other bands named. Because the interference of other penicillins usually present (K a i d F) is less a t the 1on;er wave length and the photocell charactzristics are more favorable, it was decided to utilize the 2615 A. band. It v a s also noted that the use of ethanol as a solvent enhanced the intensity of the absorption band, as shown in Table I, and therefore 95% ethanol was used as a solvent throughout, as recommended by Philpotts et al. (8). The u1travi:let absorption of the sample is measured a t 2630 and a t 2645 A. These two measurements represent a dip and corresponding peak in absorption due to the benzyl group, or penicillin G. The difference of these quantities is expressed by the symbol A E . The AE values were found to be proportional to the concentration of penicillin G Tithin the range of 0.001 to 0.01 M , in accordance with Beer’s law. Consequently, within this range, a t constant concentration of total penicillin or total sample, the A E values are proportional to the percentage of penicillin G. This is shown in Figure 3, which includes data obtained with mixtures of penicillin G and sodium acetate.
0.06
0.05
s” $ 0.04 Y
9
0.03 0.02
0.01
10
90 30 40 50 60 70 PER CENT POTASSIUM PENICILLIN G
80
90
100
Figure 3. Dependence of AE on Concentration of Penicillin G
1161
V O L U M E 20, NO. 12, D E C E M B E R 1 9 4 8
10
20
30
PEPCENT
40
50
60
70
80
POTASS'UM PENICILLIN
90
100
G
Figure 4. Calibration Curve
Cram (6, 9), which is based on the precipitation of penicillin G by N-ethylpiperidine. In Table I11 are shown results obtained by analyzing 12 random samples by the spectrophotometric and gravimetric methods. The mean values (equally weighed) are included in the last column. From the individual deviations from the mean values it was calculated that the average deviation for the gravimetric method is 1.43% and for the spectrophotometric method 1.40%, while the maximum deviation is 4.5 and 5.2%, respectively. This table shows that results by the spectrophotometric method are comparable to those by the gravimetric method when applied properly. The advantage of the method lies in economy, as a skilled analyst can perform about 50 analyses per working day. The authors wish to call attention to the excellent characteristics of the Beckman ultraviolet spectrophotometer which made the establishment of this procedure possible. The whole range of analysis lies within about 5% transmittance value and therefore a 2% precision in the analysis corresponds to a difference in transmittance of the order of 0.1%.
known sample a t a concentration to obtain rgadings of 25 to 70% transmittance in the range of 2600 to 2680 A. Take readings as above, calculate to 0.17c concentration, and plot these values. Measure the height of the absorption bands E above the base ii'ne from the graphs. Calculation.
E sample x 100 =
E standard
Table 111. Comparison of Jlethods of Determining Penicillin G
benzylpenicillin
Routine Procedure. Prepare accurately an approximately 0.003 M solution of the penicillin salt sample in 95% ethyl alcohol, and a similar solution using the penicillin standard. Place these solutions in 1-em. cuvettes and fill an additional cuvette with the 95% ethanol solvent. Transfer all cuvettes into a Beckman DU spectrophotometer, set the slit to 0.50 mm., and balance the instrument with the alcohol blank. LIeasure the absorption of the samples by reaiing the maximum and minimum absorption values of the 2645 A . band. Calculation. Calibration Curve. Divide the AE values found by the concentration of the standard (mg. per ml.) and draw a straight line, using this value for the penicillin G percentage corresponding to the composition of the standard and zero point established for the AE values of normal impurities free of penicillin G (as discussed above). Sample. Divide the AE value found for the sample by the concentration of the sample (mg. per ml.) and multiply by a factor corresponding t o the ratios of the molecular weights (according to cat'ions) of the sample divided by the standard. Read per cent G off the calibration curve. Multiply AE' found by the figure under the salt used corresponding to the cation of standard.
Spectrophotometric Method
XO.
%
Gravimetric Xethoda Lab. I Lab. I1
%
1
a
%
93.0 93.4 96.3 95.8 94.0 2 98.0 97.3 94.6 99.1 98.3 94.8 98.2 3 93.5 95.5 95.5 97.3 4 98.5 96.1 97.3 94.0 96.9 98.6 ,5 98.2 97.1 97.5 96.7 6 94.9 94.0 96.8 94.0 95.1 97.0 97 0 7 9 5.8 96 6 97.5 96.7 94.4 96.0 8 9 6.0 98.2 98.5 97.5 96.3 9 96.0 97.1 91.8 99.0 96.7 92.2 94.0 10 95.5 96.5 96.3 11 96.4 96.9 99.2 93.5 9.5,3 99.5 12 9.5, 1 97.. 2 95.0 9.5.5 98.1 100.0 In Laboratory I1 a modification of the gravimetric procedure
.\lean
% 94.5 97.5 95.2 96.6 97.7 95.3 96.5 96.7 96.3 95,2 96.8 96.8
was used.
ACKNOWLEDGMENT
Notes. In reading the E values a t around 2630 and 2645 i., The authors gratefully acknowledge the cooperation of the the best practice is: balance roughly, move the wave-length dial analytical department of Schenley Laboratories, Inc., valuable slowly back and forth while watching the null point indicator and suggestions of J. L. Wachtel, and the continued interest and thereby establishing the exact location of the minimum and encouragement received from G. E. Vard. The authors thank maximurn points, and a t these points balance with the ethanol Schenley Distillers Corporation and E. C. Williams for perblank and read the extinction value of the sample. miwion to p:thlish this material. The alcohol is considered satisfactory spectrographic grade jf it transmits S5yc or more light, a5 compared to n-ater at 2600 A. LITERATURE C I T E D It can be prepared by percolating the alcohol over Darco G-60. (1) Colon, A . A., and Frediani, H. A., BoZ. col. Q U ~ P.u e r f o Rim, The light absorption by molar ratios for sodium, potassium, ammonium, iV-ethylpiperidine salts was verified and presumably any salt of penicillin G, except those of bases containing benzyl groups, d l absorb in a molar ratio. Therefore a table of these ratios may be set up as needed. If only a few types of salts are to be analyzed it is convenient to draw separate calibration curves for the individual salts-e.g., sodium, potassium, and calcium. RESULTS
This spectrophotometric method has been in use in these laboratories for over 2 years and the results have been satisfactory in that they have been similar to the ones obtained by the somewhat lengthy gravimetric procedure of Sheehan, Mader, and
4, No. 1 (1947). (2) Committee on Medical Research, Offire of Scientific Research and Development, Science, 102, 627 (1945). (3) Ewing, G. W.. and Parsons. T.. Jr.. A s n ~ .CHEM.,20, 423 (1948). (4) Gibson, G. S., and Balcom, M. M., J . Research Nail. Bur. Standards, 38, 601 (1947). (5) Grenfell, C. T., Means, J. A., and Brown, E. V., J . B i d . Chem., 170, 527 (1947). (6) Mader, W. J., and Buck, R. R., ANAL.CHEY.,20, 284 (1948). (7) Melvin, E. H., private communication. ( 8 ) Philpotts, A . R., Thain, W., and Twigg, G. H., Nature, 159, 839 f 1947). \ - - -
I .
(9) Sheehan, J. C., Mader, W. J., and Cram, D. J., J . Am. Chem. Soc., 68, 2047 (1946). (10) Wright, N., IND. EXG.CHEM.,ANAL.ED.,13, 1 (1941). RECEIVED April 14, 1948.
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