A SPECTROPHOTOMETRIC METHOD FOR THE DETERMINATION OF PENICILLIN*

BY ROGER M. HERRIOTT

(From The Rockefeller Institute for Medical Research, Princeton)

(Received for publication, March 28, 1946)

A physicochemical method has been developed by which 5 to 75 y (8 to 120 Oxford units) of penicillin per ml. may be determined within an error of f5 per cent. The procedure requires about 20 minutes for one complete analysis, but a large number of samples may be analyzed at one time. Since crystalline penicillins G, X, K, and F have nearly the same extinction per unit weight, a single measurement of any preparation containing these penicillins in any proportion determines the total penicillin present.

Principle

Crystalline salts of pure penicillins G, X, K, and F, freshly dissolved in acetate buffer, pH 4.6, do not absorb in the region of 290 to 360 rnp (see Fig. 1 and Table II). After heating in this buffer they absorb strongly in this region, with a maximum at 322 rnp. The impurities in crude prepara- tions absorb in this region but the increase on heating is due only to penicillin (see Fig. 3). The present method consists of measurement of the change in absorption at 322 rnl.c produced by heating under controlled conditions and comparing this change to that of a similarly treated standard preparation.

The preparations used are listed in Table I.

Procedure

Two related procedures have been used: the first one, Procedure A, when ample quantities of more concentrated solutions were available; the second, Procedure B, when the concentration or quantity was low.

Procedure A-A 2 ml. aliquot of a penicillin solution containing 20 to 300 y per ml., or roughly 35 to 560 Oxford units per ml., was added to 6 ml. of 0.4 M acetate buifer,‘pH 4.6, in a 30 to 50 ml. test-tube. 4 ml. of this solu- tion were pipetted into another test-tube. One of the tubes was placed in boiling water for 15 minutes, after which it was cooled rapidly to room temperature. The absorption density of the heated and unheated aliquots was determined at 322 rnl.c in a Beckman D.U. quartz photoelectric spec- trophotometer (1).

* This work was done in consultation with the Committee on Medical Research of the Office of Scientific Research and Development.

1 The ratio of molar concentrations of sodium acetate to acetic acid is 1. 725

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726 DETERMINATION OF PENICILLIN

Procedure B-To 4 ml. of a penicillin solution containing 5 to 50 y per ml., or 8 to 80 Oxford units per ml., was added 0.25 ml. of a 5 M acetate buffer solution, pH 4.6.’ The final concentration and pH were close to that in Procedure A. The absorption density of this solution was determined before heating and then again after heating, as in Procedure A.

Notes-(a) The micrograms per ml. in the above procedures refer to pure penicillin. (6) The Et z,,,,. used later in this paper is equal to E: 2. X lo-4 and is the absorption density per microgram per cm. of depth or

TABLE I Materials

Preparation

Bioassay; Staphylococcus

aweus units per mg.

Crystalline sodium penicillin Gt (CrA-152-60)

Crystalline ammonium peni- 1750-2000$ cillin Kt (AV-73)

Crystalline sodium penicillin XP

Crystalline penicillin FII 1632 (175E-ANW-6)

Commercial, Penicillin 1006f 368 ‘I ‘I 9997 1000

Elementary analyses,* per cent

C

53.94 53.75 53.46 53.22

50.27 50.19

- -

-

- H

4.77 4.87 8.13 8.09

N T _

11.69 11.62

5.73 8.38 5.94 8.56

- -

S

8.99 8.92

Na

6.45 6.36

-

-

HZ0

3.5

* The upper reading for each preparation is the calculated value; the lower read- ing, found.

t Supplied by Dr. 0. Wintersteiner, The Squibb Institute for Medical Research, New Brunswick, New Jersey.

$ The Bacillus subtilis-Staphylococcus ratio = 0.3 to 0.4. $ Supplied by Dr. Henry Welch, Food and Drug Administration, Washington,

D. C. Ij Supplied by Dr. G. F. Cartland and Dr. A. N. Wick, The Upjohn Company,

Kalamazoo, Michigan. 7 Supplied by E. R. Squibb and Sons, New Brunswick, New Jersey.

thickness. (c) In Procedure A it was found convenient to analyze the un- heated sam.ple while the other aliquot was being heated. (d) During the time of heat.ing a small funnel in the mout.h of the test-tube served as a reflux condenser. (e) Reproducible conditions must be maintained, par- ticularly when a standard penicillin is not analyzed simultaneously. A constant volume of boiling water, the level of which was kept just above the level of t,he sample in the test-tube, has been satisfactory. Depending on the number of sam.ples heated at one time, the source of heat and volume of water should be such that introduction of the tubes causes the tempera- ture of the water to drop no more than 2-3”.

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R. M. HERRIOTT 727

Conversion of Observed Increase in Absorption Density to Penicillin Concentration

There are several related procedures for accomplishing this conversion which are worth noting for their usefulness under different circumstances. As will be discussed in another section, the change in absorption does not increase exactly linearly with increasing concentrations of penicillin; i.e. the specific extinction, E : zm,., is not constant (see Table III).z Were it not for this slight deviation, conversion would be simple.

The three following procedures may be used. Empirical curves, such as are shown in Figs. 2, A and 2, Bj have resulted

from plotting the averages of the values in Table III. It is important to have a scale large enough to permit a precise conversion, particularly in the region of low concentration (see Fig. 2, B).

A less precise, but convenient, procedure assumes no deviation from linearity; i.e., Beer’s law is obeyed. The observed increase in density is divided by the E:& = 0.022. All results obtained by this method will be correct within f 10 per cent.

Another procedure, which has certain obvious advantages, particularly if the conditions of the procedure have to be altered to any degree, is the comparison of the density of the unknown solutions with those of simul- taneously analyzed standards.

Results

Diferent Kinds of Penicillin-Table II contains the absorption densities at various wave-lengths of 25 y per ml. of crystalline sodium penicillins, G, X, K, and F, before heating, as well as after heating. It may be seen that the different penicillins show almost identical absorption densities at any wave-length between 290 and 400 mp. There is practically no absorp- tion over this range before heating, but after heating the absorption is very strong, with a maximum at 322 mp. This is shown graphically in Fig. 1 where, to eliminate confusion, only the curves of crystalline sodium penicillin G have been plotted.

The results shown in Table II indicate that the spectrophotometric analyses are independent, within the limits already stated, of the nature or proportion of the various penicillins. To determine the extent of any possible interaction or influence of one penicillin on another, particularly during heating, equal quantities of the four kinds of penicillins were dis- solved together and analyzed at different levels of concentration. In every instance the spectrophotometric analysis of this mixture agreed within 5 per cent of the correct total concentration of penicillin.

2 This inconstancy of E : J,,,, was brought to the writer’s attention by Dr. Frank H. Wiley of the Food and Drug Administration, Washington, D. C.

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728 DETERMINATION OF PENICILLIN

E$ect of Penicillin Concentration on Absorption Density-The analyses of various concentrations of crystalline sodium penicillin G are shown in Table III and Fig. 2. It may be seen that the E : &,. increases from about

TABLE II

Ultraviolet Absorption Spectra

25 y per ml. of crystalline sodium penicillinsG, X, K,* and F, before and aft,er heating 15 minutes at 100° in 0.3 M acetate buffer, pH 4.6.

mP

400 375 360 350 345 340 335 330 325 322 320 315 310 305 300 290 286 270 260 250 240

-i Absorption densities, log $

- Before heating

G

0.010 0.000 0.000 0.002 0.004 0.005 0.007 0.009 0.010 0.010 0.010 0.010 0.008 0.007 0.005 0.002 0.000 0.000 0.010 0.015 0.030

- --

-

X

0.000 0.000

0.000 0.000 0.000 0.002 0.002 0.003 0.003 0.002 0.002 0.002 0.000 0.000 0.004 0.070 0.067 0.027 0.033 0.140

F

0.005 0.003 0.003 0.002 0.001 0.002 0.001 0.002 0.004 0.005 0.005 0.005 0 .ooo 0.000 0.000 0.003 0.005 0.007 0.003 0.010 0.044

-

.-

K -

.-

-

G X F

0.003 0.000 0.001 0.002 0.005 0.009 0.010 0.012 0.015 0.015 0.016 0.016 0.013 0.013 0.010 0.007 0.004 0.004 0.007 0.018 0.044

0 .OOl 0.006 0.027 0.115 0.218 0.345 0.445 0.520 0.570 0.580 0.580 0.545 0.470 0.375 0.283 0.135 0.060 0.040 0.070 0.123 0.154

0.000 0.006

0.120 0.220 0.340 0.440 0.505 0.550 0.570 0.560 0.524 0.450 0.360 0.260 0.135 0.130 0.110 0.095 0.145 0.225

0.000 0.002 0.006 0.010 0.030 0.033 0.120 0.120 0.223 0.220 0.343 0.343 0.455 0.450 0.535 0.530 0.595 0.580 0.610 0.590 0.610 0.590 0.590 0.560 0.525 0.490 0.425 0.395 0.325 0.300 0.156 0.142 0.069 0.060 0.045 0.037 0.060 0.053 0.120 0.120 0.190 0.190

* This material was an ammonium salt containing 3.5 per cent moisture. The absorption densities have been corrected to an anhydrous material and to a sodium salt to make it comparable with the other penicillins.

1

After heating

Penicillin

0.0200 at 5 y per ml. to 0.0227 at about 25 y per ml., or about 12 per cent over this range.

Precision and Reproducibility-An examination of the results in Tables III and IV will show how readily reproducible the results are. Occasionally a value is obtained which is out of line, for which no adequate explanation can be given. This is annoying but not a frequent occurrence.

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FL M. HERRIOTT 729

It may be seen from the results in Tables III and IV that individual values deviate from the average rarely more than 5 per cent.

Commercial or Crude Preparations and Interfering Substances-Whereas the purified preparations showed practically no absorption between 290 and 400 rnp before heating, the crude or commercial preparations absorb considerably over this range (see Fig. 3). However, the increase in absorp- tion upon heating is almost directly proportional to the penicillin concen- tration (see Table IV and Fig. 3). The shape and location of the curves obtained by plotting the increase in absorption against the wave-length are very close to, if not identical with, those of pure penicillin. This is

0.6

-=I, 0.5

I-Y

2 0.4 -" 2 : 0.3 ‘G K

2 0.2 <

0.1

0

Wave lenglh - m,u

FIG. 1. Ultraviolet absorption curves of 25 y per ml. of crystalline sodium penicil- lin G, before (0) and after (A) heating 15 minutes at 100’ in 0.3 M acetate buffer, pH 4.6.

strong evidence that only penicillin is responsible for the change in absorp- tion density upon heating.

Table IV shows the results of two series of spectrophotometric analyses of five different commercial preparations carried out on different days. Bioassays3 and Scudi calorimetric analyses4 of the samples were included

3 The bioassays were obtained by a slightly modified Oxford staphylococcus cup method (2) (an unpublished modification from The Squibb Institute for Medical Research and the Biological Laboratories of E. R. Squibb and Sons). The figures are the average of four to eight single assays with six cups per assay. They were made by Miss Helen Jones of the Penicillin Potency Laboratory of E. R. Squibb and Sons, New Brunswick, New Jersey.

4 The calorimetric values (Scudi, unpublished method) represent the average of six to seven analyses of each sample. They were determined in Dr. A. P. Richard- son’s Pharmacological Division of The Squibb Institute for Medical Research.

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730 DETERMINATION OF PENICILLIN

for comparison. It may be seen that the spectrophotometric analyses are in substantial agreement with those obtained by the two other methods.

TABLE III Change in Absorption Density at X9.2 mp after Heating Various Concentrations of

Crystalline Sodium Penicillin G

I Absorption densities, log $

y per ml.

2.5 2.5 2.5 2.5 5.0 5.0 5.0 5.0 7.5 7.5

10.0 10.0 12.5 12.5 15.0 15.0 20.0 20.0 25.0 25.0 30.0 30.0 35.0 35.0 40.0 40.0 50.0 50.0 75.0 75.0

Before After A

0.000 0.053 0.053 0.0212 0.000 0.050 0.050 0.0200 0.004 0.054 0.050 0.0200 0.002 0.050 0.048 0.0192 0.000 0.105 0.105 0.0210 0.005 0.102 0.097 0.0198 0.005 0.098 0.103 0.0206 0 .ooo 0.100 0.100 0.0200 0.000 0.143 0.143 0.0191 0.005 0.157 0.152 0.0203 0.000 0.210 0.210 0.0210 0.003 0.215 0.212 0.0212 0.002 0.267 0.265 0.0212 0.003 0.270 0.267 0.0213 0.003 0.342 0.339 0.0226 0.004 0.332 0.328 0.0218 0.000 0.445 0.445 0.0223 0.004 0.449 0.445 0.0223 0.008 0.572 0.564 0.0226 0.013 0.605 0.592 0.0236 0.013 0.695 0.682 0.0227 0.018 0.700 0.682 0.0227 0.015 0.807 0.792 0.0226 0.016 0.825 0.809 0.0231 0.020 0.925 0.905 0.0226 0.025 0.938 0.913 0.0228 0.030 1.13 1.10 0.0220 0.035 1.16 1.125 0.0225 0.038 1.70 1.66 0.0221 0.048 1.78 1.74 0.0232

-

Thus far no impurity has been found whose absorption at 322 rnp is changed on heating under the conditions of the method. Crystalline or commercial impure penicillins inactivated by 0.1 N HCl or 0.1 N NaOH do not show any increase in absorption when subsequently heated and analyzed

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R. M. HBRRItYi'T 731

by the present method (see Fig. 3). Thus penicillin inactivated, but carried along during purification, will not interfere with the determination.

I.1 1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3

5 0.2 go 0.1 -Observed values

Q '0 IO 20 30 40 50 60 70 80 a Crystalline penicillin G -@/ml.

Crystalline penicillin G -&ml

FIG. 2. Effect of increasing concentrations of crystalline sodium penicillin G on the change in absorption density at 322 rnp brought about by heating under standard conditione.

A great many substances either absorb or scatter light of 322 rnp and when present in sufficient concentration prevent a precise estimation of the penicillin. Blood, urine, and certain microbiological suspensions contain such interfering substances in high concentrations. It has not been possible as yet to separate quantitatively the penicillin from these sub-

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732 DETERMINATION OF PENICILLIN

stances and thus render the spectrophotometric method applicable to such systems.

Extinction-If it is assumed that the above method produces a complete conversion of penicillin into the absorbing material, then the extinction, E : z., for this substance is about 227 (see Table III). The molecular extinction, assuming no change in molecular weight, would be about 8000.

Since the molecular weights (3) of the various penicillins as well as their extinctions (see Table II) do not differ by more than 10 per cent, the molec- ular extinctions of all these heated penicillins will be nearly the same.

350 4 Wave length (mp)

FIG. 3. The ultraviolet absorption curves of 75 7 per ml. of a crude penicillin preparation (No. 1006, about 18 per cent penicillin). l before heating; 0 after heating; 0 increase caused by heating; treated 30 minutes with 0.1 N alkali (0) or 0.1 N HCl (A), then neutralized, and heated in acetate, pH 4.6.

Variables of Method and Their E$ect on Absorption Density-Figs. 4, 5, and 6 bring out the effect of most of the variables of the method on the absorption density of different penicillin preparations. Preparation 1006 was dark red and assayed only 18 per cent penicillin. Preparation 999 was light yellow and was about 60 per cent penicillin, while the crystalline sodium penicillin G was considered 100 per cent.

Other experiments indicate that unheated solutions of penicillin in 0.3 M acetate buffer, pH 4.6, change their absorption densities at the rate of 3 to 4 per cent of the heated absorption value per hour at 26”. Thus, for

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R. M. RERRIOTT 733

TABLE IV Analysis of Commercial Penicillin Preparations

AbsorptioyOdensity,

log i Oxford units per ml. original sample

Per cent Preparation No. peni- Dilution Spectro- Bioassay,

cdlin in water* photometrict scudi hzid Heated A

‘?ipple s%p*e

I&$$ g$;;;

m:%d method

-------

1585 44 1:lOOO 0.0700.48 0.41 119,000 128,000 114,000 1 :x00 0.0500.3250.275 120,000

1612 51 1:300 0.0450.0270.23 20,000 31,800$ 15,000 1:200 0.0650.41 0.345 20,000 16,000

1614 78 1:600 0.0500.44 0.39 68,000 60,300 57,000 1: 1000 0.0380.28 0.242 70,000

1618 43 1:600 0.0600.3550.295 34,000 40,000$ 29,000 1:500 0.0440.28 0.236 34,000 31,000

1621 57 1:60 0.0650.47 0.405 7,000 6,560 5,045 1 :lOO 0.0400.29 0.25 7,256

Crystalline 9.7 mg. in 0.0070.35 0.343 sodium 150 ml. G 9.7 in mg. 0.0070.18 0.173

300 ml.

* This dilution does not include the 1:4 dilution in acetate buffer of Procedure A. t The acetate buffer used in these particular analyses was pH 4.8. $ Since this work was completed, it has been found that these two preparations

were more unstable than usual and that a number of days elapsed between perform- ance of the bioassay and the spectrophotometric analysis.

Time in minuies Time in minuies

FIG. 4. Effect of time of heating at 100’ in 0.3 M acetate buffer, pH 4.6, on the FIG. 4. Effect of time of heating at 100’ in 0.3 M acetate buffer, pH 4.6, on the absorption density measured at 322 rnp. absorption density measured at 322 rnp.

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734 DETERMINATION OF PENICILLIN

OO I I I I I I I I I I I

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 I.1 Molar concentration of acetate buffer pH 4.6

2

FIG. 5. Effect of concentration of acetate buffer on the absorption density of penicillin preparations at 322 rnp after heating 15 minutes at 100’.

0.9

0.8 .I- s 0.7L \ ,” 2 0.6-

3

.$ 0.5-

-z

8 .- 0.4- -& b

4 0.3- .c i 2 0.2- v

O.l,-

0=15,ug./ml.of crystalline G penicillin.

O= SO~ug./ml. of No.999 penicillin.

l = 75/lg./ml. of No. 1006 penicillin.

O3.0 I I I I I I I I I I I

4.0 5.0 6.0 7.0 8.0 9.0 pH of 0.3 molar acetate buffer

FIG. 6. Effect of pH on the change in absorption densities at 322 rnp produced by heating penicillin preparations 15 minutes at 100’.

at least a half hour the change is negligible. Once the samples have been heated and cooled, there is no further change for several hours.

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R. M. HERRIOTT 735

Attempts to increase the absorption density by using solvents other than sodium acetate-acetic acid buffer of pH 4.6 were without success. The materials tested at pH 4.6 were formate, chloroacetate, glycine, propionate, lactate, citrate, butyrate, borate, phosphate, sodium octyl sulfate, as well as magnesium, barium, mercuric, and potassium acetates. Substances such as sodium chloride, magnesium sulfate, ethyl alcohol, methyl cello- solve, acetic anhydride, and others were added to 0.3 M acetate, pH 4.6, but were likewise depressing in action. Propionic acid-sodium propionate buffer at pH 4.6 was just as effective as acetate buffer but no more so.

Heating for various periods of time at 80”, or allowing the samples to stand for long periods at room temperature, failed to raise the absorption value above that obtained by heating 15 minutes at 100”.

A pparutus-While any sensitive ultraviolet spectrophotometer will presumably be adequate, the Beckman (1) instrument is admirably suited for this measurement. It requires only 4 ml. of solution and has proved very reliable. The instrument was standardized against the strong hydro- gen line at 656 rnp, as recommended by the manufacturer, and in addition against 0.02 per cent benzene solution in isooctane. The three highest absorption maxima were obtained at 261, 245.5, and 249 rnp, which are within 1 or 2 rnp of the positions given in the International Critical Tables.

DISCUSSION

A discussion of the r$ative merits of the present method may not be out of place at this point. They are speed and simplicity, reliability and re- producibility, the wide range of concentrations included by a single test, the small volume and concentration of material required, the freedom from interference by normal impurities in commercial preparations, and indepen- dence of the nature or proportions of the various penicillins.

The biggest objection to the method is the cost of the instrument for measurement of the absorption density. A photoelectric calorimeter with quartz cells and a steady light source confined to 315 to 330 rnp could be used in place of the spectrophotometer.

The spectrophotometric method gives a precise value of the total peni- cillin content of any preparation. This may or may not be quantitatively related to its antibiotic activity. However, since the antibiotic activity varies with the test organism (4) and among the different penicillins (4, 5), there is no particular advantage in having the penicillin content expressed in Oxford units rather than in mg. of pure penicillin. Differential bio- assays on a variety of organisms yield useful qualitative information only when the mixture of penicillins is unusual, such as when there is a high proportion of K, X, or F penicillin. Specific and quantitative tests for each different penicillin would be preferable to any test now available.

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736 DETERMINATION OF PENICILLIN

SUMMARY

A method is described by which 5 to 75 y = about 8 to 120 Oxford units per ml., of any of the four known penicillins, pure or crude, may be de- termined in 20 minutes with an error not greater than f5 per cent.

The writer is glad to acknowledge the receipt of advice and generous quantities of crystalline, as well as crude preparations, from the individuals and organizations indicated in Table I.

BIBLIOGRAPHY

1. Car-y, H. H., and Beckman, A. O., J. Opt. Sot. America, 31, 682 (1941). 2. Abraham, E. P., Chain, E., Fletcher, C. M., Gardner, A. D., Heatley, N. G.,

Jennings, M. A., and Florey, H. W., Lancet, 2, 177 (1941). 3. Committee on Medical Research, Office of Scientific Research and Development,

Washington, and Medical Research Council, London, Science, 102, 627 (1945). 4. Schmidt, W. H., Ward, G. E., and Coghill, R. D., 1. Bact., 49, 411 (1945). 5. Coghill, R. D., and Koch, R. S., Chem. and Eng. News, 23, 2310 (1945).

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Roger M. HerriottPENICILLIN

FOR THE DETERMINATION OF A SPECTROPHOTOMETRIC METHOD

1946, 164:725-736.J. Biol. Chem. 

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