ANALYST, OCTOBER 1986, VOL. 111 . 1179

Spectrophotometric Determination of Benzyl Penicillin in Pharmaceutical Preparations Using Copper(l1) Acetate as a Complexing Agent

Utpal Saha Central Public Health and Drugs Laboratory (Drugs Wing), 2 Convent Lane, Calcutta-700 015, India

Copper(l1) acetate is proposed as a chromogenic reagent for the spectrophotometric determination of benzyl penicillin. The reagent forms a green 2 : 1 complex with benzyl penicillin at pH 6-6.8. This complex is stable for nearly 25 min after its formation. The green species has an absorption maximum at 650 nm with a molar absorptivity of 0.24 x lo3 I mol-1 cm-1. Beer’s law is valid over the concentration range 20-1000 yg ml-I of benzyl penicillin. The precision of the method is 1.89%. The reagent has been successfully used for the spectrophotometric determination of benzyl penicillin alone and in pharmaceutical preparations. Keywords: Benzyl penicillin determination; copper(l1) acetate; spectrophotometry; pharmaceutical preparations

Benzyl penicillin is a potent antimicrobial with a low toxicity and is widely used against a number of infections in many pharmaceutical preparations. Ford1 reported a method for the spectrophotometric determination of penicillin. The method is based on the fact that penicillin reacts rapidly with hydroxylamine to give a hydroxamic acid. This acid forms a purple complex with Fe3+, which can be determined spectro- photometrically. As this reaction is not specific to penicillin, many esters, anhydrides, amides, aldehydes and ketones interfere in the determination. The use of a strict timing schedule, especially for the interval between the addition of the iron(II1) chloride and the spectrophotometric determina- tion, is necessary because of the instability of the colour. Twigg2 introduced a method for the ultraviolet determination of benzyl penicillin. This determination is based on the absorption bands due to the phenyl group. The chief limitation of this method is that because it is based on the absorption of the phenyl group, it will include all phenyl-containing sub- stances, whether these are active penicillin or not. Herriott3 proposed a spectrophotometric method for the determination of total penicillin, in which an aqueous solution of penicillin was heated for 15 min in a water-bath with a 0.4 M acetate buffer (pH 4.6) and then rapidly cooled. The increase in absorption at 322 nm was measured and compared with a blank, which had been kept at room temperature. According to Herriott, Beer’s law is not obeyed and the results are not reproducible. Stock4 eliminated the above discrepancies by adding a trace amount of copper sulphate. Penicillin under- goes a controlled degradation to penicillenic acid on heating in a medium of sodium acetate - acetic acid buffer of pH 4.6 containing a trace amount of copper. However, there are three important variables in this method: the time of heating in the water-bath, the pH of the buffer solution and the molar concentration of the buffer. In addition, nearly 1 h is required to perform the analysis. Recently copper(I1) acetate has been used in this laboratory5 as a chromogenic agent for the spectrophotometric determination of salicylic acid in phar- maceutical preparations. This paper describes the use of copper(I1) acetate in the spectrophotometric determination of benzyl penicillin and its successful application to various pharmaceutical preparations. A brief report6 has been publi- shed regarding the possibility of a direct application of this method. This paper gives modifications and detailed accounts of the method, which is both simple and fast, the results being available within 20 min. The stability of the colour system within a wide pH range (6-6.8) can be considered as an advantage over the earlier methods.

Experimental Apparatus

All spectral measurements were carried out with a Hitachi Model 139 UV - visible spectrophotometer with 1-cm path length silica cells.

Reagents

The reagents used were of analytical-reagent grade and all water was doubly distilled.

Standard benzyl penicillin solution, 10 mg ml-1. Prepared by dissolving the standard potassium salt of benzyl penicillin (1600 units mg-1) in distilled water.

Copper(IZ) acetate solution, 10 mg ml-1. The solution was prepared by dissolving 500 mg of copper(I1) acetate (BDH Chemicals) in SO ml of distilled water.

Sodium acetate solution, 0.2 M. Prepared by dissolving 8.2 g of anhydrous sodium acetate in 500 ml of distilled water.

Buffer solutions. Buffer solutions of different pH values were prepared by mixing acetic acid (0.1 M) with sodium acetate (0.1 M) and adjusting the solution to the desired pH.

Acid and salt solutions. Several acid and salt solutions were prepared by dissolving the required amount of acid and salt in the appropriate volume of water. These solutions were used for interference studies.

Procedure

Different volumes (0.1-5.0 ml) of benzyl penicillin solution (10 mg ml-I) were drawn througb a pipette into a 50-ml calibrated flask. An equal volume of copper(I1) acetate solution (10 mg ml-1) and sufficient sodium acetate solution (0.2 M) were added to reach a final volume of SO ml. After waiting for 5 min the absorbance was measured within 30 min at 650 nm against a sodium acetate solution (0.2 M) blank.

Application to Various Pharmaceutical Preparations

Tablets Twenty tablets were weighed and powdered. An amount of powder equivalent to 500 mg of benzyl penicillin was weighed accurately and was transferred into a 50-ml calibrated flask. It was shaken with 40 ml of distilled water and then diluted to volume with distilled water. The solution was filtered through a Whatman No. 1 filter-paper. The first 10 ml of filtrate were discarded and the remaining portion was collected in a dry Erlenmeyer flask fitted with a glass stopper.

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ANALYST, OCTOBER 1986, VOL. 111 1180

0.4

8 0.3 m e 2 0.2 a

0.1

Fig. 1. Effect of pH on the absorbance of the green copper - benzyl Ynicillin complex. Benzyl penicillin concentration, 670 yg ml-l; and

max.7 650 nm

0.5

0.4

a,

0.3

0 ' 1 I 650 750

Wavelength/nm

Fig. 2. Absorption spectra. A, Green copper - benzyl penicillin complex in 0.2 M sodium acetate solution, measured against solvent blank. Benzyl penicillin concentration, 800 yg ml-1; pH 6.5; and A,,,,, 650 nm. B, Reagent blank measured against solvent blank

The benzyl penicillin content of the above solution was obtained by applying the standard procedure to a 4-ml aliquot of this solution mixed with 4 ml of copper(I1) acetate solution.

Injections The total mass of a vial was quantitatively transferred into a calibrated flask and dissolved in distilled water to give a final concentration of benzyl penicillin of 10 mg ml-1.

A 4-ml aliquot of this solution, admixed with an equal volume of copper(I1) acetate solution, was treated according to the standard procedure and the benzyl penicillin content was determined.

Results and Discussion Copper(I1) acetate is soluble in water, giving a light blue colour in dilute solution. It forms a green complex with benzyl penicillin at room temperature (27 "C) in sodium acetate solution (0.2 M). Although only present in small amounts, free copper(I1) acetate solution in a sodium acetate (0.2 M) medium does absorb and the solution becomes turbid after a few minutes owing to the formation of copper(I1) hydroxide, followed by hydrolysis. In order to avoid the presence of excess free copper(I1) acetate in the reaction mixture, the chromogenic reagent, copper(I1) acetate solution, is added in a 1% excess over that of the benzyl penicillin to form the green complex of copper and benzyl penicillin in the ratio 2 : 1. In order to obtain more accurate results, the absorbance of the green complex is measured against a solvent (0.2 M sodium

:: 0.2 4 1 I

2 0.1 0 1

Time/h

Fig. 3. Effect of time on the absorbance of the green copper - benzyl penicillin complex. Benzyl penicillin concentration, 500 yg ml- 1 ; pH, 6.5; and A,,,., 650 nm

0.3

a,

K m 13

v)

;; 0.2

2 0.1

L I I

0 20 40 60 80 Tern peratu re/"C

I I

Fig. 4. Effect of temperature on the absorbance of the green copper - benzyl penicillin complex. Benzyl penicillin concentration, 500 pg ml-l; pH, 6.5; and A,,,,, 650 nm

acetate) blank instead of the reagent blank, as it has been found that Beer's law is not obeyed in the presence of a reagent blank.

Effect of pH

The effect of pH on the intensity of the colour system was studied. The optimum pH range for a constant maximum absorbance was found to be between 6 and 6.8 (Fig. 1). Below pH 6 the maximum colour intensity was not obtained and above pH 6.8 the colour intensity slowly decreased. Above pH 7 turbidity was observed. Therefore, in subsequent studies the optimum pH range (6-6.8) was maintained throughout the experiment by using 0.2 M sodium acetate solution.

Absorption Spectra

The absorption spectra of the green complex and the reagent blank are shown in Fig. 2. The complex exhibited maximum absorbance at 645-655 nm. The absorption spectrum of the reagent blank showed a relatively smaller absorbance at these wavelengths. Consequently, all subsequent studies were carried out at 650 nm against a solvent blank (0.2 M sodium acetate solution).

Effects of Time and Temperature

The intensity of the colour formed reached a maximum 5 min after mixing the reagents and was stable for a further 25 min as determined by the constancy of its absorbance values. After 25 min there was a gradual decrease in absorbance (Fig. 3).

The formation of the green complex was investigated at temperatures ranging from 0 to 100 "C. The absorbance values were not affected by temperature over the range 0-35 "C. At 40 "C the absorbance decreased and at 90 "C decomposition leading to a brown colour was observed (Fig. 4).

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ANALYST, OCTOBER 1986, VOL. 111 1181 ~ ~~~

Table 1. Tests on precision and accuracy of the method

Benzyl penicillin/pg ml-1 Relative standard

Solution No. Taken Found Error, YO deviation, Yo 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5

100 100.0 97.5

100.0 95.0 97.5

500 495.0 500.0 510.0 495.0 500.0

900 885.0 910.0 900.0 885 .O 910.0

0.00 3.06 -2.50

0.00 -5.00 -2.50 -1.00 1.22

0.00 +2.00 -1.00

0.00 - 1.66 1.41 +1.11

0.00 - 1.66 +1.11

~~

Table 2. Determination of benzyl penicillin alone and in pharmaceutical preparations by the proposed and official methods

Percentage of labelled content found

Proposed Iodimetric Biological Sample Sample No. method* method method

Benzyl penicillin potassium . . . . 1 98.53 k 0.27 97.25 97.80

2 97.96k0.30 98.37 97.66 3 99.04k0.26 99.50 99.17 4 97.29k0.22 98.62 96.81 5 98.39k0.33 99.43 98.59

Tablet (250mg) . . 6 102.06 f 0.21 102.78 101.72 7 93.94f0.40 93.66 94.05 8 97.51 k0.36 98.70 96.99

Tablet (500mg) . . 9 105.35 k 0.42 106.46 106.03 10 94.12f0.29 93.20 95.18

Injection (250 mg) 11 96.46 k 0.23 97.55 96.73 12 103.54 k 0.28 102.49 103.39

Injection (500 mg) 13 100.97 k 0.39 102.06 100.28 14 98.04f0.35 99.52 99.07 15 100.17 k 0.26 101.05 100.82

* Results are means of five determinations with standard deviations.

The above method was successfully applied to raw materials and to tablets and injections. The method was compared with the two official methods, i . e . , iodimetric titrationlo and biological assay.11 The results obtained when all three methods were applied to benzyl penicillin itself and the two pharmaceutical preparations are shown in Table 2. These results indicate that the proposed method is in good agree- ment with the official methods.lOJ1 The proposed method is simpler and less time consuming than the official methodslOJ1; also, the biological method has the disadvantage that it can only be used in laboratories specialising in biological assay work.

Interferences

Phenylacetic acid, phenylacetamide and 6-aminopenicillanic acid may be present as impurities in benzyl penicillin as they are used as synthetic precursors. Streptomycin sulphate is used in some preparations of benzyl penicillin. In order to investigate the applicability of this spectrophotometric method to the determination of benzyl penicillin alone or in preparations, the effects of the presence of the above compounds and common co-existing preservatives and bases were studied. The components were added in excess of that amount used in the preparations which resulted in a solution containing 40 mg of benzyl penicillin in the final 50 ml of solution. Phenylacetic acid, 6-aminopenicillanic acid and streptomycin sulphate were found not to interfere. If calcium carbonate, sodium acetate, magnesium stearate, talc powder, starch and lactose were present, they also did not interfere in the determination of benzyl penicillin in the amounts present in some of the preparations.

The author is grateful to Dr. N. N. Chakraborty, Director of the Central Public Health and Drugs Laboratory, for making available the laboratory facilities and also Mr. Sunit Das, S.S.O. of the same laboratory, for assistance with the biological assay. The author is grateful to Dr. D. K. Roy, Emeritus Professor, Department of Pharmacy, and Prof. Amalendu Banerjee, Department of Chemistry, both of Jadavpur University, Calcutta, for their constant encourage- ment and helpful discussions.

Composition, Calibration, Sensitivity, Precision and Accuracy

Job’s method of continuous variation7 and a molar-ratio method8 were used to determine the composition of the copper - benzyl penicillin complex. The results obtained indicated the formation of a 2 : 1 complex between copper and benzyl penicillin between pH 6.0 and 6.8. A calibration graph was constructed by plotting known concentrations of benzyl penicillin against the corresponding absorbance values using a standard procedure. The resulting graph was linear and passed through the origin. Beer’s law was obeyed from 20 to 1000 yg ml-l and the molar absorptivity (E) of the complex was 0.24 X 103 1 mol-1 cm-1. Sandell’s sensitivity9 for this method is 1.555 p.p.m. of benzyl penicillin. In order to study the precision and accuracy of the method, standard solutions containing three different concentrations of benzyl penicillin were prepared and five absorbance measurements were made on each standard benzyl penicillin solution. The over-all relative standard deviation of 15 determinations was 1.89%. The results are given in Table 1.

1. 2. 3. 4. 5. 6. 7. 8.

9.

10.

11.

References Ford, J. H., Anal. Chem., 1947, 19, 1004. Twigg, G. H., Analyst, 1948, 73, 211. Herriott, R. M., J . Biol. Chem., 1946, 164, 725. Stock, F. G., Analyst, 1954, 79, 662. Saha, U., and Baksi, K., Analyst, 1985, 110,739. Saha, U., IRCSMed. Sci., 1983, 11, 91. Job, P., Ann. Chim., 1928,9, 113. Yoe, J . H., and Jones, A. L., Ind. Eng. Chem., Anal. Ed. , 1944, 16, 111. Sandell, E. B., “Colorimetric Determination of Traces of Metals,” Third Edition, Interscience, New York, 1958, p, 917. “British Pharmacopoeia 1980,” HM Stationery Office, London, 1980, p. 54. “Indian Pharmacopoeia Supplement 1975 ,” Government of India Press, Nasik, 1976, p. 206.

Paper A6/62 Received February 26th, 1986

Accepted April 25th, 1986

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