A colorimetric method has been developed for the determination of small quantities of chlorhexidine by reaction in alkaline solution with sodium hypobromite. The precipitation of the base is prevented by the use of a surface-active agent. The method is rapid and accurate and has been applied to a variety of formulated products containing chlorhexidine.THE antibacterial drug chlorhexidine (I : 6-di(N'-p-chlorophenylbiguanidide N5)hexane) may be incorporated in various formulations in a range of concentrations. When present in relatively large amounts it may usually be estimated by extraction of the base followed by titration in a non-aqueous medium. Alternatively a spectrophotometric method may be applied to a suitably purified extract. If the concentration of chlorhexidine is 1 per cent or less, however, large amounts are required for the base extraction method and it is often difficult to prepare extracts which are sufficiently free from irrelevant absorption for a spectrophotometric determination to be possible. During a search for a more suitable procedure it was noted that hypochlorous acid reacts with chlorhexidine to give a transient red colour and it was thought that this might form the basis of a colorimetric method of assay. EXPERIMENTAL A solution of chlorhexidine diacetate in dilute hydrochloric acid was prepared and diluted with water to contain 0.3 mg. of chlorhexidine per millilitre. Aliquots each of 5 ml. were treated separately with increasing amounts of a solution of sodium hypochlorite containing 1 per cent available chlorine and the effect assessed visually. Small volumes of hypochlorite gave an immediate blood-red colour which faded rapidly to pale yellow ; with increasing volumes the blood-red colour persisted but decreased progressively in intensity. This effect was found to be due to the raising of the pH, caused by the alkali present in the sodium hypochlorite solution which precipitated chlorhexidine base before the reaction was complete. The colour produced in neutral or acid solution was too transient to be of practical value and an attempt was made to prevent the precipitation of chlorhexidine base in alkaline solution by the addition of a surface-active agent. With this object 5 ml. of a 20 per cent sodium lauryl sulphate solution was added to a 5 ml. aliquot of the chlorhexidine acetate solution, together with sufficient 0-1N sodium hydroxide solution to produce an excess alkalinity of 0.5 ml. On addition of 5 ml. of sodium hypochlorite solution an immediate blood-red colour was produced which faded very slowly. A slight turbidity was present, however, which 370
A chromatographic assay for the determination of griseofulvin is described, employing a hexane : methanol : chloroform : water partition system supported on Celite. Chromatographic separation is followed by ultra-violet measurement of the eluate fractions at 291 rnp.
A spectrophotometric method originally designed for the estimation of potassium penicillin has been applied to procaine penicillin and benzathine penicillin. A solution containing the penicillin salt is heated under controlled conditions with a sodium acetate-acetic acid buffer solution of pH 4 6 to which a trace of copper sulphate has been added. The optical absorption of the resulting penicillinic acid is measured on a spectrophotometer. The method has a standard error of 3 per cent and is applicable to a variety of penicillin preparations ranging in potency from 0.5 units/mg. upwards. A method has been described by Herriotl for the determination of penicillin by its controlled degradation to penicillinic acid on heating with acetic acid-sodium acetate buffer of pH 4.6, followed by spectrophotometric measurement of the ultra-violet absorption at 322 mp. It has since been shown by Stock2 that traces of copper in the buffer solution play an important role in the reaction and that, in the absence of this element, reproducible results are unobtainable. Stock has successfully applied this method to the determination of penicillin in oral tablets. The object of this work was to study the extension of the method to formulated penicillin products other than oral tablets and to the procaine and dibenzylethylenediamine salts of penicillin, both of which are now widely used. EXPERIMENTALA standard solution was prepared containing 0.1 g./l. of Procaine Penicillin B.P. in 0.01M phosphate buffer. 20 ml. of this solution was diluted with 50 ml. acetate buffer (acetic acidsodium acetate buffer of pH 4.6 containing copper sulphate equivalent to 0.45 p.p.m. of copper). Aliquots, each of 5 ml. of the diluted solution were heated in a water bath at 100" for varying periods of time, ranging from 5 to 35 minutes, cooled and made to volume in 10 ml. stoppered cylinders with acetate buffer. The optical absorptions of each of the solutions were measured at 322 mp in 1 cm. silica cells with a spectrophotometer. A blank was done at the same time by proceeding exactly as in the preparation of the sample solution, but omitting the heating process. The results are shown in Figure 1. It was obvious from the outset that owing to the low solubility of procaine penicillin in water (0.4 per cent w/v) the method in its original form would be of little use in dealing with most procaine penicillin formulations because of the small weight of sample, and the uncertainty of securing complete extraction. 762Application to procaine penicillin.
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