180 Analyst, March, 1967, Vol. 92, $9. 180-184 The Assay of Certain Organic Bases in Aqueous Eye-drops BY P. J. COOPER AND P. W. HAMMOND (Ministry of Technology, Laboratory of the Government Chemist, Cornwall House, Stamford Street, London, S.E. 1) Methods have been devised for the assay of medicaments in eye-drop solutions containing benzalkonium chloride and chlorhexidine diacetate in which interference caused by these substances is eliminated. THE 1966 Supplement to the British Pharmaceutical Codex 1963 has specified some marked changes in the monographs for eye-drops. These changes have introduced the use of chlor- hexidine diacetate as a bactericidal and fungicidal agent and extended the use of benzalkonium chloride. Also, to meet the wider needs of hospital practice, most monographs on eye-drops now deal with solutions of a range of strengths instead of a single concentration, as previously.Before these changes were made the assay of organic nitrogenous bases depended upon precipitation of the base with a measured excess of sodium tetraphenylboron solution, and subsequent titration of the residual excess with a solution of cetylpyridinium chloride. The fact that this method, had its use been continued, would have caused bactericidal agents to be precipitated with medicaments, with consequent determination of the former as the latter, would not have mattered greatly had the concentration ranges for the medicaments not been extended. The level of bactericidal agent was formerly extremely low in relation to the level of medicament, and any interference was correspondingly small.The new monographs in the B.P.C. Supplement, however, include much lower concentrations of medicaments, and interference caused by chlorhexidine diacetate and benzalkonium chloride becomes relatively larger as levels are reduced. At the time of publication of the 1966 Supplement suitable methods of assay were not available to meet this situation, and for this reason, there are at present no standards given under eye-drops in most of the monographs in the B.P.C. The B.P.C. Formulary Standards Sub-committee A, which is responsible for the provision of standards for these formulations, has recognised that this is an unsatisfactory situation, and the work now described has been undertaken at the request of that committee.The methods described are modifications of the original tetraphenylboron assay by Johnson and King1; and they are intended for use with aqueous eye-drops containing nitrogenous bases with chlorhexidine diacetate or benz- alkonium chloride as a bactericidal agent. The use of sodium tetraphenylboron for the assay of quaternary ammonium salts and organic bases, singly and in mixtures, has been widely investigated.2 to Direct and indirect titrimetric procedures and gravimetric methods have been described. Pate1 and Anderson2 reported the assay of benzalkonium chloride by titration with sodium tetraphenylboron in a two-phase system under alkaline conditions with bromophenol blue indicator. They showed that under these conditions only quaternary ammonium salts could be titrated ; non-quaternary salts were not titratable.Uno, Miyajaima and Tsukatoni3 reported that quaternary am- monium salts could be titrated with sodium tetraphenylboron under acidic conditions with methyl orange as indicator. The acidic colour of methyl orange was not observed, except in the presence of an excess of sodium tetraphenylboron, when a pink colour was produced. The end-point in the titration was marked by the change from yellow to pink. Kaito and Kobayashi* determined organic bases in a two-phase system by direct titration with Nile blue as indicator, which they claimed was superior to bromophenol blue. Boden5 described the direct titration of quaternary ammonium salts with sodium tetraphenylboron and he, too, used a cationic indicator.In this instance the indicator, neutral red, marked the end- point by its appearance in the organic solvent layer. Cross6 combined a number of features from some of the methods mentioned to provide a neat and convenient method for the assay of mixtures of cationic surfactants. By titrating with sodium tetraphenylboron at pH 3, 10 and 13, he was able to resolve mixtures of quaternary and non-quaternary ammonium salts, and nitrogenous bases of the pyridinium and guanidinium type. He used methyl orange and bromophenol blue as indicators in a two-phase chloroform - water system.COOPER AND HAMMOND 181 In general, two-phase titration techniques are based on the transfer of indicator, with or without an associated colour change, from one phase to the other at the end-point.Whether a cationic or an anionic indicator is used, the requirements are the same for both, namely, that the indicator salt should be almost wholly soluble in one phase before the end-point, and almost wholly soluble in the other phase after it. It is also necessary that the substituted ammonium tetraphenylboron salt formed should be stable, and that the indicator should not be permanently precipitated by any of the components of the mixture being titrated. Solutions for eye-drops were found to fall short of almost all of these requirements, so that the methods described for direct titration, despite their obvious attractions, could not be applied. Chlorhexidine formed precipitates with the anionic indicators and these precipitates were insoluble in both chloroform and water.Other chlorinated solvents were no more satisfactory than chloroform in their ability to dissolve these chlorhexidine - indicator salts, and the use of solvents such as ethanol or acetone, either alone or in admixture with other solvents, simply caused a distribution of colour between the phases. Further, the medicaments themselves did not form tetraphenylboron salts that were of sufficient stability to enable a direct titration procedure to be used. It was necessary, therefore, to retain the precipitation stage in the method described for eye-drops in the B.P.C. 1963, and it was possible to apply the diphasic titration technique only to the preliminary titration of benzalkonium chloride. No satisfactory method for the direct titration of chlorhexidine diacetate could be devised and, as it displayed a considerable readiness to precipitate with a variety of reagents under various conditions, it was decided to remove it in this way.Accordingly, a method was devised based on the precipitation of chlorhexidine as sulphate. The methods for assay of eye-drop solutions are described below. ASSAY OF SOME NITROGENOUS BASES I N EYE-DROPS IN THE PRESENCE OF BENZALKONIUM CHLORIDE- The method is based on titration at pH 10 and 3.7 to determine benzalkonium chloride and medicament, respectively. The titre at pH 10 is determined in a two-phase chloroform - water system, with bromophenol blue as indicator. At the start of the titration the colour is concentrated in the chloroform layer, and the end-point is marked by the change from blue to colourless.Titration at pH 3.7 follows precipitation of the medicament and bacteri- cidal agent with excess of sodium tetraphenylboron solution. The method described has been applied to aqueous solutions containing amethocaine, atropine, carbachol, cocaine, homatropine, physostigmine and pilocarpine at concentrations of 0.5 per cent. w/v, and hyoscine at a concentration of 0-25 per cent. w/v. Table I lists results that show the absence of interference by medicaments at pH 10, and Table I1 the results obtained with prepared eye-drop solutions. TABLE I TITRATION OF BENZALKONIUM CHLORIDE (0.02 PER CENT.) AT pH 10 IN PRESENCE OF NITROGENOUS BASES Titre, ml of 0.01 M sodium tetraphenylboron Medicament, 0.1 per cent. w/v r h > (4-ml samples) Without benzalkonium chloride With benzalkonium chloride Amethocaine Atropine .. Carbachol . . Cocaine . . Homatropine Hyoscine . . Physostigmine Pilocarpine . . . . .. .. . . .. .. . . . . .. . . .. .. . . .. .. .. * . . * . . .. . . . . .. .. 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.20 0.23 0.22 0.2 1 0.2 1 0-21 0-21 - Five millilitres of a 1 per cent. w/v solution of cocaine containing 0.5 per cent. w/v of benzalkonium chloride gave a titre of 6-55 ml of 0.01 M sodium tetraphenylboron solution at pH 10; and 5 ml of 0.5 per cent. w/v benzalkonium chloride solution gave a titre of 6.6 ml a t pH 10. No titre was obtained at pH 10 with 5 ml of 1 per cent. w/v cocaine solution.182 COOPER AND HAMMOND ASSAY OF CERTAIN [Analyst, Vol. 92 TABLE I1 RECOVERY OF MEDICAMENTS FROM EYE-DROP SOLUTIONS CONTAINING 0.02 OF BENZALKONIUM CHLORIDE Titre a t pH 10, ml of 0.01 M sodium Medicament (2-ml samples) tetraphen ylboron Amethocaine Atropine .. Carbachol . . Cocaine . . Homatropine Hyoscine . . Physostigmine Pilocarpine .. . . .. .. . . . . . . .. 0.12 0.11 0.11 0.1 1 0.13 0.12 0.1 1 0.11 0.1 1 0.12 0.11 0.11 0.12 0.10 0.1 1 0.11 Concentration determined, per cent. w/v (2) -=h Without benzalkonium benzalkonium chloride chloride 0.52 0.50 0-50 0.50 0.50 0.25 0.53 0.50 0.51 0.52 0.50 0.50 0.46 0.47 0.47 0.47 0.50 0-50 0.28 0.26 0.27 0.52 0.52 0.50 0.50 PER CENT. W/V Recovery, (ii) as percentage 99 100 93 04 100 110 of (i) 98 100 METHOD REAGENTS- Appendix IIB. 1963, Appendix 8. and 6 ml of 0-25 M trisodium orthophosphate solution. Codex, Appendix 8 ; Volumetric Solutions.of water, and adjust the pH to between 8-0 and 9.0 with sodium hydroxide. amber-coloured bottle. Chloroform-Analytical-reagen t grade. PROCEDURE- Transfer by pipette 2 ml of eye-drop solution into a 100-ml glass-stoppered conical flask. Add 5 ml of pH 10 buffer solution, 5 ml of chloroform and 2 drops of bromophenol blue indicator. Shake the mixture well and titrate slowly with 0.01 M sodium tetraphenyl- boron solution, shaking it well between successive small additions, until the chloroform layer changes from blue to colourless (the chloroform layer may not turn blue until the first drop of 0.01 M sodium tetraphenylboron solution is added). To a further 2 ml of eye-drop solution in a beaker, add 5 ml of pH 3.7 buffer solution and 5 ml of 0.01 M sodium tetraphenylboron solution.Allow the solution to stand for 10 minutes and then filter it through a dry, No. 4 porosity, sintered-glass crucible. Rinse the beaker and the crucible with 5 ml of water and titrate the combined filtrate and washings with 0-005 M cetylpyridinium chloride solution, with 0-5 ml of bromophenol-blue solution as indicator. Repeat the operation, omitting the sample and the filtration stage. The difference between the titres is equivalent to the total medicament and benzalkonium chloride present. Calculate the amount of medicament from the difference between the blank titre and the sum of the titres at pH 3.7 and 10, expressed in terms of 0.01 M sodium tetraphenyl- boron solution. Bromophenol-blue solution-Prepare as described in the British Pharmacopoeia 1963, Acetate bufer solution, pH 3-7-Prepare as described in the British Pharmaceutical Codex Bufer solution, pH 10-Mix 100 ml of 0.2 M disodium hydrogen orthophosphate solution Cetylpyridinzium chloride, 0.005 M-Prepare as directed in the British Pharmaceutical Sodium tetraphenylboron, 0-01 M-Dissolve 3.422 g of sodium tetraphenylboron in 1 litre Store in an Store in an amber-coloured bottle.Standardise with analytical-reagent grade potassium chloride.March, 19671 ORGANIC BASES IN AQUEOUS EYE-DROPS 183 (Y + 2%) . loof 2 2 Percentage w/v of medicament = x -, where x is the blank titre in millilitres of 0.005 M cetylpyridinium chloride, y is the titre at pH 3.7 in millilitres of 0.005 M cetylpyridinium chloride, x is the titre at pH 10 in millilitres of 0.01 M sodium tetraphenylboron, and f is the weight of medicament equivalent to 1 ml of 0.01 M sodium tetraphenylboron.ASSAY OF SOME NITROGENOUS BASES IN EYE-DROPS IN THE PRESENCE OF CHLORHEXIDINE The method is based on the precipitation of chlorhexidine as sulphate at pH 3.7, with subsequent precipitation of medicament by using a measured excess of sodium tetraphenyl- boron and titration of the excess of reagent with cetylpyridinium chloride solution. Initially, saturated nickel sulphate solution was used as a precipitant, but while this was satisfactory when the concentration of chlorhexidine was high, it was not suitable at low concentration. The results in Table I11 were obtained by using nickel sulphate and they demonstrate that, even in the presence of a large amount of chlorhexidine diacetate, interference following sulphate precipitation is small.Chlorhexidine at the 0.25 per cent, w/v level would interfere to the extent of about 0.2 per cent. w/v, expressed as medicament, if it were not removed before precipitation with sodium tetraphenylboron. TABLE I11 ASSAY OF MEDICAMENTS IN EYE-DROP SOLUTIONS IN THE PRESENCE OF 0.25 PER CENT w/v CHLORHEXIDINE DIACETATE DIACETATE- Medicament (5-ml samples) Concentration determined, per cent. w/v Cocaine (0.5 per cent. w/v) . . . . 0.51, 0.50, 0-53, 0.51 Homatropine (0.5 per cent. w/v) . . . . 0.51, 0.54 Pilocarpine (0.5 per cent. w/v) . . . . 0.52, 0.52 Hyoscine (0.2 per cent. w/v) . . .. 0.22, 0-20 To avoid masking the bromophenol-blue indicator solution change at the end-point of the sodium tetraphenylboron - cetylpyridinium chloride titration, sodium sulphate was substituted for nickel sulphate in the final method. The results obtained by the method described are set out in Table IV.The results in Table V indicate that chlorhexidine interferes at about the 0.01 per cent. level if it is not removed. TABLE IV RECOVERY OF MEDICAMENTS FROM EYE-DROP SOLUTIONS CONTAINING 0.01 PER CENT. W/V OF CHLORHEXIDINE DIACETATE Concentration determined, per cent. w/v > (ii) With Recovery, (ii) as Medicament (2-ml samples) chlorhexidine diacetate chlorhexidine diacetate percentage of (2) Cocaine . . . . .. .. 0.10 0.11 110 0.50 0.49 9s 0.11 0.49 0.48 0.10 0.49 0.09 0.26 0.10 0.10 0.10 0-49 0.10 0.10 100 0-50 0.49 98 0.10 0.09 90 0.25 0.26 104 0.10 0.10 100 0.50 0.49 98 Homatropine .. .. . . Hyoscine.. .. .. . . Pilocarpine . . .. . .184 COOPER AND HAMMOND TABLE V ASSAY OF MEDICAMENTS IN EYE-DROP SOLUTIONS CONTAINNG 0.01 PER CEKT. w/v OF CHLORHEXIDINE DIACETATE WITH AND WITHOUT SULPHATE PRECIPITATION Concentration determined, per cent. w/v Medicament (2-ml samples) With sulphate precipitation Without sulphate precipitation Cocaine . . . . . . .. Homatropine . . .. . . Hyoscine . . .. .. .. Pilocarpine . . .. .. .. 0-49 0.49 0.26 0.49 0.50 0.5 1 0.27 0.51 METHOD REAGENTS- Those listed under Method for assay of some nitrogenous bases in eye-drops containing benzalkonium chloride. Sodium sulphate, anhydrous, granzdar-Analytical-reagent grade. PROCEDURE- Transfer by pipette 2 ml of eye-drops into a 50-ml beaker, add between 0.3 and 0.5 g of anhydrous sodium sulphate, dissolve it by swirling, and allow the mixture to stand for 10 minutes.Add 5 ml of pH 3.7 buffer solution and 5 ml of 0.01 M sodium tetraphenylboron, and allow it to stand for a further 5 minutes. Filter the solution through a dry, No. 4 porosity, sintered-glass crucible and wash the beaker twice with 1 ml of water, adding the washings to the solution in the crucible before applying suction. Titrate the solution as described in the method for eye-drops containing benzalkonium chloride. Repeat the procedure, omitting the sample and the filtration stage. The difference between the two titres, expressed in terms of 0.01 M sodium tetraphenylboron solution, is equivalent to the concentration of medicament present. The authors thank the Government Chemist for permission to publish this paper. 1. 2. 3. 4. 5 . 6. 7. 8. REFERENCES Johnson, C. A., and King, R. E., J . Pharm. PharmPzac., 1962, 14 (suppl.), 77T. Patel, D. N., and Anderson, R. A., Drug Stand., 1958, 26, 189. Uno, T., Miyajaima, K., and Tsukatoni, H., J . Pharm. SOC. Japan, 1960, 80 (l), 153. Kaito, T., and Kobayashi, S., Bull. Natn. Hyg. Lab., Tokyo, 1961, 79, 73. Boden, T., Chemist Analyst, 1963, 52 (4), 112. Cross, J. T., Analyst, 1965, 90, 315. Worrell, L., and Ebert, W., Drug Stand., 1956, 24, 153. Gautier, J. A., Renault, J., and Pellerin, F., Annls Pharm. Fr., 1955, 13, 725. Received September 22nd, 1966