Activity and safety of Bronopol Solvent front 13 t• O O O o o o o Bromonitroethanol Bronopol Origin 0 2 4 6 8 I0 16 20 25 30 Time of storage of solution (rnin) Fig•e 4. Autobiogram of Bronopol aqueous solutions (initially 10•o w/v) stored at 100 ø and maintained at pH 6 (test-organism: Pseudomonas aeruginosa) very specific and is subject to interference from breakdown products. In the absence of interfering substances, the precision of this procedure is about q-2•o. The method using t.l.c. is more specific, but the spot-comparison procedure that has been used is liable to relative errors of about 15•o errors of this magnitude may be acceptable, however, at the concentrations in which Bronopol is usually incorporated in formulations. The microbiological method had an error of about 4-10•o on aqueous solutions and about 4-10 to 20• on creams. Polarographic Assay. The base electrolyte was Mcllvaine's buffer solution containing 2•o v/v of 0.2• v/v Triton X 200 as a maximum-suppressor. Mcllvaine's buffer solution, pH 4, was prepared by mixing 12.29 parts by volume of 0.1•t citric acid solution and 7.71 parts by volume of 0'2•o disodium phosphate (Na•HPOa) solution. The test solution was prepared as follows. Aqueous solutions only required dilution with base electrolyte to a Bronopol concentration between 10 -a and 10-SM. Bronopol in gels, creams and other fatty-base formulations could be extracted with base electrolyte by warming gently on a steam-bath, after which any insoluble matter in the aqueous phase was removed by centrifuging and the aqueous phase diluted to give appropriate concentrations of Bronopol. Other formulations were more appropriately treated by dissolving in chloro- form and extracting with base electrolyte. The determination was carried out by transferring a portion of the solution in base electrolyte to the cell of a suitable polarograph. A stream of oxygen-free nitrogen was passed through the solution for 10-15 min to remove dissolved oxygen. The height of the mercury reservoir was adjusted to give a constant drop rate appropriate to the apparatus, this drop rate being identical with that used for the preparation of the calibration curve.

14 D.M. Bryce et al. The polarogram was recorded over the range 0 to -1 V relative to the quiescent mercury pool using the appropriate recorder or galvanometer sensitivity to give a suitable wave. The diffusion current at -0.8 V relative to the mercury pool was measured, and the concentration of Bronopol read from the calibration curve. Since the polarographic response was affected by the composition of the test solution, it was necessary to prepare a calibration curve for each formulation examined. Such calibration curves were obtained by adding known amounts of Bronopol to blank formulations and processing in the required manner. The following are examples of the assay method which have been used. Microbioloical Assay. Bronopol can be assayed microbiologically by agar diffusion using Ps. aeruginosa in agar of the following composition: % w/v Dextrose 0.1 Lemco beef extract 0.15 Difco yeast extract 0.•3 Sodium chloride 0.5 Difco casitone 0.08 Magnesium sulphate (7H•O) 0.004 Oxoid peptone 0.6 Davis agar 1.8 Distilled water to 100, pH adjusted to 5.3. Alternatively Difco Assay Agar No. 11 (pH 7.9) with Bacillus subtills NCIB 8054 can be used. The minimum detectable level of Bronopol in water with Bacillus subtilis is 0'005•o. A rapid diffusion method using Bacillus stearothermophilus has been described by Kabay (18). Gas-liquid Chromatographic Assay. Although Bronopol is a water-soluble compound it can be extracted from aqueous solution into diethyl ether or ethyl acetate after the addition of sodium chloride. The extract can then be evaporated to dryness, the residue acetylated and the Bronopol estimated by means of g.l.c. with electron-capture detection. This procedure offers a means of determining Bronopol in aqueous formulations and has been applied to Bronopol concentrations down to 5 ppm. In aqueous formulations containing concentrations of Bronopol down to 50 ppm, the Bronopol has been determined by a similar procedure, but using n-pentadecane as the internal standard, acetyl chloride in chloroform as an acetylating reagent, carbon disul- phide as the final solvent and flame ionization detection. The following are examples of the methods which have been used to assay Bronopol by g.l.c. Based on the acetylated material. The sample (about 0.15 g accurately weighed) was dissolved in 15 ml of chloroform with the aid of minimum heating, 5 ml of a 2•o solution of n-pentadecane (as internal standard) in chloroform was added and the solution diluted to 25 ml. To 1 ml of this solution in a vial was added 0.3 ml acetyl chloride and the vial was sealed and then heated on a steam bath for 3 h. The mixture (2 [tl) was subjected to g.l.c. in a glass column (183 cm x 3 mm) packed with 10•o of silicone JXR on Gas Chrom Z (70 to 80 mesh), operated at 150øC with nitrogen (20 ml min -x) as carrier gas and flame ionisation detection. The ratio of the product of the peak height and retention