FLUOROMETRIC DETERMINATION OF PRESERVATIVES 659 at 510 nm rs. concentration was plotted, and the concentrations of Bronopol unknowns were determined directly from this plot. lqESULTS AND DISCUSSION In preliminary experiments, varying amounts of Bronopol were mixed with a shampoo base and diluted with 10% methanol 2 ml of each solution was added to an equal volume of the acetylacetone-ammonium acetate reagent. The reaction mixtures, including formaldehyde standards, were heated at 37øC for 1 hour. They were cooled and their fluorescences were measured. Representative data are presented in Table I. Formaldehyde derived from Bronopol was determined from the calibration curve of the known formalde- hyde reaction mixtures. The concentration of preservative added to the reac- tion mixture and the per cent determined given in this and subsequent tables were calculated assuming molar stoiehiometry, although two of the com- pounds (Bronopol and Germall 115) have two methylol functional groups. Examination of the data given in Table I shows that the amount of formalde- hyde derived from BronopoI at 37øC is relatively low and variable. The Table I The Dependence of the Concentration of 3,5-Diacetyl-l,4~dihydrolutidine Formed from Two Methy]ol-containing Compounds on Reaction Temperature and pH of Cosmetic Preservative Reaction Preservative Cosmetic Preservative Temperature (C ø) Added to (1 g sample) Determined (%) Cosmetic a (mg) Bronopol 37 9.54 Bronopol 37 9.54 Bronopol 37 0.954 Bronopol 60 9.54 Bronopol 60 9.54 Bronopol 60 0.954 Bronopol 60 9.54 Bronopol 60 9.54 Hydroxymethyl- dimethylhydantoin 37 10.0 Hyclroxymethyl- dimethylhydantoin 37 1.00 Hydroxymethy]- dimethy]hydantoin 37 0.100 Shampoo 70 Shampoo 47 Shampoo 84 0 114 Shampoo 109 Shampoo 114 Basic shampoo 115 b Basic bath oil 120 • Vanishing cream 94 Vanishing cream 102 Vanishing cream 100 These solutions were diluted before performing the reaction so that the magnitude of their fluorescence peaks would be similar to those of the formaldehyde standards. b A shampoo containing Bronopol was made basic, allowed to stand for one hour pH was then adjusted to approximately 7. A bath oil containing Bronopol was made basic and allowed to stand overnight pH was then adjusted to approximately 7.

660 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS Hantzsch reaction with Bronopol was repeated with different reaction tem- peratures and pH's of the cosmetic. Reference to Table I shows that at 60øC the yield of formaldehyde from Bronopol is greater and less variable than at 37øC. The pH of the cosmetic had little effect on the amount determined. The pH of the reaction was not varied because the pH chosen is optimal for the determination of formaldehyde. Included in Table I are data which show that hydroxymethyldimethylhydan- toin can be determined reliably by the fluorometric method for formaldehyde if a reaction temperature of 37øC is used. However, since it was necessary to perform the reaction at a higher temperature to obtain better results with Bronopol, subsequent reactions with all three compounds were performed at 60øC. Some of the results of these determinations are shown in Table II. It is evident that Germall 115 and hydroxymethyldimethylhydantoin can be esti- mated in this manner. The results for Bronopol are different from those ob- tained earlier at 60øC (Table I). To overcome this daily variability, in the next series of experiments known solutions of Bronopol were included in the determinations and the amount of Bronopol found was based on a graph of standard solutions of Bronopol rather than on that of formaldehyde. The final series of experiments, the results of which are reported in Tables III and IV, were blind studies, that is, solutions of these compounds were pre- pared by a colleague and their concentrations were unknown to the present authors. Table II 3,5-Diacetyl-l,4-dihydrolutidine Derived from Three Methylol-containing Cornpounds Held at Reaction Temperature 60 ø C, pH 6, for 1 Hour Preservative Cosmetic Preservative Preservative Found Preservative (1 g sample) Added to Added to Re- (•g/ml) Determined Cosmetica action (•g/ml) I•HCHO (%) (rag) (HCHO equivalent) equivalent ) Germall 115 Bath oil 0.634 0.186 0.20 108 Germall 115 Bath oil 0.634 0.186 0.20 108 Germall 115 Shampoo 0.634 0.186 0.20 108 Hydroxymethyl- dimethylhydantoin Bath oil 0.602 0.427 0.400 94 Hydroxymethyl- dimethylhydantoin Bath oil 0.602 0.427 0.420 98 Hydroxymethyl- dimethylhydantoin Shampoo 0.602 0.427 '0.400 94 Bronopol Bath oil 0.584 0.320 0.20 63 Bronopol Bath oil 0.584 0.320 0.250 78 Bronopol Shampoo 0.584 0.320 0.220 70 These solutions were diluted before performing the reaction so that the magnitude of the fluorescence peaks would be similar to those of the HCHO standards.