J. Soc. Cosmet. Chem., 39, 85-92 (March/April 1988) Interaction between dehydroacetic acid sodium salt and formaldehyde: Structural identification of the product C. A. BENASSI, A. BETTERO, P. MANZINI, A. SEMENZATO, and P. TRALDI,* Department of Pharmaceutical Sciences, University of Padova, Via Marzolo 5, I 35131 Padova, and C.N.R.,* Corso Stati Uniti 4, 1 35100 Padova, Italy. Received September 15, 1987. Synopsis The structural identification of the product formed by the interaction between dehydroacetic acid sodium salt (Prevan ©) and formaldehyde, either free or released from some other preservatives commonly employed in the cosmetic field, has been obtained by elemental analysis, •H-NMR, mass spectrometry and confirmed by X-ray analysis. The physicochemical data lead to the identification of 3,7-dimethyl-Ill, 9H, 10H-di- pyrano[4,3-b:3' ,4'-e]pyran- 1,9-dione. INTRODUCTION Improvements in cosmetic quality control have resulted in rapid and reliable procedures for the evaluation of preservative agents in raw materials and in finished products (1-3). It is well known that a mixture of preservatives generally has a wider profile of activity against microorganisms than that of each constituent of the mixture. Furthermore, the resulting toxicity may be lower, due to the smaller amount of each constituent used, in comparison with its use as a single component. In order to test the stability of such mixtures, we recently reported a comparative study of the behavior of dehydroacetic acid sodium salt (I) (DHA.Na) (Prevan©), when used with imidazolidinylurea (Germall 115©), bromonitropropandiol (Bronopol©), and isothiazolinone (Kathon CG ©) derivatives, in preservative standard mixtures (4) and in cosmetic products (5). On that occasion we emphasized the presence of an interaction product between DHA.Na and formaldehyde released from Bronopol © and Germall 115 © . In the present paper we describe the chemical interaction between DHA.Na and free or released formaldehyde. The structural characterization of the compound which origi- nates from this reaction has been obtained by •H-NMR, mass spectrometry, elemental analysis and confirmed by X-rays (Bandoli et al., in preparation). The presence of the interaction product has been proved by HPLC methods also in cosmetic emulsions. 85

86 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS H3C O CH20 COCH 3 O- Na + 0 O DHA. Na C 13H1 005 Scheme I: The reaction of dehydroacetic acid with formaldehyde. EXPERIMENTAL MATERIALS Bronopol © (2-bromo-2-nitropropane- 1,3-diol) (Formenti, Milano, Italia) formaldehyde 40% RPE (Carlo Erba, Milano) Germall 115 © (N,N'-methylenebis[N'(1-hydroxy- methyl)2,5-dioxo-4-imidazolidinylurea)], (Medolla, Milano) Prevan © (3-acetyl-6- methyl-2H-pyran-2,4(3H)-dione sodium salt) (Formenti, Milano). STANDARD AQUEOUS SOLUTIONS DHA.Na 1.5 mg/ml formaldehyde from 0.1 to 2.0 mg/ml DHA.Na-Bronopol © 1.5 and 0.1 mg/ml, respectively DHA.Na-Germall 115 © 1.5 and 2.5 mg/ml DHA.Na- formaldehyde 1.5 and 0.2 mg/ml. SAMPLES About 1 g, accurately weighted, of each cosmetic emulsion was diluted to 10 ml with a tetrahydrofuran/water solvent mixture (THF/H20 9/1). METHODS HPLC. All experiments were carried out in reversed phase mode using a Perkin Elmer S-4 liquid chromatograph equipped with an LC-85 UV detector and a Sigma 15 data station. Chromatographic parameters: Method la: Li-NH2 (10 I•m) Merck column, 1.0 ml/min flow rate, 216 nm UV detec- tion, and acetonitrile/phosphate buffer 0.01 M, pH 4.7 (55/45%) mobile phase, 6 I•l injection volume. Method lb: Li-NH2 (10 I•m) Merck column, 1.0 ml/min flow rate, 216 nm UV detec- tion, and acetonitrile/phosphate buffer 0.01 M, pH 4.7/phosphoric acid 0.01 M (55/42/3%) mobile phase, 6 I•l injection volume. Method 2:RP-8 (10 I•m) Merck column, 1.0 ml/min flow rate, 300 nm UV detection, and acetonitrile/phosphate buffer 0.01 M, pH 4.7 (50/50%) mobile phase, 6 I•l injec- tion volume. 1H-NMR. 1H-NMR spectra were recorded on a Varian FT-80A spectrometer with the