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

REACTION OF DEHYDROACETIC ACID AND FORMALDEHYDE 87 following procedure: frequency 80 MHz mode FT lock internal from CDC13 tempera- ture 3 iøC solvent CDC13 with 1% v/v TMS as internal standard concentration about 0.1 M tube size 5 mm O.D. pulse width 58 }xs: acquisition time 1 sec spectral width 1500 Hz no. of transients 100 no. of data points 4K (16K with zero filling before FT). Coupling constants are given in Hz the relative peak areas, the decoupling experi- ments, and calculations of the chemical shifts using additivity rules were in agreement with all assignments. Mass Spectrometry. All measurements were performed on a VG ZAB 2F mass spectrom- eter operating in Electron Impact (EI) conditions (70 eV, 200 }xA). Samples were intro- duced via direct inlet system with an ion source temperature of 200øC. Metastable transitions were obtained by B/E linked scans (6). Exact mass measurements were per- formed by the peak matching technique at 10,000 resolution (10% valley definition). RESULTS AND DISCUSSION Our observation that some formulations could not prevent the development of molds over a long period of time suggested the possible degradation of the preservative system. Hence we undertook a study of the kinetics of disappearance of DHA.Na vs time, either alone or in a mixture with formaldehyde releasers (Bronopol © or Germall 115 ©) via HPLC. The results, reported in Figure la, showed that there is a strong decrease in the concen- tration of DHA.Na in the presence of Bronopol © or Germall 115 © in comparison with control samples of DHA.Na alone. It was postulated that this decrease was due to an interaction between DHA.Na and the released formaldehyde, giving rise to the forma- tion of a new product. In fact, the HPLC runs of binary mixtures showed a third product, whose concentration is time-dependent (see Figure lb). To confirm the involvement of formaldehyde in the reaction, we carried out a series of tests on aqueous solutions of DHA.Na and formaldehyde in different molar ratios. The results, reported in Figure 2, fully support our hypothesis. The observed peak due to the interaction product was confirmed by various HPLC methods to be a unique product (4) whose structure must be determined as a first necessary step for further toxicological studies. The unknown product (compound 1) is obtained as a precipitate in significant amount (1.4 g) with a 25% yield by long-time (4 weeks) reaction of aqueous equimolecular (0.08 M) solutions of DHA. Na and formaldehyde at room tem- perature. The product, recrystallized from chloroform, does not melt up to 340øC. The elemental analysis gave: C, 63.44% H, 3.99%, which is in accordance with a molec- ular formula: C13HloO 5 (Calculated: C, 63.41% H, 4.09%). The corresponding mo- lecular weight was confirmed by mass spectrometry. In fact, the 70 eV electron impact mass spectrum (Figure 3) shows the most abundant peak at m/z 246, in agreement with a molecular ion [C13HloO5] +o. Furthermore, accurate mass measurements gave a value of 246.0534 (_+0.002), in agreement with the molecular formula C•3HloO 5 (246.0525). The •H-NMR spectrum (Figure 4) shows a quartet at 8 5.88 with a coupling constant of 0.8 Hz, a broad singlet at 8 3.29 with the same unit integral, and a doublet at 8 2.26