332 JOURNAL OF COSMETIC SCIENCE ANALYSIS OF THE STABILITY OF THE PRESERVATIVE• BRONOPOL• AND IDENTIFICATION OF ITS DECOMPOSITION PRODUCTS Asira Ostrovskaya, Peter A. Landa, Anthony D. Rosalia and Daniel Maes Estee Lauder Inc., Research and Development Center, 125 Pinelawn Road, Melville, NY 11747 Introduction: Bronopol (2-bromo-2-nitropropane-l,3-diol) has broad-spectrum anti-bacterial activity and is, therefore, used as a preservative in many toiletries, pharmaceuticals, household items and cosmetics (1). Despite its common usage, the stability of bronopol has been suspect and of great concern to the industry as a whole, because of its stability issues in products under certain conditions (2). In order to stabilize bronopol in products, we investigated the kinetics and decomposition pathways of bronopol. We further compared degradation patterns of bronopol in aqueous and methanolic solutions. The decomposition products of bronopol have been previously postulated and examined by UV spectrophotometry, NMR, and HPLC (3-5). However, to fully identify and confirm bronopol's decomposition products we employed Gas Chromatography - Mass Spectroscopy (GC-MS). This led us to a better understanding of the degradation products, and to confirm the postulated mechanisms of bronopol's decomposition. Methods: The stability of bronopol was investigated by High Performance Liquid Chromatography in aqueous and methanolic solutions at concentrations of 0.04g/250mL. The method employs an Inertsil ODS-2 column with a mobile phase of acetonitrile and water at 220nm. The decomposition products of bronopol, were determined and identified via Gas Chromatography - Mass Spectroscopy (GC-MS) using a DB-1 column. This method uses an oven temperature program consisting of 40øC for 10 minutes, 5øC/min to 280øC, hold for 2 minutes. The MS collected masses 40-550 AMU at 1.4 scans/see and data were subsequently analyzed and interpreted. Results and Discussions: HPLC: The kinetics of bronopol were studied at room and elevated ten,.peratures in both aqueous and methanolic solutions. In aqueous solutions at room temperature, Bronopol degraded readily due to hydrolysis, and when solutions were heated the rate of decomposition increased. In methanolic solutions no apparent decomposition was observed even at elevated temperatures. The HPLC method is limited to the determination of bronopol levels in solutions but is not useful for determining by-products of bronopol degradation. GC-MS: To identify the decomposition products of Bronopol and determine its degradation pathways, GC-MS was employed. GC-MS chromatograms obtained for both aqueous and methanolic solutions of bronopol, initially contained one major and one minor peak. The major peak was identified as bronopol. The mass spectrum for the minor peak was identified as 2-bromo-2-nitroethanol, previously described in the literature as result of a retroaldol decomposition ofbronopol (1). Mass Spectrum of Minor Peak at 14.9min - Identified as 2-bromo-2-nitroethanol o o H-O-C-C-H • I H Br 2 -bromo-2-nttroethanol

2000 ANNUAL SCIENTIFIC SEMINAR 333 Over time, two additional peaks at 3.3 and 3.7 minutes were observed and were more prevalent in aqueous solutions. The mass spectra of these two peaks were interpreted and identified as bromo-nitro-methane and 1-bromo-l-nitro-ethene, respectively (see below,}. These secondary peaks occur as a result of further decomposition of the intermediate, 2-bromo-2-nitroethanol. Mass Spectra of Two Secondary Peaks bromo-nitro-methane • 3.3min 1-bromo-l-nitro-ethene • 3.7m in Bronopol, therefore, loses a formaldehyde molecule degrading to 2-bromo-2-nitro-ethanol. In turn, 2- bromo-2-nitro-ethanol further degrades by either one of following two pathways: 1) loss of an additional formaldehyde becoming bromo-nitro-methane or 2) loss of the hydroxyl group with the formation of a double bond between carbons becoming 1-bromo- 1-nitro-ethene. Bronopol's Initial and Secondary Decomposition Pathways in Aqueous Media o o o o \x/ \\/ H N H H N ,• i I • i H-O-C-C-C -O -H ,• H-O-C-C-H H Br H H Br O O H-C-H I bromo-nitro-m•lhane N H Bronopoi 2-bromo-2-nitroethanol Br~C=C i % H H 1 -Bromo- 1 -nitro-ethene In methanol, on the other hand, the decomposition is initiated when the lone electron pair of oxygen withdraws a hydrogen from bronopol's hydroxy group. Area ratios between these two peaks indicate that the kinetics of the retroaldol mechanism in aqueous solutions is more advantageous than the decomposition mechanism in methanolic solutions. Conclusions: By utilizing the GC-MS, we were able to identify and confirm the decomposition products of bronopol, and postulate its decomposition pathways. We were able to correlate the GC-MS data with the HPLC assay results. We found that the primary degradation mechanism of Bronopol to 2-bromo-2-nitro-etha.nol occurs readily in water and is less energetically favorable in methanolic solutions. The subsequent degradation to bromo-nitro-methane and 1-bromo-l~nitro-ethene in methanol is found at a much slower rate due to the lower availability of the intermediate, 2-bromo-2-nitro-ethanol, in solution. This study will allow us to collaborate with formulators to implement conditions that can improve the stability of bronopol in cosmetic formulations. 1. Kabara, J.J., "Cosmetic and Drug Preservation." 1, Marcel Dekker, Inc., New York, 31-62, ]984. 2. Moore, K.E. and Stretton, R.J., d. Applied Bacteriology, 51,483-494, ]98]. 3. Sanyal, A.K. et. al., d. Pharm. Biotaed. AnaL, 14, 144%1453, 1996. 4. Challis, B.C. and Yousaf, T.I., J. Chem. Soc. Perkin Trans., 2, 283-286, ]99]. 5. Lian, H.Z. et. al., J. Pharm. Biotaed. AnaL, 15, 667-671, 1997.