J. Soc. Cosmet. Chem., 45, 337-345 (November/December 1994) Analytical investigation of W/O/W emulsion stability using dihydralazine as breakdown indicator C. LAUGEL, P. CHAMINADE, A. BAILLET, M. SEILLER, and D. FERRIER, Laboratoire de Chimie Analytique HI (C.L., P.Co, A.Bo, D.F. ), and Laboratoire de Pharmacie Galgnique et de Biopharmacie, URA CNRS 1218 (M.S.), Facultg de Pharmacie, AvenueJean-Baptiste Clgment, Ch•tenay-Malabry Cedex, France. Received February 21, 1994. Synopsis This paper focuses on the analytical parameters to be determined for the choice of a suitable tracer compound introduced within a W/O/W emulsion for its stability evaluation. Physicochemical properties of dihydrala- zine respond to these requirements. This compound is then appropriate to quantify some W/O/W multiple emulsion breakdowns. The results obtained with three quantitative methods, two spectrophotometric and one chromatographic, are validated for monitoring of W/O/W emulsion stability. The stability kinetics of one multiple emulsion stored at ambient temperature and at 40øC are compared. INTRODUCTION Multiple emulsions have been described since 1925, when Seifriz (1) reported his observation of aqueous droplets inside the oil drops of an O/W emulsion. Numerous studies on multiple emulsion formulation and physicochemical control have been published over the last 20 years. Among them, stability studies in relation to diffusion of components and emulsion breakdown are of primary importance. Multiple emulsions of the W/O/W type (water/in oil/in water) can be deteriorated following several possible mechanisms of breakdown (2,3): ß Coalescence of the internal aqueous droplets ß Coalescence of the simple or multiple oil drops ß Rupture of the oil film ß Passage of water to and from internal droplets through the oil layer It has been reported that a combination of these mechanisms possibly takes place, and consequently the exact mechanism for the instability is not yet completely elucidated. Over the past few years, a number of new techniques have been developed to detect and quantify emulsion breakdown. Three main classes of methods can be distinguished: 337

338 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS ß Particle size analysis --photomicrography (3,4) --freeze-etching electron-microscope technique (5) --coulter-counter analysis ß Rheological studies (6,7) ß Use of a tracer utilized to assess the leakage and release of components entrapped in internal phase of systems [glucose (8), NaCl (6,9,10), tritium (11), fluorescent marker (12), pigments (13) . . .]. Among the preceding methods, the use of a tracer, included in the inner aqueous phase, seems to be the more suitable. However, the primary purpose of these works was to investigate the entrapment efficiency and the short term stability (two weeks) of W/O/W emulsions without a thorough examination of the analytical difficulties. The main objective of the present study was to deeply investigate the physicochemical parameters for the choice of a suitable tracer. These criteria (solubility in the inner phase, pK a, stability, non-diffusibility) have to be determined in the presence of the raw materials. They are of first importance to validate the use of a given tracer in the stability monitoring of any multiple emulsion. A W/O/W emulsion stability study was then undertaken over a larger time domain (three months) using a carefully selected UV-visible tracer to investigate the usability of several analytical techniques of procedures. Several analytical techniques or procedures were developed. Breakdown kinetic curves were modeled and compared. EXPERIMENTAL PREPARATION OF THE MULTIPLE EMULSION General formula. The lipophilic and hydrophilic emulsifiers were the mixtures of non- ionic surfactants to obtain HLB - 4.6 and HLB = 14.1, respectively. The oily phase was a mineral oil. Hydrated magnesium sulfate was used in the inner aqueous phase as a stabilizing agent. Dihydralazine sulfate was utilized as an internal tracer. The general formula was: ß Primary W/O emulsion (w/w): --Lipophilic emulsifier 4% --Mineral oil 20% --MgSO 4 0.3 % --Tracer 0,3 % --Demineralized water st 100% ß Multiple emulsion (w/w): ---Primary emulsion 70% --Hydrophilic emulsifier 4% --Demineralized water st 100% Preparation procedure. The W/O/W emulsion was prepared by the two-step emulsification procedure (14). The emulsion process for the first step, in which a single emulsion (W/O emulsion) was prepared, employed a micro-vortex with a centrifugal turbine of 30-mm diameter. The rotation speed was initially set at 1500 rpm and then decreased every 10