j. Soc. Cosmet. Chem., 37, 279-286 (July/August 1986) HPLC analysis of sanguinarine in oral health care products E. M. THORNE, R. T. BOULWARE, R. J. HARKRADER, and G. L. SOUTHARD, Vipont Laboratories, Fort Collins, CO 80524. Received March 14, 1986. Presented in part at the International Association of Dental Research, Sixty-Second General Session, Dallas, Texas, March 15-18, 1984. Synopsis Sanguinarine is a component of an alkaloid extract obtained from the plant Sanguinaria canadensis. This extract is used in a commercial toothpaste and oral rinse exhibiting anti-plaque effectiveness. Using high performance liquid chromatography, rapid methods were developed whereby methanolic sanguinarine so- lutions of the plant rhizomes, sanguinaria extract, oral rinse and toothpaste were quantitatively analyzed. The method employed a Waters HPLC and a 5-CN 10-• Radial-Pak column with a mobile phase of 84:16 (v/v) methanol:water containing 5 x 10-3M triethylamine and phosphoric acid, pH 5.6, at a flow rate of 0.5 ml/minute. A Model 440 detector with a 280-nm filter was used with a Hewlett Packard Model 440 recorder. The detection limit for sanguinarine was 0.25 •g/ml. The method was demonstrated to be useful as a quality control technique and as a means for monitoring sanguinarine levels in stability samples. For example, an acidic pH range was found to be required for sanguinarine stability in oral rinse. INTRODUCTION Sanguinarine is a component of an alkaloid extract obtained from the plant Sanguinaria canadensis. The extract is used as a component in a commercial toothpaste and oral rinse exhibiting anti-plaque effectiveness. The effectiveness of the extract is attributable to sanguinarine, which is present in the extract as the chloride salt. Sanguinaria extract has been used in a variety of medications for over 100 years in the United States and other countries (1). The extract is principally a mixture of benzo- phenanthridine alkaloids, the chief constituent alkaloid being sanguinarine (approxi- mately 33% by weight). Sanguinarine is highly fluorescent under long-wave ultraviolet light which makes it relatively easy to monitor and analyze. Various methods have been reported for the analysis of this extract. An early method reported in 1913 detailed problems resulting from the use of any volumetric processes (2) and was tedious and time consuming. Later methods used paper chromatography (3-5) and electrophoresis, but these methods gave unreliable results. In 1977 a thin layer chromatography method was developed (6), but the method lacks the precision and accuracy for routine quality control analysis. A high performance liquid chromatography (HPLC) method was developed to quantita- tively analyze for sanguinarine in the plant rhizomes, sanguinaria extract, and oral care products. The rhizomes were analyzed for sanguinarine content, enabling a quality 279

280 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS control determination prior to producing sanguinaria extract. The oral rinse and tooth- paste were analyzed for sanguinarine content, enabling the products to be quality con- trolled and to be studied for sanguinarine stability and influences of formula variables on sanguinarine stability. An earlier HPLC method has already been reported (7) by the authors to analyze for sanguinarine retention in human plaque and saliva after using oral care products containing sanguinaria extract. The structure of sanguinarine and other benzophenanthridine alkaloids is shown in Figure 1 along with the observed HPLC retention times. EXPERIMENTAL MATERIALS AND METHODS APPARATUS A High Performance Liquid Chromatograph (Waters Associates, Milford, MA) was fitted with an M-45 delivery system, a UK 6 variable volume injector, a radial compres- R 1 R2 R 3 N +Cl' CH 3 94 R 5 Relative Approximate % in Retention Benzophenanthridine Sanguinaria Time Alkaloid Structure Extract (minutes) A--Chelirubine R• •- OCH3 R 2 q- R3, 4 10.4 R 4 q- R 5 = OCH20 B--Sanguinarine R• = H R 2 + R3, 50 12.0 R 4 + R 5 = OCH20 C--Sanguirubine R• = OCH3 R2 + R 3 = OCH20 4 13.7 R4, R5 = OCH 3 D--Chelilutine R•, R2, R 3 = OCH3 2 15.4 R 4 + R 5 = OCH20 E--Chelerythrine R• = H R 2, R 3 = OCH3 25 16.6 R4 + R 5 = OCH20 F--Sanguilutine R•, R2, R 3, R4, R 5 = OCH 3 15 17.7 Figure 1. Chemical structure of benzophenanthridine alkaloids, their relative amounts in sanguinaria extract, and HPLC retention times.