282 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS disposable centrifuge tube. Thirty pieces of reagent grade lead shot were added and approximately 20 ml of methanol acidified with 5% citric acid (w/w) added. The tube was capped, vortexed for one minute, and centrifuged for five minutes at 2500 RPM. The cap was rinsed with the acidified methanol into the centrifuge tube and the super- natant liquid decanted into a 100-ml volumetric flask. Vortexing and centrifugation was repeated two times with 15 ml and 20 ml acidified methanol and the supernatant liquids transferred to the volumetric flask. The centrifuge tube cap was rinsed with the acidified methanol into the centrifuge tube, then decanted into the volumetric flask. (Complete transfer was checked by means of a long-wave UV light where fluorescence remaining in the residue left after decantation was indicative of incomplete transfer. If fluorescence was present the centrifugation and vortexing steps were repeated.) The combined rinsings in the volumetric flask were diluted to volume with methanol. Samples were injected into the HPLC and the •g of sanguinarine per gram of tooth- paste calculated. Oral rinse. No preparation was required to analyze the oral rinse. Samples were injected directly into the instrument and •xg of sanguinarine per ml of oral rinse calculated. STANDARD CURVE Standard solutions of sanguinarine of 10, 15, 20, and 25 •xl were injected into the HPLC and a regression analysis of sanguinarine chloride versus the area under each peak made. A correlation coefficient of 0.9998 or better was necessary before the curve was used. REPRODUCIBILITY Rhizomes. Reproducibility of the method was determined by taking a single sampling of full intact rhizomes and splitting into three lots. Each lot was treated in accordance with the above method. Reproducibility of the method was determined to be ___ 0.3% (Table I). Sanguinaria extract. A single lot of sanguinaria extract was divided into six portions. Each portion was analyzed according to the above method. Reproducibility of the method was determined to be + 0.3% (Table I). Oral rinse. Six bottles from a single production lot were each analyzed by direct injection into the HPLC to give a method reproducibility of +-0.1% (Table I). Toothpaste. Three tubes of an experimental toothpaste similar to the formula in Table III but without silica were each analyzed in triplicate according to the above procedure. The reproducibility of the method was determined to be + 1.2% (Table I). Table I Table of Reproducibility Sample Source No. of HPLC Runs Reproducibility Rhizome 3 -+ 0.3 % Sanguinaria Extract 6 -+ 0.3% Oral Rinse 6 -+ 0.1% Toothpaste 9 _+ 1.2%

SANGUINARINE ANALYSIS BY HPLC 283 Table II Stability of Sanguinarine in Oral Rinse (ambient temperatures) Containing 300 •g/ml Sanguinaria Extract Equivalent to Theoretical Input Levels of 90 t-tg/ml Sanguinarine Time Elapsed (months) Sanguinarine (t.tg/ml) 0 88.2 7 89.1 10 92.7 13 86.6 16 87.1 19 85.6 22 85.3 25 87.3 28 79.8 31 86.2 RESULTS AND DISCUSSION Standard solutions containing pure sanguinarine provide a clean chromatogram devoid of other benzophenanthridine alkaloids (Figure 2). Sanguinarine analysis of the dried rhizomes enable a detailed study (9) of the best growing conditions for the Sanguinaria canadensis plant. Plants were collected from over 100 sites along a 1,000-mile transect essentially along the Appalachians from northern Georgia to Vermont. The rhizomes were weighed, dried, and the water content determined. Samples were ground and analyzed according to the method described. Plants were catalogued for soil type, pH, climate, drainage, litter depth, and other parameters. The sanguinarine content was found to range from 0.63% (of the dried rhizome weight) to 6.31%. The mean san- guinarine content was found to be 2.74% + 1.18% over 100 sites. Positive correla- tions were found between sanguinarine content and slope, litter depth, soil clay con- tent, soil structure, rhizome weight, and also the time of year the plant was collected. SaCI I I I I Figure 2. Chromatogram for pure sanguinarine chloride.