298 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS with volatile sulfurous compounds and potential thiol substrates, and long-term residual levels at effective concentrations (2). Useful approaches to controlling VSC levels include the use of bactericidal agents, thiol trapping agents, such as zinc salts, and microbial metabolic inhibitors. A new anti- plaque agent, sanguinaria extract, a mixture of benzophenanthridine alkaloid salts, could be effective via any of these three mechanisms, thus making it a potentially formidable VSC control agent (5,7, 10). This paper compares four commercial oral rinses as VSC control agents using a new method of preliminary analysis. The analytical method, in contrast to gas chromatog- raphy techniques for VSC analysis, utilizes easy-to-use, inexpensive instrumentation available to most laboratories. Products screened included oral rinses containing cetylpyridinium chloride with dom- iphen bromide (D), zinc chloride (B), sanguinaria extract with zinc chloride (A), and a product containing a mixture of "essential oils" (C). The method utilized a thiol-specific colormetric reagent, 4,4'-bis(di-methylamino)di- phenylcarbinol (BDC-OH), to measure the levels of reactive sulfur present in incubated saliva. BDC-OH has historically been used as a reagent for the analysis of sulfhydryl groups in proteins based on this chemistry (6,9), a patent was recently issued for the use of BDC-OH as a general colormetric indicator of "bad breath." Under mildly acidic conditions (pH 5.1) and in the presence of certain surfactants, BDC-OH is present as CH3 CH 3 CH3 CH3 \N • NN • H + BDC-OH ----HC (• H + R-SH •, HC-SR N N /\ CH3 CH3 CH3 CH3 • max BDC + -- 612 nm • max BDC - SR = no abs. Figure 1. Chemistry of 4,4'-Bis(dimethylamino)diphenylcarbinol (BDC-OH) determination of sulphydryl groups.

ORAL VOLATILE SULFUR COMPOUNDS 299 a resonance-stabilized carbonium immonium ion (BDC + ). BDC + is the thiol-reactive form of BDC-OH and exhibits an intense blue color associated with an absorption at • max = 612 nm. The product of the reaction, BDC-SR, does not absorb at 612 nm, and thus the molar amount of reactive sulfur in a given system can be determined as a function of the decrease in absorption observed at 612 nm (Figure 1). MATERIALS AND METHODS Five females and two males ranging from 22 to 45 years of age were asked to use one of the commercial oral rinses for two consecutive 15-ml rinses of 15 seconds each at 9:00 AM of each test morning. Subjects were tested randomly and used each of the four test products five times, one product per day, in a nonconsecutive manner. Two ml of saliva stimulated by chewing orthodontic bands were collected at 8:15 AM, just prior to product use, and again 60 minutes after product usage. Saliva samples were collected in 150 X 15-mm tubes immersed in cracked ice. All subjects were asked to abstain from normal morning oral hygiene, as well as the use of tobacco products, coffee, or tea. Solutions of BDC-OH were made fresh weekly. A 0.75-ram solution of BDC-OH in 100 ml acetone was prepared and 10.0 ml of this solution diluted to 500 ml with a freshly prepared buffer/surfactant consisting of 3% disodium citrate and 7 mM sodium lauryl sulfate. The resulting solution (A) was 15 •M in BDC-OH and was stored at room temperature and protected from light. Saliva samples were mildly sonicated and incubated at 37øC for 24 hours in sealed polypropylene tubes. Samples were then centrifuged for 15 minutes at 4950 RPM and 1.0 ml of the supernatant from each sample was transferred to a cuvette containing 5.0 ml of solution A. The cuvettes were capped with rubber septa and shaken for one hour at 400 rpm at 40øC. The transmittance of each solution was then determined on a Bausch & Lomb Spectronic 20 at 612 nm against the buffer/surfactant as 100% T. Solution A was diluted with buffer/surfactant to make daily calibration standards at .12, .09, .06, .03, .015 and .0075 mM concentrations of BDC-OH and a linear regression of absorbance versus BDC + calculated. Absolute amounts of reactive sulfurs present were calculated for each saliva sample and results reported as a AR-SH with Table I Percent Reduction of Measurable Volatile Sulfur Compounds from Fermenting Saliva by Four Commercial Mouth Rinses Oral Rinse Active Concentration %AR-SH -+ S.E. A Sanguinaria Extract 0.030% Zinc Chloride 0.20% - 65.4 4.0 B Zinc Chloride 0.220% - 44.5 4.4 D Cetylpyridinium Chloride 0.045 % Domiphen Bromide 0.005 % - 43.9 5.8 C "Essential Oils" 16.4 mM - 15.6 6.0 AB•DC