Materials such information and Methods Chemicals [Pyridine-D4]nicotine, [pyridine-D4]nornicotine, and [pyridine- D4]N��-nitrosonornicotine ([pyridine-D4]NNN) were obtained from Toronto Research Chemicals (Toronto, Ontario, Canada), and [2,2��,3,4,5,6-13C]NNN ([13C6]NNN) was purchased from Cambridge Isotope Laboratories, Inc. (Andover, MA). All other chemicals were obtained from Fisher Scientific (Pittsburgh, PA). Subjects Nonsmoking volunteers were recruited at the Masonic Cancer Center, University of Minnesota, and were asked to collect 2�C5ml of their saliva via expectoration into sterile polypropylene tubes. All subjects were Caucasian, between 23 and 46 years old, and 6 out of 10 were male. Collection of samples was approved by the University of Minnesota Research Subjects�� Protection Programs Institutional Review Board: Human Subjects Committee.

Saliva Incubation and [pyridine-D4]NNN Analysis Freshly collected saliva was placed on ice and treated within 1h of collection. As an initial test of nitrosation in saliva, 1ml aliquots of pooled saliva from three nonsmokers were incubated for 30min at 37 ��C with either [pyridine-D4]nicotine or [pyridine-D4]nornicotine, with and without addition of sodium nitrite. The reaction was stopped by placing samples on ice and adding 20 ��l 10 N NaOH (Mirvish, Wallcave, Eagen, & Shubik, 1972; Rao & McLennon, 1977). After addition of [13C6]NNN (internal standard), samples were promptly loaded on ChemElut cartridges and eluted with methylene chloride. This was followed by purification on Oasis MCX cartridges and BondElut cartridges as previously described for NNN analysis in urine (Stepanov, Carmella, Briggs, et al.

, 2009). The purified samples were analyzed for [pyridine-D4]NNN by LC-MS/MS as previously described (Stepanov & Hecht, 2008), with selected reaction monitoring for m/z 182 �� m/z 152 for [pyridine-D4]NNN and m/z 184 �� m/z 154 for [13C6]NNN. Analysis of Nornicotine in Nicotine Gum and Lozenge Nornicotine content in nicotine gum (Nicorette, 4mg nicotine) and lozenges (Commit, 2 and 4mg nicotine) was determined by gas chromatography�Cmass spectrometry as previously described (Stepanov, Jensen, Hatsukami, & Hecht, 2008). Results Optimization of Sample Purification Various procedures for the postincubation sample purification were tested.

Limiting sample Entinostat purification to a single-step extraction with methylene chloride and concentration of the organic extract to dryness under a stream of N2 led to ion suppression��a phenomenon that affects analyte ionization causing a loss in response by the mass analyzer��and artifactual formation of [pyridine-D4]NNN. Ultimately, we subjected samples to the procedure described in the Materials and Methods section. No significant ion suppression was observed with this method.