A Sensitive and Quantitative Isotope-Dilution LC-MS/MS Method for Analysis of Hydrazine in Tobacco Smoke

A new isotope dilution liquid chromatography/tandem mass spectrometric method was developed for the analysis of potential hydrazine present in tobacco smoke. The sample preparation was performed via an optimized derivatization method using an aqueous buffer:methanol solution of 2-nitrobenzaldehyde (10 g/L) used as a derivatizing agent. The mainstream smoke of cigarettes was passed through a glass fiber filter pad followed by a trapping solution containing an isotopically labeled 15N2-hydrazine used as internal standard. After smoking, the filter pad was extracted with the trapping solution and then incubated for 30 minutes at 35°C. An aliquot of the extract was centrifuged and the resultant hydrazone was quantified by liquid chromatography tandem mass spectrometry (LC-MS/MS). The isotope dilution standard calibration curve demonstrated good linearity (R2 > 0.999) from 0.079 to 248 ng/mL, with limits of quantification in mainstream smoke of 0.2 and 0.4 ng/cig for ISO and Canadian Intense smoking regimens, respectively. The method recovery was assessed using samples spiked with solutions of known amounts of hydrazine. The results showed good accuracy with recoveries ranging from 98 to 111%. Although there were no detectable levels of hydrazine in the reference cigarettes used in the validation (KR3R4F), the method precision was estimated to be ~10% based on the variability observed in the spiked samples. Trapping efficiencies were assessed using a hydrazine permeation tube providing a known amount of hydrazine vapor such that the distribution between the vapor phase and particulate phase of mainstream smoke could be determined.