In the past few years, volatile aldehydes (carbonyls) have been included in the FDA’s “Draft Proposed Initial List of Harmful/Potentially Harmful Constituents in Tobacco Products”. They have routinely been analyzed in tobacco smoke for many years, but much less in smokeless tobacco. Consequently, there is now need for a reliable and high-throughput method in tobacco industry labs for analysis of STPs.

Aldehydes need to be derivatized in order to improve the sensitivity and selectivity of both GC- and UHPLC-methods. In this method, the commonly used derivatization agent 2,4-dinitrophenylhydrazine (DNPH) is used together with UHPLC-MS/MS.

The key characteristic of this rapid sample preparation method is that aldehyde extraction, derivatization, and enrichment are performed simultaneously during a 1-hour-shaking in a two-phase system, consisting of an aqueous and an organic phase. The extraction and the derivatization occur in the aqueous ammonium formate buffer, and the aldehyde-DNPH derivatives are then enriched in the organic isohexane phase. After shaking, a non-measured volume of the isohexane phase is readily transferred to an LC-vial. Besides the enrichment-effect of the derivatives in the organic phase, the most important function of isohexane is stabilization of the aldehyde-derivative concentration (at least 8 days), which otherwise has proven troublesome in aqueous tobacco extracts.

Isotopically labeled internal standards improve the precision and accuracy. UHPLC enables fast separation (5 min/sample) and MS/MS (Xevo TQ-S, Waters) gives excellent sensitivity and selectivity. LOQ for Formaldehyde, Acetaldehyde and Crotonaldehyde is 0.05, 0.5 and 0.03 ppm, respectively. The method repeatability (RSD) is 6-8%, accuracy 70-110%, and the capacity is ca 75 samples/day. Altogether, this method has the robustness, sensitivity, and capacity to meet the demands of tobacco industry labs.