Benzo[a]pyrene (B[a]P) is a polycyclic aromatic hydrocarbon, which is a by-product of incomplete combustion or burning of organic compounds. B[a]P is mutagenic and carcinogenic in nature. B[a]P is an ubiquitous compound both outdoors and indoors due to releases from multiple processes like vehicle emissions, burning of wood or coal in heating stoves or fire places. In tobacco it is formed during the curing process. Currently a method for the determination of B[a]P in tobacco is prescribed by Health Canada. It involves tedious multi-step sample cleaning with the use of a solid phase extraction cartridge (SPE) to remove matrix interference.

A quick and simple HPLC method is developed for the qualitative and quantitative determination of B[a]P residues in tobacco and snus without the use of the SPE cartridge. This method uses HPLC with a fluorescence detector. B[a]P is extracted from tobacco after refluxing with ethanolic KOH and liquid - liquid partition with a mixture of cyclohexane and chloroform and with distilled water. The cyclohexane extract is concentrated using a rota-evaporator and diluting with acetonitrile and subsequent chromatographic separation on LiChrospher RP-18e (250 mm × 5 micron × 4.0 mm) (Merck), under gradient condition using Mobile phase A- {Acetonitrile (70%), 1% iso-propyl alcohol in distilled water (30%)} and Mobile phase B- {Acetonitrile (100%)}.

The separations are monitored by fluorescence absorbance excitation at 365 nm and emission at 425 nm. The retention time for B[a]P is 14.2 ±0.5 min at a flow rate of 1.0 mL/min. The method has been validated by standard validation protocols i.e. limit of detection, limit of quantification, recovery, repeatability and reproducibility. Minimum recovery of 83% was obtained with a linear regression coefficient of 0.9999 for the range of 0.07-9.20 ppb of B[a]P. The limit of detection was 0.07 ppb. Tobacco samples of various grades and snus samples were analyzed.

This method provides several advantages such as sensitivity, speed, simple and easy sample preparation. The method has been found suitable for rapid determination of B[a]P in tobacco.