Method for determining tobacco-specific nitrosamines (TSNAs) in cigarette smoke using one-step clean-up coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS)

A method for determining tobacco-specific nitrosamines (TSNAs) in a cigarette smoke using a one-step clean-up coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS), comprising the following steps:

• collecting a particulate matter of a mainstream cigarette smoke with a Cambridge filter, mixing the particulate matter of the mainstream cigarette smoke, an internal standards solution and water in a first centrifuge tube, and vortexing a first resulting mixture at room temperature to allow an extraction;
• centrifuging and transferring an extraction solution to a second centrifuge tube, adding dichloromethane, and vortexing a second resulting mixture;
• centrifuging to collect a dichloromethane extraction solution in a lower layer to a third centrifuge tube, and placing the third centrifuge tube in a water bath to remove dichloromethane;
• dissolving a resulting extraction solution in water, and transferring a resulting solution to an autosampler vial for an LC-MS/MS analysis, wherein
• the internal standards solution is prepared as follows:
• adding 5.0 mg of N′-nitrosonornicotine (NNN), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), nitrosoanatabine (NAT), nitrosoanabasine (NAB), d4-NNN, d4-NNK, d4-NAT, and d4-NAB, respectively, to 10 mL separate volumetric flasks, and adding methanol to prepare 0.5 mg/mL standard stock solutions;
• adding 2 mL of NNN, NNK, NAT, and NAB standard stock solutions, respectively, to one 10 mL volumetric flask, and adding water to prepare a 100 μg/mL mixed standards solution;
• and adding 2 mL of d4-NNN, d4-NNK, d4-NAT, and d4-NAB standard stock solutions, respectively, to one 100 mL volumetric flask, and adding water to prepare a 10 μg/mL internal standards solution, and wherein
• the method comprises using a national standard GB/T 19609-2004 for creating the extraction solution, collecting a particulate matter of a mainstream cigarette smoke of 20 cigarettes with a 92-mm Cambridge filter pad;
• and dissolving the resulting extraction solution in 300 μL of water, and transferring the resulting solution to a 2-mL autosampler vial, and chromatographic conditions are as follows:
• separation is performed using a Waters HHS T3 column (2.1 mm×150 mm×1.8 μm) and equilibrated with a first solvent A and a second solvent;
• a flow rate of 0.25 mL min−1 with a linear gradient is used as follows:
• 10% the second solvent over the course of 1 min, 10-30% the second solvent over the course of 1-8 min, the second solvent changed to 95% over the course of 8-8.01 min, then hold for 1 min, changed to 10% over the course of 9-9.01 min, and then hold for 1 min, wherein the first solvent is water containing 0.8% formic acid, and the second solvent is methanol;
• a column temperature is 30° C. with an injection volume of 2 μL.