). The length of the impactor inlet tubing was minimized and directly connected to thenthereby the breathing zone of the nose-only chamber. The nebulizer worked as 3-jet nebulizer without modification (the above 3-to-1 jet reduction version of the nebulizer was only for droplet size distribution measurement). Before the assay, the filters (PTFE Membrane Disc Filters, Pall Co.) for the impactor were balanced in a temperature (22 ��C) and humidity (49%) controlled environment where the electronic balance (Mettler-Toledo MX5 Microbalance, Mettler-Toledo Inc.) for weighing the filters was located. The filter weight difference between pre- and post-nicotine aerosol collection is the mass collected in a preset duration, for example, 1min.
To compensate the weight loss due to vaporization of the solvent (water) in the filter, we monitored the weight change during a period equivalent to the time needed from the end of aerosol sampling to the point the filter was weighed on the balance and added this equivalent weight lost to the measured value. Arterial and Venous Blood Sample Collection and Plasma Nicotine Level Measurement For arterial blood sample collection, femoral artery precatheterized rats (8�C9-week-old) were ordered from Harlan Laboratories. For venous blood collection, local anesthetic cream was applied to the rat tail 30min prior to insertion of the catheter. The rat was put into the rat holder and the tail was warmed with warm water or an infrared light. A temporary catheter (24G) was inserted into the lateral tail vein. Warm saline (2.
5ml) was infused into the arterial or venous catheter for blood volume compensation before blood collection. Then 0.1�C0.2ml of heparin (100U/ml) was injected. The first sample as pre-nicotine control was collected. Then nicotine aerosol was generated from the nebulizer and at the same time, a timer was started. The rat was exposed to the nicotine aerosol for 2min. Blood samples (equivalent to 0.2ml plasma) were collected at a series of timepoints (see Results section) with 0.6-ml capillary blood collection tubes containing ethylenediaminetetraacetic acid-K2 (RAM Scientific Inc.). Dropping from the venous catheter was sometimes slow. Bleeding can be greatly facilitated by massaging the tail over the vein. Samples were centrifuged within 15min of collection at 1,000g (gravity) for 12min at room temperature.
Supernatant plasma was transferred to labeled eppendorf Entinostat tubes and store below ?20 ��C. The plasma samples were sent to NMS Labs for measurements of nicotine and its metabolite cotinine. The analysis was performed on a Waters TQD MS with ACQUITY UPLC system equipped with an ACQUITY HSS T3 2.1 �� 50mm, 1.8 ��m analytical column, with in-line filter and positive-ion electrospray mass spectrometry (LC-MS/MS). Each analytical run was independently calibrated at concentrations of 2.5, 5.