Free-base Nicotine Research Articles

Free-base nicotine in tobacco products. Part I. Determination of free-base nicotine in the particulate phase of mainstream cigarette smoke and the relevance of these findings to product design parameters

The free-base nicotine (FBN) content of mainstream cigarette smoke (MSS) has been discussed in the peer-reviewed literature and popular press. It has been alleged that manufacturers adjust product design features to increase the percentage of total nicotine (TN) in the MSS gas–vapor phase that is unprotonated [ P g ,nic(%)] and/or the fraction of nicotine in the MSS total particulate matter (TPM) that is unprotonated (FBN/TN). Our research showed the Health Canada Intensive smoking conditions negated the effects of blend and cigarette design features reported to raise the pH of TPM collected under ISO or US FTC conditions. Our research also showed that when additive-free Canadian cigarettes were smoked under ISO conditions, the FBN/TN ratio increased as the tar/nicotine ratio decreased. Our findings are in line with other studies that have questioned allegations of a relationship between use of ammonia and its compounds as tobacco additives and amounts of unprotonated nicotine in MSS. In addition, the experimental work demonstrated how use of solid-phase microextraction to estimate FBN can yield erroneously high results due to improper conditioning and/or smoking of the cigarettes. Our research showed that there is no longer any scientific support for regulators to require smoke pH and FBN determinations on cigarette products.

An improved headspace solid-phase microextraction method for the analysis of free-base nicotine in particulate phase of mainstream cigarette smoke

The content of free-base nicotine in cigarette smoke is a controversial subject, partly due to methodological issues. In this investigation, an improved method to measure free-base nicotine in cigarette smoke using headspace solid-phase microextraction (HS-SPME) combined with GC/MS analysis, was developed and validated for this purpose. Cigarette smoke particulate phase (PP) was collected onto a 44 mm glass fiber filter pad. The pad was cut in halves with one half used to determine the concentrations of total nicotine and water. The remaining half was analyzed by HS-SPME for free-base nicotine. The following factors were found to have a significant impact on the responses of free-base nicotine: SPME fiber type, pre-equilibrium time before HS-SPME, extraction time and temperature, PP water content, and the solvent used for the preparation of standards. It was also found that the impact of PP water content on the determination of free-base nicotine from smoke sample could be corrected by a water correction factor calculated based on an experimentally determined reciprocal model. The precision of the method was evaluated with smoke samples of reference cigarettes: Canadian flue-cured monitor and Kentucky reference 2R4F. The RSD values obtained were in the 12.8–16.8% range.

A transport model for nicotine in the tracheobronchial and pulmonary region of the lung

Nicotine in mainstream cigarette smoke is predominantly present in the particulate phase. Interestingly, however, the deposition efficiency of smoke particles in the respiratory tract is less effective than is the nicotine retention. In the literature, four nicotine deposition mechanisms are identified: (a) direct gas deposition, (b) evaporative gas deposition, (c) particle deposition with evaporation, and (d) particle deposition with diffusion. In this article we present a physically motivated fundamental model to address nicotine deposition mechanisms (b) and (c) from the vapor phase. The model incorporates nicotine mass transport through estimates for the diffusion time across the epithelial layer and the time for nicotine vapor diffusion from the gas volume to the tissue surfaces in the tracheobronchial and pulmonary regions of the respiratory tract. The model comprises four mass transfer processes for nicotine at the surface of the respiratory tract epithelium: (1) conversion of free base nicotine from protonated nicotine; (2) free base nicotine transport across the epithelium; (3) free base nicotine evaporation; and (4) diffusion of free base nicotine vapor from the surface gas layer into the airway lumen. Results of the nicotine mass transport model suggest that the principal mechanism of nicotine delivery to the lung is by direct deposition of particles to the alveolar fluid lining, followed rapidly by evaporation into the lumen and then gas diffusion back to the surface as nicotine depletes in the surface layer through its transport across the epithelium.

Possible Role of Ammonia on the Deposition, Retention, and Absorption of Nicotine in Humans while Smoking†

This perspective presents an overview of the properties of tobacco smoke aerosol and the possible effect of ammonia on the deposition location, retention and the amount and rate of nicotine absorption during cigarette smoking. Three main mechanisms describe the absorption of smoke constituents: (A) gas-phase constituents deposit directly; (B) particles deposit and the constituents then diffuse through the particle into the biological buffer and then into the tissue; and (C) particulate phase constituents evaporate from the particles and then deposit from the gas phase. Nicotine from smoking deposits and is absorbed predominately in the lungs. When particles deposit on the lung-blood interfaces, nicotine is absorbed rapidly, regardless of the acid-base nature of the particles. This is due to the buffering capacity of the lung-blood interfaces and the small mass of nicotine per puff distributed over a large number of particles depositing onto a huge lung surface. The composition of both tobacco smoke aerosol particles and the gas phase are time dependent. Ammonia in mainstream smoke evaporates faster from particles than nicotine. It is, therefore, unlikely that ammonia can significantly affect the volatility of MS smoke nicotine from particles in the smoke aerosol. It is certain that no single measurement of tobacco or of smoke, especially one made under equilibrium conditions, can adequately characterize the time-dependent properties of mainstream smoke aerosol. Thus, the fraction of nonprotonated freebase nicotine in trapped, aged smoke particulate matter has not been shown to be a useful predictor of the amount or total rate of nicotine uptake in human smokers. Similarly, "smoke pH" and "pHeff" are not useful practical parameters for providing understanding or predictability of tobacco smoke chemistry or nicotine bioavailability.

Brand differences of free-base nicotine delivery in cigarette smoke: the view of the tobacco industry documents

The recent availability of internal tobacco industry documents provides significant insight into industry knowledge and manipulation of tobacco smoke delivery. One critical area of research is the role of smoke...

HSP‐70 Expression is up regulated in Human Aortic Smooth Muscle Cells (VSMC) Exposed to Nicotine In Vitro for Six Days

Objective We have previously shown that HSP-70 is up regulated by VSMC in response to nicotine in an acute in vitro model. This phenomenon correlates with a down regulation of the apoptotic pathway. We hypothesized that chronic exposure of VSMC to free-base nicotine (FBN) in vitro, would cause sustained up regulation of HSP-70. Methods VSMC grown in vitro were exposed to 10−6 M FBN for 24(day 1), 72(day 3), and 144 hours (day 6). Control cells were exposed to growth media without FBN. Expression of HSP-70 was determined by immunocytochemistry and an intensity stain index was measured quantitatively using digital image analysis. Results In VSMC, a significant increase in HSP-70 expression was observed after 24 hours exposure to FBN when compared to control (13.6 ± 2.6 vs. 6.4 ± 1.6 p= .02). This increased expression continued at 72 hours compared to 24 hours (108.1±3.9 vs. 13.6± 1.6 p=1.9−7). However, the expression of HSP-70 did not show any significant increase from 72 hours to 144 hours (108.1±3.9 vs. 110.0±4.6 p=.7). Conclusion Chronic exposure to an inflammatory stimulus (nicotine) causes a progressively significant increase of HSP-70. These observations imply a mechanism whereby HSP-70 is involved in the hyperplasia and hypertrophy of VSMC after chronic exposure to nicotine.

TGF‐β1 Expression in Human Aortic Smooth Muscle Cells (VSMC) is Down‐regulated when Exposed to Nicotine In Vitro for Six Days

Objective Previous work from this lab has shown that exposure to nicotine causes an immediate (3 hour) increase in expression of TGF-β1. We hypothesized that chronic exposure of VSMC to free-base nicotine in vitro would cause sustained up regulation of TGF-β1. Methods VSMC grown in vitro were exposed to 10−6 M FBN for 24 (day 1), 72 (day 3), and 144 hours (day 6). Control cells were exposed to growth media without FBN. Expression TGF-β1 was determined by immunocytochemistry and an intensity stain index was measured quantitatively using digital image analysis. Results After 24 hour exposure to FBN, TGF-β1 was down regulated when compared to control (132± 7.35 vs. 152± 5.31, p=.03). After 72, and 144 hours exposure, the decrease in expression of TGF-β1, when compared to the 24 hours, continues and is significant, (114.4 ± 6.8 vs.132.8 ± 7.35, p=.05) and (95.2±3.9 vs. 132.8 ± 7.35, p=.004) Conclusion Although exposure of VSMC's to nicotine causes an acute increase of TGf-β1, the long-term impact is down regulation of TGF-β1 expression. These observations imply that TGF-β1 is only involved in the acute phase of the inflammatory response to nicotine.

Significance of ammonium compounds on nicotine exposure to cigarette smokers

The tobacco industry publicly contends that ammonia compounds are solely used as tobacco additive for purposes of tobacco flavoring, process conditioning and reduction of its subjective harshness and irritation. However, neither objective scientific reports, nor the contents of a large number of internal tobacco company documents support this contention. The present review focuses on the hypothesis that addition of ammonium compounds to tobacco enhances global tobacco use due to smoke alkalization and enhanced free-nicotine nicotine exposure. Obviously, ammonia enhances the alkalinity of tobacco smoke. Consequently, the equilibrium shifts from non-volatile nicotine salts to the volatile free base that is more readily absorbed from the airways. The observed change in the kinetics of nicotine (i.e., shorter t 1/2 and higher c max) after ammoniation is, however, predominantly due to the higher concentration of nicotine in the smoke, rather than to an increase in the absorption rate of free-base nicotine in the respiratory tract. Although several findings support the hypothesis, additional studies are required and suggested to provide a proper, objective and independent scientific judgment about the effect of tobacco ammoniation on nicotine bioavailability. Scientific and public awareness of the effects of tobacco-specific ammonia compounds may stimulate global control, legislation and restriction of their use in cigarette manufacture.

Solid-Phase Microextraction-Based Approach To Determine Free-Base Nicotine in Trapped Mainstream Cigarette Smoke Total Particulate Matter

Characterizing nicotine delivery from tobacco products is important in the understanding of their addictive potential. Most previous studies report total nicotine and have not differentiated between nicotine in its protonated or free-base form. Rather than simply determining total nicotine, the method described in this paper determines the amount of free-base nicotine associated with trapped mainstream smoke particulate matter generated using a standardized smoking machine protocol. This method quantitatively determines volatile free-base nicotine associated with the particulate phase portion of mainstream cigarette smoke using solid-phase microextraction combined with gas chromatography-mass spectrometry. The headspace above total particulate matter from mainstream cigarette smoke trapped on a Cambridge filter pad (CFP) was analyzed for free-base nicotine in 26 cigarette brands. The selected cigarette brands were chosen to cover a wide range of tar and nicotine deliveries as measured under Federal Trade Commission machine smoking conditions. In the CFP's headspace the free-base nicotine levels ranged from 0.01 to 0.08 mg/cigarette. The measured ranges of free-base nicotine were remarkably similar over the different tar and nicotine delivery categories of full-flavored, light, and ultralight cigarette brands.

Nicotine delivery capabilities of smokeless tobacco products and implications for control of tobacco dependence in South Africa

Objectives: Smokeless tobacco (SLT) use is popular among black South African women and children. The study sought to determine the nicotine delivery capability of popular industrialised and traditional SLT brands...

Nicotine Induces Mononuclear Leukocyte Adhesion and Expression of Adhesion Molecules, VCAM and ICAM, in Endothelial Cells In Vitro

The pathology of atherosclerotic cardiovascular disease (ASCVD) has been characterized as an inflammatory response to vessel injury. The initial steps of this response involve mononuclear leukocyte (MNL) attachment and infiltration into the vessel wall. Leukocyte adhesion is potentiated by expression of cellular adhesion molecules. Vascular cell adhesion molecule-1 (VCAM) and intracellular adhesion molecule-1 (ICAM) are markers of cellular activation and have the ability to attach leukocytes to the endothelium, which is an initial event in the inflammatory response in the vessel wall. Human umbilical vein endothelial cells (HUVEC) were plated in endothelial growth medium (EGM) on plastic coverslips and grown until cells were 75% confluent. Free base nicotine (FBN) was diluted in EGM to a concentration of 10(-8) M and added to experimental cells. At 3 hr, coverslips were removed and fixed. Immunohistochemical staining (IHCS) was performed using a monoclonal antibody to human ICAM and VCAM. Digital image analysis (DIA) was performed to quantify the expression of ICAM and VCAM. An intensity stain index (ISI) measuring area and intensity of stain/total cellular area was determined. Additional HUVEC grown in a similar manner were either exposed to 10(-8) M FBN in EGM or EGM control for 4 hr, then were exposed to MNL suspension for 10 min. Coverslips were removed, rinsed, and fixed. Hematoxylin and eosin staining was performed and cells examined under light microscopy. Leukocyte number per high power field (HPF) was counted and compared to controls. Data were analyzed using analysis of variants (ANOVA) and Student's t-test. Differences were considered significant if p < 0.05. ICAM and VCAM expression was absent in control cells. Nicotine exposure at 3 hr induced expression of VCAM (ISI = 30.85+/-0.77) and to a lesser extent ICAM (ISI = 16.6+/-1.39) (p < 0.001). MNL adhesion was markedly increased in cells exposed to nicotine (79.4+/-16.9/HPF) when compared to control cells (1.8+/-0.91/HPF) exposed to MNL (p < 0.01). These data show nicotine's ability to activate HUVEC as evidenced by induction of ICAM and VCAM expression in vitro. The biological effects of these adhesion molecules are demonstrated by a marked increase in MNL adhesion to HUVEC as demonstrated by leukocyte adhesion assay (LAA). MNL adhesion and subsequent migration into the intima, if occurring in vivo, may be a vital step in the pathogenesis of ASCVD associated with nicotine exposure.

Effect of nicotine on spinal disc cells: a cellular mechanism for disc degeneration.

Experimental investigation to determine the effect of nicotine on intervertebral spinal disc nucleus pulposus (NP) cells cultured in vitro. OBJECTIVES.: To evaluate the effects of nicotine on cell proliferation, extracellular matrix production, and viability of NP cells in three-dimensional alginate constructs cultured in vitro. Numerous studies confirm that smoking is a strong risk factor for back pain. The most widely accepted explanations for the association between smoking and disc degeneration is malnutrition of spinal disc cells by carboxy-hemoglobin-induced anoxia or vascular disease. Nicotine, a constituent of tobacco smoke, present in most body fluids of smokers is known to have detrimental effects on a variety of tissues. It may also be directly responsible for intervertebral disc (IVD) degeneration by causing cell damage in both the nucleus pulposus and anulus fibrosus. The effect of nicotine on IVD cells has not previously been investigated. Bovine chondrocytic intervertebral disc cells were isolated by sequential digestion of nucleus pulposus and seeded in 2% alginate. The constructs were cultured for 21 days either in growth medium containing freebase nicotine (Sigma) at concentrations found in the serum of smokers (25 nmol/L-300 nmol/L) or in standard nicotine free-medium as controls. Samples were collected at time points 3, 7, 14, and 21 days and a quantitative assay was performed for DNA, glycosaminoglycans (GAG), and hydroxyproline. Samples were also processed for qualitative histologic analysis including immunolocalization of collagen types I and II. There was both a dose- and time-dependent response to nicotine, with constructs cultured in low-nicotine concentration media demonstrating an early increase in DNA, GAG, and collagen content, while constructs cultured in high nicotine concentration media demonstrated a late decrease in these parameters. At 25 nmol/L dose of nicotine, there was a significant increase (P < 0.05) in the above parameters at day 7 compared with the controls. At higher doses, there was a significant dose-dependent decrease (P < 0.05) in these parameters compared to controls; however, this was only significant at day 14 for the 300 nmol/L group and at day 21 for the 100 nmol/L, 200 nmol/L, and 300 nmol/L groups. Adverse morphologic changes were observed on histology, which included reduced cell proliferation, disrupted cell architecture, disintegration of cells, and extracellular matrix. Immunohistochemistry revealed the presence of type I collagen in the extracellular matrix rather than the normal type II collagen seen in the controls. Nicotine has an overall detrimental effect on NP disc cells cultured in vitro. There was significant inhibition of cell proliferation and extracellular matrix synthesis. Nicotine in tobacco smoke may have a role in pathogenesis of disc degeneration.

Fraction of free-base nicotine in fresh smoke particulate matter from the Eclipse "cigarette" by 1H NMR spectroscopy.

Solution 1H NMR (proton-NMR) spectroscopy was used to measure the distribution of nicotine between its free-base and protonated forms at 20 degrees C in (a) water; (b) glycerin/water mixtures; and (c) puff-averaged "smoke" particulate matter (PM) produced by the Eclipse cigarette, a so-called "harm reduction" cigarette manufactured by R. J. Reynolds (RJR) Tobacco Co. Smoke PM from the Eclipse contains glycerin, water, nicotine, and numerous other components. Smoke PM from the Eclipse yielded a signal for the three N-methyl protons on nicotine at a chemical shift of delta (ppm) = 2.79 relative to a trimethylsilane standard. With alpha fb = fraction of the total liquid nicotine in free-base form, and alpha a = fraction in the acidic, monoprotonated NicH+ form, then alpha a + alpha fb approximately 1. (The diprotonated form of nicotine was assumed negligible.) When the three types of solutions were adjusted so that alpha a approximately 1, the N-methyl protons yielded delta a = 2.82 (Eclipse smoke PM); 2.79 (35% water/65% glycerin); and 2.74 (water). When the solutions were adjusted so that alpha fb approximately 1, the N-methyl protons yielded delta fb = 2.16 (Eclipse smoke PM); 2.13 (35% water/65% glycerin); and 2.10 (water). In all of the solutions, the rate of proton exchange between NicH+ and Nic was fast relative to the 1H-NMR chemical shift difference in hertz. Each solution containing both NicH+ and Nic thus yielded a single N-methyl peak at a delta given by delta = alpha a delta a + alpha fb delta fb so that delta varied linearly between delta a and delta fb. Since alpha fb = (delta a-delta)/(delta a-delta fb), then delta = 2.79 for the unadjusted Eclipse smoke PM indicates alpha fb approximately 0.04. The effective pH of the Eclipse smoke PM at 20 degrees C may then be calculated as pHeff = 8.06 + log[alpha fb/(1-alpha fb)] = 6.69, where 8.06 is the pKa of NicH+ in water at 20 degrees C. The measurements obtained for the puff-averaged Eclipse smoke PM pertain to the chemistry of the smoke PM as it might be initially inhaled at 20 degrees C. Upon inhalation, the volatilization of nicotine and other acid/base active compounds (as well as a warming toward a body temperature of 37 degrees C) will alter the pHeff value of the smoke PM during the time that it resides and ages in the respiratory tract.

A consideration of the role of gas/particle partitioning in the deposition of nicotine and other tobacco smoke compounds in the respiratory tract.

Tobacco smoke is an aerosol that contains both gaseous and suspended particulate material (PM). The particles are largely liquid droplets containing a wide variety of condensed organic compounds. Each compound in the smoke will partition between the gas and PM phases and will always seek a state of gas/particle equilibrium. When tobacco smoke is inhaled, a compound such as nicotine can deposit in the respiratory tract (RT) by four different mechanisms: (1) direct gas deposition (DGD) of the portion of the compound that is initially in the gas phase of the inhaled smoke; (2) evaporative gas deposition (EGD) of PM-phase compound by evaporation to the gas phase, then deposition; (3) particle deposition, evaporation from the deposited particle, then deposition from the gas phase (PDE); and (4) particle deposition with diffusion (PDD) into RT tissue. Three of the mechanisms (DGD, EGD, and PDE) involve volatilization from the PM phase. The relative importance of all the mechanisms is therefore greatly affected by the volatility of the compound from the PM phase as it is set by the compound's gas/particle partitioning constant K(p) through the compound's vapor pressure. For a largely nonvolatile compound such as benzo[a]pyrene, only PDD will likely be important. For a semivolatile compound such as nicotine, all four mechanisms can be important. Because tobacco smoke alkaloids such as nicotine can exist in protonated as well as free-base form, the fraction alpha(fb) of the compound that is in the neutral free-base form in the PM phase plays a critical, pH-dependent role in determining the relative importance of the four mechanisms. Equations are developed that can be used to ascertain the importance of the DGD and EGD mechanisms. The value of alpha(fb) for nicotine in a tobacco smoke PM is set by pH(eff), the effective pH of the PM phase. Historically, a primary method for measuring "smoke pH" has involved the direct exposure of a pH electrode to tobacco smoke. This method cannot yield direct insight into pH(eff) or alpha(fb) values because (1) problems exist in using such an electrode to measure smoke PM-phase pH, and (2) by itself, a measurement of the pH of tobacco smoke PM says nothing about the effects of PM-phase activity coefficients of protonated and free-base nicotine on the nicotine species distribution. The "acidic" values that have typically been measured for cigarette "smoke pH" by the direct pH electrode method are therefore neither reliable nor useful in determining the relative distribution of PM-phase nicotine among the protonated and free-base forms. The dependence of the volatility of nicotine from tobacco smoke PM on alpha(fb) means that measuring the gas/particle distribution of nicotine under equilibrium conditions in a tobacco smoke by denuder samplers (or by another method) can yield information about the nicotine K(p) for that smoke. Knowledge of K(p,fb), the partitioning constant for nicotine in the free-base form, then allows calculation of alpha(fb) through the relation K(p) = K(p,fb)/alpha(fb). The available data suggest that the smoke PM from some commercial cigarettes can be characterized by alpha(fb) > or = 0.4, i.e., 40% or more of the nicotine in the free-base form. This conclusion is consistent with (1) the gas-sampling denuder results obtained by Philip Morris in which significant tobacco smoke nicotine was observed to deposit in acid-coated denuder tubes, with more depositing when the cigarette tobacco blend was treated with ammonia; (2) the view that the sensory "impact" exhibited by some tobacco smokes is caused by the deposition of gaseous nicotine in the pharynx; (3) the observed throat irritation caused by nicotine inhalers; and (4) the high overall respiratory tract deposition efficiencies for nicotine of 0.9 and greater that have been reported for some cigarette smokes. The available information combines to create a picture of nicotine in cigarette smoke that contradicts the traditional view that cigarette smoke PM is typically acidic, with little free-base nicotine typically present in the smoke PM phase. Government agencies interested in establishing a framework for the testing and monitoring of nicotine delivery may wish to consider requiring the measurement and publication of the PM-phase alpha(fb) values for the cigars and cigarettes marketed in their jurisdictions.

Nicotine Induces Endothelial TNF-α Expression, Which Mediates Growth Retardation in Vitro

Purpose. Atherosclerosis is understood as the common pathologic manifestation of arterial injury caused by a variety of etiologies. One well-established etiologic agent is nicotine. We hypothesized that cytokines of endothelial origin are involved with the pathologic changes found in atherosclerosis associated with smoking. We chose to assay for TNF-α due to its many biologic actions that are similar to those found in peripheral vascular disease.Methods. Human umbilical vein endothelial cells (HUVEC) were plated in endothelial growth medium (EGM-2) on plastic coverslips until 75% confluent. Free base nicotine (FBN) was diluted in EGM-2 to a concentration of 10−8 M and added to experimental cells. At 1, 3, and 24 h, coverslips were removed and fixed. Immunohistochemical staining was performed using anti-TNF-α. Digital image analysis (DIA) was performed to quantify expression of TNF-α. An intensity stain index measuring area and intensity of stain/total cellular area was determined for each time point (n = 5). Additional HUVEC were plated in 12-well plates in EGM-2 at 2 × 103 cells/cm2 on T−2 day. FBN was diluted in medium to 10−9 M and added to wells with and without 0.9 μg/ml anti-TNF-α on T0 day. Cell counts were performed in triplicate on days T2–5 utilizing hemocytometry. Data was analyzed using Student's t test and ANOVA, with a 95% confidence interval.Results. Dose response determinations showed that the minimal concentration required to show statistically significant cell retardation is 10 –9 M. Accordingly, this concentration was used for subsequent proliferation studies. DIA showed a threefold increase in TNF-α activity at 1 h and a twofold increase at 3 h. Activity returned to baseline by 24 h. Cell growth was significantly decreased in cells exposed to nicotine when compared to controls on days T2–T5 (P < 0.05). In cells exposed to anti-TNF-α and nicotine there was inhibition of the growth retardation seen in the cells containing nicotine alone. Differences between the control group and the anti-TNF-α group were not statistically significant.Conclusion. These data demonstrate the ability of endothelial cells to secrete TNF-α in response to nicotine at levels found in serum after smoking and also shows that endothelial cell growth retardation as a consequence of nicotine exposure may be TNF-α mediated.

The relative buffering capacities of saliva and moist snuff: implications for nicotine absorption.

Commercial moist snuff products are used by placing a portion of tobacco inside the mouth between the inner cheek or lip and gum. Nicotine is absorbed into the blood stream via transfer across various oral membranes including the buccal mucosa (cheek lining). The resulting salivary pH when a given moist snuff product is placed in the mouth is an important factor for nicotine absorption because it will affect the proportion of free base nicotine that is readily available for absorption. The resulting salivary pH for a given moist snuff product will be determined in part by the relative acid-base buffering capacities of the saliva and moist snuff, as well as the pHs of the saliva and moist snuff prior to coming in contact with one another. In the current study, the acid-base buffering capacities (mu eq/g) of a series of commercial moist snuff products were determined and compared to the acid-base buffering capacity for unstimulated, whole human saliva. The buffering capacities of the moist snuff products were determined to be 10-20 times higher than the buffering capacity of human saliva. The resulting salivary pH ranges after contact between an artifical saliva and the various moist snuff products were also determined; the results were used to predict the proportion of free base nicotine that can be expected to occur in the mouth during the first few minutes of product use. These studies provide a basis for examining and understanding the effects that moist snuff product pHs and buffering capacities may be expected to have on nicotine absorption.