Abstract:
An apparatus ( 100 ) and method for measuring an extract ( 1000 ) released from a product sample ( 330 ) in vitro. The apparatus ( 100 ) comprising an in vitro environment which simulates an in vivo environment. The apparatus ( 100 ) contains a product sample ( 330 ) with the product sample ( 330 ) exposed to the in vitro environment. An extract ( 1000 ) is produced from the product sample ( 330 ) when the product sample ( 330 ) is subjected to the in vitro environment. The extract ( 1000 ) is analyzed to determine the amount of at least one element in the extract and the rate of release of at least one element in the product sample ( 330 ). The product sample is a smokeless tobacco product. An element analyzed for is nicotine.

Description:
FIELD OF THE INVENTION 
     The present invention generally relates to an apparatus and method which extracts and measures the quantity of at least one substance from a sample. Further, the apparatus and method simulates the in vivo environment, to which a sample would be subjected, collects any fluid released from the sample and measures the quantity of at least one element within the fluid collected. This application includes the use of the apparatus and method in measuring the nicotine in a smokeless tobacco product. Yet more specifically, this invention relates to an apparatus and a method of extracting and measuring, in vitro, the release of nicotine from tobacco sachets utilizing artificial saliva thus mimicking or simulating the nicotine release from smokeless tobacco in the human mouth. 
     BACKGROUND OF THE INVENTION 
     Nicotine replacement therapy as an aid to quitting smoking has become increasingly popular. Smokeless tobacco products that are on the market include chewing tobacco, oral snuff, or tobacco sachets, which deliver nicotine to the buccal mucosa. Tobacco sachets, which are especially popular in Scandinavia and the United States, contain ground tobacco in packets that are sucked or held in the mouth. 
     The patents and publications referred to herein are provided herewith in an Information Disclosure Statement in accordance with 37 CFR 1.97. 
     SUMMARY OF THE INVENTION 
     The apparatus and method simulates an in vivo environment, to which a sample would be subjected, collects any fluid released from the sample and measures at least one element within the fluid collected. 
    
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
       The foregoing and other features and advantages of the present invention will become more readily appreciated as the same become better understood by reference to the following detailed description of the preferred embodiment of the invention when taken in conjunction with the accompanying drawings, wherein: 
         FIG. 1  illustrates the apparatus ( 100 ) showing the nicotine extraction and collection apparatus ( 100 ), a reservoir ( 120 ) for storage of a fluid ( 140 ), a fluid pump ( 180 ), tubing ( 240 ), a bath ( 200 ), a water bath tank ( 210 ), a recirculating pump ( 460 ), controls ( 230 ) for maintaining a heating or cooling element ( 220 ) at a temperature. Also illustrated is tubing ( 240 ) and tubing water jacket ( 314 ). Inset A is the extraction bottle  300 . Also illustrated is a stand ( 420 ) supporting the extraction bottle ( 300 ). The bath ( 200 ) is a circulating temperature-regulating device and is usually a water bath ( 200 ). 
         FIG. 2  illustrates the extraction bottle ( 300 ) showing tubing ( 240 ), an extraction bottle top ( 302 ), an extraction bottle interior ( 304 ), an extraction bottle cap ( 305 ), an extraction bottle bottom ( 306 ), a supply tube ( 310 ), an extraction bottle water jacket ( 312 ), an extraction bottle water jacket supply inlet ( 315 ), an extraction bottle water jacket return ( 320 ), a screen mesh ( 325 ), a product sample ( 330 ) and a collection tube, ( 335 ). 
         FIG. 3  shows the graphical illustration of the measurement of nicotine from a smokeless tobacco product addressed in Table 1 with the use of artificial saliva recipe from Jayachandar, Gajendran, 1, Kraemer, J., and Knudsen, S. R. 2004. Product Performance Test for Medicated Chewing Gums. Dissolution Technologies, 2004, 11 (2):12-15). 
         FIG. 4  shows the graphical illustration of the measurement of nicotine from a smokeless tobacco product addressed in Table 2 with the use of artificial saliva recipe from Gal, I. Y., Fovet, Y., Adib-Yadzi, M. 2001. About a synthetic saliva for in vitro studies. Talanta, 53:1103-1115. 
         FIG. 5  shows the graphical illustration of the measurement of nicotine from a smokeless tobacco product addressed in Table 3 with the use of artificial saliva recipe from Abdelbary, G., Eouani, C., Prinderre, P., Joachim, J., Reynier J. P., Piccerelle, P. H. 2005. Determination of the in vitro disintegration profile of rapidly disintegrating tablets and correlation with oral disintegration. International Journal of Pharmaceutics, 292 (1-2):29-41. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       FIG. 1  illustrates the apparatus ( 100 ) showing the nicotine extraction and collection apparatus ( 100 ), a reservoir ( 120 ) for storage of a fluid ( 140 ) which would be found in the vivo environment comprising, for nicotine measurement of sachets, artificial saliva. Also seen is a fluid pump ( 180 ) which transports the pumped fluid ( 140 ) through tubing ( 240 ) through a bath ( 200 ) which maintains the fluid ( 140 ) at a desired temperature. The fluid pump ( 180 ), in the preferred embodiment, allows regulation of the pump rate. Tubing ( 240 ), in the preferred embodiment, is composed of rigid, semi-rigid or flexible tubing. The bath ( 200 ), in the preferred embodiment is generally a circulating water bath contained in a water bath tank ( 210 ) having a heating and or cooling attribute to maintain the bath at a desired temperature, a recirculating pump and temperature control ( 230 ) for maintaining the heating or cooling element ( 220 ) at a temperature required to achieve the desired temperature of the bath ( 200 ). Where the environment is the human mouth the desired temperature to be maintained is 37° C. with this accomplished by means of the bath ( 200 ) having a heating or cooling element ( 220 ) within the bath ( 200 ) tank ( 210 ). The operation and use of a bath ( 200 ) as described herein and as illustrated will be known to those of ordinary skills in the water bath arts. Illustrated is tubing ( 240 ) which transports the fluid ( 140 ) pumped by fluid pump ( 180 ) from the reservoir ( 120 ) through the bath ( 200 ) and to an extraction bottle ( 300 ). The at least one extraction bottle ( 300 ) is described fully in the Brief Description for  FIG. 2 . An extraction bottle ( 300 ) has a water jacket ( 312 ) and tubing ( 240 ) has a tubing water jacket ( 314 ) encompassing the tubing ( 240 ) when the tubing ( 240 ) is outside of the bath ( 200 ) and is flowing to the extraction bottle water jacket ( 312 ). The bath ( 200 ) is circulated, by a recirculating pump ( 460 ), from the bath ( 200 ) contained within the bath tank ( 210 ) through the tubing water jacket ( 314 ), to the extraction bottle water jacket supply inlet ( 315 ), into the extraction bottle water jacket ( 312 ), out of the water jacket ( 312 ) via the water jacket return ( 320 ) and returned to the bath ( 200 ). 
       FIG. 2  illustrating the extraction bottle ( 300 ) having an extraction bottle top ( 302 ), an extraction bottle bottom ( 306 ) and an extraction bottle interior ( 304 ). An extraction bottle cap ( 305 ) is shaped to be friction fitted at the extraction bottle top ( 302 ), the extraction bottle cap ( 305 ) having an aperture at which or into which tubing ( 240 ) is friction fitted or affixed by tubing fixing means including clamps, connectors and other methods readily known to those of ordinary skills in apparatus and chemical arts. A supply tube ( 310 ) downwardly extends, from the extraction bottle cap ( 305 ), into the extraction bottle interior ( 304 ). The fluid ( 140 ) transported via tubing ( 240 ) flows into and through the supply tube ( 310 ). Downwardly and distal to the extraction bottle top ( 302 ) and the supply tube ( 310 ) is a collection tube ( 335 ) which is downwardly extending from an extraction bottle bottom ( 306 ). A screen mesh ( 325 ) is affixed by affixing means within the extraction bottle interior ( 304 ) intermediate the supply tube ( 310 ) and the extraction bottle bottom ( 306 ). A product sample ( 330 ) is placed on or affixed to the screen mesh ( 325 ) in alignment with the supply tube ( 310 ) so that the fluid ( 140 ) from the supply tube ( 310 ) is flowed or dripped onto the product sample ( 330 ). The extract ( 1000 ) from the combination of the fluid ( 140 ) and the sample ( 330 ) is drained from the extraction bottle ( 300 ) by a collection tube ( 335 ) and is then deposited into at least one collection bottle ( 400 ). An extraction bottle water jacket ( 312 ) substantially encloses the extraction bottle ( 300 ). The extraction bottle water jacket ( 312 ) has an extraction bottle water jacket supply inlet ( 315 ) and an extraction bottle water jacket return ( 320 ). 
     Description of the Procedure 
     The nicotine extraction and collection procedure include the following steps: 
     1. Add fluid ( 140 ) to the reservoir ( 120 ). Fluid ( 140 ) is artificial saliva where the extraction is nicotine from a smokeless tobacco product; 
     2. Set the bath ( 200 ) and the heating or cooling element ( 220 ) temperature control ( 230 ) to the temperature desired as established from the in vivo environment. Where the fluid ( 140 ) is artificial saliva and, for the measurement of nicotine in a smokeless tobacco product, set the temperature to 37° C.; 
     3. Start the fluid pump ( 180 ) and set the pump to the desired pump rate. For measurement of nicotine in the preferred embodiment of this invention set the pump ( 180 ) to pump at 0.1 to 7 ml/min based on experiment requirement; 
     4. When the temperature and flow rate are stabilized, add the product sample ( 330 ) to extraction bottle interior ( 304 ) onto the screen mesh ( 325 ). Where the product sample is for a smokeless tobacco product sample ( 330 ) the product sample ( 330 ) is a smokeless tobacco sachet; the product sample ( 330 ) is placed onto the screen mesh ( 325 ) in alignment with the supply tube ( 310 ); 
     5. The fluid ( 140 ) from the supply tube ( 310 ) is flowed or dripped onto the product sample ( 330 ) producing an extract ( 1000 ) from the combination of the fluid ( 140 ) and the sample ( 330 ) which is drained from the extraction bottle ( 300 ) by a collection tube ( 335 ) and is then deposited into at least one collection bottle ( 400 ) at intervals determined by experimental design. Where the product sample ( 330 ) is a smokeless tobacco product sample ( 330 ) 1−n collection bottles ( 400 ) will used with bottles ( 400 ) changed at intervals of 1-5 min based on experimental design; 
     6. Extract the residual element from the product sample ( 330 ) by placing the product sample ( 330 ) in solvent effective for the type of product sample ( 330 ); where the product sample ( 330 ) is a smokeless tobacco product, the solvent in the preferred embodiment is 20 to 50 ml ethanol:5% NaOH mixture (9:1), and extract with ultrasound for 30 min. 
     7. Measure the target element in the extract ( 1000 ) by use of a measuring instrument suitable for the attributes of the target element in the extract ( 1000 ); for the preferred embodiment when the target element is nicotine from a smokeless tobacco product the measuring instrument is high-performance liquid chromatography (HPLC); the extract ( 1000 ) of nicotine in artificial saliva and the residual nicotine from ethanol NaOH mixture are determined using HPLC in the following conditions:
         1. Chromatograph column is a C18 reverse phase column   2. Mobile phase is methanol and 20 mmol phosphate buffer (23:77, volume to volume) with 0.2% Triethylamine, pH=6.0   3. Flow rate is 1 ml/min   4. Column temperature is 35° C.       

     Examples of Application 
     Example 1 
     Artificial saliva temperature was maintained at 37° C., pump flow rate 1 ml/min. Artificial saliva was made using the formula in Table 1, with a pH of 6.7. The artificial saliva collection intervals were at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 24, 28, 32 minutes after the extraction started. 
     
       
         
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Recipe for Artificial Saliva 
               
             
          
           
               
                   
                 Concentrations 
                   
                 Concentrations 
               
               
                 Components 
                 (g/L) 
                 Components 
                 (g/L) 
               
               
                   
               
             
          
           
               
                 NaCl 
                 1.550 
                 KHCO 3   
                 2.352 
               
               
                 KCl 
                 0.865 
                 MgCl 2   
                 0.715 
               
               
                 Na 2 HPO 4   
                 1.717 
                 CaCl 2   
                 0.358 
               
               
                 KH 2 PO4 
                 0.765 
                 NaHCO 3   
                 630.8 
               
               
                 Citric acid 
                 0.120 
               
               
                   
               
               
                 The recipe is from Jayachandar et al. (Dissolution Technologies, 2004, 11 (2): 12-15). 
               
             
          
         
       
     
     A nicotine release with extraction time curve was generated ( FIG. 3 ). Total nicotine is the sum of all collection bottles plus the residual nicotine in the tobacco. Under this extraction condition, nicotine release was rapid in the first 5 minutes, and gradually slowed down. In the first 5 minutes, the average rate of nicotine release was approximately 10%; from 6 min to 10 min, the average rate of nicotine release was approximately 4.3%; from 11 min to 20 min, the average rate of nicotine release was approximately 1%. After 20 min of extraction, over 90% of nicotine was released from tobacco, and very little nicotine was collected from the 20 to 32 min extraction period. 
       FIG. 3 . shows the extraction time and nicotine release where the X-axis is the time interval of nicotine elution solution collection and the Y-axis is the percent of accumulated nicotine release over time. 
     Example 2 
     Artificial saliva temperature was maintained at 37° C., pump flow rate 2 ml/min. Artificial saliva was made using the formula in Table 2, with a pH of 7.0. The artificial saliva collection intervals were at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 minutes after the extraction start. 
     
       
         
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Recipe of Artificial Saliva 
               
             
          
           
               
                   
                 Concentrations 
                   
                 Concentrations 
               
               
                 Components 
                 mg/L 
                 Components 
                 mg/L 
               
               
                   
               
             
          
           
               
                 NaCl 
                 125.6 
                 Na 2 SO4•10H 2 O 
                 763.2 
               
               
                 KCl 
                 963.9 
                 NH 4 Cl 
                 178 
               
               
                 KSCN 
                 189.2 
                 CaCl 2   
                 227.8 
               
               
                 KH 2 PO4 
                 654.5 
                 NaHCO 3   
                 630.8 
               
               
                 Urea 
                 200 
               
               
                   
               
               
                 The recipe is from Gal et al., (Talanta, 2001, 53: 1103-1115) 
               
             
          
         
       
     
     A nicotine release with extraction time curve was generated ( FIG. 4 ). Total nicotine is the sum of all collection bottles plus the residual nicotine in the tobacco. Under this extraction condition, nicotine release was rapid in the first 5 minutes, and gradually slowed down. In the first 5 minutes, 64% nicotine was release; from 6 min to 10 min, another 20% nicotine was released; In the first 20 min of extraction, over 95% of nicotine was released from tobacco, and very little nicotine was collected from the 20 to 30 min extraction period. The overall nicotine extraction efficiency was slightly better in example 2 compared to example 1. 
       FIG. 4 . shows extraction time and nicotine release where the X-axis is the time interval of nicotine elution solution collection and the Y-axis is the percent of accumulated nicotine release over time and where the recipe is from Gal et al., (Talanta, 2001, 53:1103-1115). 
     Example 3 
     Artificial saliva temperature was maintained at 37° C., pump flow rate 4 ml/min. Artificial saliva was made using the formula in Table 3, with a pH of 5.8. The artificial saliva collection intervals were at 2, 4, 6, 7, 8, 10, 12, 14, 16, 18, 20, 24, 28, and 32 minutes after the extraction start. 
     
       
         
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 Recipe of Artificial Saliva 
               
             
          
           
               
                   
                 Concentrations 
                   
                 Concentrations 
               
               
                 Components 
                 (g/L) 
                 Components 
                 (g/L) 
               
               
                   
               
             
          
           
               
                 NaCl 
                 0.4 
                 Na 2 SO4•10H 2 O 
                 763.2 
               
               
                 KCl 
                 0.4 
                 CaCl 2 •2H 2 O 
                 0.8 
               
               
                 NaS•9H 2 O 
                 0.005 
                 NaH 2 PO4 
                 0.78 
               
               
                 Urea 
                 1 
               
               
                   
               
               
                 The recipe is from Abdelbary, G., et. al. International journal of pharmaceutics, 292(1-2): 29-41. 
               
             
          
         
       
     
     A nicotine release with extraction time curve was generated ( FIG. 5 ). Total nicotine is the sum of all collection bottles plus the residual nicotine in the tobacco. Under this extraction condition, nicotine release was 82% in the first 6 min; 92% in the first 10 min. The rate of nicotine release was faster than in examples 1 and 2. 
       FIG. 5  shows extraction time and nicotine release where X-axis is the time interval of nicotine elution solution collection and the Y-axis is the percent of accumulated nicotine release over time. 
     While various embodiments of the present invention have been shown and described, it should be understood that other modifications, substitutions and alternatives are apparent to one of ordinary skill in the art. Such modifications, substitutions and alternatives can be made without departing from the spirit and scope of the invention, which should be determined from the appended claims. Various features of the invention are set forth in the appended claims.