ISSN: 2155-9872
Journal of Analytical & Bioanalytical Techniques
Like us on:
Make the best use of Scientific Research and information from our 700+ peer reviewed, Open Access Journals that operates with the help of 50,000+ Editorial Board Members and esteemed reviewers and 1000+ Scientific associations in Medical, Clinical, Pharmaceutical, Engineering, Technology and Management Fields.
 
Meet Inspiring Speakers and Experts at our 3000+ Global Conferenceseries Events with over 600+ Conferences, 1200+ Symposiums and 1200+ Workshops on
Medical, Pharma, Engineering, Science, Technology and Business

Simple and Fast Determination of Ammonia in Tobacco

Eugene Jansen*, Piet Beekhof, Johannes Cremers and Reinskje Talhout
Centre for Health Protection, National Institute for Public Health and the Environment, Netherlands
Corresponding Author : Eugene Jansen
Centre for Health Protection
National Institute for Public Health and the Environment
PO Box 1, 3720 BA Bilthoven, the Netherlands
Tel: +31-302742940
E-mail: eugene.jansen@rivm.nl
Received December 26, 2013; Accepted January 23, 2014; Published January 25, 2014
Citation: Jansen E, Beekhof P, Cremers J, Talhout R (2013) Simple and Fast Determination of Ammonia in Tobacco. J Anal Bioanal Tech 5: 178. doi: 10.4172/2155-9872.1000178
Copyright: © 2013 Jansen E, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Related article at
DownloadPubmed DownloadScholar Google

Visit for more related articles at Journal of Analytical & Bioanalytical Techniques

Abstract

The presence of ammonia in tobacco is an important factor for the absorption of nicotine, and for product taste. The determination of ammonia in tobacco is usually performed by ion chromatography with conductivity detection devices.

Here a new method is presented to measure the concentration of ammonia in tobacco based on an automated enzymatic method. This method is easy to perform and can be used on routine clinical analyzers. The enzymatic ammonia determination showed an intra-assay and inter-assay variation of 4-7 and 5-8%, respectively as determined with 3 brands of cigarettes. A comparison with the established HPLC-IC method gave similar results with respect to both concentrations in cigarettes and the reproducibility of the method. In one working day 50-60 samples of cigarettes or tobacco can be processed and analyzed.

Keywords
Ammonia; Enzymatic assay; Tobacco
Introduction
Ammonia is an important component of tobacco, primarily, because it is believed to facilitate the absorption of nicotine [1-4]. In addition, ammonium compounds react with sugars during tobacco processing and smoking to form flavour components that ‘improve’ the taste of tobacco smoke [5]. For these reasons, ammonia has been selected as priority component for method development for the WHO Framework Convention on Tobacco Control [6]. The determination of ammonia in tobacco and tobacco products has been subject of several publications [7,8]. All these methods are rather complicated by the use of pre-purification techniques with subsequent detection by ion chromatography coupled with conductivity detection devices which are usually not available in a normal analytical laboratory.
The present report describes a new simple, specific and fast method for the quantitative determination of ammonia in tobacco by a specific enzymatic reaction, with glutamate dehydrogenase in which NADPH is oxidized to NADP+. This assay is used routinely on clinical auto analyzers.
Materials and Methods
Materials
Ammonia was measured on a clinical auto-analyzer (Synchron LX20) with a kit (AMM reagent # 439770) both supplied by Beckman- Coulter, Woerden, The Netherlands. The ammonia kit consists of α-ketoglutarate (3.23 mmol/L), adenosine diphosphate (ADP, 1.9 mmol/L), β-nicotinamide adenine dinucleotide phosphate (NADPH, 0.22 mmol/L) and glutamate dehydrogenase (GLDH) from beef liver (>10 U/L). The ammonia reagent is stable until the expiration date printed on the label when stored unopened at +2°C to +8°C (usually at least one year). Once opened, the reagent is stable for 30 days at +2°C to +8°C. The standard ammonium sulfate was dissolved in 0.01M sulfuric acid to a final concentration of 154 μmol/L. The extraction solution consisted of 0.025 mol/L sulfuric acid (pH 1.75).
The cigarettes used in this study were Chesterfield, Belinda and Gauloises, obtained from the local shops. The reference cigarette K3R4F and the Marlboro cigarettes were obtained from Kentucky Tobacco Research & Development Center, University of Kentucky, Lexington, USA. All cigarettes were conditioned for 48 h at 20°C and 60% relative humidity prior to the analyses.
Methods
Determination of ammonia in whole tobacco: To approximate 100 mg tobacco, 10 mL of the extraction solution was added and shaken for 1 h. Then the extract solution was centrifuged for 10 min at 4000 rpm. The supernatant was diluted (1:3, v/v) with phosphate buffered saline (PBS, pH7.6). The pH must be between 7.2 and 7.6. Now the sample represents a concentration of 2.5 g tobacco/L and is ready to apply to the automatic measuring system (Synchron LX20), described above. The ammonia reagent, as described above, is used to measure ammonia by a timed endpoint method. In the assay reaction, glutamate dehydrogenase (GLDH) catalyzes the reaction of ammonia and α-ketoglutarate to glutamate with the concomitant oxidation of NADPH to NADP+. The amount of NADPH oxidized is directly proportional to the amount of ammonia in the sample. The Synchron LX20 automatically pipette the appropriate sample volume (40 μL) and reagent volumes (226 μL) into a cuvette. The system monitors the change in absorbance at 340 nm that is directly proportional to the concentration of ammonia in the sample and is used by the Synchron LX20 to calculate the ammonia concentration in the sample.
Results
Measuring principle
AMM reagent is used to measure ammonia by a timed endpoint method. In the assay reaction, glutamate dehydrogenase (GLDH) catalyzes the reaction of ammonia and α-ketoglutarate to glutamate with the concomitant oxidation of β-nicotinamide adenine dinucleotide phosphate (NADPH) to β-nicotinamide adenine dinucleotide phosphate (NADP+). The amount of NADPH oxidized is directly proportional to the amount of analyte in the sample. The chemical reaction scheme is shown below.
The Synchron LX-20 system automatically tranfers the appropriate sample and reagents into a cuvette. The ratio used is one part sample to 6 parts reagent (v/v). The system monitors the change in absorbance at 340 nm. This change in absorbance is directly proportional to the concentration of ammonia and its salts in the sample and is used by the Synchron LX-20 system to calculate and express the ammonia concentration.
Validation of the method
The extraction procedure has been adapted from a well-documented method [7]. Therefore recovery experiments related to the extraction procedure have not been performed in this study. Focus was put only on the detection method.
Samples can be measured in the analytical range from 160 μg ammonia/L (0.064 mg ammonia/g tobacco) to 17,000 μg ammonia/L (6.8 mg ammonia/g tobacco). Samples which exceeding the upper limit should be diluted with PBS buffer and reanalyzed.
Sensitivity for the ammonia determination is 160 μg ammonia/L (0.064 mg ammonia/g tobacco). Sensitivity is defined as the lowest measurable concentration that can be distinguished from zero with 95% confidence.
The reproducibility of the ammonia determination was very good with an intra-assay variation of 2.0% and an inter-assay variation of 3.0% as determined with plasma samples using the Beckman-Coulter system.
The reproducibility of the whole assay of ammonia in tobacco, including the weighing and extraction procedure was determined with three brands of cigarettes with 16 independent determinations/day during three days. The results have been depicted in Table 1.
The stability of the extracts prior to the analysis step was determined with three brands of cigarettes (N=16). The extracts were prepared and the ammonia concentrations were determined on the same day (day 1). Then the extracts have been stored at room temperature for another 2 days. At day 2 the average value was 101.8 % of the value measured at day 1. At day 3 this value was 101.1 %. So the extract can be prepared and kept at room temperature for at least several days before the final measurement.
In one working day, a well-trained technician can determine the ammonia concentration in 56 samples of tobacco from cigarettes. This includes weighing, extraction and measurement. The enzymatic measurement of ammonia with the auto-analyzer can be performed with a capacity of about 200 samples per hour.
Comparison with the HPLC-IC method
A comparison was made with the Health Canada methodology in which the ion chromatography with a suppressed ion conductivity detector was used. (Health Canada, 1999). This comparative study was performed in cooperation with Labstat International ULC. Kitchener. Ontario. Canada. The results with 2 brands of cigarettes have been summarized in Table 2.
For the Marlboro cigarettes RIVM measured an average value which is only 3% higher than Labstat ULC For the KR 3R4F cigarette both labs obtained exactly the same average value. The coefficients of variation are in the acceptable range (1.5-3.5%)
Discussion
With the present method, which uses an automated clinical chemical analyzer, the amount of total ammonia, including salts can be determined with great specificity, accuracy and reproducibility. In addition, the method is easy to perform and a large number of samples can be measured per day. The enzymatic method is specific for ammonia and can also be applied in a laboratory not equipped with an auto-analyzer by using the components of the automated assay. In our hands, however, the development of a suitable manual method for ammonia was not successful, using commercial kits and colorimetric detection devices, such as a spectrophotometer of a micro-titer plate reader. Most probably, the high accuracy of the auto-analyzer is a prerequisite to obtain the required assay performance.
Using two brands of cigarettes the ammonia content of the present method was compared with that obtained by the HPLC-IC method Based on this comparison, it was concluded that both the enzymatic and the HPLC-IC method gave similar results with high precision and reproducibility.
The present method to measure the concentration of ammonia in tobacco is a new and easy method which can be used on routine clinical analyzers. Almost all brands of these clinical analyzers have the possibility to measure ammonia with great accuracy, because of the use dedicated kits and calibration systems. Therefore this can be used in almost all research settings and tobacco control labs worldwide.
Declaration of Interest
The authors stated that there are no conflicts of interest regarding the publication of this article.
Acknowledgements
The authors are indebted to Dr. P. Joza from Labstat International ULC for the measurements of the HPLC-IC method.
This study was conducted with financial support of the Netherlands Food and Consumer Product Safety Authority (NVWA) and the Tobacco Free Initiative of the World Health Organization (WHO HQ/TFI).
 
References








Select your language of interest to view the total content in your interested language
 
Share This Article
   
 
   
 
Relevant Topics
Disc Affinity Purification
Disc Analytical Biochemistry Techniques
Disc Analytical Chemistry
Disc Analytical Chromatography
Disc Analytical Techniques
Disc Anthropology Biomedicine
Disc Applied Biomedicine
Disc Bioanalysis
Disc Bioanalysis Methods
Disc Bioanalytical Chemistry
Disc Bioanalytical Method Validation
Disc Bioanalytical Techniques
Disc Biomedical Chromatography
Disc Biomedicine and Pharmacotherapy
Disc Biomolecules
Disc Blood Biochemistry
Disc Capillary Electrochromatography
Disc Capillary Electrophoresis
Disc Carbohydrates Biochemistry
Disc Cellular and molecular Biochemistry
Disc Chemometric Analysis
Disc Chromatographic Techniques
Disc Chromatography
Disc Computational Biomedicine
Disc Deuterium Exchange Mass Spectrometry
Disc Differential Scanning Calorimetry
Disc Drug Resistance
Disc Electron Capture Dissociation Mass Spectroscopy
Disc Electrophoresis
Disc Electrospray Tandem Mass Spectrometry Newborn Screening
Disc Extraction Chromatography
Disc Filtration
Disc Fishery biochemistry
Disc Flow Injection Analysis
Disc Food Biochemistry
Disc Fourier Transform Mass Spectrometry
Disc GC-MS
Disc Gas Chromatography
Disc Gas Chromatography Mass Spectrometry
Disc Gravimetric Analysis
Disc HPLC
Disc HPTLC
Disc Heterocyclic Compounds
Disc Imaging Mass Spectrometry
Disc Immuno Affinity Chromatography
Disc Infrared Spectroscopy
Disc Inorganic biochemistry
Disc Ion-exchange chromatography
Disc LC-MS
Disc LC-MS principles
Disc Liquid Chromatography
Disc Liquid Chromatography Mass Spectrometry
Disc Liquid Liquid Extraction
Disc MALDI
Disc Marine Biomedicine
Disc Mass Spectrometry
Disc Mass Spectrometry in Medicine
Disc Mass Spectroscopy
Disc Mass Spectroscopy in Forensic Studies
Disc Medical Biology
Disc Membrane Biochemistry
Disc Method Validation
Disc Molecular Biomedicine
Disc Molecular Epidemiology
Disc NMR Applications in Biomedicine
Disc Negative Results in Biomedicine
Disc Pesticides Biochemistry
Disc Pharmaceutical Analytical Techniques
Disc Pharmaceutical Sciences
Disc Preparative Biochemistry
Disc Protein Biochemistry
Disc Protein Folding by Mass Spectrometry
Disc Protein Mass Spectrometry
Disc Protein Purification
Disc Qualitative Analysis
Disc Separation Techniques
Disc Soil Biochemistry
Disc Spectrophotometry
Disc Spectroscopy
Disc Sports Biomedicine
Disc Super Critical Fluid Chromatography
Disc Synthetic Organic Chemistry
Disc Systems Biomedicine
Disc Tandem Mass Spectrometry
 
Recommended Journals
Disc Chromatography Journal
Disc Analytical Biochemistry Journal
Disc Pharmaceutical Analysis Journal
Disc Pharmaceutical Analytical Chemistry Journal
Disc Mass Spectrometry Journal
Disc Bioanalysis Journal
  View More»
 
Recommended Conferences
Disc 2nd International Conference on Current Trends in Mass Spectrometry
July 20-22, 2016 Chicago, USA
Disc World Congress on Chromatography
September 21-23, 2016 Amsterdam, Netherlands
Disc  7th International Conference and Exhibition on Analytical & Bioanalytical Techniques
September 28-30, 2016 Orlando, Florida, USA
Disc 3rd Asia Pacific Mass Spectrometry Congress
October 10-12, 2016 Kuala Lumpur, Malaysia
Disc International Pharmaceutical Method Development and Validation Conference
November 24-25, 2016 Dubai, UAE
View More»
 
Article Tools
Disc Export citation
Disc Share/Blog this article
 
Article usage
  Total views: 11175
  [From(publication date):
February-2014 - Jun 30, 2016]
  Breakdown by view type
  HTML page views : 7444
  PDF downloads :3731
 
 

Post your comment

captcha   Reload  Can't read the image? click here to refresh

 
OMICS International Journals
 
Make the best use of Scientific Research and information from our 700 + peer reviewed, Open Access Journals
 
 
OMICS International Conferences 2016-17
 
Meet Inspiring Speakers and Experts at our 3000+ Global Annual Meetings
 
 

Contact Us

Agri, Food, Aqua and Veterinary Science Journals

Dr. Krish

agrifoodaquavet@omicsinc.com

1-702-714-7001 Extn: 9040

Clinical and Biochemistry Journals

Datta A

clinical_biochem@omicsinc.com

1-702-714-7001Extn: 9037

Business & Management Journals

Ronald

business@omicsinc.com

1-702-714-7001Extn: 9042

Chemical Engineering and Chemistry Journals

Gabriel Shaw

chemicaleng_chemistry@omicsinc.com

1-702-714-7001 Extn: 9040

Earth & Environmental Sciences

Katie Wilson

environmentalsci@omicsinc.com

1-702-714-7001Extn: 9042

Engineering Journals

James Franklin

engineering@omicsinc.com

1-702-714-7001Extn: 9042

General Science and Health care Journals

Andrea Jason

generalsci_healthcare@omicsinc.com

1-702-714-7001Extn: 9043

Genetics and Molecular Biology Journals

Anna Melissa

genetics_molbio@omicsinc.com

1-702-714-7001 Extn: 9006

Immunology & Microbiology Journals

David Gorantl

immuno_microbio@omicsinc.com

1-702-714-7001Extn: 9014

Informatics Journals

Stephanie Skinner

omics@omicsinc.com

1-702-714-7001Extn: 9039

Material Sciences Journals

Rachle Green

materialsci@omicsinc.com

1-702-714-7001Extn: 9039

Mathematics and Physics Journals

Jim Willison

mathematics_physics@omicsinc.com

1-702-714-7001 Extn: 9042

Medical Journals

Nimmi Anna

medical@omicsinc.com

1-702-714-7001 Extn: 9038

Neuroscience & Psychology Journals

Nathan T

neuro_psychology@omicsinc.com

1-702-714-7001Extn: 9041

Pharmaceutical Sciences Journals

John Behannon

pharma@omicsinc.com

1-702-714-7001Extn: 9007

Social & Political Science Journals

Steve Harry

social_politicalsci@omicsinc.com

1-702-714-7001 Extn: 9042

 
© 2008-2016 OMICS International - Open Access Publisher. Best viewed in Mozilla Firefox | Google Chrome | Above IE 7.0 version