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Simultaneous Quantification of 4-Hydroxy Coumarin, Coumarin, 6-Methyl Coumarin and Eugenol in Cigarette filler and in Main Stream Cigarette Smoke using HPLC-PDA
Poster No: SSPT POST 11 Raghu.H.S, Nalini Dilip Sangli, Sathya Gourishankar and Sharad.K.Mehta*
ITC Ltd, ITC R&D Centre, Peenya, Bangalore – 560 058, INDIA.
A new validated , rapid and sensitive HPLC Method has been
developed for the simultaneous quantification of 4-Hydroxy
Coumarin, Coumarin, 6-Methyl Coumarin and Eugenol compounds
in Cigarette filler & in Mainstream cigarette smoke.
The method allows extraction of Cigarette filler & Main stream
smoke particulate matter on a Cambridge filter pad with ethanol and
subsequent analysis using HPLC with PDA Detector. The
compounds were separated on a C18 Column with Water:
Methanol as mobile phase. excellent linearity was obtained over a
wide range of concentration(0.01-1000ug/ml) for all the
compounds and recovery varies from 96 to 102%. The limit of
detection is 0.0004mg/Cig and quantification is 0.0012mg/cig
BACKGROUND
SAMPLE PREPARATION
Cigarette filler/Smoke with Cambridge filter pad
Extraction with Ethanol for 30mins, on a shaker
Filter through a 0.45um PVDF syringe filter and inject filtered
solution
Quantify the analytes using HPLC-PDA
Essential oils have a long history of use as perfume, flavoring and
natural microbial agents. Clove oil ( Syzygium Aromaticum) is widely
used as perfume and food flavoring agent; Eugenol is the main volatile
compound of extracted oil from clove bud that is used in traditional
medicine as a bactericide, fungicide and anesthetic.
Coumarin and its derivative have long been used by the tobacco
industry as an additive in cigarettes. The taste of coumarin, which is like
“ new-mown-hay” has been considered by the tobacco industry to be an
important additive that significantly enhances the taste. Despite its flavor
enhancing properties, numerous studies ,beginning 1855 have indicated
that coumarin has toxic effects on the nervous system, heart, blood
vessels , and liver of animals as well as inducing cancerous tumours
and toxic conditions in humans. It’s usage is completely banned in foods
in some countries.
HPLC-PDA CONDITIONS LINEARITY GRAPHS
SAMPLE CHROMATOGRAM ABSTRACT
• Mobile Phase: Water: Methanol ( 50:50)
• Column : Grace Smart RP18 ( 250mmX4.6mmX5m)
• Column Temperature : 40°C
• Column flow: 0.7ml/min
• Detector Wavelength: 280nm
• Injection volume: 20 µl.
• Recovery varies from- 96 to 103%
• Repeatability in terms of RSD < 5%
• Reproducibility in terms of RSD < 7%
• Limit of Detection ( S/N) : 0.0004mg/Cigarette
• Limit of Quantification (S/N): 0.0012mg/Cigarette
STANDARD CHROMATOGRAM
1. The method is simple, sensitive and fast which does not involve
any sample cleanup unlike reported GC-MS Method.
2. Method is applied to Cigarette filler and Main stream Cigarette
Smoke.
y = 15056x - 345646 R² = 0.9998
0
5000000
10000000
15000000
20000000
25000000
0 500 1000 1500
y = 153.39x - 1655.6 R² = 0.9991
0
20000
40000
60000
80000
100000
120000
140000
160000
0 200 400 600 800 1000 1200
y = 245.47x - 87.254 R² = 0.998
0
5000
10000
15000
20000
25000
30000
0 20 40 60 80 100 120
y = 203.24x - 41.163 R² = 0.9999
0
5000
10000
15000
20000
25000
0 20 40 60 80 100 120
CONCLUSIONS
REFERENCES
Eugenol Coumarin
4-Hydroxy Coumarin 6-Methyl Coumarin
1. Food and Chemical Toxicology (2007), 45(10), 1948-1953
2. Journal of Agricultural and Food chemistry (2006), 54(22), 8580-8
3. Journal of Pharmaceutical and Biomedical Analysis (2007), 44(3),
755-762
4. J.Chromatogr B Biomed Appl.1996 Nov15; 686(2):267-73
5. Association of Official Analytical Chemists. Jan-Feb 2000; 83(1):
237-240
6. J-Chromatogr. 5 Aug 1988; 447 (1): 272-276,
OBJECTIVES
• To develop a simple, sensitive and fast analytical method to
quantify 4-Hydroxy Coumarin, Coumarin , 6-Methyl Coumarin and
Eugenol in Cigarette filler and in Main Stream Cigarette Smoke
using the same analytical conditions.
• Method should meets emerging FDA requirements as per
HPHC Listing. [ Harmful and Potentially Harmful Constituents]
METHOD VALIDATION
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