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

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0 500 1000 1500

y = 153.39x - 1655.6 R² = 0.9991

0

20000

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60000

80000

100000

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160000

0 200 400 600 800 1000 1200

y = 245.47x - 87.254 R² = 0.998

0

5000

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30000

0 20 40 60 80 100 120

y = 203.24x - 41.163 R² = 0.9999

0

5000

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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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