gHK DETERMINATION OP COUMARIN 301 TOBACCO AND ...

COUMARIK IN TOBACCO 

gHK DETERMINATION OP COUMARIN 

301 TOBACCO AND TOBACCO SMOKE 

A method for measuring the coumarin content of tobacco products has 

been developed and handed over to Analytical Services. A detailed 

description of this method is included in a separate report^* An 

amendment to the method which gives increased accuracy at low ooumarin 

levels is described in Appendix II. 

COUMARIN IN SMOKE 

OBJECTIVES 

(i) She development of a method for determining the amount of 

ooumarin in cigarette smoke, 

(ii) To determine the amount of coumarin transferred to the smoke 

of Park |riye cigarettesf 

SEASON 

Information on the transfer levels of tobacco additives to smoke is 

required to satisfy the requirements set by the Hunter Committee, 

goumarin is a component of the flavour used on Park Drive cigarettes 

and therefore meets the definition of an additivef 

LITERATURE SURVEY 

2 

A method exists for measuring coumarin in cigarette smoke* This 

involves fractionation of the condensate by thin-layer chromatography 

followed by spectrophotometry determination of the coumarin using 

Gibbs reagent (2,6 dichloroquinoner4-chloroimine), The colourimetrio 

determination of coumarin was found to be unsuccessful due to light 

absorption in the absence of coumarin. 

GC METHOD FOR COUMARIN IN SMOKE 

She method developed for smoke is based on the procedure used for the 

estimation of coumarin in tobacco* 

The condensate from 25 cigarettes containing coumarin is collected on 

a Cambridge filter pad. The pad is macerated with warm water containing 

lead acetate and celite,. After filtration the aqueous solution is 

extracted with a solventrmixture comprising 2C#> ether and 60% petrol 

(4O-6O ). The organic extracts are combined, washed with sodium 

bicarbonate and dried over anhydrous magnesium sulphate* The solution 

is reduced to email bulk and purified by passage through two chromatography 

columns. Analysis for coumarin 1B by gas chromatography using an 

external standard. 

The detailed method is described in Appendix I* 

RESULTS 

(i) SPIKING 

Park Drive cigarettes were used aB a model in the development of 

the method since they are the principal Gallaher U.K. brand 

http://legacy.library.ucsf.edu/tid/iyl97h00/pdf 

V

V_ 

v_- 


containing added oounarin* Cigarettes were prepared to normal 

Bark Drive specifications in the Experimental Factory using 

unflavoured Blend 32 filler• These were then hand spiked 

with varying amounts of coumarin. The accuracy of the spiking 

procedure was checked by carrying out the tobacco method of 

ooumarin analysis on the spiked cigarettes. The values obtained 

axe shown in Table I* Also included in this table is the 

(apparent) coumarin content of unflavoured Park Drive oigarettes. 

TABLE I. COtJMARIN XEVELS IN SPIKED CIOABETTES* 

Spiked 

Level 

(jVcig.) 

0 

5 

20 

50 

100 

200 

500 

Measured 

Level 

(ug/cig.) 

IABLE II COTJMARIN IEVEL IN PARK DRIVE 

SAMPLE 1 * 

1 

2 

3 

4 

MEAN 

COEFP, OP 

VARIATION 

3 

CIGARETTES 

b. 10 cigarette sample 

CODMARIN 

14.4 

13,0 

13.8 

16.6 

14.4 

6.796 

Shis measured coumarin level of 14*4 + 2.5p$/oig. agreeB veil 

with the calculated level of 18ng/cig. allowing for expected 

losses during flavour addition. 

(ii)'COLLECTION 

The smoke from 25 cigarettes containing coumarin was collected 

on a Cambridge filter pad. The cigarettes were smoked to a 20mm 

butt on a rotary smoking machine. The efficiency of the 

Cambridge filter pad in trapping coumarin was checked by 

attaching a dichloromethane trap after the pad when 25 

cigarettes spiked at 500ug coumarin per cigarette were smoked. 

No coumarin vaB detected in the dichloromethane solution 

Indicating complete retention of coumarin on the filter. 

(iii) EXTRACTION ** 


4 . 

&e investigation of the extraction and clean-up stages was 

undertaken using the smoke from unflavourei. Park Drive cigarettes 

spiked at exaggerated levels of coumarin (>10C^g/cig.) 

Hot water was found to be the best solvent for preferential 

extraction of coumarin from the Cambridge filter pad, A 

mixture of 2($ ether and 60% petrol (40-60 ) was found to be 

suitable for extraction of coumarin from the aqueous solution 

whilst removing a minimum of extraneous material. Recovery of 

coumarin from the aqueous layer after 4 solvent extractions 

(1 x 150, 2 x 100, 1 x 50ml) was greater than 9596.

(iv) CLEAN-UP AKD MEASUREMENT 


Using the solvent systems described in paragraph (iii) many 

impurities were still present in the organic solution. Therefore 

a careful column chromatography study vas carried out to optimise 

the conditions for separation of coumarin from impurities which 

. would interfere with the final GC analysis. 

Passage of the organic solution (reduced to small hulk) through, 

a silicic acid oolumn (height 5«.7om, diameter 1cm) using 10 ether/ 

9096 petrol (4O-6O ) as the eluent removed many impurities. The 

coumarin levels in Park Drive smoke measured alter this single 

clean-up stage are shown in Table III with a typical chromatogram 

in Figure 1* 

fable III COUMADIN LEVEL IK PARK DBIVE f * ^ ~, * f '* 

SMOKE (QBE STAGE CIEAK-UP) 

SAMPLE 

1 

2 

3 

4 

COUMARBT 

LEVEL 

(ug/cig) 

4.3 

5.1 

5.1 

5.9 

Further purification was effected by passage through a second 

Bilicic acid column (height 8.0cm, diameter 1cm) using the 

same eluent. The coumarin levels obtained after a two stage 

purification are shown in Table IV with a typical chromatogram 

in Figure II# 

Table IV COUMARIN LEVEL IK PARK DRIVE 

SMOKE, (TWO STAGE CLEAN-UP) 

SAMPLE 

1 

3 

4 

COUMABIK' 

LEVEL 

(ug/oig) 

4.0 

4.0 

4.0

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Vith reft ace to Table I the control cigr rites (made to Park 

Drive specifications using Blend }2 with \. added flavour) appear 

to have a residual coumarin level of 4*4 ± 3*4 ug/cig. A coumarin 

analysis on the smoke of 25 control cigarettes yielded the result 

shown in Table V, - 

TABLE V COUMARIN CONTENT OF 

(v) RECOVERY 

CONTROL CIGARETTES 

COUMARIN IN 

TOBACCO 

(w/oig) 

4.4 


SMOKE 

fag/cig) 

Coumarin recovery is greater than 95& 

(vi) DETECTION LIMIT 

The lower limit for detection of coumarin by this method is 

1-2 pg/cig« in the smoke which corresponds to a coumarin 

level in tobacco of approximately 5pg/cigarette. 

The measured coumarin content at low levels will be Bubject to 

interference from impurities (see Table V and Discussion). 

DISCUSSION 


(i)* The principal assumption made in using this method to measure 

coumarin is that there are no other compounds with the same 

retention time as coumarin in the final GO analysis* Comparison 

of the results in Tables III and IV shows that extraneous compounds 

contributing to the coumarin peak on the GC after one column 

chromatography step have been removed by the second column* This 

was observed experimentally by collecting fractions from the second 

column and testing for coumarin by gas chromatography* The 

coumarin comes through in the 50-75^1 fraction (by comparison with 

a standard coumarin solution passed through the column) while 

impurities with tbie same retention time as coumarin are contained 

in the first 50ml~'from the column, 

* 

(ii) When the smoke from 25 control cigarettes containing no added 

coumarin but with an apparent coumarin level of 4.4 + 3.4 Pfi/cig, 

is analysed a coumarin (?) level of 1*3pg/cig. is obtained after 

a two-stage clean-up. Allowances for this background peak can 

be made in two ways a) if the background peak is coumarin the 

measured value of tyig/cig. in the smoke of normal Park Drive 

cigarettes comprises 2.7 )ig/cig. from the coumarin added in the 

flavouring process and '\*5yg/cig, from the coumarin present in . 

Blend 32* b) if the peak in the control sample is not coumarin 

but a compound with the same retention time, the coumarin level 

in Park Drive smoke is

7* SWMAKY 

o 

In view of the low coumarin levels in Park Drive cigarette 

smoke ( < 4>»£/cig») no further resource8 are to be provided at 

this time for increasing the accuracy of the method* 

A method has been developed for measuring the coumarin content of 

cigarette smoke and iB available to be used as required. 

The coumarin level in Park Drive cigarette smoke is less than or 

equal to 4 ps/cig. 

REFERENCES 

1. The Determination of Coumarin in Tobacco - Piles B5/207/12/FD and 

B5/105/5/C 

2. E. Kroller, Z. Lebensmittelunters TJ, Porsch, 152 (4) 216-218 (1973). 

i.MWawt ml 

R. McKeivor 17,5.76. 

c.o, Belfast (full copy) 

H. White 

* V,J. Brown 

Dr. P.W. Darby 

CD. BriggB 

•Dr. D,J. Mackenzie 

V.D.E, Irwin 

File B5/207/12/PD/ 

Pile B5/105/5/C >/ 

Library 

RJ^/AP 


London (Summary) 

J.C.S. Mason 

Dr. E.S, Stem

APPENDIX I THE MEASUREMENT OF COUMARIN iff 

APPARATUS 

PARK DRIVE CIGARETTE SMOKE 

Gas chromatograph : Pye 104 vith FIB detectors. 

Columns ; Two |" 1 9' stainless steel columns packed vith 10% Carbovax 

20M-TPA on Chromosorb WAW-DMCS, 60-100 mesh. 

Top drive macerator and flask. 

Baohner funnel (9cm diameter). 

Buchner flask (300ml) 

Cambridge filter pads (9cm diameter) 

Graduated cylinders (1 x 23ml, 2 x 50ml,, 1 x 100ml) 

Conical flask (11) vith stopper. 

Beakers (1 x 50ml, 2 x 50ml) 

Glass funnelB (1 large, 1 small). 

Separatory funnel (500ml) vith stopper. 

Round bottomed flask (1 x 1l). 

Rotary evaporator. 

Chromatography columns (Two, height 30cm, diameter 1cm). 

Pear shaped flasks (3 x 50ml) 

Volumetric flasks, 

REAGENTS 

Lead acetate (E & V GPR, 8% solution). 

Celite Analytical Filter Aid' (BDH, supplied by Hyde and Entvhistle), 

Dichloromethane (H & W GPR)..; 

Petroleum Spirit 40-60° (May & Baker). 

Diethyl Ether (H & W GPR) 

Solvent mixture A (20% ether/80% petroleum spirit) 

Solvent mixture B (10% ether/90% petroleum spirit) 

Sodium bicarbonate (H & V Analar, 4% solution). 

Magnesium sulphate, anhydrous (H & W) 

Silicic acid (for resolution of basic or neutral mixtures, Sigma Chemical Co.) 

http://legacy.library.ucsf.edu/tid/iyl97h00/pdf

Coumarin standard solutions in dichloromethane (0.0015%i 0,0025%, 0,0050%, 

0,0075%.) 

GC CONDITIONS 

Nitrogen flow : ^Omls/min. 

Column.temperature : 220 isothermal 

Attenuation : 50 x 1 

Chart speed : 2,5 min,/cm, 9 

PROCEDURE 

1. Calibration of standard solutions 

2ul of each of the standard solutions are injected into the gas 

ohromatograph. Attenuation is 50 x 1, A graph of peak height vs 

ug/ul of coumarin is plotted. This calibration graph is used to 

obtain the approximate concentration of coumarin in the sample. 

It is recommended that the calibration graphB are checked each day 

and that the standard solutions are kept in a refrigerator and renewed 

weekly* 

2. Treatment of Sample 

'. (a) The Cambridge filter containing the smoke from 25 cigarettes is 

placed in a macerator flask, Celite (5g)» lead acetate (20ml, 

Q% solution) and hot water (200ml, approx. 50 C) are added and 

the mixture is macerated for 3 minutes. The macerator is then 

turned off and the residue is washed off the lid and Bides of 

the flask into the body of the solution with distilled water 

(approx, 10ml) Maceration is continued for a further 2 minutes. 

.The macerator is then switched off and the residue adhering to 

the lid and sides of the flask is washed into the solution with 

more distilled water (approx, 10ml), 


(b) The mixture is filtered by suction through a Cambridge filter 

pad into a Buchner flask. The residue on the filter pad is 

washed with two 50ml portions of warm water. 

(o) The aqueous filtrate (plus washings) 1B cooled to room temperature 

by running cold water over the surface of the Buchner flask. 

The contents of thG.-JBuchner flask are then transferred to a 50ml 

separatory funnel |nd washed with four successive portions of 

solvent mixture A (1 x 150, 2 x 100, 1 x 50ml),, the upper organic 

layer being retained on each occasion. The organic extractions 

are combined and washed with sodium bicarbonate (40ml, 4% solution) 

in the same separatory funnel. The lower bicarbonate layer is 

drawn off into a 50ml beaker. The upper organic layer is then 

drawn off into a conical flask (11) containing anhydrous magnesium 

sulphate (approx, 30-40g). The bicarbonate layer is washed with 

an additional 40mle of solvent mixture A, The lower aqueous 

bicarbonate layer is discarded and the organic layer is added to 

the contents of the conical flask. The flask is stoppered and 

the solution is left to dry for a minimum of 45 minutes.

* 

(d) Meanwhile two chromatography solumns (height 30cm, diameter 1cm, 

each containing a glass wool plug) are prepared by pouring a Blurry 

of silicic acid in solvent mixture B to heights of 5.7cm and 8.0cm 

respectively, 

(e) The dry organic solution from (o) is filtered by suotion through a 

Cambridge filter pad into a Buchner flask* The magnesium sulphate 

residue is washed with two 30ml portions of solvent mixture A, 

(f) The filtrate (plus washings) is transferred to a 11 round bottomed 

flask and concentrated to a volume- of less than 50ml and on a 

rotary evaporator using a water bath at room temperature. 

(g) The Bolution in the round bottomed flask is quantitatively transferred 

to a 50ml pear shaped flask. This solution is concentrated to small 

bulk (approx. 4-5ml) on a rotary exaporator UBing a water bath at 

room temperature. 

(h) The concentrated organic solution is quantitatively transferred by 

Pasteur pipette to the smaller silioic acid column (height 5*7cm). 

Bie pear shaped flask is rinsed with small quantities (3 x 1ml) of 

solvent mixture £ and these washings are transferred to the column. 

(i) The column is eluted with solvent mixture B. The first JOBUB are 

discarded and the 30-60 ml fraction is collected in a graduated 

cylinder. 

(j) The 30-60 ml fraction is transferred to a 50ml pear shaped flask 

and the solution is evaporated to dryness on a rotary evaporator 

at room temperature. 

(k) The residue in the pear shaped flask iB dissolved in a small volume 

of solvent mixture B (approx. 3 m l)» This is quantitatively transferred 

by Pasteur pipette to the second silicic acid column (height 8.0cm). 

The flask is rinsed with small quantities (2 x 1ml) of solvent mixture 

B«nd the washings are transferred to the column. 

(l) The column is eluted with solvent mixture B. The first 43ml are 

discarded and the 4&-75ml fraction is collected in a graduated 

cylinder. 

(m) The 48-75ml fraction is transferred to a 50ml pear shaped flask 

1 and the solution is evaporated to dryness on a rotary evaporator 

at room temperature. 

(n). The residue in the f£ask is dissolved in 2ml dichloromethene (added 

by a pipette). 2ul of this sample is injected into the gas 

chromatography The approximate concentration of aoumarin in the 

sample can be obtained from the calibration graph. A standard of 

approximately the same coumarin concentration is then injected. 

The volume of the standard solution injected is chosen to give a 

coumarin peak of approximately the same height as the Bame peak. 


It is recommended that two sample injections are made i.e. the 

sequencet sample—> standard—* sample —* standard should be 

followed.

3* CALCULATION OF RESULT 

AMOUNT OP 


SMOKE (^g/cig) 

t mjtoog,, 

R*» McKeivor 17.5.76 

m/xp 

COUMARIN CONTENT OF 

= STANDARD SOLUTION (uq/fil) 

1 

HEIGHT OF 

COUMARIN PEAK 

IN SAMPLE 

HEIGHT OP 

STANDARD PEAK 


PINAL VOL, 

OF SAMPLE 

SOLN. (jul) 

VOL. OF SAMPLE 

INJECTED (^Jl) 

VOL. STANDARD 

INJECTED {/up 

NO. OF CIGARETTES 

SMOKED

APPENDIX II AMENDMENT TO THE METHOD OF DETERMINING 

THE COUMARIN CONTENT OF TOBACCO PRODUCTS 

The following additional step in the method of measuring the coumarin 

content of tobacco products is suggested for use when a more accurate 

result (than would normally he obtained) is required for materials with 

a low coumarin content (

1 

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