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Final Report Of Project on Laboratory Testing of Smokeless tobacco products

Research · February 2018

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 Final Report

Of

Project on Laboratory Testing of Smokeless tobacco products

Dr Prakash C. Gupta,
Vice-President,
Action Council against Tobacco - India

Dr Sreevidya S,
Project Coordinator,
Epidemiology Research unit
Tata memorial Hospital
Parel, Mumbai.

APW – Sticker no. SE/02/232816

Allotment no: SE IND TOB [Link].02.

1
 INDEX
1. Objectives 4

2. Justification 5

3. Smokeless tobacco products: An overview 6

4. Smokeless Tobacco constituents and their importance 7

5. Activities 9

6. Analytical techniques: An Overview 11

7. Procedures and Results by constituent

7a. Determination of pH 12

7b. Estimation of ammonia by u.v. spectrometry 13

7c. Estimation of magnesium carbonate. 14

7d. Estimation of nicotine 16

7e. Estimation of alkaloids 18

7f. Determination of nitrosamines 21

7g. Estimation of benzo[a]pyrene 23

7h. Estimation of nitrate 25

7i. Estimation of heavy metals 27

7j. Determination of Eugenol 33

7k. Determination of Sorbic Acid 36

7l. Determination of Triacetin 39

7m. Determination of Sodium Propionate 41

7n. Estimation Procedure for Humectants 43

7o. Determination of moisture content 45

2
8. Conclusions, recommendations and limitations 46

9. Bibliography 47

Annexes

1. Smokeless tobacco brands tested 49

2. Techniques used for the measurement of tobacco constituents 51

3. Calibration curves used for estimations 53

4. Photographic documentation of smokeless tobacco samples

3
1. Objectives

1. To catalogue different categories of tobacco products from different parts of

India

2. To collect sample of these products by purchasing them and documenting

purchases

3. To photographically document the tobacco product samples

4. To do chemical analysis of these products with primary aim of getting nicotine

content

4
2. Justification

Smokeless tobacco products are tobacco products without combustion or pyrolysis at

the time of use. Tobacco induced disease is a major international public health
challenge of which Smokeless tobacco induced disease is a significant part.
Governments and health workers sometimes ignore the actual and potential health
damage caused by Smokeless tobacco use. The prevalence of smokeless tobacco use
is relatively high in South Asia. Approximately 100 million people in India and
Pakistan use smokeless tobacco, mainly as a constituent of traditional chewing
mixtures.

In India a vast array of smokeless tobacco products are used. All these products
reveal adverse health consequences. Oral tobacco use can lead to nicotine
dependence and addiction. There is conclusive scientific evidence that the use of
smokeless tobacco causes cancer in humans. The evidence is strongest for cancers of
the oral cavity, but the risk of cancers of the pharynx, larynx, oesophagus, pancreas
and urinary tract are also increased. Smokeless tobacco use also increases the
incidence of periodontal disease and causes oral leukoplakia, which in some cases
may become malignant, and may contribute to cardiovascular disease, and peptic
ulcer. A few cohort studies from India demonstrate significant excess of all cause
mortality among smokeless tobacco users. Several studies of smokeless tobacco use
by pregnant women in India demonstrate adverse reproductive outcomes, especially
low birth weight. In India, as in many parts of South East Asia, oral cancer is a
leading type of malignancy. Oral leukoplakia and oral submucous fibrosis, assumed
to be precursors of oral cancers are also highly prevalent and increasing in the young.

During the Global Youth Tobacco Survey that has been completed in 13 states and
ongoing in eight more, several new smokeless tobacco products that are in use in
different parts of the country have been discovered. They include a large variety of
manufactured pan masala and gutka products, red tooth powders, gul and toibur
(nicotine water). In fact some of them for example red tooth powders, do not even
mention that they contain tobacco. We got some samples of red tooth powders tested
for their nicotine content in a laboratory and the results showed substantive nicotine
content. It is obvious that they do contain tobacco. These results combined with
general ignorance amongst the lay public as well as scientists working on tobacco
control clearly underscore a need for conducting a systematic study of different
smokeless tobacco products

5
3. Smokeless tobacco products: an overview.

Chewing tobacco in India is made from Nicotiana rustica and N tabacum. The
tobacco leaves are harvested when they turn yellow and brown spots start appearing:
the leaves remain in the field and are turned over to achieve uniform drying. They are
then tied in bundles and moistened by sprinkling with water; the bundles are stacked
for fermentation for a couple of weeks, separated and dried again. The leaves are cut
into various sizes. Tobacco is chewed in betel quid and may sometimes be chewed
as such. Chewing tobacco in betel quid being the commonest form of smokeless
tobacco use in India. The tobacco used in the quid however varies; it may be
processed (Zarda, Kiwam) or unprocessed (Hogesoppu, Kadippudi in Karnataka).

Tobacco is commonly used with lime, (Khaini), and placed in the mouth, in one or
both cheeks or in the mandibular groove. This mixture is available in various pouches
or may also be prepared fresh. Mawa, popular among teenagers especially in Gujarat,
contains thin shavings of areca nut with some sun-dried tobacco and slaked lime. A
similar product used in Maharashtra is called Kharra.

Zarda, which is produced and used in India, is also exported to countries across the
world. Tobacco leaf, broken into small pieces is boiled in water with lime and spices
until evaporation. The residual particles are then dried and colored with vegetable
dyes to produce Zarda. It is typically flavoured with cardamom and saffron. Zarda is
usually chewed mixed with finely cut areca nut and spices, or is placed in the betel
quid.

Gutka, a dry preparation commercialised since 1975, containing areca nut, slaked
lime, catechu, condiments and powdered tobacco, was originally available custom
mixed from pan-vendors. For the last couple of decades, Gutka has been available in
several brands. A similar packaged mixture without tobacco, often with identical
brand name is called pan masala. These products have become especially popular
among teenagers and young adults in many states of India. Moist Swedish snus is
now being marketed in India under the brand name Click.

Mishri is a form of tobacco used in India as a substitute for chewing tobacco. It is a

roasted or half-burnt tobacco, prepared by baking tobacco on a hot metal plate until it
becomes uniformly black. It is then powdered and used primarily for cleaning teeth.
However its use frequently becomes habitual, and a user may apply and retain mishri
in the mouth (usually along the teeth and sulcus) several times a day. Bajjar and Gul
are used as dentifrice in Gujarat and eastern parts of India. Gudakhu is tobacco paste
made with molasses. Creamy snuff or tobacco toothpaste advertised as being
antibacterial is popular in western parts of India.

6
4. Smokeless tobacco constituents and their importance.

Natural tobacco contains at least about 3050 compounds. Among the tumorigenic
agents identified in smokeless tobacco are volatile aldehydes, N-nitrosamines,
lactones, polynuclear aromatic hydrocarbons, certain metals and radioactive
polonium. Furthermore, smokeless tobacco may be enhanced by flavouring agents,
added in the form of plant extracts, or chemicals. The chemical structures of these
can be found in Annex-2.

Nicotine-Nicotine, an alkaloid, is an extremely powerful drug. The US Surgeon

General has affirmed that the way in which nicotine causes addiction is similar to
drugs such as heroin and cocaine. Nicotine is absorbed by the body very quickly,
reaching the brain within about seven seconds. It stimulates the central nervous
system, increasing the heart beat rate and blood pressure, leading to the heart needing
more oxygen. Nicotine also functions as a neurotransmitter in the brain and interferes
with cholinergic stimulation.

Tobacco-specific N-nitrosamines (TSNA)- are the most abundant and strong

carcinogenic compounds in smokeless tobacco. These are formed by N-nitrosation of
nicotine and of minor nicotiana alkaloids during tobacco harvesting, curing,
fermentation and ageing. The major precursor of TSNA is nitrate, which is reduced to
nitrite, the nitrosating agent for the tobacco alkaloids. Of the seven TSNAs identified
in smokeless tobacco, N’-Nitroso-nornicotine (NNN) and 4-methyl-nitrosamino-l-(3-
pyridyl)-1-butanone (NNK), are the predominant carcinogens. Endogenous
nitrosation may also contribute to TSNA formation.

Benzo[a]pyrene (BaP)- this is the major representative of the class of polynuclear

aromatic hydrocarbons (PAH) in tobacco. These are enzymatically activated to
electrophiles that form covalently bound PAH-DNA adducts, that lead to mutation
and oncogene activation. Tobacco contains ten or more other carcinogenic PAH in
addition to BaP.

Heavy metals: - the heavy metals viz. Lead, Cadmium, Selenium, Chromium,
Arsenic and Nickel are capable of causing serious human disease, as they are toxic
even at trace levels. Great deal is known about overwhelming exposure through
poisoning or industrial exposure; but our knowledge of the consequences of the
chronic low-level trace metal exposure, as occurs with tobacco use, is still inadequate.
The levels of these metals accumulate cumulatively with many years of use and lead
to increasing body burdens of these metals. There is evidence of subtle brain damage
like mentation difficulties, deficits in intelligence and memory, impaired psychomotor
and visual function with chronic lead exposure. Chronic arsenic exposure is
associated with cutaneous lesions, alopecia, liver disease and skin cancers. Chronic
cadmium exposure may result in emphysema, proteinuria, or olfactory nerve damage.
Nickel is considered a potent respiratory tract carcinogen. Hepatic dysfunction,
diffuse dermatitis and gastrointestinal disturbances have been observed in selenium

7
toxicity. Chromium exposure is associated with lung and certain upper respiratory
tract cancers and also with renal tubule dysfunction.

pH:- pH is an important factor that influences nicotine delivery, (determines the

absorption rate of nicotine into the body). Nicotine is protonated and occurs in the
particulate form in weakly acidic environment, whereas it occurs in the more toxic
unprotonated form, and also in the vapour phase in alkaline environment.

Additives to smokeless tobacco products:-

Hundreds of ingredients are used in the manufacture of tobacco products. Additives

make smokeless tobacco more acceptable to the consumer —control nicotine
delivery, improve taste, flavour and aroma, and prolong shelf life. Many products are
highly engineered, exquisitely designed “nicotine delivery devices”.

Sodium carbonate and ammonium carbonate: - Increase the level of "free"

nicotine by raising the pH level. Unprotonated (free) nicotine is the chemical form of
nicotine that is most readily absorbed through the mouth into the bloodstream.
Therefore, increases in pH can increase the user’s nicotine absorption rate. Studies
with nicotine and other addictive drugs suggest that the absorption rate of drugs into
the body is an important determinant of their addiction potential.

Products with low nicotine content and pH levels have a smaller proportion of free
nicotine. In contrast, products with high nicotine content and pH levels have a higher
proportion of free nicotine

Ammonia: - increasing ammonia levels increase the pH thereby possibly enhancing

nicotine delivery.

Eugenol and menthol: numb throat and facilitate tobacco use.

Sorbic acid: - added to tobacco as an anti-microbial agent.

Triacetin: added to tobacco as a flavorant.

Sodium propionate: - added to tobacco as a mold preventative or fungicide.

8
5. Activities

Major categories of smokeless tobacco products were identified. Commonly used

Smokeless tobacco products from different parts of India were purchased with the
help of friends and colleagues, mainly from stores in Gujarat, Karnataka and Mumbai.
These products were purchased from regular retail shops and date of purchase, place
of purchase and batch number of each purchase recorded (Annex-1). Gutka has been
banned in the state of Maharashtra; Pan masala and Zarda of the same brand are
available in separate pouches, which when mixed by the consumer is equivalent to
Gutka. Some Gutka brands were also purchased from Gujarat.

As a first step towards chemical analysis of the Smokeless tobacco products, a

thorough literature search was undertaken and a comprehensive list of toxigenic,
mutagenic and carcinogenic components in smokeless tobacco products were
identified and listed. Important additives to smokeless tobacco products were also
listed. Though it was initially decided that only the Nicotine content and TSNA
(tobacco specific Nitrosamines) content of each of these products would be measured,
a host of other important ingredients were subsequently included for measurement.
This dictated that the number of samples tested would be lesser, but would offer
greater comprehensiveness.

The major components identified for testing were the alkaloids including nicotine, the
tobacco specific nitrosamines, Benzo-a-pyrene, ammonia, nitrates and nitrites, heavy
metals, triacetin, sodium propionate, humectants, sorbic acid, Eugenol, and animal
hemoglobin.

Several laboratories that would possibly carry out these tests were contacted. The
laboratory, which was finally identified as being the most credible and offering high
quality assurance for its testing processes for most constituents identified, was the
Indian Institute of Environmental Medicine, Mumbai. Dr. Rohini Chowgule is the
Founder-Director, with the laboratory headed by Dr R. N. Khandekar, a retired
scientist from the Bhabha Atomic Research Centre. Dr Khandekar was involved with
testing of constituents of cigarette smoke, and was very much interested in proceeding
with the proposed project. The list of constituents for which testing would be
required was discussed with Dr Khandekar. The only one they were unable to do was
animal hemoglobin.

The costs involved were negotiated; the cost for testing for all of the constituents for
each of the Smokeless tobacco samples was negotiated at Rs. 13500 provided the
testing was done in a batch of minimum of 20 smokeless tobacco products totalling
Rs. 270000. This cost was 9 times higher than the cost we originally estimated and
budgeted.

20 commonly used smokeless tobacco products were submitted to the Institute of

Environmental Medicine, Mumbai. This included a range of products including the
Gutkas, khainis, some brands of chewing tobacco, toothpastes, toothpowders, tobacco
water and Click. Annex-1 lists the products tested.

9
The lab In contrast to 30 g of each sample that had been initially required by the lab
for completing all of the testing processes, it was later realised that 300g would be
required. 500g of each product was purchased and the extra 200g that remained after
testing was retained in our office for future use and verification.

Smokeless tobacco products are available in pouches of various weights, ranging from
1-13g each, depending on the brand. A weighing scale with an error of 2g (max 500g,
min 20g) was purchased for weighing of the smokeless tobacco products before
dispatch to the laboratory. Few pouches of each product were opened and the
contents weighed without the wrapper. It was found that all products contained
approximately the same quantity as reported on the wrapper.

300g of each of the 20 products were handed over to the lab in their original pouches.
Thus the lab was not blinded to the brands being tested. This was with the intention
of not contaminating the products while transferring to a different pouch, preserving
their shelf life, and for the ease of laboratory testing, in which small quantities are
used at time.

10
6. Analytical techniques: An overview

In general, the Canadian standards for testing smokeless tobacco constituents were
followed.

Weighing of tobacco samples was done accurately by means of Metlar analytical

balance (0.1mg). All reagents used are recognised as analytical reagent grade in
quality. Cleaned and dried glassware was used for testing purposes, so that
contamination from glassware does not occur. Health and safety practices have been
established prior to testing according to existing applicable regulatory requirements.

All analyses were done in duplicate. The control sample in each test served as a
quality control measure. Each analytical run also included a laboratory reagent blank
to evaluate the extent of any interference due to glassware, reagents and analyser
effects. Each measurement was made against a blank preparation.

Procedures for estimation of the constituents was as follows.

The sample was first ground and sieved on a 20micron sieve. It was then extracted
with known quantity of a suitable solvent and by appropriate physical means; use of
ultrasonic bath, mechanical shaker, or after appropriate distillation as for triacetin.

The tobacco extract was subjected to analysis by most sensitive instrument and
conditions of analyses for each constituent. A tabulation of the method of analysis
used for the various constituents can be found in Annex-3.

pH was determined with a pH meter and magnesium carbonate by the titrimetric

method. Quantification for most other constituents was achieved by internal or
external standard calibration procedures where the relative response of the samples
was compared against calibration with known standards.

A calibration curve was prepared by plotting the concentration of the standards versus
their respective peak areas. The response factor was determined from the calibration
curve. The calibration curves developed for each of the constituents are presented in
Annex-4.

11
7a. DETERMINATION OF pH

This method is used to determine the pH of 10% w/v suspension of sample in water
by means of a pH meter. The method utilizes a combination electrode and
potentiometer standardized by buffer solutions to measure the pH.

Two g of homogenized tobacco from a freshly opened sample is extracted into 20 ml

of degassed water on a mechanical shaker for 30 minutes. The sample is allowed to
stand in dark for 1 hour. The supernatant is decanted into a 10 ml disposable
polystyrene beaker.

The electrode and auto-temp probe are then inserted into the beaker containing the
supernatant. The beaker is gently moved in swirling motion to create movement of
the supernatant passing by the electrode.

A buffer of known pH (ph 6 – 7) is measured and the value obtained should be within
0.10 pH units of the expected value. The pH of the tobacco sample extract is then
read. The pH in duplicate for the 20 samples are as presented under.

pH analysis
Sr. no Product Name
pH1 pH2
Sanket 999Jarda + Sanket No. 1 Supari
1 8.9 8.89
Mix
Moolchand Superb Jarda +Moolchand
2 8.56 8.55
Superb Supari
3 Shimla Jarda + Shimla Supari Mix 8.8 8.79
4 Goa 1000 Zarda + Goa 1000 Supari 8.8 8.81
5 Gutkha Pan Parag 8.63 8.62
6 Gutkha Manikchand 8.75 8.74
7 Click Eucalyptus 6.95 6.94
8 Baba Zarda 120 5.21 5.22
9 Dentobac Creamy Snuff 7.52 7.5
10 Lime Mix - Miraj Tobacco 10.1 10.11
11 Shahin Mishri 6.53 6.54
12 Dabur Red Tooth Powder 6.76 6.75
13 Baidhyanath Red Tooth Powder 5.74 5.75
14 Gai Chhap Zarda 5.96 5.95
15 Raja Khaini 8.45 8.46
16 Gutkha Tulsi Mix 9.25 9.24
17 IPCO Creamy Snuff 8.35 8.34
18 Kuber Gutkha 8.92 8.93
19 Vimal Gutkha 9.06 9.07
20 Tuiber Tobacco Water 9.26 9.25

12
7b. ESTIMATION OF AMMONIA BY U.V. SPECTROMETRY

One gram of sample was mixed with 200 ml of D.D.W. and transferred to the
distillation flask of Kjeldahl’s apparatus. Few antibumping granules were also added
to the flask to promote regular ebulliation in the subsequent distillations.

100 ml of standard 0.1 M HCl was placed in a receiver and the flask was adjusted so
that the end of the condenser just dips into the acid. 100 ml of NaOH was placed in
the funnel and run into the flask opening the tap. The tap was closed as soon as the
alkali had entered. The flask was heated so that the contents boiled gently and the
distillation was continued for 40-50 minutes by which time ammonia passed over into
the receiver.

Absorbance was measured at 420 nm following the Vogel’s procedure.

[Link] Product Name Ammonia µg/g

1 2
Sanket 999Jarda + Sanket No. 1
1 6.05 6.15
Supari Mix
Moolchand Superb Jarda
2 4.42 4.45
+Moolchand Superb Supari
Shimla Jarda + Shimla Supari
3 12.88 12.32
Mix
4 Goa 1000 Zarda + Supari 3.8 3.92
5 Gutkha Pan Parag 16.6 16.42
6 Gutkha Manikchand 11.7 11.4
7 Click Eucalyptus 3224.8 3288
8 Baba Zarda 120 8.6 8.34
9 Dentobac Creamy Snuff 4.33 4.46
10 Lime Mix - Miraj Tobacco 7.76 7.92
11 Shahin Mishri 3387.1 3320
12 Dabur Red Tooth Powder 4.42 4.34
13 Baidhyanath Red Tooth Powder 4.04 4.12
14 Gai Chhap Zarda 4827.2 5280
15 Raja Khaini 12.43 12.31
16 Gutkha Tulsi Mix 163 152
17 IPCO Creamy Snuff 12.8 12.54
18 Kuber Gutkha 7.39 7.26
19 Vimal Gutkha 0.7 0.77
20 Tuiber Tobacco Water 31.9 30.96

13
 7c. ESTIMATION OF MAGNESIUM CARBONATE.

1. Ground tobacco sample is extracted with 50 ml of double distilled water on a wrist

action rotary shaker for 60 minutes.
2. The extract is then filtered. Colour if present, is removed by adding Aluminium
Hydroxide solution. Magnesium Carbonate is determined by EDTA titration
3. To the extract 1 ml of ammonia buffer and Eriochrome Black T indicator is added. In
alkaline conditions EDTA reacts with Ca and Mg ions to form a soluble chelated
complex. Ca and Mg ions develops wine red colour with the indicator under alkaline
condition. When EDTA is added as a titrant Ca and Mg ions get complexed resulting in
sharp colour change from wine red to blue. Which indicates the end point of the
reaction. The pH for this reaction has to be maintained at pH10.0. Record Burette
reading as Total Ca + Mg (x ml)
4. To the extract two ml of 2N NaOH and Murexide indicator is added. At higher pH i.e.
about 12.0 magnesium ion precipitates and only calcium ions remain in the solution. At
this pH Murexide Indicator forms a pink colour with calcium ion. When EDTA is
added calcium gets complexed resulting in colour change from pink to purple
indicating end point. Record Burette reading as Total Ca (y ml)
5. MgCO3 content in the solution is found out by subtracting calcium concentration from
total of calcium and magnesium (x – y ml).
6. This method is sensitive upto 1 ppm.

Calculations:

0.01 N ( EDTA ) X x ( ml )
1 ) Total Ca + Mg = ---------------------------------------
50 ml

2 ) ppm MgCO3 = (x – y ml ) X 4.861

14
Estimation of Total Carbonate, Calcium Carbonate and Magnesium Carbonate
Total
CaCO3 µg/g MgCO3 µg/g
Sr. carbonateµg/g
Product Name
no
1 2 1 2 1 2

Sanket 999Jarda +
1 540 580 293 315 247 265
Sanket No. 1 Supari
Moolchand Superb Jarda
2 500 560 272 304 228 256
+Moolchand Superb Supari
Shimla Jarda +
3 440 480 239 261 201 219
Shimla Supari Mix
4 Goa 1000 Zarda + Goa Supari 520 540 283 293 237 247
5 Gutkha Pan Parag 600 580 326 315 274 265
6 Gutkha Manikchand 520 560 283 304 237 256
7 Click Eucalyptus 620 560 337 304 283 256
8 Baba Zarda 120 2040 2000 1109 1087 931 913
9 Dentobac Creamy Snuff 660 620 359 337 301 283
10 Lime Mix - Miraj tobacco 1480 1440 804 783 676 657
11 Shahin Mishri 1760 1820 957 989 803 831
12 Dabur Red Toothpowder 160 140 87 76 73 64
13 Baidhyanath Red Toothpowder 220 180 120 98 110 82
14 Gai Chhap Zarda 1840 1900 1000 1016 840 884
15 Raja Khaini 380 420 207 228 173 192
16 Gutkha Tulsi Mix 620 600 337 326 283 274
17 IPCO Creamy Snuff 560 600 304 326 256 274
18 Kuber Gutkha 800 720 435 391 365 329
19 Vimal Gutkha 700 720 380 391 320 329
20 Tuiber Tobacco Water 140 160 76 87 64 73

15
7d. ESTIMATION OF NICOTINE IN SMOKELESS TOBACCO SAMPLES:

Nicotine was extracted from tobacco by using chloroform, and tobacco extracts were
analysed by gas chromatography to determine the nicotine content.

Extraction:

One gram of sample is soaked in 15 ml distilled water and 5 ml of 2% NaOH.

Soaking is done for one hour with intermittent shaking. Decant the supernatant and
extract with 20 ml Chloroform in a separating funnel. Collect the chloroform solution
separately. Extract the aqueous phase two more times with 15 ml of chloroform each
time. Collect all the three extracts together. Proceed for the estimation of Nicotine on
a Gas Chromatograph.

Analytical Conditions:

Column: 3 % ov – 101.

Detector: FID.

Oven Temp. : 175 degree C.

Injector port Temp. : 220 degree C.

Detector Port Temp: 250 degree C.

Carrier gas: Nitrogen.

Attenuation: 8.

Injection volume: 1 ul.

Calculation of nicotine concentration was using the following equation:

Area of Sample Conc. of Standard

Nicotine ug / g= -------------------- X -----------------------
Area of standard 1

16
Nicotine content of the samples tested.

[Link] Product Name Nicotine ug/g

1 2
Sanket 999Jarda +
1 1778 1765
Sanket No. 1 Supari Mix
Moolchand Superb Jarda
2 5224 4691
+Moolchand Superb Supari
Shimla Jarda + Shimla Supari
3 3296 3277
Mix
4 Goa 1000 Zarda + Goa Supari 3702 3344
5 Gutkha Pan Parag 3277 3494
6 Gutkha Manikchand 1021 1082
7 Click Eucalyptus 3315 3042
8 Baba Zarda 120 8443 9650
9 Dentobac Creamy Snuff 9856 10160
10 Lime Mix - Miraj Tobacco 3832 4346
11 Shahin Mishri 2670 2790
12 Dabur Red Tooth Powder 4455 4479
13 Baidhyanath Red Tooth Powder 5051 5134
14 Gai Chhap Zarda 2579 2631
15 Raja Khaini 1242 1325
16 Gutkha Tulsi Mix 703 708
17 IPCO Creamy Snuff 5267 5978
18 Kuber Gutkha 1792 2089
19 Vimal Gutkha 1460 1662
20 Tuiber Tobacco Water <0.0001 <0.0001

17
7e. ESTIMATION OF ALKALOIDS IN SMOKELESS TOBACCO SAMPLES:

The alkaloids were extracted from tobacco by using potassium hydroxide in methanol,
and the tobacco extracts were analysed by gas chromatography to determine the
alkaloid content.

Extraction: 1 ml of extraction solution i.e. 0.05N KOH in Methanol was added to 25

mg of tobacco Sample. The sample was sonicated in the ultrasonic bath for one hour.
The sample was removed from the bath after one hour to vortex, swirling the tube to
get all the tobacco into the solvent, and returned to the sonicator for another 2 hours.
After sonication was complete, the tubes were centrifuged for five minutes at low
speed to separate the tobacco from the solvent. The supernatant was transferred to a
sampler vial to be analysed on the GC.
Estimated time for extraction: 3 hrs 25 min per sample.
Estimated time for analysis: 35 min per sample.

Analytical Conditions:

Column: BP 5. 50 m X 0.22 I.D. X 1.0 um.

Detector: : Thermionic Ionisation Detector.(TID)

Oven Temp. : 110 degree C for 1 minute.

Rate : 5 Degrees per minute hold for 5 minutes.

Total Run Time : 29 minutes

Injector port Temp. : 220 degree C.

Detector Port Temp : 300 degree C.

Carrier gas : Helium at 15 psi.

Attenuation : 4.

Injection volume : 1 ul.

Calculations:

Area of Sample Conc. of Standard ml of Extract

Alkaloids ug / g= -------------------- X ---------------------- X ------------------
Area of standard 1 Wt. of Sample

18
Nornicotine and anabasine concentrations in smokeless tobacco samples

Nornicotine ug/g Anabasine ug/g

[Link] Product Name
1 2 1 2
Sanket 999 Jarda + Sanket
1 <0.0001 <0.0001 <0.0001 <0.0001
No. 1 Supari Mix
Moolchand Superb Jarda
2 0.01 0.12 <0.0001 <0.0001
+Moolchand Superb Supari
Shimla Jarda + Shimla
3 <0.0001 <0.0001 <0.0001 <0.0001
Supari Mix
Goa 1000 Zarda + Goa
4 <0.0001 <0.0001 <0.0001 <0.0001
Supari
5 Gutkha Pan Parag <0.0001 <0.0001 <0.0001 <0.0001
6 Gutkha Manikchand <0.0001 <0.0001 <0.0001 <0.0001
7 Click Eucalyptus <0.0001 <0.0001 <0.0001 <0.0001
8 Baba Zarda 120 0.21 0.23 0.006 0.009
9 Dentobac Creamy Snuff 0.032 0.028 <0.0001 <0.0001
10 Lime Mix - Miraj Tobacco <0.0001 <0.0001 <0.0001 <0.0001
11 Shahin Mishri <0.0001 <0.0001 <0.0001 <0.0001
12 Dabur Red Tooth Powder <0.0001 <0.0001 <0.0001 <0.0001
Baidhyanath Red Tooth
13 <0.0001 <0.0001 <0.0001 <0.0001
Powder
14 Gai Chhap Zarda 0.016 0.014 <0.0001 <0.0001
15 Raja Khaini <0.0001 <0.0001 <0.0001 <0.0001
16 Gutkha Tulsi Mix <0.0001 <0.0001 <0.0001 <0.0001
17 IPCO Creamy Snuff 0.046 0.044 <0.0001 <0.0001
18 Kuber Gutkha <0.0001 <0.0001 <0.0001 <0.0001
19 Vimal Gutkha <0.0001 <0.0001 <0.0001 <0.0001
20 Tuiber Tobacco Water 0.13 0.11 <0.0001 <0.0001

19
Myosmine and anatabine concentrations in smokeless tobacco samples

Myosmine ug/g Anatabine ug/g

[Link] Product Name
1 2 1 2
Sanket 999 Jarda + Sanket
1 0.066 0.079 <0.0001 <0.0001
No. 1 Supari Mix
Moolchand Superb Jarda
2 <0.0001 <0.0001 <0.0001 <0.0001
+Moolchand Superb Supari
Shimla Jarda + Shimla
3 <0.0001 <0.0001 <0.0001 <0.0001
Supari Mix
Goa 1000 Zarda + Goa
4 <0.0001 <0.0001 <0.0001 <0.0001
Supari
5 Gutkha Pan Parag <0.0001 <0.0001 <0.0001 <0.0001
6 Gutkha Manikchand <0.0001 <0.0001 <0.0001 <0.0001
7 Click Eucalyptus <0.0001 <0.0001 <0.0001 <0.0001
8 Baba Zarda 120 <0.0001 <0.0001 <0.0001 <0.0001
9 Dentobac Creamy Snuff <0.0001 <0.0001 <0.0001 <0.0001
10 Lime Mix - Miraj Tobacco 0.027 0.03 0.01 0.02
11 Shahin Mishri <0.0001 <0.0001 <0.0001 <0.0001
12 Dabur Red Tooth Powder <0.0001 <0.0001 <0.0001 <0.0001
Baidhyanath Red Tooth
13 <0.0001 <0.0001 <0.0001 <0.0001
Powder
14 Gai Chhap Zarda 0.009 0.01 0.028 0.026
15 Raja Khaini <0.0001 <0.0001 <0.0001 <0.0001
16 Gutkha Tulsi Mix <0.0001 <0.0001 <0.0001 <0.0001
17 IPCO Creamy Snuff <0.0001 <0.0001 <0.0001 <0.0001
18 Kuber Gutkha <0.0001 <0.0001 <0.0001 <0.0001
19 Vimal Gutkha <0.0001 <0.0001 <0.0001 <0.0001
20 Tuiber Tobacco Water <0.0001 <0.0001 <0.0001 <0.0001

20
7f. DETERMINATION OF NITROSAMINES IN TOBACCO SAMPLES:

Nitrosamines have been recognized as extremely potent chemical carcinogens and

there has been considerable interest in the determination of nitrosamines by a number
of analytical techniques. Differential Pulse Polarography (DPP) is a very sensitive
analytical technique for the determination of nitrosamines and their metabolite by-
products.

Extraction Procedure:

Weigh 1 g of tobacco sample in a conical flask. Add 25 ml methylene chloride.

Extract for l hour on wrist action shaker, centrifuge if necessary. Collect the
extract in another flask. Add 25 ml methylene chloride into the sample. Extract
for another hour on wrist action shaker. Collect the extract and pool both the
extracts. Centrifuge the extract and collect the supernatant in a beaker. Evaporate
to dryness by Nitrogen gas. Reconstitute the residue with 10 ml of 0.1 M HCl and
analyse the analyte on DPP.

Time for extraction: - 3.15 min for each sample.

Time for estimation: - 20 min for each sample.

Estimation by Differential Pulse Polarography:

Instrument: EG&G Princeton Applied Research

Integrator: Model No. 394.
PARC: Model No. 303 A SMDE
Stirrer: Model No. 305.

Ref. Electrode: SCE.

Ref. Value: 0.2415
Working electrode: HMDE
Drop size: Small.
Electrolyte: 0.1 M HCl.
Purge Time: 50 seconds.
Initial potential: - 0.04 V.
Final potential: -1.1 V.
Scan rate: 2 mV/second.
Scan increment: 2 mV.
Drop time: 1 second.
Electrolyte area: 1 sq. cm.
Pulse height: 50 mV.

21
Concentrations of N-Nitrosonornicotine (NNN), 4(N-methyl-N-Nitrosamino)1-(3-
pyridyl)-1-butanol (NNK), N-nitrosoanabasine (NAB), N-nitrosoanatabine
(NAT), in the smokeless tobacco samples.

NAB NAB NAT NAT NNK NNK NNN NNN

Sr. no Product Name µg/g µg/g µg/g µg/g µg/g µg/g µg/g µg/g

Sanket 999Jarda + Sanket No. 1

1
Supari Mix
4.57 3.6

Moolchand Superb Jarda

2
+Moolchand Superb Supari
4.9 5.13

Shimla Jarda + Shimla Supari

3
Mix
11.58 12.58 11.58 9.54
4 Goa 1000 Zarda + Goa Supari 1.92 2.45
5 Gutkha Pan Parag 10.68 11.51
6 Gutkha Manikchand 5.75 6.89
7 Click Eucalyptus * * * * * * * *
8 Baba Zarda 120 6.55 7.36
9 Dentobac Creamy Snuff * * * * * * * *
10 Lime Mix - Miraj Tobacco * * * * * * * *
11 Shahin Mishri 4.02 4.77
12 Dabur Red Tooth Powder * * * * * * * *
13 Baidhyanath Red Tooth Powder * * * * * * * *
14 Gai Chhap Zarda * * * * * * * *
15 Raja Khaini * * * * * * * *
16 Gutkha Tulsi Mix 1.87 2.15
17 IPCO Creamy Snuff 4.88 4.38
18 Kuber Gutkha 6.53 6.27 5.73 6.01
19 Vimal Gutkha 5.03 4.74
20 Tuiber Tobacco Water 20.12 19.65
*These samples contain Nitrosamines other than the four Nitrosamines mentioned
above

22
7g. ESTIMATION OF BENZO [a] PYRENE IN SMOKELESS TOBACCO
SAMPLES:

Extraction: Weigh 1g of tobacco sample; add 25 ml of hexane and mix on a wrist

action rotary shaker for 60 minutes. Sit the sample for 30 minutes. Decant the clear
supernatant into the beaker after centrifugation. Repeat extraction with 25ml hexane,
and pool the extracts. Evaporate the extract to total dryness by passing Nitrogen gas.
Reconstitute the residue with Acetonitrile. Proceed to analyse the sample on Gas
Chromatograph. Time for extraction: 3 hrs 30 min per sample.
Time for analysis: 45 min per sample.

Analytical Conditions:

Column: BP5, Capillary column, 50 m X 0.22 mm X 1.0 um.

Detector: Flame Ionisation Detector. (FID)
Attenuation: 2, Range 10.
Autozero : On.
Carrier: Helium 1 ml / min.
Air: 300 ml/min.
Hydrogen 420 ml/min.

Temperature Programme:
Oven Temp.: 130 degree C.
Time: ---- min.
Rate: 4.0 degree / min. to 290 degree C.
Total Run Time: 40 minutes.
Injector Temp: 200 degree C.
Detector Temp: 250 degree C.

Polarity: Negative.
Back off: Negative.
X 10.

Standards: Standards of different known concentrations of benzo[a]pyrene were run

and the calibration curves of the concentration against peak area were plotted.

Calculations:
Area of sample Conc. of Standard 50
Concentration ug / g = -------------------- X ---------------------- X ------
Area of standard 1 1

23
 Concentration of Benzopyrene in smokeless tobacco samples

Benzo[a]pyrene ug/g
Sr. no Product Name
1 2

1 Sanket 999Jarda + Sanket No. 1 Supari Mix <0.0001 <0.0001

Moolchand Superb Jarda +Moolchand Superb
2 0.46 0.44
Supari
3 Shimla Jarda + Shimla Supari Mix 0.27 0.29
4 Goa 1000 Zarda + Goa Supari <0.0001 <0.0001
5 Gutkha Pan Parag <0.0001 <0.0001
6 Gutkha Manikchand 0.61 0.62
7 Click Eucalyptus <0.0001 <0.0001
8 Baba Zarda 120 <0.0001 <0.0001
9 Dentobac Creamy Snuff 0.88 0.79
10 Lime Mix - Miraj Tobacco <0.0001 <0.0001
11 Shahin Mishri <0.0001 <0.0001
12 Dabur Red Tooth Powder <0.0001 <0.0001
13 Baidhyanath Red Tooth Powder <0.0001 <0.0001
14 Gai Chhap Zarda <0.0001 <0.0001
15 Raja Khaini 0.5 0.56
16 Gutkha Tulsi Mix 0.45 0.47
17 IPCO Creamy Snuff 0.94 0.89
18 Kuber Gutkha 0.45 0.47
19 Vimal Gutkha 0.64 0.61
20 Tuiber Tobacco Water <0.0001 <0.0001

24
7h. ESTIMATION OF NITRATE

The ground tobacco sample is extracted with 50 ml of 5 % acetic acid on a wrist

action rotary shaker for 60 minutes and then filtered. The nitrate ion in the filtrate is
reduced upon reaction with hydrazine sulphate at 370C in the presence of copper at a
pH of 10.2. The nitrite formed is reacted with sulphanilamide under acidic conditions
to yield a diazo compound, which then mixed with N- (1-Napthyl) – ethylenediamine
which forms a red colour complex.

The absorbance is measured with a colorimeter with 550 nm filter. Standards of

different known concentrations of Nitrate are run and the calibration curves of the
concentration against absorbance are plotted, and from this the concentration of
nitrate in unknown sample is calculated. Time for extraction: 60 min per sample.
Time for analysis: 30 min per sample.

Calculations:
ppm Nitrogen (µg/ml ) X 50 ( ml )
Concentration µg / g = -----------------------------------------------
0.5 g sample

25
 Nitrate µg/g
Sr.
Product Name
no Nitrate 1 Nitrate 2

1 Sanket 999 Jarda + Sanket No. 1 Supari Mix < 0.1 < 0.1
Moolchand Superb Jarda +Moolchand Superb
2 6.68 8.01
Supari
3 Shimla Jarda + Shimla Supari Mix < 0.1 < 0.1
4 Goa 1000 Zarda + Goa Supari < 0.1 < 0.1
5 Gutkha Pan Parag < 0.1 < 0.1
6 Gutkha Manikchand < 0.1 < 0.1
7 Click Eucalyptus 8.01 5.34
8 Baba Zarda 120 12.02 10.68
9 Dentobac Creamy Snuff 8.01 9.35
10 Lime Mix - Miraj Tobacco 13.85 10.68
11 Shahin Mishri 13.35 5.34
12 Dabur Red Tooth Powder < 0.1 < 0.1
13 Baidhyanath Red Tooth Powder < 0.1 < 0.1
14 Gai Chhap Zarda 4.01 4.01
15 Raja Khaini 12.02 9.35
16 Gutkha Tulsi Mix < 0.1 < 0.1
17 IPCO Creamy Snuff 9.35 9.35
18 Kuber Gutkha 5.34 5.34
19 Vimal Gutkha < 0.1 < 0.1
20 Tuiber Tobacco Water < 0.1 < 0.1

26
7i. ESTIMATION OF HEAVY METALS IN SMOKELESS TOBACCO
SAMPLES

The concentration of the heavy metals is very low (few micrograms to nanograms) as
compared to the other constituents in tobacco. Reliable estimation thus requires an
ultrasensitive method of detection and measurement. After studying some of the
sensitive techniques capable of analysis of ultra-trace levels, differential pulse
stripping voltammetry was chosen due to its ability to analyse simultaneously many
metals at ultra-trace levels even without prior chemical separation in addition to high
sensitivity, inherent reliability and accuracy being added advantages.

Apparatus and Reagents:

Voltammetric measurements were carried out with Princeton Applied Research

electrochemical trace analyser model No 394 and Voltammograms were recorded on
computer. A static mercury drop electrode assembly (PARC-303 A) was used with
PARC-305 stirrer.

All chemicals used were Merck, Suprapur, Analar or electronic grade. Standard stock
solutions (0.01 M) of Ni, Pb, Cd, Cr, As, Se and Hg were prepared and necessary
dilutions were made as and when required.

Pretreatment:
In view the low concentrations of the trace metals generally expected in tobacco
samples, extreme care was taken to avoid contamination. One gram of each sample
was digested with nitric acid (1ml) and perchloric acid (0.5ml), for about 2 hours.
The residual was taken in 10 ml of 0.25% Nitric acid (Electronic grade) and taken into
the electrolytic cell.

An aliquot of digested solution (0.1-0.5) was added to 10 ml of supporting electrolyte

(5 ml of 0.25% HNO3 + 5 ml of 0.15 M ammonium acetate). The solution was
deaerated for 8 minutes with highly purified nitrogen gas. The initial potential was
kept -1.2 volt. The solution was stirred and electrolysis was carried out for 1.0
minute. After a rest period of 30 seconds the stripping voltammogram was recorded.
The concentrations of metals were calculated with the help of calibration curves as
well as by the method of standard solutions.

Quality assurance:
Analysing the standard reference materials has checked the reliability of the
procedure of estimation of the heavy metals in the tobacco samples.

The values have also been independently checked with measurements using other
sensitive techniques such as atomic absorption spectrophotometry. One of the
important factors for achieving reliability has been our endeavour to maintain clean
blanks with extremely low background concentrations for heavy metals. The precision
for replicate analysis is usually better than + 8.0%.

27
Measurement procedure for estimation of Lead:

After nitrogen purging Lead is electrodeposited for one minute on Hanging Drop
Mercury Electrode with initial potential of - 0.6 V.

Rest period 30 seconds.

Voltamogram recorded with instrumental settings as under:

Scan rate: 5 mV/sec.

Pulse repetition time 0.5 seconds.
Modulation amplitude 50 mV.
Lead peak obtained at - 0.42 V.

Measurement procedure for estimation of Cadmium:

After nitrogen purging cadmium is electrodeposited for one minute on Hanging Drop
Mercury Electrode with initial potential of - 0.6 V.

Rest period 30 seconds.

Voltammogram recorded with instrumental settings as under:

Scan rate: 5 mV/sec.

Pulse repetition time 0.5 seconds.
Modulation amplitude 50 mV.
Cadmium peak obtained at - 0.6 V.

Measurement procedure for estimation of Selenium:

After nitrogen purging Selenium is electrodeposited for one minute on Hanging Drop
Mercury Electrode with initial potential of - 0.8 V.

Rest period 30 seconds.

Voltamogram recorded with instrumental settings as under:

Scan rate: 2 mV/sec.

Pulse repetition time 0.5 seconds.
Modulation amplitude 50 mV.
Selenium peak obtained at - 0.6 V.

28
Measurement procedure for estimation of Arsenic:

After nitrogen purging Arsenic is electrodeposited for one minute on Hanging Drop
Mercury Electrode with initial potential of - 0.4 V.

Rest period 30 seconds.

Voltammogram recorded with instrumental settings as under:

Scan rate: 2 mV/sec.

Pulse repetition time 0.5 seconds.
Modulation amplitude 50 mV.
Arsenic peak obtained at - 0.5 V.

Measurement procedure for estimation of Nickel:

After nitrogen purging Nickel is electrodeposited for one minute on Hanging Drop
Mercury Electrode with initial potential of - 1.6 V.

Rest period 30 seconds.

Voltammogram recorded with instrumental settings as under:

Scan rate: 2 mV/sec.

Pulse repetition time 0.5 seconds.
Modulation amplitude 50 mV.
Nickel peak obtained at - 0.9 V.

Measurement procedure for estimation of Chromium:

Chromium standard solution was prepared from K2Cr2O7. Stock standard solution of
50 ppb was prepared. Different standard solutions were prepared by making suitable
dilutions.

Calibration was done by 100ul addition from 50 ppb standard in 6 ml

(Cell volume) of background electrolyte.

Chromium Peak Potential = - 1.262 V

Initial Potential = - 1.0 V
Final Potential. = - 1.5 V

29
Preparation of background electrolyte:-
Background electrolyte was prepared in D.D.W.

1) Sodium Acetate ( 0.2 mol/L)

2) DTPA [Diethylene Triamine Penta Acetic Acid] (0.05mol / L)
3) NaNo3 (2.5 mol/L)

Finally pH was adjusted to 6.2 in 30 % NaOH.

Sample preparation:

Sample is digested in HNO3 and HCl 4, evaporated to dryness and finally taken in
background electrolyte as per required volume from this.
6 ml of sample was taken, pH was adjusted to 6.2 in 30% NaOH and analysed by
anodic stripping voltammetry after calibration.

Estimation procedure for Mercury :

Mercury is analysed by Cold Vapour AAS procedure.

Apparatus and Reagents:

Mercury estimations are carried out on MA 5640 Atomic Absorption Spectrometer.

All chemicals used are of Merck, Suprapur, Analar or electronic grade.

Pretreatment:

Sample: 100g + Acid 250ml. Keep for wet digestion till white residue is obtained.

Add 25 ml of 0.25% Nitric acid and take it for analysis.

Measurement Procedure:

Switch to HOLD mode operation.

Switch ON the absorbance switch ( %T/Abs)
Mercury free air is purged through the reaction vessel.
Note the absorbance as early as possible.
Switch back to Normal Mode. The meter indication should be back to 100% T.
Switch OFF the lamp and stirrer.
Adjust 0% and 100 just before each measurement.

30
[Link] Product Name Cadimum µg/g Lead µg/g nickel ug/g Arsenic
µg/g
Cd1 Cd2 Pb1 Pb2 Ni 1 Ni 2 As 1 As 2
Sanket 999Jarda +
1 Sanket No. 1 Supari 0.07 0.07 0.15 0.15 0.02 0.04 0.72 0.64
Mix
Moolchand Superb
2 Jarda +Moolchand 0.27 0.25 0.34 0.27 0.01 0.01 1.94 1.86
Superb Supari
Shimla Jarda +
3 Shimla Supari Mix
0.19 0.2 0.2 0.22 0.07 0.08 0.19 0.2

4 Goa 1000 Zarda + Supari 0.19 0.2 0.043 0.02 0.02 0.03 0.3 0.38

5 Gutkha Pan Parag 0.17 0.17 0.14 0.11 < 0.005 < 0.005 0.6 0.54

6 Gutkha Manikchand 0.19 0.19 0.23 0.22 0.02 0.02 0.17 0.18

7 Click Eucalyptus 0.03 0.03 0.08 0.07 < 0.005 < 0.005 0.08 0.07

8 Baba Zarda 120 0.5 0.51 0.94 0.98 0.04 0.03 0.43 0.36

9 Dentobac Creamy Snuff 0.14 0.16 0.56 0.61 0.02 0.04 0.73 0.56

10 Lime Mix - Miraj Tobacco 0.07 0.07 0.14 0.17 < 0.005 < 0.005 0.78 0.88

11 Shahin Mishri 0.11 0.12 0.29 0.31 < 0.005 < 0.005 1.53 0.1

12 Dabur Red Tooth Powder 0.17 0.19 4.85 4.97 < 0.005 < 0.005 0.16 0.1

13 Baidhyanath Red Tooth 0.25 0.22 5.14 5.04 0.01 0.01 0.25 0.21

14 Gai Chhap Zarda 0.23 0.23 0.53 0.53 0.04 0.05 0.39 0.35

15 Raja Khaini 0.04 0.04 0.31 0.35 0.05 0.05 0.11 0.17

16 Gutkha Tulsi Mix 0.11 0.12 0.18 0.2 0.02 0.01 0.3 0.22

17 IPCO Creamy Snuff 0.07 0.07 0.13 0.13 0.06 0.05 0.84 0.74

18 Kuber Gutkha 0.08 0.08 0.24 0.25 < 0.005 < 0.005 1.11 1.08

19 Vimal Gutkha 0.011 0.01 0.02 0.03 0.03 0.02 0.14 0.13

20 Tuiber Tobacco Water 0.011 0.012 0.015 0.018 0.03 0.01 1.08 1.02

31
Sr.n
Product Name Mercur Mercur Selenium Selenium Chromium µg/g
o
y µg/g y µg/g
µg/g µg/g µg/g
Sanket 999Jarda +
<0.000
1 Sanket No. 1 Supari <0.0002<0.0002 <0.0002 <0.0002 <0.0002
2
Mix
Moolchand Superb
<0.000
2 Jarda +Moolchand <0.0002<0.0002 <0.0002 <0.0002 <0.0002
2
Superb Supari
Shimla Jarda + Shimla <0.000
3 <0.0002<0.0002 <0.0002 <0.0002 <0.0002
Supari Mix 2
Goa 1000 Zarda + <0.000
4 <0.0002<0.0002 <0.0002 <0.0002 <0.0002
Supari 2
<0.000
5 Gutkha Pan Parag <0.0002<0.0002 <0.0002 <0.0002 <0.0002
2
<0.000
6 Gutkha Manikchand <0.0002<0.0002 <0.0002 <0.0002 <0.0002
2
<0.000
7 Click Eucalyptus <0.0002<0.0002 <0.0002 <0.0002 <0.0002
2
<0.000
8 Baba Zarda 120 <0.0002<0.0002 <0.0002 <0.0002 <0.0002
2
Dentobac Creamy <0.000
9 <0.0002<0.0002 <0.0002 <0.0002 <0.0002
Snuff 2
Lime Mix - Miraj <0.000
10 <0.0002<0.0002 <0.0002 <0.0002 <0.0002
Tobacco 2
<0.000
11 Shahin Mishri <0.0002<0.0002 <0.0002 <0.0002 <0.0002
2
<0.000
12 Dabur Red Tooth <0.0002<0.0002 <0.0002 <0.0002 <0.0002
2
<0.000
13 Baidhyanath Red <0.0002<0.0002 <0.0002 <0.0002 <0.0002
2
<0.000
14 Gai Chhap Zarda <0.0002<0.0002 <0.0002 <0.0002 <0.0002
2
<0.000
15 Raja Khaini <0.0002<0.0002 <0.0002 <0.0002 <0.0002
2
<0.000
16 Gutkha Tulsi Mix <0.0002<0.0002 <0.0002 <0.0002 <0.0002
2
<0.000
17 IPCO Creamy Snuff <0.0002<0.0002 <0.0002 <0.0002 <0.0002
2
<0.000
18 Kuber Gutkha <0.0002<0.0002 <0.0002 <0.0002 <0.0002
2
<0.000
19 Vimal Gutkha <0.0002<0.0002 <0.0002 <0.0002 <0.0002
2
<0.000
20 Tuiber Tobacco Water <0.0002<0.0002 <0.0002 <0.0002 <0.0002
2

32
7j. Determination of Eugenol

Summary:

This method is used to separate and identify Eugenol in the ethanol extract of the
sample. The extract is filtered and analysed using reversed phase/isocratic High
Performance Liquid Chromatography with ultra violet detection. For standards
external standard calibration procedure is observed. 20 ul of each standard is injected
into HPLC and analysed. A calibration curve is prepared by plotting concentration of
Eugenol vs. peak area. The response factor is determined from the calibration curve.

Preparation of Standards:
1) Prepare primary Eugenol stock solution by accurately taking 190 ul of pure
Eugenol liquid into 100 ml of Ethanol.
2) Six working standards are prepared (ranging from 2 ug/ml to 1000 ug/ml) by
diluting appropriately with ethanol.
3) Eugenol calibration standards are prepared afresh every five working days, which
are stored in dark and at 4 °C until analysed.

Sample Preparation: (Extraction of sample)

1) The sample removed from the original package is inspected for any extraneous
material.
2) Tobacco sample is ground / pulverized to pass through a 20 mesh screen.
3) The sample is conditioned.
4) Weigh 2g of ground tobacco into 250 ml conical flask.
5) Add 50 ml of ethanol to the sample and seal the cap with parafilm.
6) Extract in a shaker for 2 hours at 50 °C.
7) Centrifuge at 1200 rpm for 10 minutes.
8) Allow cooling to room temperature.
9) Filter the aliquot into a 50 ml amber colour bottle (in duplicate) using a
syringe filter.
10) Cap and store in dark and at 4 °C. until analysed.

** Estimated time of extraction for one sample is 3hrs 10min.

Reversed Phase HPLC Analysis. (Chromatographic conditions)

1) Columns: RP 18e column.
2) Column Temp.: 30 °c.
3) Mobile Phase : Solvent A: Methanol.
Solvent B: Type I water
4) Sample wash: Solvent.

5) Mobile phase gradient

33
Flow rate: 0.7 ml / minute.

Time: Min. Composition:

0.0 80% A 20% B 0% C
20.0 80 % A 20% B 0% C
Method end action: 80% A 20% B 0% C
(Equillibrate 10 min.)

** Estimated time for each sample to analyse is 20min.

Sample Analysis:
1) 20 ul of each sample vial is injected onto HPLC column and analysed.
2) Identification of peaks is done by comparison of retention times with standards and
the spiking of the samples.

Calculations and Quantification:

Determination of Eugenol deliveries in ug / g:

Peak Area ml of solution

Eugenol (ug/g) = ----------------- x ---------------------
[Link] Wt (g) of tobacco

By entering correct multiplier (overall volume of original sample in ml) and divisor
(original sample wt. in grams) the concentration of eugenol is calculated in ug/g.
To convert this conc. in a percentage (%) the ug/g result is divided by 10,000.
The results are expressed on an “as conditioned” basis. These may be expressed on a
dry matter basis using the appropriate moisture result.

Normative References:
[Link] Society for Testing and Materials (ASTM)
D 1193-77-Standard Specifications for Reagent Water.
Version 1977.

2. Health Canada Test Method. T-115.

Determination of Tar, Water, Nocotine and Carbon Monoxide
in Mainstream Tobacco Smoke, 1999-12-31.
Health Canada – Official Methods.

34
 Eugenol ug/g
Sr. no Product Name
Eugenol 1 Eugenol 2
Sanket 999 Jarda + Sanket No. 1
1 71.83 74.93
Supari Mix
Moolchand Superb Jarda +Moolchand
2 303.06 300.33
Superb Supari
3 Shimla Jarda + Shimla Supari Mix 875.67 867.3
4 Goa 1000 Zarda + Goa Supari 96.89 92.78
5 Gutkha Pan Parag 1164.95 1122.4
6 Gutkha Manikchand 405.94 403.65
7 Click Eucalyptus < 0.00005 < 0.00005
8 Baba Zarda 120 20348.8 20261.1
9 Dentobac Creamy Snuff 25373.53 25706.13
10 Lime Mix - Miraj Tobacco 13.97 12.82
11 Shahin Mishri 6345.45 6094.77
12 Dabur Red Tooth Powder 6648.43 6606.2
13 Baidhyanath Red Tooth Powder 1220.55 1250.61
14 Gai Chhap Zarda < 0.00005 < 0.00005
15 Raja Khaini < 0.00005 < 0.00005
16 Gutkha Tulsi Mix 273.73 265.51
17 IPCO Creamy Snuff 165.56 161.22
18 Kuber Gutkha 1439.14 1370.72
19 Vimal Gutkha 8.91 9.38
20 Tuiber Tobacco Water 41.72 42

35
7k. Determination of Sorbic Acid

Summary:

This method estimates the amount of Sorbic Acid by reversed phased high
performance liquid chromatography (HPLC) and UV detection. External standards are
used in this method.

Preparation of Standards:

Weigh 100 g of Potassium Sorbate into 100 ml volumetric flask.

Dilute to volume with Type I water.
This is the Primary Stock Standard Solution.

Prepare different working standards ranging the sorbate concentration from 0.0 ug/ml
to 100 ug/ml. (0, 5, 10, 25, 50 and 100 ug/ml).

Sorbic Acid calibration standards are prepared afresh every five working days which
are stored at 4 °C until analysed.
20 ul of each standard is injected into HPLC and analysed.
A calibration curve is prepared by plotting concentration of Sorbic Acid vs. peak area.
Determine response factor from calibration curve.

Sample Generation:

Weigh accurately 1g finely chopped (20 mesh) tobacco into a 50 ml glass beaker.
Add 30 ml hot (50 °c) Type I water to the sample and sonicate for 30 minutes at 50
°c.
Centrifuge at 1200 rpm for 10 min and filter the aliquote.
Decant the water into a 100 ml standard flask.
Similarly extract another 1g of tobacco and collect the extract resulting from 2g of
tobacco.
Allow to cool and make the volume to 100 ml.
Mix well, filter and transfer the aliquot into a test tube for HPLC analysis.

** Estimated time of extraction for one sample is one and half-hour.

Sample Analysis:

Chromatographic Conditions:

Column Temp: 30 °c.

Mobile Phase : Solvent A – Methanol, filter and degas.
Solvent B – 2 litres of 1% IPA adjusted to pH 2.3 with phosphoric
acid.
Sample wash: Solvent A.

36
Mobile phase : Gradient.
20 ul of each sample is injected onto HPLC.

** Estimated time for analysing each sample: 35 min.

Calibration and Calculations:

A calibration curve is plotted with concentrations vs respective peak areas.

Peak area ml of solution

Sorbitol : ( g/g) = ---------------------------- X -----------------------
Resp. Factor Wt of Tobacco

Normative References:

1) American Society for Testing and Materials D 1193 – 77.

Standard specification for Reagent water, Version 1977.

2) Health Canada Test Method T – 115, 1999 – 12 – 31.

37
[Link] Product Name Sorbic Acid ug/g
1 2
Sanket 999 Jarda + Sanket No. 1 Supari
1 < 0.00005 < 0.00005
Mix
Moolchand Superb Jarda +Moolchand
2 < 0.00005 < 0.00005
Superb Supari
3 Shimla Jarda + Shimla Supari Mix < 0.00005 < 0.00005
4 Goa 1000 Zarda + Goa Supari < 0.00005 < 0.00005
5 Gutkha Pan Parag < 0.00005 < 0.00005
6 Gutkha Manikchand < 0.00005 < 0.00005
7 Click Eucalyptus < 0.00005 < 0.00005
8 Baba Zarda 120 0.59 0.53
9 Dentobac Creamy Snuff 1315.43 1284.42
10 Lime Mix - Miraj Tobacco < 0.00005 < 0.00005
11 Shahin Mishri < 0.00005 < 0.00005
12 Dabur Red Tooth Powder < 0.00005 < 0.00005
13 Baidhyanath Red Tooth Powder < 0.00005 < 0.00005
14 Gai Chhap Zarda < 0.00005 < 0.00005
15 Raja Khaini < 0.00005 < 0.00005
16 Gutkha Tulsi Mix < 0.00005 < 0.00005
17 IPCO Creamy Snuff < 0.00005 < 0.00005
18 Kuber Gutkha < 0.00005 < 0.00005
19 Vimal Gutkha < 0.00005 < 0.00005
20 Tuiber Tobacco Water < 0.00005 < 0.00005

38
 7l. Determination of Triacetin

Extraction: Weigh 2 g of the tobacco sample in a flask and add to it 150 ml aq. 0.5
H2SO4. Steam distill this mixture. Collect the distillate in a flask under
approximately 25 ml of Distilled water. Collect approximately 200 ml of the
distillate. Transfer the distillate to a 500 ml separating funnel and add to it 50 ml of
methylene chloride. Shake the separating funnel. Drain off and collect extracted
methylene chloride fraction into a 250 ml conical flask. The extraction is carried for
two more times, thus collecting total of 150 ml of the extract. This 150 ml extract is
evaporated to 1 ml. This l ml extract is taken for the analysis in Gas Chromatograph.

Gas Chromatograph Configuration:

Gas Chromatograph: Chemito 8610.

Injector: Splitless mode.
Column: BP 5 ; Fused silica capillary column.
Detector: Flame Ionization Detector (FID)
Channel A, 1 Volt full scale.
Range: 12
Atten.: 8
Auto Zero: On.
Carrier : He at 40.0 psi, flow+ 1.3 ml / minute @ 70 degree
C.

Gas Chromatograph Operating Condition:

Makeup gas: Nitrogen; 25 ml/min.

Injector: 220 degree C.
Detector: 250 degree C.
Start Temp.: 70 degree C; Hold for 2 minutes.
Rate: 5 degree C/min to 220 C; Hold for 3.0 min.
Total Run Time: 35.00 minutes.

Estimated time for analysis: - 45 minutes.

Calculation: A calibration curve of Triacetin is prepared by plotting the concentration

of the standards versus the respective peak area.

Calculations:

Area of Sample Conc. of Standard ml of Extract

Triacetin ug / g= ------------------- X --------------------- X ---------------
Area of standard 1 Wt. of Sample

39
 Triacetin ug/g
Sr. no Product Name
1 2
1 Sanket 999Jarda + Sanket No. 1 Supari Mix <0.0001 <0.0001
Moolchand Superb Jarda +Moolchand Superb
2 0.04 0.06
Supari
3 Shimla Jarda + Shimla Supari Mix 0.06 0.05
4 Goa 1000 Zarda + Goa Supari <0.0001 <0.0001
5 Gutkha Pan Parag <0.0001 <0.0001
6 Gutkha Manikchand 0.033 0.036
7 Click Eucalyptus <0.0001 <0.0001
8 Baba Zarda 120 0.01 0.03
9 Dentobac Creamy Snuff <0.0001 <0.0001
10 Lime Mix - Miraj Tobacco <0.0001 <0.0001
11 Shahin Mishri <0.0001 <0.0001
12 Dabur Red Tooth Powder 0.021 0.024
13 Baidhyanath Red Tooth Powder <0.0001 <0.0001
14 Gai Chhap Zarda 0.31 0.28
15 Raja Khaini 0.033 0.038
16 Gutkha Tulsi Mix <0.0001 <0.0001
17 IPCO Creamy Snuff <0.0001 <0.0001
18 Kuber Gutkha 0.034 0.036
19 Vimal Gutkha <0.0001 <0.0001
20 Tuiber Tobacco Water <0.0001 <0.0001

40
7m. Determination of Sodium Propionate

Preparation of extraction solution:

Take 35 g of CaCl2 and dissolve it in about 1 litre of Dist. Water.

Add 0.54 ml of Conc.H2SO4. Make the total volume to 2 liters.

Extraction:

Weigh 10 g of the tobacco sample in 125 ml flask. Add 20 ml of the aqueous

extraction solution. Moisten the tobacco on rotary shaker until all the solution is
absorbed by tobacco. Add 80 ml of methanol. Seal the flask with the parafilm.
Shake the sample flask for 30 minutes. Let the sample sit for 1 hour. Shake the
sample again for 30 seconds. Decant liquid into centrifuge tubes. Centrifuge at low
speed. Take 2 ml of the supernatant and add 2 ml methanol to it. Mix thoroughly for
10 seconds. Centrifuge at medium speed. Take the supernatant for analysis on Gas
Chromatograph.

Estimated time for extraction: - 2 hours per sample.

Sample Analysis – Gas Chromatography :

Gas Chromatograph Configuration:

Injector: Splitless.
Detector : Flame ionization (FID)
Column: BP 5 ; fused silica capillary column.
Carrier: Heat 12.0 psi; flow=2.3 ml/min. @ 70 degree C.
Makeup gas: Nitrogen gas; 25 ml / minute.

Gas Chromatograph – Operating Condition:

Injector: 225 degree C.
Detector: 230 degree C.

Column Oven Temperature Profile:

Start Temp.: 70 degree C Hold for two minutes.
Rate: 7.5 deg. C/minute to 155 deg. C . Hold for 3.07 minutes.
Rate: 7.5 deg. C/minute to 205 deg. C. Hold for 4.0 minutes.
Total Run time: 25 minutes.

Calculations: A calibration curve of Sodium Propionate is prepared by plotting the

concentration of the standards versus the respective peak area.

Calculations:

Area of Sample Conc. of Standard ml of Extract

Na-propionate ug / g= ------------------- X --------------------- X ---------------
Area of standard 1 Wt. of Sample

41
 Na-propionate
ug/g
Sr. no Product Name
1 2

1 Sanket 999Jarda + Sanket No. 1 Supari Mix <0.0001 <0.0001

Moolchand Superb Jarda +Moolchand Superb
2 0.35 0.37
Supari
3 Shimla Jarda + Shimla Supari Mix <0.0001 <0.0001
4 Goa 1000 Zarda + Goa Supari <0.0001 <0.0001
5 Gutkha Pan Parag <0.0001 <0.0001
6 Gutkha Manikchand <0.0001 <0.0001
7 Click Eucalyptus 0.004 0.005
8 Baba Zarda 120 <0.0001 <0.0001
9 Dentobac Creamy Snuff <0.0001 <0.0001
10 Lime Mix - Miraj Tobacco <0.0001 <0.0001
11 Shahin Mishri <0.0001 <0.0001
12 Dabur Red Tooth Powder 0.44 0.46
13 Baidhyanath Red Tooth Powder <0.0001 <0.0001
14 Gai Chhap Zarda <0.0001 <0.0001
15 Raja Khaini 0.0007 0.0005
16 Gutkha Tulsi Mix 0.0002 0.0003
17 IPCO Creamy Snuff 0.0003 0.0005
18 Kuber Gutkha 1.01 1
19 Vimal Gutkha <0.0001 <0.0001
20 Tuiber Tobacco Water <0.0001 <0.0001

42
 7n. Estimation Procedure for Humectants
(Glycerol, Propylene Glycol and Triethylene Glycol)

Extraction: Weigh 4 g of tobacco sample in a 250 ml conical flask. Add 50 ml warm

( 50 degree C.) double distilled water into the flask. Cover the n\mouth of the flask
with parafilm. Mix on a wrist action rotary shaker for 60 minutes. Sit for 30 minutes.
Decant the clear supernatant into the sample bottle. (Centrifuge if necessary) Proceed
to analyse the glycols on Gas Chromatograph.

Chromatographic conditions:

Chromatograph : Model Chemito – 8610.

Injector: Split with split flow 15 ml/min.
Column: BP5, Capillary column, 50 m X 0.22 mm X 1.0 um.
Detector: Flame Ionisation Detector.
Attenuation: 2, Range 10.
Autozero : On.
Carrier: Helium 30 ml / min.
Air: 333 ml/min.
Hydrogen 420 ml/min.

Temperature Programme:

Oven Temp.: 110 degree C.

Time: 1 min.
Rate: 8.0 degree / min. to 220 degree C. , hold for 2 min.
Total Run Time: 17 minutes.
Injector Temp: 250 degree C.
Detector Temp: 250 degree C.
Polarity: Negative.
Back off: Negative.
X 10.

Identification & Quantification: Locations of the peaks for Glycerol, Propylene

Glycol and Triethylene Glycol were ascertained by running the standards of each of
the glycol individually. Standards: Standards of different known concentrations
of each of the glycol were run and the calibration curves of the concentration against
peak area were plotted.

Calculations: Individual Hemectant (Glycerol or Propylene Glycol or Triethylene

Glycol)
Area of sample Conc. of Standard 50
Concentration ug / g = -------------------- X ---------------------- X ------
Area of standard 1 4

43
 Triethylene
[Link] Product Name Propylene glycol µg/g µg/g
glycol µg/g Glycerol
TEG 1 TEG 2 PG 1 PG 2 GLY 1 GLY2
Sanket 999 Jarda + Sanket
1 <0.0001<0.0001 <0.0001 <0.0001 <0.0001 <0.0001
No. 1
Moolchand Superb Jarda
2 <0.0001<0.0001 <0.0001 <0.0001 <0.0001 <0.0001
+Moolchand
Shimla Jarda + Shimla Supari
3 651.38 647.53 <0.0001 <0.0001 <0.0001 <0.0001
Mix
4 Goa 1000 Zarda + Supari <0.0001<0.0001 <0.0001 <0.0001 <0.0001 <0.0001
5 Gutkha Pan Parag <0.0001<0.0001 <0.0001 <0.0001 <0.0001 <0.0001
6 Gutkha Manikchand <0.0001<0.0001 211.77 214.84 <0.0001 <0.0001
7 Click Eucalyptus <0.0001<0.0001 <0.0001 <0.0001 <0.0001 <0.0001
8 Baba Zarda 120 <0.0001<0.0001 <0.0001 <0.0001 <0.0001 <0.0001
9 Dentobac Creamy Snuff <0.0001<0.0001 <0.0001 <0.0001 <0.0001 <0.0001
10 Lime Mix - Miraj <0.0001<0.0001 <0.0001 <0.0001 <0.0001 <0.0001
11 Shahin Mishri 1174.81 1120.94 <0.0001 <0.0001 <0.0001 <0.0001
12 Dabur Red Tooth Powder 6250.61 6270.26 5870.08 5774.16 <0.0001 <0.0001
13 Baidhyanath Red tooth powder 316.75 322.69 <0.0001 <0.0001 <0.0001 <0.0001
14 Gai Chhap Zarda <0.0001<0.0001 <0.0001 <0.0001 <0.0001 <0.0001
15 Raja Khaini 269.45 262.56 <0.0001 <0.0001 <0.0001 <0.0001
16 Gutkha Tulsi Mix <0.0001<0.0001 <0.0001 <0.0001 <0.0001 <0.0001
17 IPCO Creamy Snuff <0.0001<0.0001 <0.0001 <0.0001 <0.0001 <0.0001
18 Kuber Gutkha <0.0001<0.0001 <0.0001 <0.0001 <0.0001 <0.0001
19 Vimal Gutkha <0.0001<0.0001 382.5 370.69 <0.0001 <0.0001
20 Tuiber Tobacco 2399.72 2321.42 <0.0001 <0.0001 <0.0001 <0.0001

44
7o. Determination of Moisture Content

Accurately weigh and record the weight of an appropriately labeled, dry 20 ml glass
vial with airtight cap.
Transfer the tobacco (2g) into the pre-weighed 20 ml glass vial with airtight cap.
Accurately weigh and record the total weight of the capped vial with tobacco.
Place the samples in the oven at 110ºc with the caps loosened to allow the moisture to
be driven off from the tobacco.
After a minimum of 24 hours, remove the vials and tighten the caps immediately to
prevent re-absorption of moisture from the air. Keep the samples for 2 hours in a
desiccator for cooling.
Accurately weigh and record the total weight of the capped glass vial with tobacco
after heating and calculate the % Moisture content.
% Moisture content = weight of tobacco and glass vial – weight of tobacco and glass vial X 100
(Weight before heating) (Weight after heating) 2

[Link] Product Name Moisture Content

on percentage basis
1 Sanket 999 Jarda + Sanket Supari 7.72
Moolchand Superb Jarda +
2 Moolchand Supari 7.47
3 Shimla Jarda+ Shimla Supari Mix 5.03
4 Goa 1000 Zarda + Goa Supari 6.53
5 Gutkha Pan Parag 4.99
6 Gutkha Manikchand 4.86
7 Click Eucalyptus 16.87
8 Baba Zarda 120 11.25
9 Lime Mix - Miraj Tobacco 24.34
10 Shahin Mishri 10.15
11 Dabur Red Tooth Powder 5.74
12 Baidhyanath Red Tooth Powder 5.24
13 Gai Chhap Zarda 11.33
14 Raja Khaini 24.52
15 Gutkha Tulsi Mix 5.29
16 Kuber Gutkha 5.64
17 Vimal Gutkha 7.97

45
8. Conclusions, recommendations and limitations

Substantive quantities of nitrosamines, BaP and heavy metals exist in most smokeless
tobacco products. Nicotine contents are very high in some and lower in others.
It is possible that those with lower nicotine content are targeted towards children; as
“starter” products. It is alarming that Red Tooth powders which are not marketed as
tobacco products also contain substantive quantities of nicotine.

These findings underscore the need for intensive efforts to prevent men women and
children from using any smokeless tobacco product.

It is recognized that the currently marketed tobacco products have not been subjected
to adequate regulation. All smokeless tobacco products should be subjected to review
based on procedures applicable to other consumer products intended for human
consumption. The incorporation of non-tobacco ingredients into smokeless tobacco
products should also be regulated, for their potential for harm independently or by
interaction with tobacco. Health warnings and labelling should reflect the known
adverse health effects of the smokeless tobacco product.

The findings in this report are subject to at least two limitations. First, the analysis did
not use a sales-weighted or representative sample of all brands or manufacturers; the
products tested were 20 leading products. Second, the findings for any specific brand
could have been affected by factors unique to the sample delivered to each city
surveyed, such as the retailers' duration and conditions of storage (e.g., humidity and
temperature) and manufacturing dates.

46
Bibliography

1. World Health Organization. Tobacco or Health, A Global Status Report. Geneva,

WHO, 1997.

2. Centers for Disease Control and Prevention, Office on Smoking and Health,
Smokeless Tobacco Fact Sheets, 2002.

3. World Health Organization: Smokeless Tobacco Control, Technical Report Series,

773. WHO, Geneva 1988.

4. US Department of Health & Human Services: "The Health Consequences of Using

Smokeless Tobacco". A report of the Surgeon General, Washington 1986.

5. Department of Health and Human Services. Reducing Tobacco Use: A Report of the
Surgeon General. Atlanta: U.S. Department of Health and Human Services, Centers for
Disease Control and Prevention, 2000.

6. Centers for Disease Control and Prevention. Determination of nicotine, pH, and
moisture content of six U.S. commercial moist snuff products — Florida, January–
February 1999. MMWR 1999; 48:398-401.

7. International Agency for Research on Cancer: Tobacco Habits Other Than Smoking:
betel, quid and areca-nut chewing; and some related nitrosamines. Vol. 37. Lyon, IARC
l985.

8. Hatsukami, D., Nelson, R., Jensen, J.: Smokeless Tobacco: Current Status and Future
Directions. British Journal of Addiction, Vol. 86 No. 5, 1991.

9. Huhtasaari F, Lundberg V, Eliasson M, Janlert U, Asplund K. Smokeless tobacco as a

possible risk factor for myocardial infarction: a population-based study in middle-aged
men. Journal of the American College of Cardiology, 1999, 34: 1784-1790.

10. Gupta PC, Bhonsle RB, Mehta FS, Pindborg JJ. Mortality experience in relation to
tobacco chewing and smoking habits from a 10-year follow-up study in Ernakulam
District, Kerala. International Journal of Epidemiology, 1984, 13: 184-187.

11. Gupta PC, Mehta HC. Cohort study of all-cause mortality among tobacco users in
Mumbai, India. Bulletin of the World Health Organization, 2000, 78: 877-883.

12. West, R., Krafona, K.: Oral Tobacco: Prevalence, Health Risks, Dependence
Potential and Public Policy. British Journal of Addiction, Vol. 85 No. 9, 1990.

13. Bhonsle RB, Murti PR, Daftary DK, et al. Regional variations in oral submucous
fibrosis in India. Community Dent Oral Epidemiol 15:225-229, 1987.
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Dental Research, 1997, 11: 330-5.

14. Tomar SL, Henningfield JE. Review of the evidence that pH is a determinant of
nicotine dosage from oral use of smokeless tobacco. Tobacco Control, 1997, 6: 219-225.

47
15. Brunnemann KD, Prokopczyk B, Djordjevic MV, Hoffmann D. Formation and
analysis of tobacco-specific N-nitrosamines. Critical Reviews in Toxicology, 1996, 26:
121-137.

16. MDPH - 08/21/01 - Massachusetts Department of Public Health Issues Challenge to

US Snuff Makers and Warns Consumers of High Cancer Risk Tied to Smokeless
Tobacco. ([Link]

17. Food and Drug Administration. 21 CFR Part 801, et al. Regulations restricting the
sale and distribution of cigarettes and smokeless tobacco to protect children and
adolescents; final rule. Federal Register, 61 (168), 44396-45318, 1996.

18. Gupta PC, Ray SR. Tobacco and youth in the South East Asia Region. Indian
Journal of Cancer, 2002, 39: 3-34.

19. Djordjevic MV, Fan J, Bush LP, Brunnemann KD, Hoffmann [Link] of storage
conditions on levels of tobacco-specific N-nitrosamines and N-nitrosamino acids in U.S.
moist snuff. Journal of Agricultural and Food Chemistry, 1993, 41: 1790-1794.

20. Agrawal P, Chansoriya M, Kaul KK. Effect of tobacco chewing by mothers on

placental morphology. Indian Pediatrics, 1983, 20: 561-565.

21. Krishna K. Tobacco chewing in pregnancy. British Journal of Obstetrics and

Gynaecology, 1978, 85: 726-768.

22. Krishnamurthy S, Joshi S. Gender differences and low birth weight with maternal
smokeless tobacco use in pregnancy. Journal of Tropical Pediatrics, 1993, 39(4): 253-
254.

23. Verma RC, Chansoriya M, Kaul KK. Effect of tobacco chewing by mothers on fetal
outcome. Indian Pediatrics, 1983, 20: 105-111.

24. Mehta AC, Shukla S. Tobacco and pregnancy. Journal of Obstetrics and
Gynaecology of India, 1990, 40: 156-160.

25. The Global Youth Tobacco Survey Collaborative Group. Tobacco use among
youth: a cross country comparison. Tobacco Control, 2002, 11: [Link]-1:

26. PC Gupta, PR Murti, RB Bhonsle. Epidemiology of cancer by tobacco products and

the significance of TSNA. Critical reviews in toxicology 1996; 26(2): 183-98.

48
 Annex-1: Details of smokeless tobacco brands tested:

place of
No PRODUCT NAME BATCH Date of purchase purchase

1 Sanket 999 Jarda + J 22-Oct 2003 Mumbai

Sanket No 1 Supari Mix J

2 Moolchand Superb Jarda+ 004, 006 22-Oct 2003 Mumbai

Moolchand Superb Supari batch 006

3 Shimla Jarda+ MSY 22-Oct 2003 Mumbai

Shimla Supari Mix MSY

4 Goa 1000 zarda+ A-9 22-Oct 2003 Mumbai

Goa supari A-9

5 Gutka Pan parag 8-Nov 2003 Gujarat

6 Gutka Manikchand B 8-Nov 2003 Gujarat

7 Click Eucalyptus 351090 19-Oct 2003 Bangalore

8 Baba zarda 120 Barcode no.89018370406068 4-Nov 2003 Mumbai

9 Dentobac Barcode no>8904027801041 22-Oct 2003 Mumbai

10 Lime mix - Miraj tobacco AA9 22-Oct 2003 Mumbai

11 Shahin mishri RC [Link]/CH-24/2/95 22-Oct 2003 Mumbai

12 Dabur red tooth powder 2432 22-Oct 2003 Mumbai

13 Baidhyanath red tooth powder 1423-1416-1552 22-Oct 2003 Mumbai

14 Gay chap tobacco 6 22-Oct 2003 Mumbai

15 Raja khaini Regn. No. ADUPG810FXM002 22-Oct 2003 Mumbai

16 Gutka Tulsi mix 08CI23 22-Oct 2003 Gujarat

49
17 IPCO creamy snuff 8-Nov 2003 Gujarat
Regn.
No.AAEFA6022AXM001
JULY02
Regn.
No.AAEFA6022AXM001
FEB03
Regn.
No.AAEFA6022AXM001
JUNE03

18 Kuber gutka 0.8 8-Nov 2003 Gujarat

19 Vimal gutka 8-Nov 2003 Gujarat

20 Tuiber tobacco water

50
Annex-2: Techniques used for the Measurements of chemical constituents of
Smokeless Tobacco

Name of Make of Detectors Column Tobacco Chemicals

Instruments Instruments
Gas Toshnival Flame Capillary Glycerol,
Chromatograph Instrument Ionization Column (BP- Propylene Glycol,
Dual Port (India) ltd. Detector 5) Triethylene Glycol.
Model no. (FID) 50 m X 0.22 Benzo(a)pyrene
8610. I.D. X 1.0 Triacetin
um. Sodium Propionate

Packed Alkaloids:
Column (3% Nicotine
ov-101)
Thermal Capillary Alkaloids:
Ionization Column (BP- Nornicotine,
Detector 5) Anabasine,
(TID) 50 m X 0.22 Myosmine,
I.D. X 1.0 Anatabine.
um.
Electro Chemical EG&G Techniques: Heavy Metals:
Trace Analyser. Instruments Differential Nickel,
Polaragraphic & Princeton Pulse Anodic Lead,
Voltammetric Applied Stripping Cadmium,
Techniques. Research. Voltametric Chromium,
Model no. (dpasv) Arsenic,
303A/394/305 Differential Selenium.
Pulse
Cathodic
Stripping
Voltametric
(dpcsv)
Differential
Pulse square
Voltametric
(dpsv)
Differential Electrolyte: Nitrosamines:
Pulse 0.1 % Hcl N-nitrosonornicotine,
Polorography 4-(N-
(DPP) nitrosomethylamino)-
1-(3-pyridyl)-1-
butanone, N-
nitrosoanatabine,
N-nitrosoanabasine.

51
High Performance Hitachi, Liquid – C-18 column Sorbic Acid,
Liquid Merck : Liquid Phase Eugenol [2-Methoxy-
Chromatograph L-6220/L- 4-(2-propenyl)-
7400/L-7350 phenol]
Double beam Toshnival Wavelength: Ammonia
Ultra-Violet Instrument 420nm Nitrate.
Spectrophotometer (India) ltd. 550nm
Spectrascan
Model no:
2600
Digital Mercury Electronics Mercury
Analyser with Corporate of
Vapour India.
Generating system Model no:
MA 5840
MS 500.
pH meter Testronix pH value
Model no:
511
Titrimeteric Magnesium
Method Carbonate (MgCo3)

52
Annex-3: Calibration curves used for estimations:

53
54
55
56
57
58
 59

V i e w p u b l i c a t i o n s t a t s