investigation of some biological and … · investigation of some biological and biotoxicological...

UNIVERSITY OF MEDICINE AND PHARMACY

„GRIGORE T. POPA” IASI

FACULTY OF PHARMACY

INVESTIGATION OF SOME BIOLOGICAL AND

BIOTOXICOLOGICAL PARAMETERS IN THE CHRONIC

EXPOSURE TO TOBACCO SMOKE

PhD THESIS SUMMARY

Scientific Coordinator

Prof. Dr. Elena BUTNARU

PhD Student Doina ŞPAIUC

IAŞI 2013

PhD thesis contains:

• 179 pages (Current stage of knowledge: 53 pages, personal

contributions: 110 pages)

• 87 figures

• 80 tables

• 200 references

• Approval Study

• Consent

• Three scientific articles -1 B+; 1 ISI; 1C

This summary keeps the PhD thesis' way of numbering for

contents, tables, figures and references.

Keywords: nicotine, cotinine, haematological parameters,

biochemical parameters, tobacco smoke.

Doctoral thesis abstract

1

TABLE OF CONTENTS

Pag.

1. Introduction. Target objectives 1

A. CURRENT STAGE OF KNOWLEDGE

2. TOBACCO. GENERAL AND HISTORICAL

CONSIDERATION

4

2.1. The discovery of tobacco and its properties. Tobacco

penetration in Europe

2.2. Tobacco penetration in Romania

4

5

3. COMPOSITION AND HARMFULNESS OF TOBACCO

SMOKE

5

3.1. Tobacco products and consumption mode 9

3.2. Nicotine. Chemical structure. Physico-chemical properties 10

3.2.1. Chemical structure 10

3.2.2. Physical properties 10

3.2.3. Chemical properties 11

3.2.4. Nicotine biosynthesis 12

3.2.5. Pharmaco-toxicological profile of nicotine 13

3.2.5.1. Toxicokinetic particularities 13

3.2.5.2. Toxicodynamic particularities 20

3.2.5.3. Toxic effects of nicotine 21

3.2.6. Acute intoxication with nicotine. Treatment 22

3.2.7. Chronic intoxication with nicotine. Tabagism 22

3.3. Carbon monoxide 26

3.4. Carcinogens 26

3.5. Additive substances 27

3.6. Tar

28

4. SMOKING EFFECTS ON HEALTH 29

4.1. Respiratory diseases 29

4.2. Cardiovascular diseases 30

4.3. Digestive diseases 30

4.4. Neoplasia with different localizations 30

4.5. Smoking and neuropsychiatric diseases 31

4.6. Smoking effects on drug action 32

4.7. Passive smoking 35


2

4.8. Withdrawal syndrome. Treatment 36

4.8.1. Treatment of the nicotine withdrawal 37

4.8.2. Replacement therapy of the nicotine from cigarettes 37

4.8.3. Drugs to combat nicotine withdrawal. Non-nicotine

therapy

41

4.8.4. Complementary methods to quit smoking 43

4.9. Fight against tobacco. The pharmacist's role in the fight

against smoking

44

5. COTININE, BIOMARKER OF THE INTOXICATION

WITH NICOTINE

46

5.1. Cotinine. Chemical structure. Physico-chemical properties.

Synthesis.

46

5.1.1. Physical properties 46

5.1.2. Chemical properties 46

5.1.3. Cotinine synthesis 47

5.2. Toxicokinetic particularities 48

5.2.1. Cotinine metabolism 49

5.2.2. Renal excretion of cotinine 49

5.3. Toxicodynamic particularities 50

5.4. Cotinine, biomarker of the intoxication with nicotine 50

5.4.1. Nicotine from food, source of cotinine

50

6. METHODS OF EXTRACTION AND ANALYSIS OF

NICOTINE AND COTININE

52

6.1. Methods of nicotine extraction from tobacco 52

6.2. Methods of nicotine and cotinine extraction from biological

fluids

53

6.3. Chromatographic and spectrophotometric methods used to

determine the nicotine and the cotinine

54

6.3.1. TLC, analysis method of nicotine 54

6.3.2. Methods for the determination of nicotine and cotinine

through high performance liquid chromatography (high-

performance liquid chromatography, HPLC).

54

6.3.2.1. HPLC 55

6.3.2.2. HPLC - mass spectrometry 55

6.3.3. Gas-chromatographic method (GC) 55

6.3.3.1. Gas chromatography 56

6.3.3.2. Gas chromatography-mass spectrometry 56

6.4. UV-VIS spectrophotometric methods 57


3

B. PERSONAL CONTRIBUTION

7. THE VARIATION OF THE BIOLOGICAL PARAMETERS IN

SMOKING PATIENTS FROM THE NEUROPSYCHIATRY

CLINIC WITHIN THE MILITARY CLINICAL EMERGENCY

HOSPITAL “DR. IACOB CZIHAC” IASI DURING 2006-2007

58

7.1. Introduction 58

7.2. Method 59

7.3. Results 59

7.4. Discussions 65

7.5. Conclusions

66

8. THE ANALYSIS OF THE BIOTOXICOLOGICAL

PARAMETERS: NICOTINE AND COTININE FROM

BIOLOGICAL FLUIDS

67

8.1. Contributions to the quantitative determination of nicotine in

biological fluids through UV spectrophotometry

67

8.1.1. Introduction 67

8.1.2. Materials and methods 68

8.1.2.1. Determination of nicotine in urine samples with internal

standard

73

8.1.2.2. Determination of nicotine in serum samples with internal

standard

74

8.1.2.3. Determination of nicotine in biological samples from

smokers

77

8.1.3. Results 78

8.1.4. Discussions 78

8.1.5. Conclusions 79

8.2. Development and validation of a method for simultaneous

quantitative determination of nicotine and cotinine in biological

samples by gas chromatography coupled with mass spectrometry

80


8.2.2. Materials and methods 80

8.2.3. Results from the validation of the method for determination

of nicotine and cotinine in biological fluids by GC-MS

81

8.2.3.1. Specificity 86

8.2.3.2. Linearity 87

8.2.3.3. The limit of detection (LD) and the limit of quantification

(LQ)

93

8.2.3.4. Accuracy 93

8.2.3.5. Accuracy (Exactness) 96

8.2.4. Conclusions regarding the determination of nicotine and 96


4

cotinine in serum by GC-MS

8.3. Application of the GC-MS method for quantitative determination

of nicotine and cotinine in biological fluids

98


8.3.2. Materials and method 99

8.3.3. Results 100


8.3.5. Statistical analysis of nicotine and cotinine concentrations

using ANOVA test

108

8.3.6. Results obtained by using similar methods mentioned in the

specific literature

109


8.4. Study regarding the influence of tobacco smoke exposure on

biological parameters

113


8.4.2. Method 114

8.4.3. Results 114


8.4.5. Conclusions

118

9. CHEMICAL-TOXICOLOGICAL STUDIES TO DETERMINE

THE TOBACCO NICOTINE BY GAS CHROMATOGRAPHY

COUPLED WITH MASS SPECTROMETRY

119

9.1. Development and validation of a method for determination of

nicotine in tobacco by gas chromatography coupled with mass

spectrometry (GC-MS)

119


9.1.2. Material and method 119

9.1.3. Results from validation of the method of determination of

nicotine in tobacco by GC-MS

120

9.1.3.1. Method development 120

9.1.4. Method Validation 123

9.1.4.1. Linearity 123

9.1.4.2. The limit of detection and the limit of quantification 124

9.1.4.3. Accuracy 124

9.1.4.4. Accuracy (Exactness) 126


9. 2. Application of the method of determination of nicotine in

tobacco samples by GC-MS

128


9.2.2. Materials and method 128

9.2.3. Results 128

9.2.3.1. Determination of nicotine content in cigarettes 128


5

9.2.3.2. Determination of nicotine content in cigars and pipe

tobacco

131

9.2.3.3. Determination of the nicotine content in tobacco leaves 131

9.2.4. Discussion 132

9.2.5. Conclusions

135

10. STATISTICAL INTERPRETATION OF THE DATA FROM

THE PROGRAM “STOP SMOKING” DEVELOPED BY THE

PNEUMOLOGY HOSPITAL IASI, JANUARY 2009 - FEBRUARY

2012

136

10.1. Introduction 136

10.2. Method 137

10.2.1. Presentation of the statistical parameters from the program

“Stop Smoking” conducted by the Pneumology Clinical Hospital,

during January 2009 - February 2012

137

10.2.2. The target objective: determination of the factors that

influence the treatment for nicotine abstinence.

138

10.3. Results 141

10.3.1. Centralization of the statistical data from the program “Stop

smoking” during January 2009 - February 2012.

141

10.3.2. The distribution of the patients from the program “Stop

Smoking” by groups (group A and group B); the structure of the

groups A and B according to: sex (M/F), age (years), marital status,

children, health of patients.

141

10.3.3. Data regarding the patients' motivation to quit smoking and

the evaluation of the level of nicotine dependence.

146

10.3.4. Recommendations for the antinicotine treatment, adverse

reactions to the recommended treatment, final status of the patients.

150

10.3.5. Values of the carbon monoxide concentrations determined

during the initial and final visits of the patients, as a toxicological

biomarker of exposure to tobacco smoke.

157

10.4. Discussions regarding the statistical interpretation of the data

from the program “Stop Smoking”

158

10.5. Conclusions 167

11. CONCLUSIONS 168

12. REFERENCES 172

Annex 1

Study approval

Consent

List of the works that were published from the doctoral thesis


6


7

INTRODUCTION. TARGET OBJECTIVES

Tabagism is a widespread phenomenon due to its multi-

factorial character, affecting all age groups, predominantly the age

category between 14 and 35 years; it constitutes a major public

health issue because it has significant repercussions on the

individual and the domestic levels. The tobacco intoxication is the

most common of all known drug addictions.

The World Health Organization states in the report “Women

and the tobacco epidemic - Challenges for the 21st Century”, that

there are around 4 million deaths worldwide, every year, due to

smoking. By 2020, it is estimated that this number will increase

up to 8.4 million deaths, 70% of these will appear in the

developing countries. The same WHO report shows that at a

global level, 12% of all women smoke, compared to 48% of men,

but the percentage of women who smoke is increasing.

Smoking registers the second level of the prevalence of drug

use related to the Romanian population; 62.1% of the respondents

had smoked at least once throughout their lifetime, 31.3% of them

declared that they smoked daily. The study on the statistical

relation between smoking and age, revealed that the segment of

people between 25 and 34 years old, show the highest percentage

for the three indicators of prevalence: 70.5% for lifelong use, 46%

both for recent and current use.

The tobacco addiction (tabagism), according to the criteria

adopted by the World Health Organization in the International

Classification of Diseases, fits into the category: “Mental and

behavioral disorders due to the tobacco consumption” and it is

encoded with the disease code F.17.

Nicotine, the main alkaloid in the tobacco leaves, produces

addiction 6 to 8 times higher than alcohol, as high as heroin, 95 to

100% of smokers being addicted. The nicotine penetrates the body

via the respiratory system, cutaneous system, through the mucosal

and digestive systems; absorption is extremely fast. It is

biotransformed in hepatocytes (80-90%) through oxidation at the

level of the pyrrolidine nucleus to cotinine, the major metabolite


8

with a toxic action.

The cotinine is a toxicological biomarker in the intoxication

with nicotine, in the case of smokers of cigarettes, of smokers of

other tobacco products and also in the case of persons exposed to

tobacco smoke in the environment.

In the treatment of tobacco use, the necessity of

therapeutical intervention is asserted, following a well-established

plan coordinating and fitting the treatment measures to the

particularities of every individual.

The objectives of the thesis

The purpose of this thesis is to perform a complex

epidemiological, clinical and experimental study, that respond to

the topical questions related to the study of biological and

biotoxicological parameters in the case of chronic exposure to

tobacco smoke:

Variation of the biological parameters (hematological and

biochemical) in smoking patients

Study of the biotoxicological parameters: nicotine and

cotinine in biological fluids in smoking patients. In this

study we proposed:

- to develop a method of dosing the nicotine in biological fluids

by UV spectrophotometry

- to develop and validate a method for simultaneous determination

of nicotine and cotinine in biological fluids by gas

chromatography coupled with mass spectrometry (GC-MS)

- to apply the GC-MS validated method to the quantitative

determination of nicotine and cotinine in biological fluids in

smoking patients and the study regarding the influence of

exposure to tobacco smoke on biological parameters.

The development and the validation of a method for

quantitative determination of the nicotine content in

tobacco by GC-MS and the application of the method to

determine the nicotine content in different samples of

tobacco

The statistical analysis of the data from the program “Stop

Smoking”, has pursued:


9

- the identification of the level of nicotine addiction in patients

enrolled in the program

- information regarding the patients' motivation to quit smoking

- emphasizing the decisive role of treatment and medical

counseling to succeed in giving up smoking

- the rate of the patients who become abstinent after the

administered treatment.

The paper is structured in two parts: “The current state of

knowledge” (chapters 1-6) and “Personal contributions”

(chapters 7-10).

A. The current state of knowledge includes general and

historical considerations regarding tobacco and its properties,

information on the composition and harmfulness of tobacco

smoke, the toxicokinetic and toxicodynamic properties of the

nicotine and cotinine (marker of the intoxication with nicotine),

the disorders caused by the use of tobacco products, methods of

therapy for the nicotinic withdrawal, and methods of extraction

and analysis of nicotine and cotinine, according to the literature.


10

B. PERSONAL CONTRIBUTIONS

7. THE VARIATION OF THE BIOLOGICAL PARAMETERS IN

SMOKING PATIENTS FROM THE NEUROPSYCHIATRY CLINIC

WITHIN THE MILITARY CLINICAL EMERGENCY HOSPITAL “DR.

IACOB CZIHAC” IASI DURING 2006-2007

Introduction

The study aimed to establish possible correlations between the

variation of biological parameters in smoking subjects as opposed

to non-smoking subjects. The evaluation of the results obtained by

comparing the two groups (smokers / non-smokers) refered to: the

average values, the results of the ANOVA test and the deviations

of the values of these biological parameters as opposed to the

biological interval of reference.

Method

We reviewed retrospectively the charts of the patients

hospitalized in the Neuropsychiatry Clinic from the Military

Clinical Emergency Hospital “Dr. Jacob Czihac” Iasi, during

2006-2007.

The information about the patients was noted, including:

sex, age, occupation, diagnosis, duration of hospitalization,

smoker / non-smoker / alcohol consumer, administered treatment,

hematological parameters (ESR, WBC, RBC) and biochemical

parameters (GOT, GPT, GGT, glycemia, triglycerides and

cholesterol). The data of the study were introduced into an Excel

database and analyzed using the tools menu for data analysis

(ANOVA tests and descriptive statistics) to establish whether

there were statistically significant differences between the

hematological and the biochemical parameters of the two groups

composed by smokers and respectively, non-smokers.


11

Results

During 2006-2007, in the Clinic of Neuropsychiatry of the

Clinical Emergency Hospital Military “Dr. James Czihac”, Iasi,

89 patients were hospitalized, out of which 59 were smokers

(66.30%) and 30 were non-smokers (33.70%). Both groups were

formed of mostly male persons: 93.22% and respectively 73.34%.

When they were checked in, the patients were performed

hematological tests: ESR, WBC, RBC (ABX Pentra 60 C+

analyzer), and biochemical tests: GOT, GPT, GCT, glycemia and

cholesterol (VITALOB Flexor E analyzer). The structure of the

set of tests varied according to the diagnosis. The statistical results

for the hematological and biochemical parameters are summarized

in Tables 7.III and 7.IV.

Table 7.III. Statistical analysis of the hematological parameters

Statistical data ESR WBC RBC

The biological interval of reference

(IBR) 5-15 mm/1h

4.0-10.0x103

/mmc

3.80-5.60x106

/mmc

Smokers (59 subjects) (F)

Number of cases 56 52 52

Minimum 2 6.30 3.76

Maximum 42 9.30 5.2

Average 12.38 7.28 4.51

The average of absolute deviation as

opposed to the arithmetic average ±4.71 ±0.45 ±0.38

Standard deviation 6.64 0.62 0.44

Values IBR 13 0 0

Values IBR 3 0 1

Values in IBR 40 (71.43%) 52 (100%) 51 (98.08%)

Non-smokers (30 subjects) (NF)


Minimum 2.00 5.2 3.9

Maximum 42.0 11.5 5.1

Average 13.77 7.47 4.37

The average of absolute deviation as

opposed to the arithmetic average ±7.22 ±0.86 ±0.30


Values IBR 8 2 0

Values IBR 4 0 0

Values in IBR 18 (69%) 26 (92.86%) 28 (100%)

Total group (89 subjects)


Minimum 2.0 5.2 3.76

Maximum 42.0 11.5 5.2

Average 12.86 7.34 4.46

The average of absolute deviation as ±5.67 ±0.60 ±0.36


12

opposed to the arithmetic average


Values IBR 21 2 0

Values IBR 7 0 1

Values in IBR 58 (67.44%) 78 (97.50%) 79 (89.75%)

Table 7.IV. Statistical analysis of the biochemical parameters

Statistical data GOT GPT GGT Glycemia Cholesterol

The biological interval

of reference (IBR)

2.0 –

48.0 U.I.

2.0 –

40.0 U.I. 5-36 U.I.

74.0 – 110.0

mg/dL

0.0 – 200.0

mg/dL

Smokers (59 subjects) (F)

Number of cases 46.00 46.00 18.00 41.00 41.00

Minimum 3.60 2.60 4.61 52.00 109.00

Maximum 83.00 97.00 402.00 152.00 329.00

Average 30.23 38.51 97.96 94.85 216.71

The average of absolute

deviation as opposed to

the arithmetic average

12.12 15.41 39.26 10.89 34.53

Standard deviation 16.35 20.94 81.93 16.52 46.02

Values IBR 8 13 16 5 26

Values IBR 0 0 0 1 0

Values in IBR 38

(82.61)

33

(71.74)

2

(11.11%) 35 (85.37) 15 (36.59)

Non-smokers (30 subjects) (NF)

Number of cases 25.00 24.00 7.00 19.00 19.00

Minimum 9.00 2.70 35.00 76.00 142.00

Maximum 71.00 112.00 156.00 102.00 256.00

Average 28.56 33.86 88.14 90.32 196.32




15.12 16.28 25.27 4.16 24.44




Values in IBR 21

(84.00)

19

(79.17)

1

(14.29%) 18 (94.74) 10 (52.63)

Total group (89 subjects)

Number of cases 71.00 70.00 25.00 60.00 60.00

Minimum 3.60 2.60 4.61 52.00 109.00

Maximum 83.00 112.00 402.00 152.00 329.00

Average 29.65 36.92 95.21 93.42 210.25




13.22 15.67 35.34 8.93 32.18


Values IBR 12 18 22 6 35


Values in IBR 59

(83.10)

52

(74.29)

3

(12.00%) 53 (88.33) 25 (41.67)


13

Discussions

According to the information presented in Table 7.III, we

note that the average values of the ESR for the group of smokers

(12.38 mm/1h) is slightly lower than for the group of non-smokers

(13.77 mm/1h) and than those for the total group (12.86 mm/1h).

For the erythrocytes (RBC), we obtained an average value which

was similar for all the three groups (F = 4.51, NF = 4.37 and the

total group = 4.46 x 106/ mmc) and for the leukocyte (WBC), the

average values were also similar (F = 7.42, NF = 7.47 and for the

total group = 7.37 x103/mmc).

According to the information presented in Table 7. IV, we

notice that the average values of GOT and GPT in the group of

smokers and non-smokers fall within to the biological interval of

reference; 7 subjects from the smokers’ group, were found

increased values for both parameters, and the TGP / TGO report

was supra-unitary which indicates liver damage.

The GGT test was performed on 18 smokers (30.5%) from

the total of 59 subjects, registering 16 values over the normal

values (5-36 IU), out of which six values over 100 I.U. The test of

a 49 year old patient, who was an alcohol consumer, registered the

value of 402 I.U.

The glycemia determined to smokers ranged between 52 and

152 mg / dL.

The total cholesterol was determined for 41 smokers,

registering a minimum value of 109.0 mg / dL and a maximum

value of 329.0 mg / dL, the average value being 216.71 mg / dL.

Values over the normal range (0.0 to 200 mg / dL) were registered

in the case of 28 smokers (68.3%).

Conclusions

• The data of the statistical study hasn’t highlighted

correlations between the hematological parameters (ESR, WBC,

RBC) and smoking, the average values either for the non-smokers

or for the smokers are significantly close.

• The biochemical tests (SGOT, SGPT, GGT, glycemia) for

the subjects of this study haven’t showed significant statistical


14

differences. The cholesterol level has recorded higher average

values for the smokers as compared to non-smokers.

• The statistical study conducted on a relatively small group,

for a period of only one year, showed no changes of the biological

parameters for the smokers as compared to the non-smokers.


15

8.THE ANALYSIS OF THE BIOTOXICOLOGICAL PARAMETERS:

NICOTINE AND COTININE FROM BIOLOGICAL FLUIDS

Nicotine and cotinine are the main biotoxicological

parameters that determine the acute and chronic intoxications

(tabagism) to persons who smoke or to those who use different

other treatments to substitute nicotine (chewing gum, patch, nasal

spray, oral inhaler, sublingual tablets).

8.1. Contributions to quantitative determination of nicotine in

biological fluids by UV spectrophotometry

Introduction

This study aimed to establish the methodology of

spectrophotometric UV determination of the nicotine. In the

toxicological research of nicotine, an important step is represented

by the isolation from the biological material (urine, serum).

Materials and method

Chemicals and reagents

• Nicotine (Merck) - density 1.009 g/mL

• Nicotine standard solution I: the nicotine was dissolved in

distilled water at 60°C. The stock standard solution = 2.176

mg/mL.

• Standard solution II:

a. the acid solution (50 g/mL): 1.15 mL of the nicotine

standard solution I is diluted with sulfuric acid 0.1 N to

volumetric flask of 50 mL.

b. the alkaline solution (50 g/mL): 1.15 mL of the nicotine

standard solution I was diluted with sodium hydroxide 0.1 N to

volumetric flask of 50 mL.

• Chloroform P.A. - Loch-Ner

• Absolute methanol - Chimopar S.A.

• Diethyl ether - Sigma Reidel - de Haën

• H2SO4 (Merck)

• NaOH Chemical Company


16

Equipment and glassware

• analytical balance SCALTEC, precision 0.0001 g

• water bath, Memmert WBU 45

• magnetic stirrer VEPL

• centrifuge

• separating funnels, filter funnels, pipettes, flasks,

cylinders, porcelain capsules, glass rods.

• spectrometer UV/VIS, T90/T90 type, Zuzi brand

Experimental data were evaluated using the software of the

spectrophotometer UVVIN 5.0, on Microsoft Windows

98/2000/XP program.

Procedure

Initially we recorded the absorption spectra of the nicotine

in the acid and alkaline medium, in the range of 200-300 nm; the

background correction of the apparatus/equipment was performed

with distilled water (Figures 8.2, 8.3).

Fig. 8.2. Spectrum of absorption of nicotine (50 g / mL) in acid medium

Note: The base line - H2SO4 0.1 N as opposed to distilled water

The acidic solution - 1.15 mL of the nicotine standard solution (2.176 mg / mL in

distilled water) + H2SO4 0.1 N up to 50 mL, reading as opposed to H2SO4 0.1 N

Peak: =260 nm, Abs. = 1.7641

0

4

1

2

3

2 00 4 003 00

A b s

W a v e le n g t h [n m ]


17

Fig. 8.3. Spectrum of absorbtion of nicotine (50 g/mL)

in an alkaline medium

Note: The base line - NaOH 0.1 N as opposed to distilled water

The alkaline solution - 1.15 mL of the nicotine standard solution (2.176 mg / mL in

distilled water) + NaOH 0.1 N up to 50 mL, reading as opposed to NaOH 0.1 N

Peak: =261 nm, Abs.=1.1617 Taking into account the allure and peak positions, we chose

the maximum of absorption to be = 260 nm for the quantitative

analysis of the nicotine. These spectra were recorded on

spectrophotometer UV / VIS Jasco V-550.

The calibration curve was performed between 10 and 50

g/5 mL. The extinctions were read at = 260 nm as opposed to

H2SO4 0.1 N

The calibration curve complies with the Bouguer Beer-

Lambert's law for the established field of concentrations (Figure

8.4).

0

2 .3

1

2

2 00 4 003 00

A b s

W a v e le n g t h [n m ]


18

Fig. 8.4. Calibration curve for nicotine

The nicotine was determined in the urine and serum

samples with internal standard (nicotine). The extraction yield of

the nicotine was better for the samples extracted in the alkaline

medium both in chloroform and in diethyl ether.

Determination of the nicotine in biological samples from

smokers

Nicotine was determined in 10 serum samples and 5 urine

samples from smoking subjects hospitalized in the Emergency

Military Hospital “Dr. James Czihac”, Iaşi. The separation of the

nicotine from the biological samples was performed by liquid-

liquid extraction of alkalinized samples (pH 9), using diethyl ether

as extraction solvent. The samples were worked in the same

conditions as the samples with internal standard. Absorbances

were read at spectrophotometer UV-VIS T90/T90 + as opposed to

0.1 N sulfuric acid at = 260 nm.

The results were expressed in g/mL.

In Table 8.VIII we synthetically presentated: the age, sex,

number of cigarettes smoked daily, nicotine concentrations in the


19

serum and urine samples ( g/mL), data regarding the provenance

of the biological samples (the initials of the patients’ name).

Table 8.VIII. The nicotine concentrations ( g / mL) in serum and urine

from smokers

Nr.

crt. N/P Sex Age

Number of

cigarettes

daily

smoked

The nicotine

concentrations

( g/mL)

Serum Urine

1 C.F. F 53 18 60.240 8.6630

2 G.E. F 50 15 25.642 3.7956

3 V.M. F 32 10 29.160 3.2972

4 C.M. F 56 15 41.768 6.0534

5 S.A. F 42 20 71.382 5.1738

6 S.S. M 36 18 26.522 -

7 L.C. M 38 18-20 26.522 -

8 C.D. M 51 25 109. 206 -

9 G.P. M 48 20 82. 524 -

10 H.P. M 43 18 45.872 -

Discussions

The group of patients comprised 10 people, five women

aged between 32 and 56 years old, with a daily consumption

between 15 and 20 cigarettes, and five men aged between 36 and

51 years old, with a daily consumption between 18 and 25

cigarettes. We had also urine samples from the female subjects.

In the case of the group of female subjects, the nicotine

concentrations ranged between 25.6420 and 71.382 g/mL in

blood samples, and between 3.2972 and 8.6630 g/mL in urine

samples. In the case of the group of male subjects, the nicotine

concentrations varied between 26.522 and 109.206 g/mL in

blood samples.

The serum values are high, which means that at the

absorption peaks of the nicotine ( = 260 nm) other substances

from the serum composition are interfering, such as the DNA

protein, with the same UV maximum absorption.


20

In the literature this method was quoted for the

determination of nicotine in different tissues of the Nicotiana

rustica plant, objects belonging to smokers (contact lenses) [130,

131, 132, 133].

Conclusions

In the case of the studies of analytical toxicology performed

by UV absorption spectrophotometry, we developed a method for

the quantitative determination of nicotine in biological samples

(serum, urine).

The UV spectrophotometric method is based on the

determination of nicotine using the absorption band at 260 nm.

In the research phase of the applicability of the

spectrophotometric method for the nicotine determination in

biological samples, the following experimental parameters were

studied and settled :

• nicotine is isolated from biological fluids (serum, urine)

through the liquid-liquid extraction at pH - alkaline (pH = 9);

• the optimum extraction solvent is diethyl ether;

• the linearity range investigated (10-50 g/ 5mL);

• in women, nicotine concentrations ranged between

25.6420 and 71.382 g/mL in serum and in urine between 3.2972

and 8.6630 g/mL;

• in men, the nicotine concentrations varied between 26.522

and 109.206 g/mL in blood samples;

• the nicotine concentrations varied depending on the

number of cigarettes consumed daily;

• the method could also be applied to the determination of

nicotine in cigarettes, in pharmaceutical products with nicotine

(chewing gum, drops, sublingual tablets, nicotine patches, nasal

sprays, or inhalation).


21

8.2. Development and validation of a method of simultaneous

quantitative determination of nicotine and cotinine in

biological samples by gas chromatography coupled with mass

spectrometry (GC-MS)

Introduction

We aimed to establish the experimental conditions for the

quantitative determination of nicotine and cotinine by GC-MS

method that we can subsequently use for the nicotine and cotinine

dosing in biological fluids collected from smokers.

In the development of this method we have started from the

already existent data in the literature and we considered the

physico-chemical properties of nicotine and cotinine [137, 138,

139, 140].

Materials and methods

Reagents

• Nicotine - C 99%, density 1.009 g/mL, Merck Germany

• Cotinine - 98% Sigma - Aldrich – Germany

• Methanol - Sigma - Aldrich – Germany

• Dichloromethane – Promochim

Standards

• Standard of nicotine in methanol: 500 g/mL

• Standard of cotinine in methanol: 500 g/mL

Mixing 0.1 mL methanolic solution of nicotine + 0.1 mL

methanolic solution of cotinine + 4.8 mL serum/urine. The

concentration of nicotine and cotinine is 10 g/mL.

Sample preparation

To 1 mL of serum/urine, we added 0.5 mL of 5%

ammonium sulphate solution and 1 mL of dichloroethane:

methanol (1: 1).

Energic stirring for five minutes at 1000 vibrations/min, we

added 0.5 mL dichloromethane and we stirred again for five

minutes. The mixture was centrifuged at 4000 rotations/min for 10

minutes. The organic phase was separated, evaporated to dryness


22

and it was repeated with 0.5 mL mixture of dichloromethane /

methanol (1: 1).

Apparatus

• Agilent Technologies 7890 gas chromatograph equipped

with Agilent Technologies 7683B auto-injector and mass

spectrometer detector Agilent Technologies 5975 inert MSD;

• mixer Vortex IKA VIBRAX VXR

• centrifugal

Chromatographic conditions

The analyses were performed on a chromatographic

column DB 5 MS (30 m x 0.25 mm, 0.25 µm film thickness) with

mobile phase consisting of helium with a flow rate of 1 mL/min;

the injector’s temperature was 250 ºC, the MSD quadrupole’s

temperature was 150 ºC. The temperature gradient in the column

compartment was initially 100 °C, followed by a ramp of

temperature – 10 °C/min up to 250 °C and immediately, a second

ramp - 20 °C/min up to 280° C and finally the temperature was

kept constant for 3.5 minutes; the analysis lasts for 20 minutes and

the last approximately 8 minutes are required to remove any

volatile components extracted simultaneously with nicotine and

cotinine in serum, as in the conditions described above. The

volume of solution injected was 0.5 µL, at a split ratio of 1/5.

Detection was performed by mass spectrometry in SIM mode

(single ion monitoring) (m/z for nicotine: 84 - basic peak, 133 and

162 - molecular peak; m/z for cotinine: 98 - basic peak, 118 and

176 molecular peak).

The nicotine and cotinine identification was performed by

spectral comparison with the Wiley spectra library for a standard

solution, then by comparing the retention times.

Interpretation of results: Agilent Technologies ChemStation

software.


23

Results achieved at the validation of the method for

determination of nicotine and cotinine in biological fluids by

GC-MS

To validate the method of simultaneous quantitative

determination of nicotine and cotinine in serum and urine by GC-

MS, the following parameters were monitored: specificity/

selectivity, linearity, limit of detection (LOD), limit of

quantification (QL), precision (system precision, method precision

and intermediate precision), accuracy [142, 143, 144, 145].

To determine the specificity of the method for the

simultaneous determination of nicotine and cotinine in serum, a

blank sample was processed in the mentioned conditions. The

sample thus prepared was analyzed by GC-MS in the specified

operating conditions. From the analysis of the obtained

chromatogram it can’t be noticed the apparition of any

chromatographic peak at the values of the retention times

corresponding to the nicotine (6.24 minutes) and cotinine (10.32

minutes) (Figure 8.20).

Fig. 8.20. Chromatograms for a blank sample

containing nicotine and cotinine


24

the linearity of the function of response was studied (the

peak area modification was monitored according to the sample

concentration). The function of response is linear in the studied

range, from 12.18 to 500 ng/mL for nicotine and 3.39 to 500

ng/mL for cotinine.

The equations of the obtained regression lines were

calculated by the method of the smallest squares:

Nicotine: Area = 2153.6 x Concentration (ng/mL) + 185788.3

Cotinine: - for the 1-100 ng/mL

Area = 3821.8 x Concentration (ng /mL) + 46638.4

- for the 100-500 ng/ml

Area = 491.84 x Concentration (ng/mL) + 383712

the linearity of the results was studied, between the

theoretical and the calculated concentration, there is a very good

correlation (Figure 8.23 and Figure 8.25).

Fig. 8.23. The calibration curve for nicotine in

the concentrations range of 1-500 ng/mL


25

Fig. 8.25. The calibration curve for cotinine in

the concentrations range of 100-500 ng / mL

the limits of detection were calculated (LOD)

• nicotine - 4.02 ng/mL (concentration range 1-500 ng/mL)

• cotinine -1.12 ng/mL (concentration range 1-100 ng/mL)

• cotinine - 13.29 (concentration range 100-500 ng/mL)

the limits of quantification (LQ), using the estimation of

these limits based on the standard deviation and of the regression

curve slope.

• nicotine -12.18 ng/mL (concentration range 1-100 ng/mL)

• cotinine - 3.39 n /mL (concentration range 1-100 ng/mL)

• cotinine - 40.27 ng/mL (concentration range 100-500 ng/

mL)

the working range was established: 12.18 to 500 ng/mL

for nicotine and 3.39 to 500 ng / mL for cotinine

to estimate the precision, it was determined:

• the repeatability of injection (the system precision) for a

number of 10 determinations, the values of relative standard

deviation (RSD) being:


26

- nicotine - 0.2505% (concentration range 1-100 ng/mL)

- cotinine - 0.28935% (concentration range 1-100 ng/mL)


• the repeatability of analysis (the method precision) for

three independent solutions, at three different concentration levels,

for which the relative standard deviation (RSD), in the range 10-

50 ng / mL are:




• the intermediate precision for three independent solutions

at three different concentration levels, for which the relative

standard deviation (RSD), in the range 10-50 ng / mL are:




to estimate the exactness, the retrieval for a number of

three samples was determined for three different concentration

levels, thus obtaining an average retrieval of 100.7% (minimum

97.9%, maximum 103.6%) for nicotine, 99.97 % and 100.5%

(minimum 97.9%, maximum 101.3% and minimum 98.1%,

maximum 101.8%) for cotinine, in the range of 10-50 ng / mL.

The advantage of this established and validated method

consists in the extraction of the analytes in a single step, reduced

volume of reagents, reducing the analysis time, sensitivity and

specificity of the method.


27

8.3. Application of the GC-MS method for the quantitative

determination of nicotine and cotinine in biological fluids

Introduction

The gas chromatographic method coupled with mass

spectrometry (GC/MS) developed and validated for the

determination of nicotine and cotinine in biological fluids has

been applied to a lot of 40 smoking patients hospitalized in the

Internal medicine clinic of Emergency Clinical Military Hospital

“Dr. James Czihac”, Iasi, in September-November 2011. The

study was performed comparatively with 40 non-smoking patients

hospitalized in the same clinic. The objective of the study was to

correlate the levels of nicotine and cotinine in biological samples

of smokers with the number of cigarettes smoked daily and the

level of addiction to nicotine determined by the Fagerström test.

For the non-smoking patients, we had in view the

correlation of the nicotine and cotinine concentrations and the

level of exposure to tobacco smoke.


Reagents and biological samples

• Methanol - Sigma - Aldrich - Germany

• Dichloromethane - Promochem

• Ammonium sulphate P.A. – Sellnord

• serum and urine from smokers and non-smokers

Sample collection and preparation

The volunteer patients who participated in this study signed

an information form on the study protocol. The level of addiction

to nicotine of smoking patients was determined by the Fagerström

test (score 1-4: mild addiction, score 5-8: moderate addiction,

score 9-12: severe addiction). The non-smoking patients specified

whether they were exposed to environmental tobacco smoke.

Biological samples were collected in the morning, before

eating. The venous blood was collected in tube without


28

anticoagulant or separator gel. The blood was allowed to clot at

room temperature and then it was centrifuged to separate the

serum.

Urine samples were collected in special sealed disposable

containers.

The biological samples were frozen at - 20˚C until

processing.

The preparation of the biological samples was presented in

Chapter 8.2.

Apparatus and chromatographic conditions

• The biological samples were analyzed with Agilent

Technologies 7890 gas chromatograph equipped with Agilent

Technologies 7683B autoinjector and mass spectrometer detector

Agilent Technologies 5975 inert MSD used to validate the

method, in the same chromatographic conditions.


• centrifugal

Results

The information of the patients was centralized for the two

studied groups: sex, age, occupation, diagnosis. Men formed the

majority (80%), average age of patients for the two groups was

approximately 48 years. We mention that 22 patients in the group

of smokers and 15 patients in the group of non-smokers were

active military personnel who carried out annual medical

examinations who were performed the control tests; these patients

had no clear diagnosis, reason why the diagnosis influence was

not taken into account as the information was incomplete. Most of

the subjects were employed.

In Figure 8.28 there are graphic representations of the

average values of the nicotine and cotinine concentrations in

serum and urine, determined for the patients in the two studied

groups.


29

Fig. 8.28. The average concentrations of nicotine and cotinine in serum and

urine for smokers and non-smokers

Discussions

The information presented in figure 8.28 shows much higher

concentrations (about 3-10 times higher) of nicotine and cotinine

in serum and urine in the smokers’ group, compared with the non-

smokers’ group. The data confirm the level of nicotine addiction

(Fagerström test).

The average values of nicotine in serum were 135.18 ng/mL

in the smokers’ group and 13.87 ng/mL in the non-smokers’

group; in urine, the nicotine average value was 258.97 ng / mL for

smokers, and 23.04 ng/mL for non-smokers. Cotinine presented

an average value of 185.80 ng/mL in serum and an average 51.01

ng/mL in urine to the group of smokers. The group of non-smoker

135,18

258,97

185,80

51,01

13,87 23,04 19,03 18,81

0

50

100

150

200

250

300

serum urine serum urine

Nicotine concentration (ng/mL)Cotinine concentration (ng/mL)

ng

/mL

smokersnon-…


30

averaged a value of cotinine in serum of 19.03 ng/mL and 18.81

ng/mL in urine.

To highlight the correlation between the number of daily

smoked cigarettes respectively with the level of nicotine

addiction, and the nicotine and cotinine concentrations

determinated from the biological fluids, the smoking patients were

distributed into three groups, depending on the nicotine addiction

level (mild, moderate, severe). The average values of these

biotoxicological parameters are very close for patients with severe

nicotine addiction and for those with moderate nicotine addiction,

but higher than average values were identified for patients with

mild nicotine addiction (Figure 8.29).

Fig. 8.29. The variation of the average concentrations

of nicotine and cotinine in serum and urine in smokers depending on

nicotine addiction

Statistical analysis of the nicotine and cotinine

concentrations using ANOVA test

The values of the nicotine and cotinine concentrations from

biological samples (serum and urine) determined for smokers and

non-smokers (40 subjects from each group), presented in the table

8.XXVII, were compared using ANOVA test to determine if there

Mild

Moderate

Severe

0,00

50,00

100,00

150,00

200,00

250,00

300,00

350,00

400,00

450,00

500,00

serumNicotine

urineNicotine

serumCotinine

urineCotinine

76,41

150,27

68,83

34,42

149,04

284,80

210,87

54,37

246,50

463,73

421,80

85,58

ng/mL


31

are significant differences between the average values of the two

groups.

Table IV. The average values of the nicotine and cotinine concentrations in

serum and urine of the studied groups and ANOVA test (alpha = 0.5 and F

critical 3.96)

Concentration

Average values ANOVA Test

Co

mp

ari

son

of

the

av

erag

e of

gro

up

s

(sig

nif

ica

nt

/

insi

gn

ific

an

t)

Sm

ok

ers’

gro

up

(40

su

bje

cts

)

No

n-s

mo

ker

s’

gro

up

(40

su

bje

cts)

Th

e fu

ncti

on

va

lue

F

Prob

ab

ilit

y (

P)

Nicotine

(ng/mL serum) 135.18 13.87 174.18 << 0.01

Averages of the

two groups

differ

significantly

Nicotine

(ng/mL urine) 258.97 23.04 188.45 << 0.01

Averages of the

two groups

differ

significantly

Cotinine

(ng/mL serum) 185.80 19.03 60.54 << 0.01

Averages of the

two groups

differ

significantly

Cotinine

(ng/mL urine) 51.01 18.81 125.01 << 0.01

Averages of the

two groups

differ

significantly

Comparing the value of the function F with the value of the

Fcritic, the results are discussed as it follows: the value of the

function F for alpha = 0.5 being much higher than the value

Fcritic = 3.96 rejects the hypothesis of homogeneity of the sample

averages, therefore it results that the factor of grouping the

lots/groups had an important influence on the sample averages

(influence is very significant), ie P is 0.01, so the probability of

a significant influence is higher than 99.99%.

Results obtained by similar methods mentioned in the

literature

The results obtained for the quantitative determination of

nicotine and cotinine in biological fluids by the GC-MS method


32

are comparable to the results obtained by similar methods in the

literature (Table 8.XXVIII) [149, 159, 160, 161].

Table 8.XXVIII. Summarized situation of the minimum and maximum

concentrations of nicotine and cotinine

in various studies

Study

Nicotine

(minimum-maximum

concentration ng/mL)

Cotinine

(minimum-maximum concentration

ng/mL)

Serum/Plasma Urine Serum/Plasma Urine

No

n-s

mo

ker

s

Sm

ok

ers

No

n-s

mo

ker

s

Sm

ok

ers

No

n-s

mo

ker

s

Sm

ok

ers

No

n-s

mo

ker

s

Sm

ok

ers

Shin H.S. et.

al. 6-498 0-1590 4-96 0-2986

Moriya F and

Hashimoto Y

4.4-

62.1

158-

314

49.9-

217

68.9-

300

Massadeh AM

et. al. 76-700

181-

3702 36-890

1364-

1972

34.9-

65.19 21-4420 37-223 6-3946

Culea M 50 425-

1197 122

929-

6132

Our study 2.95 261.86 11.93 493.07 9.00 431.80 9.31 91.29

Conclusions

• The nicotine and cotinine concentrations were determined

in biological fluids (serum and urine) for 40 smoking and 40 non-

smoking patients hospitalized in the internal medicine clinic of the

Emergency Military Hospital “Dr. James Czihac”, Iasi, using a

validated GC-MS method.

• Statistical results show much higher concentrations (about

3-10 times) of nicotine and cotinine in serum and urine in the

smoking patients compared with the non-smoker patients.

• The nicotine and cotinine concentrations in serum and

urine determined in smoking patients ranged between: 47.99 and

439.07 ng/mL for nicotine and between 27.35 and 431.80 ng/mL

for cotinine.

• The nicotine and cotinine concentrations in serum and

urine determined in non-smoking patients ranged from: 2.95 and

36.13 ng/mL for nicotine and between 9.00 and 30.76 ng/mL for

cotinine.


33

• For the smoking patients a correlation was established

between the level of nicotinic addiction (mild, moderate, severe),

the number of cigarettes smoked daily, and the nicotine and

cotinine concentrations in serum and urine. The data confirms the

level of addiction to nicotine (Fagerström test).

• The patients with moderate and severe nicotinic addiction

had close average values of nicotine and cotinine concentations in

biological fluids, but higher compared to the values of these

parameters for the patients with mild nicotinic addiction.

• The non-smoking patients exposed to environmental

tobacco smoke at home and/or at work, showed no significant

differences of the average values of nicotine and cotinine

concentrations in biological fluids compared to the patients who

were not exposed to environmental tobacco smoke and for this

reason we considered that this group is homogeneous.

• The results obtained from the quantitative determination of

nicotine and cotinine in biological fluids from smokers and non-

smokers, comply with the results obtained by gas

chromatographic methods quoted in the specific literature.

8.4.The study regarding the influence of the exposure to

environmental tobacco smoke on the biological parameters

Introduction

In this chapter we have studied the correlation between the

concentrations of the nicotine and cotinine in biological fluids

determined by the validated GC-MS method, the level of

addiction to nicotine and the biological parameters

(haematological and biochemical parameters) and how they are

influenced in chronic exposure to tobacco smoke.

Method

We analyzed the hematological parameters (ESR, RBW,

RBC) and the biochemical parameters (GOT, GPT, glycemia,

triglycerides, cholesterol) of the smokers group compared with the


34

control goup, non-smoker patients, in the context of the nicotine

and cotinine concentrations determined in serum and urine.

From the 40 smokers, we selected 25 subjects consuming

more than 10 cigarettes/day (moderate and severe dependence);

from the non-smokers’ group we selected 25 patients, eliminating

the patients who were exposed to environmental tobacco smoke.

We also removed the subjects who didn't have all the determined

hematological and biochemical parameters.

Results and Discussion

The data presented in Table 8.XXXI and Figure 8.33 shows

the average values of the nicotine and cotinine concentrations in

the biological fluids significantly higher for the smoking patients

compared to those of the non-smokers, confirming the statistical

results and conclusions presented in Chapter 8.3.

Table 8.XXXI. The average concentrations of the nicotine and cotinine

concentrations in serum and urine for selected groups and the ANOVA

test (alpha = 0.5 and Fcritic = 4.04)

Co

nce

ntr

ati

on

Average value ANOVA Test

Comparison of

average

concentrations

determined for the

groups of 25 subjects

(significant /

insignificant)

Sm

ok

ers

No

n-s

mo

ker

s

Th

e a

ver

ages

rati

o

Lo

t S

mok

er /

no

n-s

mo

ker

Va

lue

of

the

fun

ctio

n F

Prob

ab

ilit

y

(P)

Nicotine

(ng/mL

serum)

158.20 13.71 11.54 255.09 < 0.01 The averages differ

significantly

Nicotine

(ng/mL

urine)


significantly

Cotinine

(ng/mL

serum)


significantly

Cotinine

(ng/mL

urine)


significantly


35

Fig. 8.33. The average of the nicotine and cotinine concentrations in serum

and urine

The values of the hematological and biochemical parameters

for the smoker and non-smoker groups were compared using the

ANOVA test to determine if there are significant differences

between the two groups and if the exposure to tobacco smoke,

evidenced by the nicotine and cotinine concentrations in

biological fluids, influence these parameters. Results of the

statistical analysis are shown in Table 8.XXXII and Figure 8.34.

158,2

305,25

225,63

57,16

13,71 22,05 18,77 17,280

50

100

150

200

250

300

350

serum urine serum urine

Nicotine concentration Cotinine concentration

ng

/mL

smokers


36

Table 8.XXXII. Centralization of the results of the statistical analysis using

the ANOVA test, regarding the influence of the nicotine and cotinine

concentrations on the hematological and biochemical parameters (alpha =

0.5; Fcritic = 4.04)

Hematological

and biochemical

parameters

Average value ANOVA Test The influence of

smoking on

biological

parameters

(significant /

insignificant)

Sm

ok

er

gro

up

s

(25

pa

tien

ts)

No

n-s

mo

ker

gro

up

s

(25

pa

tien

ts)

Va

lue

of

the

fun

ctio

n F

Prob

ab

ilit

y

(P)

ESR (mm/h) 14.40 11.96 0.55 0.46 insignificant

WBC (x103 /mmc) 7.73 6.23 5.67 0.02 insignificant

RBC (x106 /mmc) 4.94 4.88 0.22 0.64 insignificant

GOT (U.I.) 23.32 24.12 0.11 0.75 insignificant

GPT (U.I.) 25.36 29.30 0.64 0.43 insignificant

Glycemia (mg/dL) 97.55 96.21 0.10 0.75 insignificant

Triglycerides

(mg/dL) 141.23 115.28 0.26 0.61 insignificant

Cholesterol

(mg/dL) 189.96 187.64 0.04 0.85 insignificant

• The value of the F function is lower than the Fcritic for most

of the biological parameters, so that the hypothesis of the

homogeneity of the samples’ averages is accepted, and so, the

grouping factor of the patients by groups had no influence on the

samples’ averages (the influence is insignificant).

• For a better probability the ANOVA calculations were

resumed for alpha = 0.4 and Fcritic= 4.46, the results being

identical. For WBC the result was P = 0.02, the conclusion being

that the influence of the grouping factor of patients into lots is

insignificant.


37

Fig. 8.34. The variation of the average values of the hematological and

biochemical parameters

Conclusions

The exposure to tobacco smoke (evidenced by the nicotine

and cotinine concentrations in biological samples of smokers), as

a factor of influence of the hematological parameters- ESR, RBC,

WBC and biochemical parameters - GOT, GPT, glycemia,

triglycerides, cholesterol is insignificant, evidenced both in

patients with moderate and severe nicotine addiction.

The average values of the hematological parameters (ESR,

WBC, RBC) and biochemical parameters (glucose, triglycerides,

cholesterol) were highlighted, as slightly increased in the case of

smokers compared with those in the case of the non-smokers. The

results obtained in this study were correlated with the data from

the literature [111, 116, 121, 122, 175, 176].

VSH (mm/h)WBC (x1000/

mmc)

RBC

(x1000000/mm

c)

GOT (U.I.) GPT (U.I.)Glycemia

(mg/dL)

Triglycerides

(mg/dL)

Cholesterol

(mg/dL)

smokers 14,4 7,73 4,94 23,32 25,36 97,55 141,23 189,96

non-smokers 11,96 6,23 4,88 24,12 29,3 96,21 115,28 187,64

0

20

40

60

80

100

120

140

160

180

200


38

9. CHEMICAL AND TOXICOLOGICAL STUDIES OF NICOTINE

DETERMINATION IN TOBACCO BY GAS CHROMATOGRAPHY

COUPLED WITH MASS SPECTROMETRY

9.1. Development and validation of a method for nicotine

determination in tobacco by gas chromatography coupled

with mass spectrometry (GC-MS)

Introduction

In this chapter we aimed to develop and validate a method

for the quantitative determination of nicotine in tobacco by GC-

MS.

By the gas chromatographic analysis of tobacco samples

we monitored the nicotine separation from a tobacco organic

extract for the identification and quantitative determination. Due

to the high variability of the substances found in tobacco, in order

to achieve the separation, identification and quantitative

determination of nicotine by gas chromatography coupled with

mass spectrometry, we performed originally a series of tests in

order to determine the optimal experimental conditions for the

analysis. [179, 180, 181, 182].

The following parameters were monitored: specificity/

selectivity, linearity and linearity range, limit of detection and

limit of quantification, precision (repeatability), accuracy

(retrieval coefficient).


Reagents

• Nicotine-density 1.009 g/mL (Merck)

• Methanol chromatographic purity (Merck)

• Dichloromethane chromatographic purity (Merck)

• Solvent: methanol/dichloromethane (1/1)

The standard nicotine solution - concentration 201.8 g/mL.


39


Agilent Technologies gas chromatograph 7890 A, mass

spectrometer coupled with an Agilent Technologies 5975C inert

MSD as detection system.

• Mobile phase: helium at a flow rate of 1 mL/minute

• Stationary phase: gas chromatographic column DB 5 MS

(30 mx 0.25 mm, 0.25 μm)

• The injector’s temperature was 250 C. The temperature

gradient in the column compartment: increased from 100 C to

190 C with a growth rate of 10 C /min, followed by an increased

of 20 C/min to 280 C where the temperature was kept constant

for another 5 minutes.

• MSD source temperature was 230 C, MSD quadrupole

temperature was 150 C. Acquisition mode spectra: SCAN

(tracing all the lines in the mass spectrum);

• the followed table field was : 10-450 atomic mass units.

Interpretation of results: Agilent Technologies ChemStation

software.

The results obtained from the validation of the method

of nicotine determination from tobacco by GC-MS

We validated a method for the nicotine analysis by gas

chromatography coupled with mass spectrometry, establishing the

working conditions [142-145].

the volume of the solution injected was 0.3 L and split

ratio of 1/10 to the input port of the injector which has a

temperature of 250 C;

detection was performed by mass spectrometry, MSD

source temperature of 230 C and the MSD quadrupole

temperature of 150 C;

nicotine identification was performed by comparing

spectra with Wiley spectra library.

the linearity of the function of response was studied (the

peak area modification depending on the sample concentration


40

was monitored). The function of response is linear in the studied

range (1.01 to 201.8 g/mL) (Figure 9.6). The equation for the

regression slope obtained was calculated by using the method of

least squares: Area = 4877.4 x Concentration ( g/mL) +

1569486.174;

Fig. 9.6. Calibration curve for the nicotine determination

in tobacco by GC-MS

the limit of detection (LOD = 3.6 g/mL) and the limit of

quantification (LQ = 10.8 g/mL) were calculated using the

estimation of these limits based on the standard deviation of the

regression slope.

To estimate the precision we determined:

the repeatability of the injection (precision system) for a

number of 5 determinations, the relative standard deviation (RSD)

being 0.3763%;

the repeatability of the analysis (method precision) for

three independent solutions at three different concentration levels,

the relative standard deviation (RSD) is 0.6562% in the range of

70.63 -131.17 g/mL;


41

the intermediate precision for three independent solutions

at three different concentration levels for which the relative

standard deviation (RSD) is 0.7121% in the range of 70.63-

131.17 g/mL.

To estimate the exactness, the recovery was determined

for a number of three samples at three different concentration

levels obtaining an average recovery of 100.28% over the range of

99.08 to 100.96 g / mL.

The GC-MS validated method was applied to determine

nicotine in various tobacco samples.

9.2. Application of GC-MS method to the quantitative

determination of nicotine in tobacco samples

Introduction

The developed GC-MS method for the dosage of nicotine

was used for its quantitative determination from various tobacco

samples: different types of cigarettes, cigars, pipe tobacco and

tobacco leaves.


Reagents

• methanol chromatographic purity (Merck)

• dichloromethane chromatographic purity (Merck)


• centrifugal

• laboratory glassware


The analyses were performed on the same equipment used

in the validation of the method, under the same experimental


42

conditions used for the GC-MS determinations in order to validate

the method.

The nicotine was identified by comparing the retention peak

times corresponding to the chromatograms of tobacco extracts

with the chromatogram of a standard solution containing nicotine,

and by comparing the mass spectra.

Using the peak areas corresponding to nicotine, the nicotine

concentration was calculated using the equation of the calibration

curve obtained from the linearity study of this method.

Tobacco samples

A number of 39 samples of tobacco were analyzed, namely:

- 31 samples from different cigarettes types,

corresponding to 15 brands, purchased from local

retail poits

- five samples of cigars

- one sample of pipe tobacco

- two samples of tobacco leaves, from two geographical

regions (Timisoara and Iasi)

Preparation of samples

The samples of tobacco (1.0-1.5 g) were extracted in a

mixture of methanol/dichloromethane (1/1, v/v) by stirring for one

hour at 500 vibrations/min. After filtration, the solution was

diluted at 50 mL, followed by another dilution of 1 mL to 4 mL

with the same solvent; 0.3 µL of these solutions were injected in

the GC/MS instrumentequipment in the mentioned conditions.

For each sample, three measurements were made.

The nicotine content was expressed in mg/g tobacco.

The statistical evaluation of the results was performed using

Microsoft Excel.

Results and discussion

After the GC/MS analysis, the peak of nicotine was

identified and the peak area was measured.


43

In the figure 9.7 as an example, there is the chromatogram

for the Jewels vanilla cigar sample, at the retention time of 6.23

minutes.

Figure 9.7. Gas chromatogram for Jewels vanilla cigar sample

In the conditions of the proposed method (extraction in

methanol/dichloromethane (1/1, v/v) and analysis by GC - MS,

there are no additional peaks at retention times close to that of

nicotine (approximately 6.2 minutes). Furthermore, by comparing

the mass spectrum of the peak corresponding to nicotine with

those from spectral libraries (Wiley) a high purity is obtained

(over 98%). These arguments demonstrate that other components

present in the samples of tobacco (alkaloids or other compounds)

do not interfere.

In table 9.XIII we tabulated the minimum, average and

maximum measurements of nicotine content in tobacco samples

that were analyzed.


44

Table 9.XIII. The minimum, average and maximum measurements of

nicotine content from cigarettes, cigars, pipe tobacco and tobacco leaves

mg nicotine/

g tobacco Cigarettes

Cigars and pipe

tobacco Tobacco leaves

Minimum 10.14 13.15 -

Average 15.35 22 26.79

Maximum 21.15 28.38 -

Fig. 9.11. The average values of nicotine determined in tobacco samples

Figure 9.11. compares the average content of the nicotine

from different tobacco samples studied.

Conclusions

The content in nicotine from 39 tobacco samples was

determined by using a validated GC/MS method.

For the 31 tobacco samples corresponding to 15 cigarette

brands, the variation limits of the nicotine content were more

restricted (10.14-21.15 mg/g) in comparison with the 6 samples of

cigars and pipe tobacco for which the nicotine content varied in a

larger range of values, 13.15-28.38 mg/g. For the two samples of

tobacco leaves, the nicotine’s concentration registered very close

values.

According to the average values, the highest nicotine

content was registered in the case of the tobacco leaves (26.79

15,35

22

26,79

0

5

10

15

20

25

30

Cigarettes Cigars and pipe tobacco Tabacco leavesmg

nic

oti

ne/g

to

bacco


45

mg/g), followed by cigars and pipe tobacco (22 mg/g), and by the

cigarettes (15.35 mg/g).

The obtained results are in accordance with the information

identified in the specific literature and confirm the method’s

applicability [187, 191, 192, 194].


46

10. STATISTICAL INTERPRETATION OF THE DATA FROM THE

PROGRAM “STOP SMOKING”, CONDUCTED BY THE

PNEUMOLOGY HOSPITAL IASI, DURING JANUARY 2009 -

FEBRUARY 2012

The purposes of the statistical interpretation of the data from

the program “Stop Smoking” provided by the Pneumology

Hospital Iaşi were:

• identifying the level of the addiction to nicotine of the

patients enrolled in the program

• identifying the reasons that underlie the intent to quit

smoking

• emphasizing the decisive role of the treatment and medical

advice in succeeding in quitting smoking

The study was performed on 190 patients divided into two

groups as it follows: group A, patients who presented associated

diseases - 81 patients (42.63%) and group B, patients without

other associated diseases - 109 patients (57.37%).

Figure 10.10 shows that in both groups, the largest share is

represented by the patients with moderate nicotine addiction (5-8

Fagerström scale), 71.60% in group A and 75.23% in group B.


47

Fig. 10.10. The proportion of the patients from group A and group B,

according to level of addiction to nicotine

The recommended treatment for nicotine addiction was:

nicotine patches concentration 15 mg, Zyban 150 mg tablets,

Champix 0.5 mg and 1 mg tablets.

55 patients (30.86% in group A and 27.52% in group B)

were treated with nicotine patches; 27 patients were treated with

Zyban (9.88% group A and 17.43% group B), 104 patients were

treated with Champix (58.02% in group A and 52.29% in group

B), three patients received an associated therapy with nicotine

patches/Zyban (2.75% group B), one patient received no

treatment.

Most adverse reactions were reported by the patients treated

with Champix (31.25% group A and 25.69% group B) and in low

proportion by the patients treated with nicotine patches (7.5%

group A and 3.67% group B) and Zyban (6.25% group A and

4.59% group B) (Figure 10.23).

20,99

15,60

71,6075,23

7,419,17

0,00

10,00

20,00

30,00

40,00

50,00

60,00

70,00

80,00

Group A Group B

%

Mild

Moderate


48

Fig. 10.23. The share of patients who presented adverse effects according

to the treatment administered

Following the treatment, 130 patients (68.42%) became

abstinent, 60 patients (31.58%) reduced the number of cigarettes

smoked per day.

This study emphasizes the role of the National Program

“Stop Smoking” in helping patients determined to quit smoking

through medical counseling and appropriate medication.

0,00

10,00

20,00

30,00

40,00

50,00

60,00

70,00

80,00

90,00

Mild Moderate Severe Total number of patients

8,224,11 2,74

15,070,00 10,96 1,37

12,33

2,74

60,27

9,59

72,60

%

Champix


49

GENERAL CONCLUSIONS. ORIGINAL CONTRIBUTIONS.

PERSPECTIVES OF RESEARCH

GENERAL CONCLUSIONS

The research undertaken within this thesis led to the

following conclusions:

1. The statistical study of the hematological parameters

(ESR, WBC, RBC) and biochemical parameters (GOT, GPT,

GGT, glycemia and cholesterol) conducted on a relatively small

group, 89 patients, that included 59 smokers and 30 non-smokers

for a period of one year, revealed no significant changes in the

biological parameters in smoking patients compared to non-

smoking patients.

2. The spectrophotometric method in UV can be applied for

the nicotine determination in serum and urine, using the

absorption band from 260 nm, and the following experimental

parameters: nicotine is isolated from the biological material by

liquid-liquid extraction at pH - alkaline, the ethyl ether as

optimum extraction solvent, the linearity was investigated

between the limits: 10-50 g/5 mL. The nicotine’s concentration

ranged between 25.6420 and 71.382 g/mL in serum, and

between 3.2972 and 8.6630 g/mL in urine, in the case of the

group of women. In the case of men, the nicotine’s concentration

varied between 26.522 and 109.206 g/mL in serum.

3. The quantitative determination of the nicotine and

cotinine in biological samples by the validated gas

chromatographic method coupled with mass spectrometry (GC-

MS), required the validation and subsequently the application of

the protocol. To perform the analysis we used a gas

chromatograph Agilent Technologies 7890A, equipped with

autoinjector Agilent Technologies 7683B and mass spectrometer

detector Agilent Technologies 5975 inert MSD; the

chromatographic separation was achieved on a DB 5 MS column

(30 mx 0.25 mm, 0.25 μm), using helium as the mobile phase with


50

a flow rate of 1 ml / min with the following temperature gradient

in the column compartment: initially the column temperature was

100 ° C followed by a temperature ramp - 10°C / min up to 250°C

and immediately a second ramp - 20 °C/min up to 280°C and

finally the temperature is kept constant for another 3.5 minutes.

The validation parameters studied were: specificity,

linearity, limit of detection and limit of quantification, system

precision, method precision, intermediate precision and accuracy.

Linearity range was 12.18 to 500 ng/mL for nicotine and 3.39 to

500 ng/mL for cotinine.

4. The validated GC-MS method was applied to determine

the concentrations of nicotine and cotinine in biological fluids

(serum and urine) from 40 smokers and 40 non-smokers

hospitalized in the internal medicine clinic of the Clinical

Emergency Military Hospital “Dr. James Czihac”, Iaşi. In the case

of smokers, the nicotine and cotinine concentrations in serum and

urine ranged between 47.99 and 439.07 ng/mL for nicotine and

between 27.35 and 431.80 ng/mL for cotinine. In the case of non-

smokers, the determined nicotine and cotinine concentrations in

serum and urine were ranged between 2.95 and 36.13 ng/mL for

nicotine and between 9.00 and 30.76 ng/mL for cotinine. The data

suggests much higher concentrations (about 3 to 10 times higher)

of the nicotine and cotinine in serum and urine in the group of

smokers compared to the non-smokers.

For the smokers a correlation was established between the

level of nicotinic addiction (mild, moderate, severe), the number

of cigarettes smoked daily, and the nicotine and cotinine

concentrations in serum and urine. The data confirms level of

nicotinic addiction (Fagerström test).

The patients with moderate and severe nicotinic addiction

had close average values of nicotine and cotinine concentations in

biological fluids, but higher compared to the values of these

parameters in the case of patients with mild nicotinic addiction.

The non-smokers exposed to environmental tobacco smoke

at home and/or at work, showed no significant differences of the

average values of nicotine and cotinine concentrations in


51

biological fluids compared to the patients who were not exposed

to environmental tobacco smoke and for this reason we considered

that this group is homogeneous.

The results obtained to the quantitative determination of

nicotine and cotinine in biological fluids from smokers and non-

smokers, are in accordance with the results obtained by gas

chromatographic methods quoted in the specific literature.

5. The exposure to tobacco smoke (evidenced by the

nicotine and cotinine concentrations in biological samples of

smokers) as an influencing factor of haematological parameters-

ESR, RBC, WBC and biochemical parameters- GOT, GPT,

glycemia, triglycerides, cholesterol, is insignificant, evidenced

both in patients with moderate and severe level of nicotine

addiction.

There were highlighted average values of haematological

parameters (ESR, WBC, RBC) and biochemical parameters

(glucose, triglycerides, cholesterol) that are slightly increased in

smokers compared to non-smokers.

6. The quantitative determination of the nicotine in tobacco

by gas chromatography coupled with mass spectrometry was

performed using gas chromatograph Agilent Technologies 7890A,

by applying the GC-MS validated method. The validation

parameters were specificity, linearity, limit of detection and limit

of quantification, system precision, method precision,

intermediate precision and accuracy.

The concentration of nicotine in various samples of tobacco

was determined by the GC-MS validated method using a DB 5

MS column and helium as the mobile phase. Nicotine was

extracted from 39 tobacco samples with methanol/

dichloromethane (1/1, v/v). For the 31 tobacco samples

corresponding to 15 cigarette brands, the variation limits of the

nicotine content were more restricted (10.14-21.15 mg/g) in

comparison with the 6 samples of cigars and pipe tobacco for

which the nicotine content varied in a larger range of values,

13.15-28.38 mg/g. For the two samples of tobacco leaves, the

nicotine’s concentration registered very close values.


52

According to the average values, the highest nicotine

content was registered in the case of the tobacco leaves (26.79

mg/g), followed by cigars and pipe tobacco (22 mg/g), and by the

cigarettes (15.35 mg/g).

The obtained results are in accordance to the information

identified in the specific literature and confirm the method’s

applicability.

7. The statistical analysis of all the information from the

program “Stop Smoking” provided by Pulmonary Hospital Iasi

from January 2009-February 2012, intended to highlight the

importance of the anti-nicotine treatment and medical counseling

in quitting smoking. The study was performed on 190 patients

divided into two groups as it follows: group A, patients who had

associated diseases - 81 patients (42.63%) and group B, patients

without other associated diseases- 109 patients (57.37%).

The recommended treatment for nicotine addiction was:

nicotine patches with 15 mg concentration, Zyban 150 mg tablets,

Champix 0.5 mg and 1 mg tablets; following the treatment, 130

patients (68.42%) became abstinent, 60 patients (31.58%) reduced

the number of cigarettes smoked per day. Out of the 189 patients

who received medical treatment, 140 (77.04%) had moderate

nicotine addiction, 33 mild nicotine addiction (17.46%) and 16

patients (8.46%) had severe nicotine addiction.

55 patients were treated with nicotine patches (30.86% in

group A and 27.52% in group B), 27 patients were treated with

Zyban (9.88% group A and 17.43% group B), 104 patients were

treated with Champix (58.02% in group A and 52.29% in group

B), three patients received a combined treatment with nicotine

patches/Zyban (2.75% group B), one patient received no

treatment. Most adverse reactions were reported by the patients

treated with Champix (31.25% group A and 25.69% group B) and

in low proportion by the patients treated with nicotine patches and

Zyban. The study emphasizes the important role of the program

“Stop Smoking” in helping patients to quit smoking.


53

ORIGINAL CONTRIBUTIONS

The studies conducted within the thesis “Investigation of

some biological and biotoxicological parameters in chronic

exposure to tobacco smoke”, is reflected in the following original

contributions:

• The statistical analysis of the hematological parameters

(ESR, WBC, RBC) and biochemical parameters (GOT, GPT,

GGT, glycemia and cholesterol), in the case of smoking patients

compared to those in the case of non-smoking patients, using the

Anova test.

• The development of a quantitative method for the

determination of nicotine in biological samples (serum, urine) by

UV spectrophotometry using the absorption band from 260 nm.

• The development and the validation of a method for

simultaneous quantitative determination of the nicotine and

cotinine in biological samples (serum and urine) by gas

chromatography coupled with mass spectrometry (GC-MS) and

the application of this method in the the determination of nicotine

and cotinine in biological fluids, prelevated from smoking

subjects, compared to non-smoking subjects.

• The analysis regarding the influence of exposure to

tobacco smoke on hematological and biochemical parameters

correlated with the concentrations of nicotine and cotinine in

biological fluids determined by GC-MS, and the levels of

addiction to nicotine (Fagerström test).

• The development and the validation of a method for the

quantitative determination of nicotine in tobacco by GC-MS and

the evaluation of nicotine concentrations in different tobacco

products through this method.

• The statistical interpretation of the data from the program

“Stop Smoking” and the emphasis on the role of the anti-nicotine

therapy and medical counseling for successful smoking cessation.


54

PERSPECTIVES OF RESEARCH

The results obtained in this thesis provide the premises for

some new research directions:

• The monitoring of the hematological and biochemical

parameters in epidemiological studies on smoking subjects

compared to non-smoking subjects, in order to evaluate the effects

of smoking on health.

• The study on the rate of metabolism and elimination of the

nicotine and cotinine by analyzing biological samples prelevated

at different moments of a day, e.g. morning and evening.

• The analysis of the levels of nicotine and cotinine in

biological samples of smokers, depending on the number of

cigarettes smoked daily, on the brand of cigarettes and on the

nicotine content determined in the tobacco smoke for the brands

used.

• The analysis of other biotoxicological markers:

thiocyanates, carboxyhemoglobin, which may be used to assess

the level of exposure to smoke.


55

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