CHEMICAL AND CLINICAL TOXICOLOGICAL RESEARCH … · university of medicine and pharmacy „grigore t. popa” iaȘi chemical and clinical toxicological research of antiallergic drugs

UNIVERSITY OF MEDICINE AND PHARMACY

„GRIGORE T. POPA” IAȘI

CHEMICAL AND CLINICAL

TOXICOLOGICAL RESEARCH OF

ANTIALLERGIC DRUGS

- PhD Thesis Abstract -

SCIENTIFIC COORDINATOR PhD Prof. Elena BUTNARU

PhD STUDENT

Asist. Prof. Ioana-Cezara GRIGORIU

(CABA)

Invest in people !

Project co-funded by European Social Fund through the Sectoral Operational Programme Human Resources

Development 2007 - 2013

Priority Axis 1 "Education and professional training in support of economic growth and development of a

knowledge-based society"

Key Area of Intervention 1.5 ”Doctoral and post-doctoral programs supporting the research”

Project title: ”Strategic partnership for increasing the quality of scientific research in medical universities through

doctoral and postdoctoral scholarships – DocMed.Net_2.0”

Contract no: POSDRU/159/1.5/S/136893

Beneficiary: University of Medicine and Pharmacy ”Iuliu Hatieganu” Cluj-Napoca Partener

P4: University of Medicine and Pharmacy “Grigore T. Popa” Iași

2016

CONTRIBUȚII LA CERCETAREA CHIMICO-TOXICOLOGICĂ ȘI CLINICĂ

A UNOR MEDICAMENTE ANTIALERGICE

The PhD thesis contains 176 pages (10 pages of annexes), 67

tables, 97 figures and 174 references.

In this abstract the numbering of the figures, tables and the table

of contents are kept in the same form like in the PhD thesis.

Keywords: allergy, H1 antihistamines, loratadine,

desloratadine, cetirizine, chlorpheniramine, UPLC-QTOF/MS, LC-

MS/MS, toxicity.

The scholarship received between April 2014 to September 2015

in the project entitled: “Strategic partnership for increasing the quality

of scientific research in medical universities through doctoral and

postdoctoral scholarships – DocMed.Net_2.0”, no.

POSDRU/159/1.5/136893, contributed to the studies undertaken in

the period of the thesis and also to the obtained results.



ACKNOWLEDGMENT

Gratitude to Mrs. PhD Prof. Elena Butnaru, scientific supervisor

of the work, for encouragement to become a PhD student, for professionalism

and scientific guidance and for all wise advices and support during the

doctoral studies.

I want sincerely thank to Mrs. Dean PhD Prof. Lenuța Profire and

entire team of project management POSDRU 159/1.5/S/136893, "Strategic

partnership for increasing the quality of scientific research in medical

universities through doctoral and postdoctoral scholarships –

DocMed.Net_2.0", which gave me the status of Fellow during April 2014 to

September 2015 and supported steps for conducting a training for a period of

international mobility of 3 months.

Special thanks to Mrs. PhD Prof. Ruth Maria Fernandez-Torrez

from the University of Seville, Faculty of Chemistry, Spain, for

professionalism, seriousness, generosity and trust that showed me during 3

months of internship transnational mobility, especially for my initiation into

the "secrets" of high performance liquid chromatography with detection

QTOF/MS.

Also I wish to thank for all the recommendations provided by the

guidance committee members: PhD Prof. Anca Miron, PhD Prof. Antonia

Poiată, PhD Assoc. Prof. Luminita Agoroaei and PhD Assoc. Prof.

Cornelia Mircea.

Warm thanks to Mrs. PhD Assoc. Prof. Veronica Bild, PhD Prof.

Walther Bild and staff from the Department of Pharmacology and

Experimental Pharmacodynamics, for their support in conducting pharmaco-

toxicological studies on experimental animals.

I also thank PhD Lecturer Adrian Spac and PhD Chem Bogdan

Cioroiu for scientific support in quantitative determination of H1

antihistamines by liquid chromatography.

I want to thank Mrs PhD Prof. Evelina Moraru from Emergency

Hospital for Children and PhD Prof. Georgeta Siniţchi from "Atopy"

Medical Center of Allergology and Immunology for collaboration agreement

to achieve statistical surveys.

I also want to thanks all my teachers who have contributed to my

professional evolution and, not least, I want to thank my family for moral

support, permanent support and understanding.



TABLE OF CONTENTS

GENERAL PART

CHAPTER 1 1

ALERGY – GENERALITY. DEFINITION. PHYSIOPATHOLOGY 1

1.1. Process of allergic sensitization 2 1.2. Antigen – antibody interaction 3

1.3. Types of allergic reactions 3

1.4. Immunological mechanisms of type I hypersensitivity 5

1.4.1. Primary exposure to allergens 5

1.4.2. Secondary exposure to allergens 6

1.4.3. Released mediators and symptoms 7 1.5. Description of allergic diseases 8

1.5.1. Anaphylaxis 8

1.5.2. Allergic rhinitis 8 1.5.3. Hives and angioedema 9

1.5.4. Food allergies 9

1.5.5. Mastocytosis 10

CHAPTER 2 11

TREATMENT OF ALLERGIC DISORDERS 11 2.1. Specific treatment 11

2.2. Nonspecific treatment 12

2.3. H1 antihistamine drugs registered in Romania 13

CHAPTER 3 14

HISTAMINE 14 3.1. Short history of histamine 14

3.2. Biosynthesis, storage and metabolism of histamine 15

3.3. Endogenous functions of histamine. Histamine receptors. Physiological and pathological properties of histamine 16

3.4. Pharmacokinetics and pharmacodynamics of histamine 17

CHAPTER 4 18

H1 ANTIHISTAMINES – H1 RECEPTOR ANTAGONISTS 18 4.1. Short history of antiallergic antihistamines 18

4.2. Mechanism of action of antihistamines 19

4.3. Classification of H1 antihistamines 19 4.3.1. First generation H1 antihistamines 20

4.3.1.1. Phisiopathology. Mechanism of action. Therapeutic effects 20

4.3.1.2. Adverse effects. Drug interactions. Overdose 20 4.3.1.3. Chlorpheniramine – toxicity 22

4.3.2. Second generation H1 antihistamines 22

4.3.2.1. Mechanism of action 22

4.3.2.2. Toxicokinetic and toxicodynamic of second generation

antihistamines 22

4.3.2.3. Clinical cases: adverse events following the administration of H1 antihistamines 22

4.3.3. LORATADINE 28



4.3.3.1. Chemical structure. Physico-chemical properties 28 4.3.3.2. Toxicokinetics and toxicodynamics 28

4.3.3.3. Pharmacotherapy and pharmacography 29

4.3.3.4. Adverse effects of therapy with loratadine 29 4.3.3.5. Contraindications 30

4.3.3.6. Drug interactions 30

4.3.4. DESLORATADINE 30 4.3.4.1. Chemical structure. Physico-chemical properties 30

4.3.4.2. Toxicokinetics and toxicodynamics 31

4.3.4.3. Farmacoterapie și farmacografie 31

4.3.4.4. Adverse effects of therapy with desloratadine 31

4.3.4.5. Contraidications 32 4.3.4.6. Drug interactions 32

4.3.5. CETIRIZINE 32

4.3.5.1. Chemical structure. Physico-chemical properties 32 4.3.5.2. Toxicokinetics and toxicodynamics 33

4.3.5.3. Farmacoterapie și farmacografie 33

4.3.5.4. Adverse effects of therapy with cetirizine 33 4.3.5.5. Contraindications 34

4.3.5.6. Drug interactions 34

CHAPTER 5 35

METHODS FOR THE ANALYSIS OF SECOND GENERATION

H1 ANTIHISTAMINES, REPORTED IN THE LITERATURE 35 5.1. Analytical methods for loratadine and desloratadine 35

5.2. Analytical methods for cetirizine 37

5.3. Analytical methods for simultaneous determination of H1

antihistaminic compounds 38

CHAPTER 6 39 GENERAL ASSESSMENT OF CHRONIC AND ACUTE TOXICITY 39

6.1. Toxic effect. Acute toxicity. Chronic toxicity 39

6.2. Graded response. Quantal response 40

6.3. In vivo and in vitro methods for toxicity determinination 40

PERSONAL PART

CHAPTER 7 41

MOTIVATION FOR CHOOSING THE THEME AND OBJECTIVES 41

CHAPTER 8 43

INCIDENCE OF ALLERGIC DISEASES AND H1 ANTIHISTAMINES CONSUMPTION 43

8.1. Statistical study on the incidence of allergic diseases and consumption of

H1 antihistamines in patients from “Atopia” Medical Center 43

8.1.1. Introduction 43

8.1.2. Material and method 43

8.1.3. Results and discussions 44 8.1.4. Conclusions 47

8.2. Statistical study on the incidence of allergic diseases and consumption of



H1 antihistamines in patients from “Sf. Maria” Clinical Emergency Hospital for Children 48


8.2.2. Material and method 48 8.2.3. Results and discussions 49

8.2.4. Conclusions 55

CHAPTER 9 56

ANALYSIS OF LORATADINE BY RP-HPLC-UV 56

9.1. Development and validation of RP-HPLC-UV method for the

determination of loratadine 56

9.1.1. Introduction 56 9.1.2. Material and method 56

9.1.2.1. Equipment 56

9.1.2.2. Reagents 56 9.1.2.3. Method 56

9.1.3. Results and discussions 57

9.1.3.1. Determination of optimal detection wavelength 57 9.1.3.2. Identification of loratadine`s peak 57

9.1.3.3. Liniarity 59

9.1.3.4. Detection limit and quantification limit 61 9.1.3.5. Precision 62

9.1.3.6. Accuracy 65

9.1.4. Application of HPLC-UV method for the quantitative determination of loratadine in pharmaceutical products 66


CHAPTER 10 70

BIOANALYTICAL METHOD FOR QUANTITATIVE DETERMINATION

OF H1 ANTIHISTAMINES IN HUMAN PLASMA BY LC-MS/MS 70 10.1. Introduction 70

10.2. Material and method 70

10.2.1. Equipment 70

10.2.2. Reagents 70

10.2.3. Chromatographic method 71

10.2.4. Preparation and processing of samples 72 10.2.4.1. Standard stock solutions 72

10.2.4.2. Biological samples 72

10.3. Results and discussions 72 10.3.1. Specificity and selectivity study of the method 72

10.3.1.1. Specificity 72

10.3.1.2. Selectivity 77 10.3.2. Residual effect 81

10.3.3. Linearity response 81

10.3.4. Detection limit and quantification limit 88

10.3.5.Accuracy and precision 91

10.3.6. Stability of the samples 94

10.4. Application of LC-MS/MS method for quantitative determination of loratadine, desloratadine and cetirizine in human plasma 96

10.5. Conclusions 98



CHAPTER 11 100 SIMULTANEOUS QUANTITATIVE DETERMINATION OF LORATADINE,

DESLORATADINE AND CETIRIZINE FROM BIOLOGICAL SAMPLES BY

UPLC-QTOF/MS METHOD 100 11.1. Introduction 100

11.2. Material and method 100

11.2.1. Equipment 100 11.2.2. Reagents 101

11.2.3. Chromatographic separation parameters 101

11.2.4. QTOF/MS detection parameters 102

11.2.5. Preparation of stock solutions 102

11.2.6. Statistical analysis 102 11.3. Results and discussions 103

11.3.1. Optimisation of simultaneous cuantification of loratadine,

desloratadine and cetirizine by UPLC-QTOF/MS method 103 11.3.2. Validation of UPLC-QTOF/MS method for loratadine, desloratadine

and cetirizine dosing in human plasma 104

11.3.2.1. Specificity of the method 104 11.3.2.2. Linearity and linearity range 104

11.3.2.3. Detection limit and quantification limit 107

11.3.2.4. Precision 108 11.3.2.5. Accuracy 109

11.4. Conclusions 110

11.5. Aplications of UPLC-QTOF/MS method for simultaneous determination of loratadine, desloratadine and cetirizine in human plasma samples 111

11.5.1. Optimization method of extraction of H1 antihistamines 111

11.5.2. Reproductibily of the extraction method 113

CHAPTER 12 115

PHARMACO-TOXICOLOGICAL EVALUATION OF H1 ANTIHISTAMINES BY IN VIVO / IN VITRO STUDIES 115

12.1. Chronic toxicity studies of loratadine 115


12.1.2. Material and method 115

12.1.2.1. Drugs and auxiliary 115

12.1.2.2. Experimental animals 115 12.1.2.3. Experimental mehods 116

12.1.3. Results and discussions 117

12.1.3.1. Evaluation of eating behavior and body weight 117 12.1.3.2. Macroscopic and microscopic evaluation of harvested organs 119


12.2. Studies regarding H1 antihistamines influence on the behavior and skeletal muscle in mice 126

12.2.1. Introductions 126

12.2.2. Material and methods 126

12.2.2.1. Joulou-Courvoisier test (traction test) 126

12.2.2.2. Rotation axis test 127

12.2.2.3. Foot-print test 128 12.2.2.4. Boissier test 128

12.2.2.5. Hypotonic action on skeletal muscle in mice 128



12.2.2.6. Analysis and interpretation of experimental data 129 12.2.3. Test results after administration of H1 antihistamines 130

12.2.3.1. Results – Chlorpheniramine 130

12.2.3.2. Results – Cetirizine 133 12.2.3.3. Results – Loratadine 135

12.2.3.4. Results – Desloratadine 138

12.2.4. Discussions 139 12.2.4.1. Chlorpheniramine 139

12.2.4.2. Cetirizine 140

12.2.4.3. Loratadine and desloratadine 141


12.3. In vitro studies for evaluation of H1 antihistamines action on vascular reactivity 144


12.3.2. Material and method 144 12.3.2.1. Drugs and auxiliary 144

12.3.2.2. Experimental animals 145

12.3.2.3. Experimental methods 145 12.3.3. Results and discussions 145

12.3.3.1. H1 antihistamines effects on tracheobronchial muscle 145

12.3.3.2. H1 antihistamines effects on smooth muscle - thoracic aorta 150 12.3.3.3. H1 antihistamines effects on digestive tract muscles 153


CHAPTER 13 155

GENERAL CONCLUSIONS 155

CHAPTER 14 157

ORIGINAL CONTRIBUTIONS. RESEARCH PERSPECTIVE 157

14.1. Original contributions 157 14.2. Research perspective 157

REFERENCES 158

APPENDICES 167

Appendices 1 167 Ethics Commission Research Agreement 167

“Sf. Maria” Clinical Emergency Hospital for Children Agreement 169

Informed consent 171 Appendices 2 173

List of published works 173

2.1. Papers published in ISI journals 173 2.2. Papers published in B+ journals indexed 173

2.3. Papers presented at national and international congresses and symposia 174



ABBREVIATIONS

ACN Acetonitrile

ALT Alanine aminotransferase AMPc Adenosine monophosphate

AST Aspartate aminotransferase

ATP Adenosine triphosphate C Separation column

CMC-Na Sodium carboxymethylcellulose

CP Tablet CP FILM ELIB MODIF Modified release tablet

CP ORODISP Dispersible tablets

CPDA Citrate / Phosphate / Dextrose / Adenine CPS Capsule

CTZ CetirizinE

CV Coefficient of variation D Flow

DA Adult dose

DA50 50% activity level DAD Diode array detector

DAM50 Atrophy myocardial dose for activity level of 50%

DCI International Common Name DH50 Hypotonic dose level for activity level of 50%

DMSO Dimethylsulfoxide DSL Desloratadine

E Elution

ECF-A Eosinophilic chemotactic factors of anaphylaxis EMP Maximum possible effect

ES standard error

ESI Electrospray Ionisation Fc Calculated factor

Fcrit Critic factor

Fdet Determined factor

FM Mobile phase

Ft Theoretical factor

GC Gas Chromatography GDP Guanosine diphosphate

GTP Guanosine triphosphate

HPLC-UV High performance liquid chromatography with UV detection IgE Immunoglobulin E

IL Interleukin

IMAO Moniamine oxidase inhibitors ITS Specific immunotherapy

IUPAC International Union of Pure and Applied Chemistry

LC-MS/MS Liquid chromatography with tandem mass spectrometric detection

LIDC Lower limit of quantification

LIDD Lower limit of detection LIOF Lyophilized



LMD Minimum detection limit LOD Detection limit

LOQ Limit of quantification

LOR Loratadine LSDC Upper limit of quantification

m.c. Bodyweight

MeOH Methanol MS Mass spectrometer

NANC Non-adrenergic non-cholinergic

NF Nucleus fastigiata

NI Nucleus interposed

NP-HPLC High performance liquid chromatography on normal phase p Statistical probability of the null hypothesis

PAF Platelet activating factor

pic oft Eye drops pKa Dissociation constant

QTOF Quadrupole time of flight

RP-HPLC High performance liquid chromatography with Reverse phase rpm Rotations per minute

RSD Relative standard deviations

Rt Retention time SCE Electrical field stimulation

SD Standard deviation

SI Internal standard SKH Krebs Henseleit Solution

SM Mass spectrometry

sol Solution SPE Solid phase extraction

SRS-A Slow reactive substance of anaphylaxis

SSF Physiological saline solution T1/2 Half-life

TA Analysis time

Th1 T helper cell type 1

Th2 T helper cells type 2

TSLP Thymic stromal lymphopoietin



1

CHAPTER 7

MOTIVATION FOR CHOOSING THE THEME, AIM AND

OBJECTIVES

Allergies represents a major health issue in modern

industrialized societies and throughout the world. In recent decades,

epidemiological data have reported an increased incidence of allergic

diseases by 10-30% of the affected population. Allergic disease is the

most common chronic illnesses in developed countries and their

incidence and prevalence increase in industrialized countries,

resulting in significant morbidity and loss of quality of life in affected

individuals.

Treatment of allergies has been revolutionized by

introducing in therapy of the H1 antihistamines. Second generation H1

antihistamines are very effective in the treatment of allergic diseases,

including seasonal and perennial allergic rhinitis, allergic rashes and

dermatitis. H1 antihistamines are a heterogeneous group of

compounds with significantly different chemical structures with a

spectrum of antihistaminic properties, half-life, tissue distribution,

different metabolism and various degrees of anti-inflammatory

effects.

New generation H1 antihistamines are administered with or

without a prescription in most countries in Europe. These are among

the most frequently prescribed drugs, also among the safest in the

world, but not without side effects. They can be selected and used

improperly and may become a source of morbidity.

From this perspective, the main purpose of personal research

was the analysis of chemical-toxicological and clinical of the most

commonly used antihistaminic compounds for the treatment of

allergic disorders.

This paper has proposed a comprehensive epidemiological

and experimental study, to answer some topical questions regarding

safety concern of H1 antihistaminic compounds from first and second

generation.

The objectives that were the basis achievement of personal

research were:



2

Statistical study on the incidence of allergic manifestations,

of the consumption of H1 antihistamines and of adverse reactions

frequency following administration of H1 antihistamine compounds.

Part of the objectives of this work was focused on analytical

toxicology studies: development of advanced analytical

methods to determine plasma levels of drug substances and

metabolites useful in clinical toxicology laboratories or the

forensic medicine. For reliable results, it is essential that

used bioanalytical methods to be completely validated and

documented. Full validation of a method is required

regardless of whether it is new or if it is based on data from

the literature.

Qualitative (detection) and quantitative (quantify) analysis

of H1 antihistamines substances proposed in this thesis are subsequent

researchers to continuous improvement efforts, bringing new, more

sensitive, faster, better and more precise:

Development and validation of an RP-HPLC-UV method

for the quantitative determination of loratadine in

pharmaceuticals.

Development and validation of an analytical method for the

determination of loratadine, its major metabolite,

desloratadine and cetirizine in human plasma by LC-MS /

MS.

Simultaneous quantitative determination of loratadine,

cetirizine and desloratadine from biological samples by

UPLC -QTOF / MS method (development and validation of

UPLC-QTOF / MS method quantification of H1

antihistamines and optimization of extraction method).

Pharmaco-toxicological evaluation of H1 antihistamine

compounds from first and second generation by in vivo and in vitro

studies:

Experimental research of chronic toxicity

following po administration of loratadine, in Swiss-type



3

mice, male (behavioral observations, histological analysis of

heart, stomach, liver and kidney sections).

Experimental research for acute toxicity of antihistaminic

compounds using in vivo studies by ataxia testing,

supplemented by complementary motility tests / agility.

In vitro experimental research of effects on vascular

reactivity of H1 antihistamines circular sections of the aorta,

trachea, bronchia, harvested from Wistar rat, male. This

study was aimed to identify the organs on which H1

antihistamines substances could have toxic effects.

CHAPTER 8

INCIDENCE OF ALLERGIC DISEASES AND H1

ANTIHISTAMINES CONSUMPTION

8.1. Statistical study on the incidence of allergic diseases

and consumption of H1 antihistamines in patients from “Atopia”

Medical Center

A clinical and pharmacotoxicological retrospective trial was

conducted, on a mixed group of 365 patients diagnosed and treated at

"Atopia" Medical Center for a period of one year (2012-2013).

Allergic disease diagnosis was made after clinical history and allergy

skin tests performed.

Analyzed group includes 365 patients of which 64% women

and 36% men (Fig. 8.1). Patients with allergic diseases registered at

"Atopia" Medical Center come from urban areas (82%) and rural

(18%) (Fig. 8.2). Patient age was between 2 years and 90 years, the

average age was 35.98 years lot of at women 39.55 years and 29.74

years for men.



4

Fig. 8.1. Gender distrinution of

patients

Fig. 8.2. The distribution of patients

in relation to the area of origin

The prevalence of allergic diseases in the studied group is

shown in figure 8.4.

Fig. 8.4. The prevalence of allergic diseases

In the studied group (365 patients) the prevalcence of

allergic diseases has been urticaria (21%), allergic bronchitis (14%),

allergic rhinoconjunctivitis (14%), allergic asthma (12%) and other

allergic symptoms (19%).

I followed the incidence of major adverse effects noted in

the literature also. Of all patients (365) receiving levocetirizine or

desloratadine (5 mg / day) a percentage of 2.74% experienced side

effects (dry mouth, headache, fatigue, drowsiness, fatigue and muscle

pain). We noted in a 61 years patient, old female, urban diagnosed

with bronchitis and allergic rhinoconjunctivitis, the following side

36%

64%

Bărbaţi

Femei

82%

18%

Urban

Rural

12%

21%

14%14%

39% Astm bronşic alergicUrticarieBronşita alergicăRinoconjunctivita alergicăAlte manifestări alergice



5

effects: fatigue, headache, myalgia, following administration of

desloratadine (5 mg / day).

8.2. Statistical study on the incidence of allergic diseases

and consumption of H1 antihistamines in patients from “Sf.

Maria” Clinical Emergency Hospital for Children

The study objectives were:

• The statistical study on the prevalence of allergic diseases in

Moldova area for 2010-2014.

• Assessment of demographic and clinical characteristics of children

with allergic diseases hospitalized in the Clinic of Allergology and

Immunology from “Sf. Maria” Clinical Emergency Hospital for

Children.

• The frequency of adverse reactions following treatment with

antiallergic medication.

The group included 1567 patients aged 0 to 18, for a period

of 5 years, between 2010-2014.

After statistical processing, in the period 2010-2014 it

appears that for all five studied years, the most children from the study

group were predominantly presented allergic rhinitis, atopic

dermatitis and allergic asthma. Contact dermatitis and other

unspecified allergic are cases fewest recorded each year throughout

the studied period (Fig. 8.8).

Fig. 8.8. Distribution of the number of cases in 2010-2014 period

159

179

139

80

98

41

7562

36

121130

149

116

101110

23 23 20 21 179 3 10 9 35

2010 2011 2012 2013 2014

Rinită alergică Astm bronșic alergic

Dermatită atopică Dermatită alergică de contact

Alergie nespecificată



6

Figure 8.10 characterized the study group by age: 189

patients (49.5%) were aged 0-4 years, 108 patients (28.3%) were 5-9

years old, 44 patients (11.5 %) 10-14 years old and 41 patients

(10.7%) 15-18 years old.

Fig. 8.10. Percentage distribution by age

The correlation regarding average age and the incidence

of allergic disorders

Using t test, it was calculated the average age for each

disease: those with drug allergy 4.58 (SD = 4.583; p = 0.413), for

allergy unspecified 15.54 (SD = 1.197; p < 0.001); 14 for the

dermatitis due to the ingestion of food (SD = 0; p = 0.003); 1.82 atopic

dermatitis (SD = 2.678; p < 0.001); 13 for the non-specific dermatitis

(SD = 0.707; p < 0.001); 6.23 for allergic rhinitis (SD = 3.319; p =

0.193).

The average age for the 98 patients who suffered from

allergy as the primary diagnosis, had a statistically significant average

age of 4.69 (SD = 3.873; p = 0.021).

The average age assessed by gender, did not reach statistical

significance, the difference is not large: 5.54 for males (SD = 5.108;

p = 0.546) compared to 5.85 for women (SD = 4.687; p = 0.546).

The correlation on the incidence of symptoms of allergic

diseases and sex of patients

Table 8.5. presented the percentage of allergic

manifestations by the patient's sex.

49,5 %

28,3 %

11,5 %

10,7 %

0-4 ani

5-9 ani

10-14 ani

15-18 ani



7

Table. 8.5. The prevalence of allergic manifestations depending on the sex

of the study group

Unspecified

allergy

Contact

dermatitis -

ingestion of food

Contact

dermatitis -

unspecified

reason

Atopic

dermatitis

Allergic

asthma

Allergic

rhinitis

Drug

allergy

M

(%)

F

(%)

M

(%)

F

(%)

M

(%)

F

(%)

M

(%)

F

(%)

M

(%)

F

(%)

M

(%)

F

(%)

M

(%)

F

(%)

10,5 7,6 1 0,6 2,4 4,7 31,4 25 32,4 43 27,6 25 0,5 1,2

p = 0,375 p = 0,575 p = 0,175 p = 0,102 p = 0,049 p = 0,323 p = 0,425

The most common allergic manifestations were asthma,

allergic rhinitis and atopic dermatitis. Allergic contact dermatitis,

allergies and drug allergies declared as specified observation sheets

had the lowest rate of occurrence. The frequency of allergic diseases

is relatively balanced in terms of gender, 55% of a child being male

and 45% female. A percentage of 49.5%, falling in the age group 0-4

years, 28.3% were aged 5-9 years, 11.5% between 10-14 and only

10.7% are 15-18 years old.

CHAPTER 9

ANALYSIS OF LORATADINE BY RP-HPLC-UV

9.1. Development and validation of RP-HPLC-UV

method for quantitative determination of loratadine

To determine loratadine, a method of analysis by high

performance liquid chromatography was developed, using a Agilent

Technologies type 1100 liquid chromatograph equipped with

Multidiode detector. After establishing of the optimal conditions for

analysis (stationary phase, mobile phase flow, wavelength detection),

the method was validated, pursuing the following parameters:

loratadine identification, linearity response function, linearity results,

detection limit, quantification limit, precision (system precision,

method, intermediate precision) and accuracy.



8

Fig. 9.2. Chromatogram of loratadine - standard solution and sample solution

The linearity of the response function was studied.

Response function is linear in the studied range (0.1 to 50

µg/mL, regression coefficient r2 = 0.9995).

The calibration line equation is:

Area = 34,31 x Concentration(g/mL) + 0,753

Fig. 9.4. Fitted regression line of loratadine



9

Table 9.2. Statistical data range from 0.1 to 50 µg/mL

Statistical regression calculation

Correlation coefficient ( r) 0,9997

Regression coefficient ( r2 ) 0,9995

Standard error (ES) 11,0169

Intercept 0,753

Slope 34,310

There were calculated the limit of detection (LOD = 1.06

µg/mL) and the limit of quantification (LQ = 3.21 µg / mL) by

estimation of these limits based on standard deviation and regression

slope.

To estimate the precision were determined:

injection repeatability (precision system) for a total of 10

determinations, RSD value being 0.5276%;

analysis repeatability (precision of the method) for three

independent solutions at three different concentration levels for which

the RSD value is 0.8276% with a reliable interval of the average value

in the range of 101.84% - 103.14%;

intermediate precision for three independent solutions at

three different concentration levels for which RSD value is 0.8943%.

In order to estimate the accuracy of the determined recovery

it was worked a total of three different samples at three concentration

levels (in the range 70-130%), the recovery average obtained was

103.97% on the 102.76 to 105.79% range.

9.1.4. Application of HPLC-UV method for the

quantitative determination of loratadine in pharmaceutical

products

RP-HPLC method was applied for the determination of

loratadine from three different types of tablets that are found in the

community pharmacies, each containing 10 mg of active substance.

The obtained results expressed in mg loratadine / tablet and recovery

percentage values calculated was in accordance with the European

Pharmacopoeia, 8th edition.



10

CHAPTER 10

BIOANALYTICAL METHOD FOR QUANTITATIVE

DETERMINATION OF H1 ANTIHISTAMINES IN HUMAN

PLASMA BY LC-MS/MS

This chapter is experimental protocol that was followed to

validate the LC-MS/MS method for determination of loratadine,

cetirizine and desloratadine from biological samples. The parameters

investigated to ensure the analytical performance and truthfulness

was selectivity, the limit of detection and quantification, area of

linearity, precision, interference, accuracy, and stability matrix effect

of analytes in biological matrix.

10.2.4. Preparation and processing of samples

10.2.4.1. Standard stock solutions

For stock standard solutions, the substances were dissolved

in methanol, the amount of each component was 10 mg.

10.2.4.2. Biological samples

Solvent extraction was carried out by a clean up procedure

consisting of trichloroacetic acid precipitation and centrifugation

using a centrifuge-type Hetrich.

10.3. Results and discussion

10.3.1. Specificity / selectivity study of the method

10.3.1.1. Specificity

The study was conducted by monitoring the specificity of

the parameters on the efficiency of separation by column

chromatography.

The analytical method differentiated the analytes

(desloratadine, loratadine and cetirizine) and internal standard from

endogenous matrix components or other components from the

sample.

In the table 10.3 are listed areas obtained from the injection

blanks and areas signals obtained by injecting samples with



11

concentrations in the upper limits of quantification, using the 6

matrix.

Table 10.3. Specificity method of LC-MS/MS method

(DSL Rt = 0,82; LOR Rt = 1,97; CTZ Rt = 1,38)

Source

PM Rt

Areas

(50 ppb)

Area

PM

C% PM

(ppb)

%

interference

(compared

with LIDC)

S1

0,82 10277 0 0,00 0,00

1,97 16314 0 0,00 0,00

1,38 29051 0 0,00 0,00

S2

0,82 7130 10,9 0,048 1,936

1,97 11459 0 0,00 0,00

1,38 16605 0 0,00 0,00

S3

0,82 8356 25,36 0,262 10,460

1,97 7677 0 0,00 0,00

1,38 19934 1,41 0,000 0,004

S4

0,82 5334 10,3 0,043 1,729

1,97 11640 0 0,00 0,00

1,38 21070 6,34 0,002 0,071

S5

0,82 7130 0 0,00 0,00

1,97 11459 0 0,00 0,00

1,38 16605 9,98 0,004 0,175

S6

0,82 7130 0 0,00 0,00

1,97 11459 0 0,00 0,00

1,38 16605 9,5 0,004 0,159

SI

S1 1,3 254157 118 0,017 0,674

S2 1,3 289388 0 0,00 0,00

S3 1,3 496217 498 0,071 2,846

S4 1,3 579197 0 0,00 0,00

S5 1,3 545449 619 0,088 3,537

S6 1,3 421914 141 0,020 0,806



12

Representative chromatograms are shown in figure 10.1.

Fig. 10.1. Sample at the upper limit of quantification

(desloratadine 311–>259, loratadine 383 –> 337, cetirizine 389 –>201)



13

10.3.3. Liniarity

The regression coefficient determined when designing

concentrations depending the areas on linearity range 2.5 to 150 had

values of 0.9911, 0.9949 and 0.9953 for desloratadine, loratadine and

cetirizine respectively.

Correlogram (right calibration) for each compound (Fig.

10.6 - 10.8) shows a linear relation between sample concentration and

peak area, thus predicting peaks area will be done using linear

regression equation:

Fig. 10.6. Correlogram corresponding to desloratadine tests

Fig. 10.7. Correlogram corresponding to loratadine tests



14

Fig. 10.8. Correlogram corresponding to cetirizine tests

10.4. Application of LC-MS/MS method for quantitative

determination of loratadine, desloratadine and cetirizine in

human plasma

Method LC MS / MS developed and validated was tested in

patients who received treatment with H1 antihistamines to allergic

manifestations different method LC-MS / MS validated.

For the determination of plasma levels of loratadine,

desloratadine and cetirizine, the biological sample was processed

according to the method, and then analyzed by LC-MS / MS. The

peak areas were determined corresponding compounds, the areas of

the internal standard, amitriptyline and using calibration curves

determined from the linearity study, concentrations were calculated.

Amitriptiline (SI) Desloratadine

Loratadine Cetirizine

Fig. 10.18. The chromatograms of extracted compounds from human

plasma samples



15

Table 10.17 presents the results obtained: plasma

concentrations calculated the minimum, maximum and average

values of loratadine, cetirizine and desloratadine concentrations.

Table 10.17. Concentrations of loratadine, desloratadine and cetirizine

in human plasma

Compound

Patient data Plasmatic

C% (µg/L)

Average

plasmatic

C% (µg/L)

C%

min

(µg/L)

C%

max

(µg/L) Gender

(M/F) Age

DSL

F 23 4,72

5,51 4,72 6,66

F 25 5,40

M 30 5,86

F 28 4,94

F 60 6,66

LOR

M 31 6,09

5,17 3,84 6,09

F 28 5,67

F 23 3,84

F 28 4,97

M 60 5,26

CTZ

F 23 27,70

23,05 16,74 27,70

M 30 25,54

F 61 21,03

F 29 24,25

F 29 16,74

Determined concentrations in human plasma at one hour

after administration of therapeutic doses of the 3 antihistamines,

ranged from: 4.72 to 6.66 µg / L to desloratadine, from 3.84 to 6.09

µg / L to loratadine and 16.74 to 27.70 µg / L to cetirizine.

The results are comparable to those from reported studies of

kinetic methods from literature.

Conclusion

The main characteristics evaluated of the LC-MS / MS

method, to ensuring acceptability and truthfulness of analytical

results were: selectivity, linearity of response, lower limit of

quantification, the matrix effect, accuracy, precision, stability of the

analytes in biological matrix. Thus:



16

Chromatographic separation was performed on a column

(2,1 mm x 50 mm, 1,9 µm), using a mobile phase consisting of FMA

(formic acid solution) and FMB (acetonitrile with 0.1% formic acid),

isocrate elution (65:35), with a flow of 0,25 mL/min;

The injected volume was 10 L;

Detection was carried out by MS/MS.

Selectivity study was conducted by investigating the

interference due metabolites, and degradation compounds due to

concomitant administration.

Linearity of response was studied. Correlogram are linear

in the studied range (2,5 - 120 g/L), regression coefficients

determined were 0,9911 (DSL), 0,9949 (LOR), 0,9953 (CTZ).

There were calculated lower limits of quantification (3,41

µg/L – DSL; 3,48 – LOR; 4,6 µg/L – CTZ) and lower detection

limits (1,13 µg/L – DSL; 1,15 – LOR; 1,52 µg/L – CTZ), the

estimation being performed with the standard deviation and

regression slope.

The results obtained from the quantitative determination of

loratadine, desloratadine and cetirizine from human plasma,

argue that the LC-MS / MS method may be used for determining

the therapeutic range of H1 antihistaminic compounds or in the

event of overdose.

CHAPTER 11

SIMULTANEOUS QUANTITATIVE DETERMINATION OF

LORATADINE, DESLORATADINE AND CETIRIZINE

FROM BIOLOGICAL SAMPLES BY

UPLC-QTOF/MS METHOD

The experimental research described in this chapter were

conducted in the Department of Analytical Chemistry of the Faculty

of Chemistry in Seville, Spain.

The purpose of the study was to develop and validate for the

first time, an analytical UPLC-QTOF / MS method for simple and

rapid determination of second generation H1 antihistamines,

loratadine, cetirizine and desloratadine from human plasma.



17

11.3. Results and discussion

11.3.1. Optimisation of simultaneous cuantification

method of loratadine, desloratadine and cetirizine by UPLC-

QTOF/MS

The optimization method of determining the H1

antihistaminic compounds was based on the data available in the

literature regarding the selected column, the type of elution to obtain

an effective separation of the substances and as high as possible

intensity signals.

The substances were identified based on the accurate mass.

ESI + parameters have been established for the determination of

loratadine, desloratadine and cetirizine, and the identification was

carried out based on the accurate mass (m / z) that are highlighted in

the MS spectra (Fig. 11.2).

Fig. 11.2. MS spectra of cetirizine (a), desloratadine (b) and

loratadine (c) - 100 µg/L

Figure 11.3 shows the chromatograms obtained from the

analysis of a 100 µg/L concentration solution of the three compounds.

a

)

b

)

c

)



18

Fig. 11.3. Chromatograms for loratadine (a), desloratadine (b)

and cetirizine (c) - 100 µg/L

11.3.2. Validation of UPLC-QTOF/MS method for

loratadine, desloratadine and cetirizine dosing in human plasma

According to the guideline validation of analytical methods

were studied parameters such as selectivity / specificity, linearity,

limit of detection and quantification limits, precision, accuracy.

11.3.2.1. Specificity of the method

The selectivity of the chromatographic method for separated

compounds is carried out by QTOF / MS detection allowing accurate

determination of exact mass (m / z) of each analyte, as shown in

Figure 11.3.

11.3.2.2. Linearity and linearity range

Preparation of stock solutions: Stock solutions of loratadine,

desloratadine and cetirizine were prepared in methanol at a

concentration of 100 mg / L (100 ppm) .Any dilution that occurs in

the preparation of the standard solutions is carried out with MilliQ

grade water. The linearity of the method has been demonstrated by

graphic visualization of the areas variation with concentrations and

a

b

)

c

)



19

the concentration range for which the variation is linear was

determined.

Figures 11.4 – 11.6 presented the calibration curve obtained

for linearity study of quantitative determination method of loratadine,

desloratadine and cetirizine on the area of work.

Fig. 11.4. Calibration curve of loratadine by

UPLC-QTOF/MS

Fig. 11.5. Calibration curve of desloratadine by

UPLC-QTOF/MS

Fig. 11.6. Calibration curve of cetirizine by

UPLC-QTOF/MS



20

11.3.2.3. Detection limit and quantification limit

The following values were calculated:

- for loratadine: LOD = 5,33 µg/L; LOQ = 15,75 µg/L,

- for desloratadine: LOD = 4,43 µg/L; LOQ = 14,77 µg/L,

- for cetirizine: LOD = 6,66 µg/L; LOQ = 22,2 µg/L.

11.5. Aplications of UPLC-QTOF/MS method for

simultaneous quantitative determination of loratadine,

desloratadine and cetirizine in human plasma samples

11.5.1. Optimization method for extraction of H1

antihistamines

Figure 11.7 described in detail the optimized extraction

method of loratadine, cetirizine and desloratadine in plasma samples:

Fig. 11.7. Extraction scheme of loratadine, desloratadine and cetirizine

0,5 mL plasma

+

1 mL acetonitrile

|

stirring

|

centrifugation 15 min, 9000 rpm

|

taking supernatant

|

evaporation under a stream of nitrogen

|

the residue taken up with 0.25 mL purified

water|

stirring

|

centrifugation 5 min, 5000 rpm

|

microfiltration, 2 times

|

UPLC-QTOF/MS analysis



21

11.5.2. Reproductibily of the extraction method

The method of simultaneous extraction of loratadine,

desloratadine and cetirizine in human plasma samples were checked

for reproducibility.

Table 11.10. The results of reproducibility extraction from plasma samples

Theoretical

plasma

concentration

(µg/L )

Peak

area

Recovered

concentration

(µg/L )

Recovery

(%)

LORATADINE

10 12963,9 10,8733824 108,7338

10 12747 10,6913887 106,9139

50 41100,8 34,4821254 68,96425

50 37469,6 31,4353055 62,87061

250 201502,6 169,069976 67,62799

250 184184,6 154,539014 61,81561

Statistical data

Media 79,48769

SD 22,124

RSD% 27,83324

DESLORATADINE

10 4438,3 9,031366 90,31366

10 3949,9 7,907444 79,07444

50 15646,9 34,82495 69,64989

50 15647,5 34,82633 69,65265

250 125653,9 287,9765 115,1906

250 123740,9 283,5742 113,4297

Statistical data

Media 89,55182

SD 20,63937

RSD% 23,04741

CETIRIZINE

10 6874,9 11,29525 112,9525

10 6799,3 11,10665 111,0665

50 21938,1 48,87339 97,74679

50 21537 47,87277 95,74554

250 100066,7 243,7807 97,51229

250 107110,2 261,3521 104,5409

Statistical data

Media 103,2607

SD 7,435384

RSD% 7,200592



22

Samples were prepared from 0.5 mL of spiked plasma with

small volumes of stock solutions of the three compounds at three

different concentration levels according to the protocol described

above. Each sample was chromatographic determined, finally, the

percent of recovery for the three compounds was calculated. Using

the peaks area, the recovered concentrations and recovery

percentages were calculated from the equations of regression curve

(table 11.10).

CHAPTER 12

PHARMACO-TOXICOLOGICAL EVALUATION OF

H1 ANTIHISTAMINES BY IN VIVO / IN VITRO STUDIES

12.1. Chronic toxicity studies of loratadine

The aim of this study was to evaluate in experimental

conditions, pharmacotoxicological potential of loratadine, a second

generation antihistamine, based on data presented in the literature

regarding the liver, renal or cardiovascular toxic impairment after

loratadine receiving.

In this study it was administered in mice a single-dose of

loratadine in geometric progression, between 5-20 mg / kg for 28

days, and it was followed the assessment of chronic toxicity in mice

by eating habits and body weight evaluating, macroscopic and

microscopic evaluation of harvested organs at the end of treatment.

12.1.3.1. Evaluation of eating behavior and body weight

Experimental data for assessing eating behavior and body

weight of the animal groups from the study (day I of treatment and

after 28 days of treatment) have been accumulated, recorded,

analyzed as averages and represented in tables 12.1 - 12.2 and figures

12.1 - 12.2.



23

Fig. 12.1. Evaluation of body weight and feeding behavior after day I

treatment

Fig. 12.2. Evaluation of body weight and feeding behavior after 28 days of

treatment

12.1.3.2. Macroscopic and microscopic evaluation of

harvested organs

By anatomopathological studies was performed

histopathological examination of liver, kidney, heart and stomach

samples harvested by microsurgical technique from experience

0

510

15

20

25

30

35

Martor LOR 20

mg

LOR 10

mg

LOR 5

mg

Consum apă Consum hrană Greutate

0

10

20

30

40

consum apa consum hrana greutate

Martor Lor 20 mg lor 10 mg lor 5 mg



24

animals (Swiss white mice), weight 18-30 g, which received doses of

loratadine in geometric progression, orally, for a period of 28 days.

A B

C D

E F

G H

Fig. 12.6. Anatomopathological aspects in mice which received doses of 20 mg/kg

body weight / day

(A) heart (hematoxylin - eosin, x 200); (B) liver (hematoxylin - eosin, x 200); (C)

liver (hematoxylin - eosin, x 200); (D) liver (hematoxylin - eosin, x 400); (E)

liver (hematoxylin - eosin, x 200); (F, G) liver (hematoxylin - eosin, x 200); (H) stomach (hematoxylin - eosin, x 100)



25

The anatomopathological analysis of organs harvested from

mice that received a dose of 20 mg/kg body weight daily in a single

dose showed suggestive liver toxicity issues. Figure 12.6 B are

highlighted impaired liver areas, with congestion, centrilobular

hepatic vein stasis, port space and sinusoidal. Figure 12.6 C are

highlighted the congestion infiltrate bleeding, presence of red blood

cells in sinusoid, centrilobular stasis in large vein, with no

inflammation and microvesicular steatosis. Kidney section showed in

Figure 12.6 F shows small areas of tubular atrophy, such as in Figure

12.6 G and vacuolation in the tubular cells. The stomach was

examined for all the samples harvested for this dose and no

pathological changes were characterized, as well as the other doses.

12.1.4. Conclusions

During the 28 days of loratadine administration in Swiss-

mice, it was evaluated the eating behavior and weight body influence.

Deviations in weight between groups were small (4-18%) and it can

be interpreted as physiological. Thus, loratadine does not influence

the feeding behavior. On histopathological analysis, have been

observed:

The phenomenon of stasis, vasodilatation in all veins.

Gastric Impaired was slightly evidenced by the small portions of

inflammatory infiltrates in the submucosal esogastric passage.

Isolated cases of ischemic myocardic suffering was illustrated by

alternating of small myocardial cells anucleate with large

myocardial fibers.

Kidney impairment was poor.

Liver toxicity was the most pronounced, but it were not

highlighted several portions of hepatic necrosis, rarely described

in the literature also. A single slightly cell liver necrosis was

observed to be at a dose of 5 mg / kg body weight per day.

Clear correlations regarding relations between the doses

administered and gastric, kidney or heart toxicity could not made.

It can be said, however, that the liver toxicity is greater at the 20

mg / kg body weight per day, which is argued by the appearance

of hepatic steatosis more pronounced at this dose.



26

12.2. Studies regarding H1 antihistamines influence on

the behavior and skeletal muscle in mice

The objective of this study is to analyze , using in vivo

experimental conditions, the pharmacotoxicological potential of

some first and second generation H1 antihistamines to Swiss mice,

due to the fact that the Ist generation presents, in addition of

antiallergic activity, based on blocking of the histamine receptors,

other actions that are not related to this mechanism, such as influence

on the nervous system. Another reason that led to such a study was

linked to the controversial appearance in pharmaceutical products

prospectuses with second generation antihistamines containing, as

they are / are not or are relatively sedatives.

In this study single and repeated dose have performed for

chosen substances work the following sets of experiments were

performed: evaluation of the behavior spontaneously, motor

coordination and hypotonic action on skeletal muscle, after taking H1

antihistamines using: Joulou-Courvoisier test, shaft rotation test,

footprint test, Boissier test and Fleury Frommel technique.

12.2.3. Test results after administration of H1

antihistamines

12.2.3.2. Results – Cetirizine

Foot-print test results for cetirizine are represented in figures

12.17 – 12.20.

Fig. 12.17. Foot-prin test – left step variation at the 3 doses of cetirizine

co

ntr

ol-

pas s

tg

Cet-

28 m

g

Cet-

14 m

g

Cet-

7 m

g

0

2

4

6

8

lun

gim

e/c

m

co

ntr

ol-

pas s

tg

Cet-

28 m

g

Cet-

14 m

g

Cet-

7 m

g

0

2

4

6

8

1 0



27

Fig. 12.18. Foot-prin test – right step variation at the 3 doses of cetirizină

Fig. 12.19. Foot-prin test – back support base variation at the 3 doses of

cetirizine

Fig. 12.20. Foot-prin test – front support base variation at the 3 doses of

cetirizine

Doses of 7 mg/kg and 14 mg/kg present statistic

semnification (p = 0,0007; t = 4,852; df = 10, for 14 mg/kg dosis and

p < 0,0001; t = 9,167; df = 10, for 7 mg/kg dosis).

lun

gim

e/c

m

co

ntr

ol-

pas d

r

Cet-

28 m

g

Cet-

14 m

g

Cet-

7 m

g

0

2

4

6

8

lun

gim

e/c

m

co

ntr

ol-

pas d

r

Cet-

28 m

g

Cet-

14 m

g

Cet-

7 m

g

4

5

6

7

8

lati

me

/cm

co

ntr

ol-

baza s

pate

cet-

28

Cet-

14

Cet-

7

0

1

2

3

4

lati

me

/cm

co

ntr

ol-

baza s

pate

cet-

28

Cet-

14

Cet-

7

1 .5

2 .0

2 .5

3 .0

3 .5

co

ntr

ol-

baza f

ata

Cet-

28

Cet-

14

Cet-

7

0 .0

0 .5

1 .0

1 .5

2 .0

2 .5

lati

me

/cm

co

ntr

ol-

baza f

ata

Cet-

28

Cet-

14

Cet-

7

1 .2

1 .4

1 .6

1 .8

2 .0

2 .2

2 .4

* *

* *

* *



28

- Cetirizine study results show a correlation between dose

and time of ataxia installation. Animals exhibited agitation, tremor,

hyperexcitability and paradoxical phenomena of muscle strength

increased to 120 minutes.

The foot print test for cetirizine showed a tendency to

noticeably laterality, shorter visible steps, particularly to the right.

Thus, we can say that cetirizine had a possible effect of the nigro-

striatal receptor type dystonic reactions or laterality.

- Experimental data show an ataxia installation time for

chlorpheniramine 83.97 minutes statistically significant for 20 mg

dose and 50 minutes for a dose of 5 mg. It could not made a dose-

effect relationship for the activity level of 50%, which suggests the

placing of this side effect as a secondary toxicodynamics mechanism

for studied the therapeutic range.

Referring to the action on skeletal muscle hypotonic dose for

level of activity of 50% (DH50) was determined. Statistical

parameters argue the graded dose-effect relationship, which define

this side effect as toxic dose-dependent type, which involve

toxicodynamics mechanism.

Step lengths variation between the left and right observed in

the footprint test to chlorpheniramine, are markers of right left failure

coordination, a clear sign of neuronal intoxication.

- Analysis of regression curve of loratadine demonstrated

with statistical significance that the range of occurrence of ataxia

phenomena is located between 83.94 to 107 minutes. DA50 values

were calculated for all the study periods: 30, 75, 120 minutes.

For desloratadine, the estimated value for DA50 is 12.8 mg

/ kg 75 minutes. This value supports the hypothesis that the DA50

value calculated for loratadine at 30 minutes is due to its effect and

not due to its metabolite.

Loratadine can be considered to have the lowest systemic

toxic effect of antihistamines substances studied. Loratadine had the

fewest central effects compared with other substances.



29

12.3. In vitro studies for evaluation of H1 antihistamines

action on vascular reactivity

In view of the utility, frequency of use and the risk of

intoxication presented by substances antihistamines, it has been

desired to investigate the effects of vascular and visceral smooth

muscle. For all the in vitro experiments were used Wistar adult male

rats, weighing 180-220 g, kept under the same conditions as used in

previous experiments mice.

12.3.3.1. H1 antihistamines effects on tracheobronchial

muscle

The preparations used in this study were lower rat tracheal

rings or rings of main bronchia. After sacrifice and exsanguination

the trachea preparations were taken from the chest and placed in

Krebs Henseleit serum. After 5-10 minutes, when the preparation has

reached room temperature, the tracheo-bronchial tree was dissected

out, cleaned of connective tissue and tracheal rings were assembled

in organ baths.

1. Has the test substance per se effect (contracting or

relaxing) on smooth muscle preparation?

Fig. 12.30. The effect of per se administration of the H1-antihistamines

substances on the tracheal ring into the bath

Preparations showed no muscular constriction effects on

tracheal ring preparation, which is in accordance with the literature.

10078.57142857 92.85714286 94.155

1 2 3 4

con

trac

ție

car

bac

ol 1

0-5

CARBACOL CETIR CLORFENIRAMIN DESLORAT



30

2. Has the studied substance relaxing effects on a

precontracted preparation?

There was used a mioconstrictor compound, different for

each type of organ and the receptors that are found at this level. For

tracheal smooth muscle, cholinergic stimulation was carried out with

carbachol. Increasing doses administered of carbachol cumulative

allowed tracing a curve-effect dose for tracheal smooth muscle.

Routinely used doses ranged from 10-9 M to 10-5 M, in which was

obtained the maximum contractile effect.

Working substances used were cetirizine, loratadine and

chlorpheniramine. Their administration was performed in the same

sequence as carbachol dose.

Fig. 12.31. The effect of H1 antihistamine agents on carbachol-induced

contraction of trachea-rings

As can be seen from Figure 12.31, the administration of H1

antihistamine agents with cholinergic contraction produced slight

inhibitory effects on it.

In conclusion, on the tracheal ring preparation,

administration of H1 antihistaminic agents doesn’t have any

significant effect, results that are according with the literature, even

if the anticholinergic effect of these agents is known.

Working technique using electrical field stimulation (EFS)

Electrical stimulation has as its essential mechanism

depolarization of the nerve endings present in the smooth muscle of

the organ under study.

0

20

40

60

80

100

120

5 6 7 8 9 10%

co

ntr

acți

e ca

rbac

ol

log doza carbacolcarb cetir



31

The formulation was being tested. After equilibration a

frequency response curve (FRC) was drawn. Each administration

used the initially FRC as a witness effect and the effect was measured

in milligrams / force.

Fig. 12.33. The effect of 10-6 M loratadine administration on EFS

contraction (p = 0,0392; R2 = 0,6038)

As shown in fig. 12.33, loratadine significantly inhibited the

contraction induced by electrical stimulation of bronchial smooth

muscle. This result is in accordance with the literature. Unlike

loratadine, chlorpheniramine administration of 10-6 M produced

slight increase in contractility effects, even under conditions of non-

electrical or pharmacological stimulation had no effect at any of the

doses. This increase in contractility was not statistically significant

due to the variability of results. This effect is possible characteristic

of chlorpheniramine, but further studies are needed to clarify the

causes (fig. 12.34).

Fig. 12.34. The effect of chlorpheniramine on the contraction induced by

electrical stimulation (p = 0,3567; R2 = 0,3305)

Regarding cetirizine, the results show the same increase in

F re c v e n ta s t im u la r e (H z )

% c

on

tra

cti

e E

FS

7 0

8 0

9 0

1 0 0

1 1 0

2 4 8 1 2 1 6

F re c v e n ta s t im u la re (H z )

% c

on

tra

cti

e E

FS

2 4 8 1 2 1 6

6 0

8 0

1 0 0

1 2 0

1 4 0

1 6 0


% c

on

tra

cti

e E

FS

2 4 812

16

0

5 0

1 0 0

1 5 0



32

contractility to electrical stimulation, but variations on the averages

groups working are not large enough to achieve statistical

significance (fig. 12.35).

Fig 12.35. The effect of cetirizine on contraction induced by electrical

stimulation

12.3.4. Conclusions

Conclusions on the effect of the administration of H1 receptor

blocking agents on the tracheobronchial smooth muscle:

Cetirizine seems to have the largest musculoskeletal relaxant per

se effect. Cetirizine affinity for H1 receptor is approximately 500 times

greater than the affinity for the H2 and H3.

Conclusions regarding the effect of the administration of H1

receptor blocking agents on the aorta:

No significant results were obtained following the administration

of loratadine, cetirizine and chlorpheniramine, which shows that

histamine mediator, at least in rat aorta does not interfere with

adrenergic mediating.

Conclusions regarding the effect of the administration of H1

receptor blocking agents over the muscles of the digestive tract:

The presented results confirm the hypothesis that at least at the

cholinergic mediation of digestive tract H1 antihistamines substances

and H1 receptor blockade does not interfere by pharmacodynamic

mechanism with contractile mechanism. Although histamine is an

important mediator of inflammation at intestinal level, with no

digestive inflammation it seems that the effects are not significant.


% c

on

tra

cti

e E

FS

2 4 812

16

0

5 0

1 0 0

1 5 0

2 0 0



33

CHAPTER 13

GENERAL CONCLUSIONS

Scientific research activities of the doctoral thesis make

important contributions for the treatment of allergies that has been

revolutionized by the introduction in the therapy of H1 antihistamines.

The research covered the chemical-toxicological and clinical analysis

of the H1 antihistamine compounds commonly administered with or

without prescription in allergy therapy, disease with prevalence and

continuous rising incidence and loss of quality of life in affected

individuals.

Thus, statistical studies conducted at the "Atopia" Medical

Center, sintetized the incidence of allergic diseases: urticaria

(21%), allergic bronchitis (14%), allergic rhinoconjunctivitis

(14%), allergic asthma (12%) and other allergic manifestations

(19%). Of all patients treated with desloratadine and

levocetirizine 2.74% experienced side effects: dry mouth,

headache, fatigue, drowsiness and muscle pain.

The statistical study conducted at the "Sf. Maria" Regional

Emergency Hospital, Iasi, Department of Allergology and

Clinical Immunology, followed the same issues: the most

common allergic manifestations were allergic asthma, allergic

rhinitis and atopic dermatitis, the incidence of allergic disorders

decreased with age. The incidence of allergic asthma and allergic

rhinitis is high in children 5-9 years old, comparing 10-14 years

children.

Quantitative analysis of loratadine was performed by a simple

and rapid HPLC method with UV detection was easily applied to

determine the loratadine from pharmaceuticals.

For analytical toxicological study of new generation H1

antihistamines has been developed and validated of a LC-

MS/MS bioanalytical method for determination of loratadine, its

main metabolite, desloratadine, and cetirizine from human

plasma. Validation was done by checking the parameters of

validation and determining the lower limits of quantification and



34

lower detection limits. The method has been applied to biological

samples (human plasma) taken from patients under treatment

with loratadine, desloratadine and cetirizine. Concentrations

obtained are in agreement with the results from the similar liquid

chromatographic methods of the literature cited.

An UPLC-QTOF/MS method for simultaneous determination of

loratadine, cetirizine and desloratadine was developed and

validated for the first time being a quick and simple method, that

does not involve high costs in toxicological analysis to quantify

human plasma.

The chromatographic separation was carried out by column

chromatography Acquity UPLC BEH C18

Detection was carried out by QTOF/MS based on the exact

mass m/z 383,1526 Da (loratadine), 311,1314 Da (desloratadine)

and 389,1630 Da (cetirizine).

The method was validated being studied validation

parameters imposed by the guidelines, limits of quantification

and detection of compounds being favorable for applicability in

toxicokinetic studies.

It was optimized a simple method of extraction with

acetonitrile, the yield of 79.48% to loratadine, desloratadine

89.55% and 103.26% to cetirizine, which can be easily applied

in determining H1 antihistamines substances in human plasma.

Pharmaco-toxicological evaluation of H1 antihistamine

compounds using experimental animals addressed several lines

of research: in vivo studies for evaluation of chronic toxicity of

loratadine and influence of H1 antihistamines on behavior and

skeletal muscle in mice and in vitro studies that evaluated the

influence of H1 antihistamine compounds on rat vascular

reactivity.

Evaluation of chronic toxicity in mice after a month's time

administration, of progressive doses of loratadine was done by

evaluating eating behavior and histopathological study of

harvested organs (liver, kidney, heart, stomach) through

microsurgical technique, which highlighted: pronounced liver



35

toxicity at 20 mg / kg body weight per day, slightly gastric

impaired, isolated cases of ischemic myocardic suffering, weak

kidney damage.

Evaluation of the influence of the H antihistamines on skeletal

muscle behavior in mice was carried out by using a battery of

tests: Joulou-Courvoisier test, the axis of rotation test, the

footprint and Fleurry-Frommel technique. There has been

observed step lengths variation between the left and right

observed in the footprint test to chlorpheniramine, sign of

neuronal intoxication, egarding the action of cetirizine, animals

exhibited agitation, tremor, hyperexcitability after cetirizine

administration and a noticeably tendency to laterality, by shorter

visible steps, particularly to the right. Thus, we can say that

cetirizine had a possible effect of the nigro-striatal receptor type

dystonic reactions or laterality. Loratadine can be considered to

have the lowest toxic effect of systemic antihistamines

substances studied. She had the fewest central effects compared

with other substances.

Central toxicity of H1 antihistamines is reduced, the doses that

alter the parameters of muscle strength, locomotor behavior and

muscle strength are probably greater than the dose ranges used

in the present experiments. Footprint test is presented in the international literature for the

study of drugs that act directly on the nervous system. After

consulting the databases we noticed that using this test represents

a national premiere. Using the test to a group of substances whose

main action is not for nervous system (from both generation H1

antihistamines: chlorpheniramine, loratadine, cetirizine and

desloratadine) is a novelty factor.

For in vitro research for assessing the H1 antihistamines influence

on the vascular reactivity have been used Wistar rats, adults.

Tests were conducted using isometric transducers vertical system

(vertical model Miograf Ugo Basile) and horizontal

(Experimetria, Budapst). The effects of H1 antihistamines were

analyzed on the tracheobronchial muscle, the tracheal rings or



36

bronchia rings, effects on smooth muscle, the thoracic aorta and

effects on the digestive tract using rat terminal ileum. From the

results, cetirizine appears to have the largest musculoskeletal per

se effect. The administration of antihistamine agents produced

slight inhibitory effects of cholinergic contraction, without

reaching the level of statistical significance except for cetirizine.

Loratadine significantly inhibited the induced contraction of

bronchial smooth muscles by SCE.

CHAPTER 14

ORIGINAL CONTRIBUTIONS. RESEARCH PERSPECTIVE

14.1. Original contributions

The original elements of the thesis are highlighted by the

results obtained in experimental research.

o Chromatographic methods of analysis presented in the

doctoral research are original and provide the ability to

determine analytes in a wide range of concentrations.

The high sensitivity of the developed method is useful in

the chromatographic analysis in the field of toxicology

and toxicokinetic studies or bioequivalence when

determining very low concentrations. The methods allow

the detection of the metabolites as desloratadine and 3-

hydroxydesloratadine; these compounds can be

determined even after a period after intake of the parent

compound loratadine.

o A novelty of the thesis is the in vivo / in vitro study

pharmaco-toxicological evaluation of chlorpheniramine,

loratadine, desloratadine and cetirizine; foot-print test

conducted on experimental animals (Swiss type white

mice) for detecting ataxia phenomena manifested after

administration of H1 antihistamines.



37

14.2. Research perspective

The experimental results obtained in the doctoral studies can

be the starting point for new research directions, such as:

Concentration determination of active substances in biological

matrices such as serum, plasma, urine, is an important aspect in

characterizing a drug. Performance validated analytical methods

can be applied in toxicokinetic studies or studies on the

interaction of H1 antihistamines with other compounds.

Due to intense metabolism of loratadine, screening studies of

metabolites can be developed.

Study on evaluation of H1 antihistamine compounds on behavior

and skeletal muscle in mice open new research directions:

- Further in vivo studies with localized administration of

compounds: the cerebellum or hypothalamus, reducing the

variability that can result from crossing the blood-brain barrier,

metabolism, plasma protein binding and other toxicokinetic

characteristics.

- Studies on histamine-dopaminergic interactions at central level,

very important and interesting to watch, given the nigro-striatal

dopaminergic system involvement in neuromuscular

degenerative diseases that are currently intensively studied.



38

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Urticaria caused by antihistamines: report of 5 cases. J Investig Allergol Clin

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10. Jurawan R, Smith A. Severe hepatitis in a primary sclerosing cholangitis

patient receiving recent cetirizine therapy. N Z Med J 2010; 123: 106–107.

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Boca Raton, Fla.: CRC, 2008.

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(Claritin) in dried blood spot (DBS) samples collected by subjects in a clinical

research study. J Chromatogr B 2015; 983-984: 117–124.

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method for simultaneous quantification of loratadine and desloratadine in

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90: 384–390.

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Universitară ‘Iuliu Hatieganu’, 2002.

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2220–2235.

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List of published works

2.1. 1. Papers published in ISI journals

- principal author

1. Ioana-Cezara Grigoriu, Bogdan-Ionel Cioroiu, Anca-Monica

Strugaru, Luminiţa Agoroaei, Cristina Dehelean, Dana Stoian, Elena

Butnaru, Preliminary Impurity Profile Study of Desloratadine Used in

Toxicological Studies, Rev. Chim. (Bucharest), 2015; 66(7): 1064-1067

(ISSN 0034-7752, Factor de impact 0,81).

2. Adrian Florin Șpac, Ioana-Cezara Grigoriu*, Constantin Ciobanu,

Luminița Agoroaei, Anca Monica Strugaru, Elena Butnaru, Validation

and Application of a RP - HPLC Method with UV Detection for

Loratadine Determination, Rev. Chim. (Bucharest), 2016; 67 (6): 1227-

1231 (ISSN 0034-7752, Factor de impact 0,956).

- co-author

1. Anca-Monica Strugaru, Cornelia Mircea, Luminița Agoroaei, Gina

Botnariu, Ioana-Cezara Grigoriu, Teodora Daniela Marti, Elena

Butnaru, Quantitative Determination of Metformin by Capillary

electrophoresis with UV Detection, Rev. Chim. (Bucharest), 2015;

66(9): 1448-1451 (ISSN 0034-7752, Factor de impact 0,81).

2. Luminița Agoroaei, Nela Bibire, Mihai Apostu, Monica Strugaru, Ioana

Grigoriu, Elena Butnaru, Content of Heavy Metals in Tobacco of

Commonly Smoked Cigarettes in Romania,

Rev. Chim. (Bucharest), 2014; 65(9): 1026-1028 (ISSN 0034-7752,

Factor de impact 0,677).

2.2. 2. Papers published in B+ journals indexed

- principal author

1. Ioana-Cezara Grigoriu, Georgeta Sinițchi, Luminița Agoroaei, Anca-

Monica Strugaru, Elena Butnaru, Statistical study on the incidence of

allergic diseases treated with desloratadine and levocetirizine at

"Atopia" Allergology Medical Center, Iași, Romania, Rev. Med. Chir.

Soc. Med. Nat. (Iași), 2013; 117(4): 1021-1027 (ISSN 0048-7848).

http://www.revistadechimie.ro/pdf/GRIGORIU%20%20I.pdf%207%2015.pdf

http://www.revistadechimie.ro/pdf/GRIGORIU%20%20I.pdf%207%2015.pdf

http://www.umfiasi.ro/Cercetare/sr/Publications/Pages/sp2013.aspx

http://www.umfiasi.ro/Cercetare/sr/Publications/Pages/sp2013.aspx



41

- co-author

1. Anca-Monica Strugaru, Gina Botnariu, Luminița Agoroaei, Ioana-

Cezara Grigoriu, Elena Butnaru, Metformin Induced Lactic Acidosis –

Particularities and Course, Rev. Med. Chir. Soc. Med. Nat. (Iași), 2013;

117(4): 1035-1042 (ISSN 0048-7848).

2. Anca-Monica Strugaru, Gina Botnariu, Cristina Tuchiluș, Ecaterina

Anisie, Luminița Agoroaei, Ioana-Cezara Grigoriu, Elena Butnaru,

Low Levels of Serum Cyanocobalamin in a Metformin-Treated Patient.

Case Report and Comparison with Literature Data, Rev. Med. Chir.

Soc. Med. Nat. (Iași), 2016; 120(2): 464-468 (ISSN 0048-7848).

3. Ştefania Corina Mahu, Monica Hăncianu, Luminiţa Agoroaei, Ioana-

Cezara Grigoriu, Anca-Monica Strugaru, Elena Butnaru, Fixed dose

combinations with selective beta-blockers: quantitative determination

in biological fluids, Rev. Med. Chir. Soc. Med. Nat. (Iaşi), 2014: 119(2):

585-591 (ISSN 0048-7848).

2.3. 3. Papers presented at congresses and symposia

1. Ioana-Cezara Grigoriu, Elena Butnaru, Julia Kazakova, Miguel Angel

Bello López, Rut Fernández-Torres

UPLC-MS/QTOF Method for the Quantitative Determination of Second

Generation H1-Antihistaminic Drugs: Loratadine, Desloratadine and

Cetirizine (poster – XV Reunion del Grupo Regional Andaluz de la

Sociedad Espanola de Quimica Analitica, 30 iunie-1 iulie 2016,

Almeria, Spania)

2. Ioana-Cezara Grigoriu, Adrian-Florin Spac, Luminita Agoroaei,

Anca-Monica Strugaru, Elena Butnaru

Implementarea unei metode lichid cromatografice performante de

determinare a loratadinei

(poster – Zilele Medicamentului, Ediția a XXIV-a, 3-4 decembrie 2015,

Iași)

3. Ioana-Cezara Grigoriu, Bogdan-Ionel Cioroiu, Luminița Agoroaei,

Anca-Monica Strugaru, Elena Butnaru

Solid phase extraction and LC-MS/MS method for the analysis of

cetirizine in human plasma

(poster – Primul Congres de Toxicologie cu participare internațională,

cu tema “Toxicologia la confluența dintre domenii”, 16-18 octombrie

2015, București)

4. Ioana-Cezara Grigoriu, Luminița Agoroaei, Anca-Monica Strugaru,

Elena Butnaru

Istoria descoperirii histaminei și antihistaminicelor – H1

http://europepmc.org/abstract/med/26204671





42

A XXIV-a Reuniune Naţională Anuală a Societăţii Române de Istoria

Farmaciei, Sibiu, 11-13 iunie 2015, Lucrări in extenso, Nr. 24/2015, Ed.

Sitech – Pharmakon, p. 178-184 (ISSN 2457-3027)

5. Ioana-Cezara Grigoriu, Bogdan Cioroiu, Luminița Agoroaei, Anca-

Monica Strugaru, Elena Butnaru

Preliminary Study of Desloratadine’s Impurity Profile Used in

Toxicological Studies

(poster – Workshop: Integrarea Școlilor doctorale în Rețele Europene,

27-28 martie 2015, Timișoara)

6. Ioana-Cezara Grigoriu, Luminiţa Agoroaei, Anca-Monica Strugaru,

Elena Butnaru

Metode analitice pentru determinarea unor compuşi antihistaminici din

fluide biologice

(e-poster - Congresul Național de Farmacie din România, cu participare

internațională, Ediția a XV-a, cu tema “Viziune și inovație în practica

farmaceutică – Orizont 2020” (24-27 septembrie 2014, Iași)

7. Anca-Monica Strugaru, Gina Botnariu, Luminiţa Agoroaei, Ioana-

Cezara Grigoriu, Elena Butnaru

Patologia asociată cazurilor de diabet zaharat tratate cu metformin și

glimepirid, în cadrul Spitalului Clinic Județean de Urgențe “Sf.

Spiridon”, Iași

Associated Pathology of Diabetes Cases Treated with Metformin and

Glimepiride, in the “Sf. Spiridon” Clinical Emergency Hospital, Iași

(e-poster – Congresul Național de Farmacie din România, cu participare

internațională, Ediția a XV-a, cu tema “Viziune și inovație în practica

farmaceutică – Orizont 2020”, 24-27 septembrie 2014, Iași)

8. Luminiţa Agoroaei, Ioana-Cezara Grigoriu, Anca-Monica Strugaru,

Elena Butnaru

200 de ani de la primul tratat de toxicologie modernă - autor: Mateo

José Bonaventura Orfila

200 Years since the First Treaty of Modern Toxicology - Author: Mateo

José Bonaventura Orfila

(comunicare orală – Congresul Național de Farmacie din România, cu

participare internațională, Ediția a XV-a, cu tema “Viziune și inovație în

practica farmaceutică – Orizont 2020” (24-27 septembrie 2014, Iași)

9. Anca-Monica Strugaru, Cornelia Mircea, Luminița Agoroaei, Gina

Botnariu, Ioana-Cezara Grigoriu, Ștefania Mahu, Elena Butnaru

Development and Validation of a Method for Quantitative

Determination of Metformin by Capillary Electrophoresis with UV

Detection

(poster – Workshop: Integrarea Școlilor doctorale în Rețele Europene,

27-28 martie 2015, Timișoara)



43

10. Anca-Monica Strugaru, Elena Butnaru, Ioana-Cezara Grigoriu,

Luminiţa Agoroaei

Repere din istoria dopingului




11. Luminiţa Agoroaei, Anca-Monica Strugaru, Ioana-Cezara Grigoriu,

Elena Butnaru

Familia de farmacişti ieşeni Lochman




12. Luminiţa Agoroaei, Anca-Monica Strugaru, Ioana-Cezara Grigoriu,

Elena Butnaru

Mateo José Bonaventura Orfila – părintele toxicologiei moderne




13. Elena Butnaru, Ioana Cezara Grigoriu, Anca Monica Strugaru,

Claudia Călinescu, Luminiţa Agoroaei

Iuliu Orient (1869-1940) – 75 de ani de la trecerea în eternitate




14. Anca-Monica Strugaru, Gina Botnariu, Cristina Tuchiluș, Ecaterina

Anisie, Luminița Agoroaei, Ioana-Cezara Grigoriu, Elena Butnaru

Comparative presentation of three cases of cyanocobalamin low serum

levels during treatment with metformin

(poster – Primul Congres de Toxicologie cu participare internațională,

cu tema “Toxicologia la confluența dintre domenii”, 16-18 octombrie

2015, București)

15. Anca-Monica Strugaru, Gina Botnariu, Cristina Tuchilus, Luminita

Agoroaei, Ioana-Cezara Grigoriu, Elena Butnaru

Influența metforminului asupra nivelului seric de vitamină B12

(comunicare orală – Zilele Medicamentului, Ediția a XXIV-a, 3-4

decembrie 2015, Iași)

16. Anca-Monica Strugaru, Luminița Agoroaei, Ioana-Cezara Grigoriu,

Elena Butnaru

Originile naturale ale metforminului

A XXV-a Reuniune Naţională Aniversară de Istoria Farmaciei, Cluj-

Napoca, 30 iunie-2 iulie 2016, Lucrări in extenso, Nr. 25/2016, Ed.




44

17. Luminița Agoroaei, Ioana-Cezara Grigoriu, Anca-Monica Strugaru,

Elena Butnaru

Profesorul Marțian Cotrău (1923-1998) și istoria farmaciei




18. Elena Butnaru, Claudia Călinescu, Anca Monica Strugaru, Ioana

Cezara Grigoriu, Luminița Agoroaei

Repere din activitatea SRIF-Secția Iași (2002-2015)




19. Ioana-Cezara Grigoriu, Anca-Monica Strugaru, Elena Butnaru,

Luminița Agoroaei

Cazuri celebre de intoxicații cu monoxid de carbon




20. Luminița Agoroaei, Anca-Monica Strugaru, Ioana-Cezara Grigoriu,

Elena Butnaru

Mina Minovici (1858-1933) – farmacist, toxicolog, medic legist,

profesor, cercetător

Mina Minovici (1858-1933) – pharmacist, toxicologist, forensic

physician, professor, researcher

(comunicare orală – Congresul Național de Farmacie din România,

Ediția a XVI-a, cu tema “Farmacia – centru al interdisciplinarității

științelor vieții” (28 septembrie-1 octombrie 2016, București)

Documents

CHEMICAL AND CLINICAL TOXICOLOGICAL RESEARCH … · university of medicine and pharmacy „grigore t. popa” iaȘi chemical and clinical toxicological research of antiallergic drugs