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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
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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.
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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.
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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
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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
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H1 antihistamines in patients from “Sf. Maria” Clinical Emergency Hospital for Children 48
8.2.1. Introduction 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
9.1.5. Conclusions 68
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
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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
12.1.1. Introduction 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
12.1.4. Conclusions 125
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
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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
12.2.5. Conclusions 143
12.3. In vitro studies for evaluation of H1 antihistamines action on vascular reactivity 144
12.3.1. Introduction 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
12.3.4. Conclusions 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
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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
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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
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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:
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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
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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.
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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
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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
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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
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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.
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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
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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.
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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
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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
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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)
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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
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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
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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:
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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.
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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
)
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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
)
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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
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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
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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
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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.
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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
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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)
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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.
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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
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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
* *
* *
* *
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- 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.
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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
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car
bac
ol 1
0-5
CARBACOL CETIR CLORFENIRAMIN DESLORAT
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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
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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
F re c v e n ta s t im u la re (H z )
% c
on
tra
cti
e E
FS
2 4 812
16
0
5 0
1 0 0
1 5 0
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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.
F re c v e n ta s t im u la re (H z )
% c
on
tra
cti
e E
FS
2 4 812
16
0
5 0
1 0 0
1 5 0
2 0 0
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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
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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
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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
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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.
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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.
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6. Dumoulin M, Martin K, Titier K et al. Cardiotoxicité des antihistaminiques
de deuxième génération. Rev Fr Allergol Immunol Clin 2006; 46: 392–401.
7. Dermengiu D, Gorun GŞ. Toxicologie medico-legală. Bucureşti: Viaţa
Medicală Românească, 2006.
8. Rodríguez-Gómez SJ, Zamora-Martínez T, Bailador-Andrés C et al.
Colestasis intrahepática grave asociada a la ingesta de cetiricina.
Gastroenterol Hepatol 2009; 32: 383–384.
9. Rodríguez del Río P, González-Gutiérrez ML, Sánchez-López J et al.
Urticaria caused by antihistamines: report of 5 cases. J Investig Allergol Clin
Immunol 2009; 19: 317–320.
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.
11. Leikin JB, Paloucek FP, eds. Poisoning and toxicology handbook. 4th ed.
Boca Raton, Fla.: CRC, 2008.
12. Mabrouk M. Simultaneous determination of loratadine and
pseudoephedrine sulfate in pharmaceutical formulation by RP-LC and
derivative spectrophotometry. J Pharm Biomed Anal 2003; 33: 597–604.
13. Li W, Doherty J, Moench P et al. LC–MS/MS bioanalysis of loratadine
(Claritin) in dried blood spot (DBS) samples collected by subjects in a clinical
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14. Patel BN, Sharma N, Sanyal M et al. LC-MS-ESI for the determination
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15. Srinubabu G, Patel RS, Shedbalkar VP et al. Development and validation
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method for simultaneous quantification of loratadine and desloratadine in
human plasma. J Chromatogr B 2007; 860: 202–208.
16. Emara S, El-Gindy A, Mesbah MK et al. Direct injection liquid
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antihistaminic drugs and their main metabolites in serum. J AOAC Int 2007;
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to human studies revisited. FASEB J Off Publ Fed Am Soc Exp Biol 2008;
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26. Olasińska-Wiśniewska A, Olasiński J, Grajek S. Cardiovascular safety of
antihistamines. Adv Dermatol Allergol 2014; 3: 182–186.
27. Liu S-C, Chu Y-H, Kao C-H et al. Steroids and antihistamines synergize
to inhibit rat’s airway smooth muscle contractility. Eur Arch
Otorhinolaryngol 2015; 272: 1443–1449.
28. Jalbani GA, Aamir K, Shaikh AM et al. To evaluate and compare the
effects of first generation anti-histamine (chlorpheniramine maleate) and
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al. Endothelium-dependent sensory non-adrenergic non-cholinergic
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30. Kim H, Dwyer L, Song JH et al. Identification of histamine receptors and
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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).
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- 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
CONTRIBUȚII LA CERCETAREA CHIMICO-TOXICOLOGICĂ ȘI CLINICĂ
A UNOR MEDICAMENTE ANTIALERGICE
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)
CONTRIBUȚII LA CERCETAREA CHIMICO-TOXICOLOGICĂ ȘI CLINICĂ
A UNOR MEDICAMENTE ANTIALERGICE
43
10. Anca-Monica Strugaru, Elena Butnaru, Ioana-Cezara Grigoriu,
Luminiţa Agoroaei
Repere din istoria dopingului
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. 370-375 (ISSN 2457-3027)
11. Luminiţa Agoroaei, Anca-Monica Strugaru, Ioana-Cezara Grigoriu,
Elena Butnaru
Familia de farmacişti ieşeni Lochman
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. 31-35 (ISSN 2457-3027)
12. Luminiţa Agoroaei, Anca-Monica Strugaru, Ioana-Cezara Grigoriu,
Elena Butnaru
Mateo José Bonaventura Orfila – părintele toxicologiei moderne
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. 36-40 (ISSN 2457-3027)
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
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. 91-96 (ISSN 2457-3027)
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.
Sitech – Pharmakon, p. 313-317 (ISSN 2457-3027)
CONTRIBUȚII LA CERCETAREA CHIMICO-TOXICOLOGICĂ ȘI CLINICĂ
A UNOR MEDICAMENTE ANTIALERGICE
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17. Luminița Agoroaei, Ioana-Cezara Grigoriu, Anca-Monica Strugaru,
Elena Butnaru
Profesorul Marțian Cotrău (1923-1998) și istoria farmaciei
A XXV-a Reuniune Naţională Aniversară de Istoria Farmaciei, Cluj-
Napoca, 30 iunie-2 iulie 2016, Lucrări in extenso, Nr. 25/2016, Ed.
Sitech – Pharmakon, p. 27-33 (ISSN 2457-3027)
18. Elena Butnaru, Claudia Călinescu, Anca Monica Strugaru, Ioana
Cezara Grigoriu, Luminița Agoroaei
Repere din activitatea SRIF-Secția Iași (2002-2015)
A XXV-a Reuniune Naţională Aniversară de Istoria Farmaciei, Cluj-
Napoca, 30 iunie-2 iulie 2016, Lucrări in extenso, Nr. 25/2016, Ed.
Sitech – Pharmakon, p. 84-97 (ISSN 2457-3027)
19. Ioana-Cezara Grigoriu, Anca-Monica Strugaru, Elena Butnaru,
Luminița Agoroaei
Cazuri celebre de intoxicații cu monoxid de carbon
A XXV-a Reuniune Naţională Aniversară de Istoria Farmaciei, Cluj-
Napoca, 30 iunie-2 iulie 2016, Lucrări in extenso, Nr. 25/2016, Ed.
Sitech – Pharmakon, p. 176-180 (ISSN 2457-3027)
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)