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- PhD Thesis Abstract –

CHEMICAL AND CLINICAL

TOXICOLOGICAL STUDY OF ORAL

ANTIDIABETIC DRUGS

SCIENTIFIC COORDINATOR

Prof. Elena BUTNARU, PhD

PhD CANDIDATE

Univ. Assist. Anca-Monica STRUGARU

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

Acknowledgment

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

work, for professionalism and scientific guidance, for her support during the

doctoral studies.

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

committee members: Prof. Anca Miron, PhD, Prof. Antonia Poiată, PhD, Assoc.

Prof. Luminita Agoroaei, PhD, and Assoc. Prof. Cornelia Mircea, for support in

capillary electrophoresis determinations.

Equally, I want to thank Mrs. Prof. Ruth Maria Fernandez Torres, PhD,

professor at the University of Seville, Faculty of Chemistry, Spain, for her

unconditional support during the transnational mobility and Mrs. Prof. Lenuţa

Profire, PhD, for the support with this research stage.

Genuine thanks to Assoc. Prof. Cristina Tuchiluş, PhD, and Lecturer

Gina Botnariu, PhD, for collaboration on laboratory analyzes and clinical study.

I thank the entire staff of the Department of Toxicology for moral and

scientific support provided during the period of doctoral studies.

Lastly, I thank my family for their patience, understanding and support

throughout these years.

The doctoral thesis includes:

• 125 pages

• 51 tables

• 66 figures

• 164 bibliographical indications

This summary respects numbering for contents, tables, figures and references of

the thesis.

Keywords: diabetes, metformin, glimepiride, UPLC-QTOF/MS, side effects,

vitamin B12.

Studiul chimico- și clinico- toxicologic al unor medicamente antidiabetice orale

1

Contents

A. CURRENT STATE OF KNOWLEDGE

Chapter 1

DIABETES MELLITUS. GENERAL NOTIONS 1

1.1. Type 2 diabetes 1

1.1.1. Type 2 diabetes epidemiology 1

1.1.2. Type 2 diabetes causes 3

1.1.3. Type 2 diabetes chronic complications 4

1.1.3.1. Diabetic nephropathy 4

1.1.3.2. Diabetic retinophathy 4

1.1.3.3. Diabetic neuropathy 4

1.1.3.4. Cardiovascular diseases 5

1.2. Oral therapy in type 2 diabetes 5

1.2.1. Sulphonylurea 5

1.2.2. Biguanide 5

1.2.3. Meglitinides 5

1.2.4. Thiazolidinediones 6

1.2.5. Gliptine 6

1.2.6. Other structures 6

1.2.6.1. Alpha-glucosidase inhibitors 6

1.2.6.2. Aldoreductase inhibitors 7

Chapter 2

METFORMIN 8

2.1. Short history 8

2.1.1. Natural origins of metformin – Galega officinalis Linn 8

2.1.2. Biguanide derivatives of synthesis 9

2.1.3. Metformin history 9

2.2. Physico-chemical characteristics of metformin 10

2.3. Metformin mechanism of action in the treatment of diabetes 11

2.4. Other therapeutic actions of metformin 13

2.5. Toxicokinetic features of metformin 14

2.6. The adverse effects of therapy with metformin 15

2.7. Contraindications 15

2.8. Drug interactions 15

Chapter 3

CHARACTERISTIC TOXIC EFFECTS IN METFORMIN THERAPY 17


2

3.1. Digestive disorders associated with metformin therapy 17

3.2. Lactic acidosis 17

3.2.1. Definition. Characterization 17

3.2.2. Metformin-induced lactic acidosis 18

3.3. Influence of metformin on vitamin B12 19

3.3.1. Chemical structure and coenzyme forms of vitamin B12 19

3.3.2. Distribution and sources of B12 19

3.3.3. Kinetic features for vitamin B12 20

3.3.4. Enzymatic role for vitamin B12 21

3.3.5. Causes of vitamin B12 deficiency 22

3.3.6. Clinical manifestations of vitamin B12 deficiency 22

3.3.7. Metformin and its influence on vitamin B12 23

3.3.7.1. Influence of metformin on serum levels of vitamin B12 - human studies

23

3.3.7.2. Proposed mechanisms for the influence of metformin on serum levels of

vitamin B12 23

3.3.7.3. General precautions 23

Chapter 4

GLIMEPIRIDE 25

4.1. Physico-chemical characteristics of glimepiride 25

4.2. Mechanism of action 26

4.3. Glimepiride comparing to other sulfonylurea in diabetes treatment 27

4.4. Toxicokinetic particularities of glimepiride 27

4.5. Drug interactions 28

4.6. Adverse effects of therapy with glimepiride 29

4.7. Glimepiride poisoning and treatment of intoxications 29

Chapter 5

METFORMIN - GLIMEPIRIDE DRUG COMBINATION 30

5.1. Antidiabetic therapy with metformin and glimepiride 30

5.2. Toxicity aspects of drug therapy metformin - glimepiride 30

5.3. Analytical methods for extraction, separation and dosing of metformin and

glimepiride 31

5.3.1. Analytical methods for metformin 31

5.3.2. Analytical methods for glimepiride 31

5.3.3. Simultaneous analytical methods for metformin and glimepiride 32

Chapter 6

CLINICAL CASES REPORTED OF ACUTE OR CHRONIC TOXICITY FOR

METFORMIN AND GLIMEPIRIDE 34


3

6.1. Clinical cases associated with metformin therapy 34

6.1.1. Metformin-induced lactic acidosis - comparative presentation of 13 cases

34

6.1.2. Vitamin B12 deficiency in patients treated with metformin 38

6.1.3. Metformin-induced hypomagnesemia – case report 39

6.2. Clinical cases associated with glimepiride therapy 39

6.3. Hypoglycemia when combining metformin - glimepiride: presentation of two

clinical cases 40

B. ORIGINAL CONTRIBUTIONS

Chapter 7

MOTIVATION FOR CHOOSING THE THEME AND OBJECTIVES 42

Chapter 8

STATISTICAL STUDY ON INCIDENCE OF ASSOCIATED PATHOLOGY

AND ANTIDIABETIC MEDICATIONS IN PATIENTS WITH TYPE 2

DIABETES FROM THE CENTER OF FAMILY MEDICINE IAȘI 44

7.1. Introduction 44

7.2. Material and method 44

7.3. Results and discussions 44

7.4. Conclusions 47

Chapter 9

QUANTITATIVE DETERMINATION OF METFORMIN BY CAPILLARY

ELECTROPHORESIS WITH UV DETECTION AND APPLICATIONS IN

HUMAN SERUM 49


9.2. Theoretical principles of capillary electrophoresis 49

9.3. Method development and validation for metformin determination by capillary

electrophoresis with UV detection 50

9.3.1. Material and method 50

9.3.2. Method optimization for electrophoretic determination 51

9.3.2.1. The influence of the pH buffer 52

9.3.2.2. The influence of buffer type 52

9.3.2.3. Determination of optimal detection wavelength at UV 53

9.3.2.4. Results and discussions 53

9.3.2.5. Conclusions 54

9.3.3. Method validation for electrophoretic determination 55

9.3.3.1. Selectivity 55


4

9.3.3.2. Linearity 56

9.3.3.3. Detection limit and quantification limit 57

9.3.3.4. Precision 58

9.3.3.5. Accuracy 60

9.3.4. Conclusions 61

9.4. Application of the determination method of metformin by capillary

electrophoresis with UV detection in human serum 61


9.4.2. Optimization method of extraction of metformin in human serum samples

62

9.4.3. Results and discussions 65


Chapter 10

SIMULTANEOUS QUANTITATIVE DETERMINATION OF METFORMIN

AND GLIMEPIRIDE BY UPLC-QTOF/MS WITH APPLICATIONS IN

HUMAN SERUM 67


10.2. Theoretical principles 67

10.3. Method development and validation for simultaneous determination of

metformin and glimepiride by UPLC-QTOF/MS 69


10.3.2. Method optimization for simultaneous determination of metformin and

glimepiride by UPLC-QTOF/MS 71

10.3.2.1. Material and method 71

10.3.2.2. Results and discussions 72


10.3.3. Method validation for simultaneous determination of metformin and

glimepiride by UPLC-QTOF/MS 75

10.3.3.1. Specificity 75

10.3.3.2. Linearity 76

10.3.3.3. Detection limit and quantification limit 79

10.3.3.4. Precision 79

10.3.3.5. Accuracy 82


10.4. Application of the simultaneous determination method of metformin and

glimepiride by UPLC-QTOF/MS in human serum 82



5

10.4.2. Optimization method of extraction of metformin and glimepiride in human

serum samples 83



10.5. Metformin determination in a group of patients hospitalized in Diabetes,

Nutrition and Metabolic Diseases Clinic – In-patient Unit, Emergency County

Hospital "St. Spiridon " Iași 91




Chapter 11

CLINICAL STUDY OF PATIENTS DIAGNOSED WITH DIABETES

HOSPITALIZED IN DIABETES, NUTRITION AND METABOLIC

DISEASES CLINIC, EMERGENCY COUNTY HOSPITAL "ST. SPIRIDON "

IAȘI 94

11.1. Presentation of clinical cases regarding precautions of treatment with

metformin 94

11.1.1. Introduction 94

11.1.2. Presentation of clinical cases 94

11.1.2.1. Case 1 94

11.1.2.2. Case 2 95



11.2. Influence of metformin treatment on serum levels of vitamin B12 96



11.2.2.1. Study design 96

11.2.2.2. Working protocol 97

11.2.2.3. Equipment 98

11.2.3. Characterization of patients groups 99

11.2.4. Analysis of the influence of metformin treatment on serum vitamin B12

104

11.2.4.1. Data on vitamin B12 deficiency in patients treated with metformin 104

11.2.4.2. The influence of treatment duration of metformin on serum levels of

vitamin B12 107

11.2.4.3. The influence of the duration of diagnosed diabetes on serum levels of

vitamin B12 for patients who have not received treatment with metformin 109


6

11.2.4.4. Influence of treatment with metformin on serum levels of vitamin B12,

independent of the duration of treatment with metformin 111

11.2.4.5. Influence of treatment with metformin on uric acid 114

11.2.4.6. Analysis of correlation between serum concentration of metformin and

vitamin B12 serum levels 117


11.3. Cardiovascular risk assessment in patients with type 2 diabetes 119





Chapter 12

GENERAL CONCLUSIONS. ORIGINAL CONTRIBUTIONS. RESEARCH

PERSPECTIVE 123

General conclusions 123

Original contributions 124

Research perspective 125

REFERENCES 126

APPENDICES 135

Ethics Commission Research Agreement 135

Emergency County Hospital "St. Spiridon "Agreement 136

Informed consent 137

List of published works 139


7

ABBREVIATIONS

APCI – Atmospheric pressure chemical ionization

BNF – British National Formulary

DDP-4 – dipeptidyl peptidase 4

DZ – Diabetes

EC – capillary electrophoresis

ES – standard error

ESI – Electrospray ionization

GLUT – Glucose transporter

HbA1c – Glycosylated hemoglobin A1c

HC – Haptocorrin

HDL – High density lipoprotein

HPLC – High-performance liquid chromatography

HPTLC – High performance thin layer chromatography

HTA – Hypertension

ICH – International Conference on Harmonisation

IF – Intrinsic factor

IMC – Body mass index

IUPAC – International Union of Pure and Applied Chemistry

LC – Liquid chromatography

LD – Limit of detection

LDL – Low density lipoprotein

LQ – Limit of quantification

MS – Mass spectrometry

OCT1 – Organic cation transporter 1

QTOF/MS – Quadrupole Time-of-Flight / Mass Spectrometry

RP – Reversed phase

RSD – Relative standard deviation

SD – Standard deviation

TGO – Glutamic oxalacetic transaminase

TGP – Glutamic-pyruvate transaminase

TLC – Thin layer chromatography

UKPDS RE – UK Prospective Diabetes Study Risk Engine

UPLC – Ultra high performance liquid chromatography

VLDL – Very low density lipoprotein


8

MOTIVATION FOR CHOOSING THE THEME, AIM AND

OBJECTIVES

Diabetes is a complex pathology with high frequency among patients

globally, but also territorial. Epidemiological data point to the large number of

people diagnosed with diabetes (366 million globally in 2011), which is expected

to increase in the next period (4). In Romania, by comparing first semester of 2015

to that of 2014, it also shows an increase in patients with diabetes. For Iaşi, for

example, there were 1286 new cases of year 2015 compared to 982 cases for 2014.

The percentage of those diagnosed with diabetes is 4.1% in 2015 against 4.0% in

2014 (5).

The onset and progress of diabetes is attributed to a series of complex

factors, including lifestyle conditions (lack of exercise, sedentary lifestyle,

smoking and alcohol consumption) or other pathologies (obesity, hypertension,

metabolic syndrome and others) encountered also at lower population ages.

Antidiabetic drug therapy present oral and injectable classes,

pharmacodynamic different and with complementary mechanisms in order to

achieve optimal glycemic control and the therapeutic efficiency of a large number

of patients with diverse pathology and clinical manifestations and private

evolution. Antidiabetic therapy has a dynamic, tailored to each patient compliance

and safety profile in pharmacotoxicological terms.

Among the oral antidiabetic drugs, including first introduced in therapy

classes are sulfonylureas and biguanides. Accordingly to these therapeutic groups,

with extensive prescribing for patients with diabetes are metformin (biguanide

compound) and glimepiride (sulfonylurea).

Metformin is currently the drug of choice in oral antidiabetic therapy

initiation in overweight patients, in the absence of specific contraindications.

Toxicological profile include gastrointestinal side effects and, in particular,

metformin is incriminated in lactic acidosis and vitamin B12 malabsorption.

Glimepiride is a second generation sulphonylurea administered alone or

in combination with other antidiabetic compounds, especially metformin, with

different mechanisms of action. From a toxicological point of view, glimepiride

has the characteristics of the new generation sulphonylurea, with a low incidence

of hypoglycemia. For this compound are reported cases of hepatic cholestasis and

hypoglycaemia, if there are favoring drug associations.

The aim of this paper was to complete characterization of the

toxicological profile of the two compounds through clinical and chemicals studies.

The objectives underlying personal research are:

Statistical analysis of patients diagnosed with type 2 diabetes,

hospitalized in the Center of Family Medicine Iasi, identifying the


9

following characteristics: clinical and laboratory parameters, associated

pathology and medication.

Development and validation of a method for the quantitative

determination of metformin by capillary electrophoresis with UV

detection and the method application for analyzing samples of human

serum.

Simultaneous quantitative determination of metformin and glimepiride

by ultra performance liquid chromatography with QTOF associated mass

spectrometry detection with the following steps:

o UPLC-QTOF/MS method development and validation.

o Optimization method of extraction in human serum samples.

o Method application on samples from patients hospitalized in

Diabetes, Nutrition and Metabolic Diseases Clinic

Clinical study of patients hospitalized in Diabetes, Nutrition And

Metabolic Diseases Clinic, with:

o Descriptive analysis of the group of patients.

o Study of the influence of metformin treatment on serum levels

of vitamin B12.

o Cardiovascular risk assessment in patients diagnosed with type

2 diabetes.

In obtaining scientific results of doctoral studies, an important role was

the internship mobility from the program POSDRU 159 / 1.5 / S / 136 893, at the

Faculty of Chemistry, Department of Analytical Chemistry, University of Sevilla,

Spain.

Personal research skills were the result of my work at the Department of

Toxicology, Faculty of Pharmacy, in interdisciplinary collaborations within the

University of Medicine and Pharmacy "Grigore T. Popa" and at Microanalysis

Laboratory of Seville.


10

Chapter 8

STATISTICAL STUDY ON INCIDENCE OF ASSOCIATED

PATHOLOGY AND ANTIDIABETIC MEDICATIONS IN

PATIENTS WITH TYPE 2 DIABETES FROM THE CENTER OF

FAMILY MEDICINE IAȘI

Diabetes mellitus increased incidence IN global population, but also in

Romania, with a frequency of 4.7% of the population (5). Descriptive /

retrospective study of patients with type 2 diabetes was conducted at the Centre

for Family Medicine in Iasi in the period July 2013 - December 2014 and it has

characterized the patients in terms of: antidiabetic medications, oral, injectable

and adjuvant, chronic complications of type 2 diabetes, associated pathology,

associated cardiovascular drug therapy.

8.3. RESULTS AND DISCUSSIONS

The statistical study was conducted on a group of 221 patients. 63.3%

are female, with a mean age of 64 years.

Table 8.I summarizes the results obtained by statistical processing of

clinical and laboratory data of patients.

Table 8.I. Characteristics of the group of patients analyzed

Parameter

Sex

Female Male

Mean Vmin1 Vmax

2 Mean Vmin1 Vmax

2

Number of patients 140 81

Age (years) 64 36 83 63 30 86

BMI (kg/m2) 32.79 19.53 48.12 31.21 18. 94 47.32

Systolic blood pressure

(mmHg) 138.09 90 185 144.23 101 214

Diagnostic DZ3 (ani) 10 1 27 9 1 35

Smoker (%) 9.28 23.46

Family history

antecedents 33.57% 25.92%

HbA1c (%)4 7.0 4.8 12.1 6.9 4.9 12

Total cohlesterol

(mg/dl) 196.9 105.9 506 181.6 83 304

HDL (mg/dl) 48.86 18 109 46.67 18.3 132

Report

total cholesterol:HDL 4.46 1.68 16.89 4.42 1.98 12.08

Patients diagnosed with type 2 diabetes have chronic complications, with

the highest frequency for neurological complications (33.65% of cases).


11

As pathology associated, dyslipidemia presents a high frequency

(62.89%). Cardiovascular diseases associated are shown in figure 8.3. The

incidence of these diseases is higher in female patients and by age, the highest

frequency have:

- 45-64 years: hypertension, chronic ischemic heart disease,

- 65-74 years: myocardial infarction, chronic ischemic heart disease,

- ≥75 years: atrial fibrillation.

*HT – hypertension

Fig. 8.3. Cardiovascular diseases associated with diabetes mellitus type 2

In terms of drug therapy, table 8.II summarizes antidiabetic treatment,

primary and adjuvant antihyperglycemic therapy. It notes that biguanides

(76.01%) and sulfonylureas (31.67%) had the highest frequency of prescribing.

Table 8.II. Antidiabetic therapy

Primary therapy Number of cases

Quick-acting insulins 16

Intermediate-acting insulins 13

Insulins and analogues with long duration of action 40

Biguanides 168

Sulfonylureas 70

4-dipeptidyl peptidase inhibitors (DPP-4) 4

Alpha-glucosidase inhibitors 15

Other oral hypoglycaemic drugs 7

Adjuvant therapy Number of cases

Alpha lipoic acid 74

The combination thiamin / pyridoxine hydrochloride /

cyanocobalamin 43

0

50

100

HT Chronic

ischemic

heart disease

Chronic heart

failure

Atrial

fibrillation

83.71

20.87.69 6.33%


12

Chapter 9

QUANTITATIVE DETERMINATION OF METFORMIN BY

CAPILLARY ELECTROPHORESIS WITH UV DETECTION

AND APPLICATIONS IN HUMAN SERUM

Capillary electrophoresis is a new method that combines mechanisms of

electrophoresis separation performance with chromatography automation.

Because of the polar character, metformin can be determined also by capillary

electrophoresis. This method was not mentioned in literature in local laboratories

for metformin analysis.

The study aims to develop and validate the method of metformin

determination by capillary electrophoresis with UV detection and optimize

methods for metformin extraction from biological products.

9.3.3. Method validation for electrophoretic determination

The method was validated by checking the parameters: selectivity,

linearity, limit of detection and quantification limit, precision, accuracy.

9.3.3.1. Selectivity

By comparing overlapping electrophoregrams for metformin and control

solution of phosphate buffer (concentration of 60 mM, pH 4.0), made under the

same conditions, there is no additional peaks (fig. 9.8).

Fig. 9.8. Electrophoregrams for metformin (600 µg/mL)

and phosphate buffer solution

9.3.4. Conclusions

There were established the optimum analysis conditions:

electrophoretic separation was performed on a Bare fused-silica capillary

column,


13

ions movement was performed in phosphate buffer, 60 mM, pH = 4.0,

injection of the sample was performed for 3 seconds at a pressure of 0.5

psi,

the wavelength for detection was 200 nm.

The method was validated by determining the parameters of validation:

linearity in the range of 5-1000 mg/mL, with a correlation coefficient r =

0.9991,

detection limit LD = 12,782 µg/mL and limit of quantification LQ =

38,734 µg/mL,

precision, with system precision (RSD = 4,42%), method precision (RSD

= 3,69%) and intermediate precision determination (RSD = 3,45%),

accuracy (mean recovery 100,39%, RSD = 2,40%).

9.4. APPLICATION OF THE DETEERMINATION METHOD OF

METFORMIN BY CAPILLARY ELECTROPHORESIS WITH UV

DETECTION IN HUMAN SERUM

9.4.2. Optimization method of extraction of metformin in human

serum samples

The procedure for extraction of metformin involves precipitation with

acetonitrile, filtration and analysis of the supernatant obtained.

Analyzing the results obtained, the method applied to isolate metformin

in serum samples is optimal. The mean recovery is 98.14% and relative standard

deviation (14.05%) is within the permissible limits below 15%.

In addition, the presence of the acetonitrile does not affect the analysis

of metformin (retention time or peak area) (fig. 9.11).

Fig. 9.11. Electrophoregram for serum samples spiked with metformin at

concentration of 500 µg/mL


14

Chapter 10

SIMULTANEOUS QUANTITATIVE DETERMINATION OF

METFORMIN AND GLIMEPIRIDE BY UPLC-QTOF/MS

Metformin and glimepiride show in the literature simultaneous assay

methods, but the determination by UPLC-QTOF/MS (Ultra Performance Liquid

Chromatography - quadrupole Time-of-Flight / Mass Spectrometry) has not been

previously applied.

The present study aims to develop and validate simultaneous

determination method of metformin and glimepiride by UPLC-QTOF/MS, and

method application for the analysis of the two compounds from biological samples

of human serum consecutive of establishing and optimizing a simple and efficient

extraction method.

10.3.3. Method validation for simultaneous determination of

metformin and glimepiride by UPLC-QTOF/MS

10.3.3.1. Specificity

Because of the detection system (MS/QTOF), specificity of the method

for chromatographically separated compounds is ensured by accurate mass

determination (m/z). Figure 10.7 shows chromatograms obtained for the masses

m/z 130.105 Da (metformin) and 491.2311 Da (glimepiride), from the analysis of

a solution of concentration 100 µg/L of the two compounds.

Fig. 10.7. Chromatograms for: a. Metformin (100 µg/L),

b. Glimepiride (100 µg/L)

a.

b.


15

10.3.3.6. Conclusions

The method developed for simultaneous determination of metformin and

glimepiridum by UPLC-QTOF/MS was validated by establishing parameters:

specificity: the detection was carried out using the system QTOF/MS by

determining the accurate masses (m/z) for the two compounds: 130.105

(metformin), 491.2311 (glimepiride),

linearity in the domain 0.5 – 100 µg/L (r = 0.9980), 100-1000 µg/L (r =

0.9917) for metformin and in the domain 1-100 µg/L (r = 0.9986), 100-

1000 µg/L (r = 0.9954) for glimepiride,

limits of detection (LD = 2.98 µg/L for metformin and LD = 2.52 µg/L

for glimepirid) and limits of quantification (LQ = 9.03 µg/L for

metformin and LQ = 7.64 µg/L for glimepiride),

precision, with system precision (RSD = 1.31% for metformin și RSD =

3.92% for glimepiride) and method precision determination (RSD1 =

5.01%, RSD2 = 4.44% for metformin and RSD1 = 5.57%, RSD2 = 9.99%

for glimepirid).

10.4. APPLICATIONS OF THE SIMULTANEOUS DETERMINATION

METHOD OF METFORMIN AND GLIMEPIRIDE BY UPLC-QTOF/MS

IN HUMAN SERUM 10.4.2. Optimization method of extraction of metformin and

glimepiride

Choosing the internal standard

We have used two internal standards, propranolol and glibenclamide in

order to obtain an effective measurement of the two compounds, respectively

metformin and glimepiride.

Optimize extraction method results for metformin and glimepiride

For the established method, the characteristics obtained for the studied

compounds and internal standards are shown in table 10.VIII.

Table 10.VIII. Identifying characteristics of the analyzed compounds

Metformin Glimepiride Propranolol Glibenclamide

Rt (minutes) 0,41 6,51 1,09 6,45

Exact mass (Da), [H+] 130,1089 491,2353 260,1654 494,1499

UPLC-QTOF/MS analysis of the four compounds is shown by the

chromatograms obtained after the separation (fig. 10.15) and MS detection spectra

(fig. 10.16).


16

Fig. 10.15. Chromatograms for a. Metformin (100 µg/L), b. Propranolol (50 µg/L), c.

Glimepiride (100 µg/L), d. Glibenclamide (50 µg/L)

Fig. 10.16. MS spectra for: a. Metformin (100 µg/L), b. Propranolol (50 µg/L),

c. Glimepiride (100 µg/L), d. Glibenclamide (50 µg/L)

a.

b.

c.

d.

a.

d.

c.

b.


17

10.4.4. Conclusions

It was developed and optimized a simple and effective method for

extracting metformin and glimepiride in human serum:

- It was used the method with acetonitrile precipitation of serum proteins.

- The method was adjusted through the introduction of internal standards:

o propranolol for metformin,

o glibenclamide for glimepiride.

- Metformin and glimepiride determination using internal standards iss

specific and linear (correlation coefficient r = 0.9947 for metformin and

r = 0.9907 for glimepiride).

- The extraction efficacy obtained was 43.96% for metformin and

102.83% for glimepiride.

- The method is reproducible and the use of internal standards corrects

possible determination errors.

- Applying the HPLC-QTOF/MS can be performed on samples of human

serum, the detection and quantification limits allowing the determination

of therapeutic concentrations of the two compounds:

o metformin determination:

LD = 6.601 µg/L,

LQ = 20.003 µg/L.

o glimepiride determination:

LD = 10.621 µg/L,

LQ = 32.185 µg/L.

10.5. METFORMIN DETERMINATION IN A GROUP OF PATIENTS

HOSPITALIZED IN DIABETES, NUTRITION AND METABOLIC

DISEASES CLINIC – IN-PATIENT UNIT, EMERGENCY COUNTY

HOSPITAL “ST. SPIRIDON” IAȘI

The method for simultaneous determination of metformin and

glimepiride by UPLC-QTOF/MS was applied to biological products of human

serum.

Enrolled patients were hospitalized in the Clinic of Diabetes, Nutrition

and Metabolic Diseases – In-patient Unit of Clinical Emergency Hospital "St.

Spiridon" Iași. The main diagnosis was type 2 diabetes and included antidiabetic

metformin oral administration.

10.5.2. Results and discussions

The results obtained from measurements are summarized in table 10.XI.


18

Table 10.XI. The metformin analysis results of the human serum samples

10.5.3. Conclusions

We have analyzed 15 serum samples from patients under treatment with

metformin.

The method was specific for metformin, which has been detected on the

basis of accurate mass m/z 130,11.

The results have shown that the method is applicable for determining the

therapeutic and toxic serum concentrations of metformin.

Crt.

no.

Date pacient Metformin analysis results in serum

Sex

(M/F)

Weight

(kg)

Dosage treatment

with metformin

Metformin area /

propranolol area

Metformin

concentration in

serum (µg/L)

Mean metformin

concentration in

serum (µg/L)

1 F 72 1000 mg x

2/day

0.287967 236.1139 268.94

0.363758 301.7678

2 M 109 1000 mg x

3/day

0.748479 635.0358 681.65

0.856098 728.2624

3 M 85 1000 mg x

1/day

2.662109 2292.734 2346.857

2.787065 2400.979

4 F 74 1000 mg x

3/day

0.059234 37.97137 34.62979

0.051519 31.28821

5 M 100 1000 mg x

2/day

1.133538 968.5965 967.4488

1.130888 966.301

6 M 91 1000 mg x

2/day

0.755026 640.7073 571.3429

0.594879 501.9784

7 F 76 1000 mg x

3/day

0.801739 681.1726 661.0527

0.755286 640.9327

8 F 84 1000 mg x

2/day

1.077564 920.1091 818.597

0.843195 717.0848

9 F 90 850 mg x 3/day 0.647171 547.2767

551.1829 0.656189 555.0891

10 M 105 1000 mg x

3/day

0.173869 137.2749 227.732

0.382714 318.189

11 F 93 850 mg x 2/day 0.134509 103.1791

116.4951 0.165253 129.8111

12 M 96 1000 mg x

3/day

0.49577 416.125 515.7181

0.725709 615.3111

13 F 69 1000 mg x

2/day

1.108472 946.883 1039.28

1.321796 1131.677

14 F 124 1000 mg x

3/day

1.020204 870.4204 871.7543

1.023284 873.0881

15 M 70 1000 mg x

1/day

0.405487 337.9159 426.1115

0.609111 514.307


19

Serum concentrations obtained for metformin is within the range 34.629

to 2346.857 mg / L, corresponding to the values reported in the literature

(30).

Chapter 11

CLINICAL STUDY OF PATIENTS DIAGNOSED WITH

DIABETES HOSPITALIZED IN DIABTES, NUTRITION AND

METABOLIC DISEASES CLINIC, EMERGENCY COUNTY

HOSPITAL ”ST. SPIRIDON” IAȘI

11.1. PRESENTATION OF CLINICAL CASES REGARDING

PRECAUTIONS OF TREATMENT WITH METFORMIN


The cases presented are two clinical situations in which antidiabetic

metformin therapy is interrupted. The causes of this medical decisions is

associated adverse effects of metformin therapy:

- Gastrointestinal disorders

Described in case 1, digestive disorders led to discontinuation of

metformin. Gastrointestinal impairment is the most common side effects

associated with metformin therapy, especially when initiating therapy (2). For the

patient in case 1, the intensity of gastrointestinal events was strong and it can be

associated with older age (80 years), so continuing treatment with low doses of

metformin was not a viable option.

- Lactic acidosis

Considered a particularly adverse effect of metformin, lactic acidosis

rarely occurs (1), but the consequences can be serious (2). According to the cases

presented in the literature, lactic acidosis associated with metformin therapy is

favored especially by particular situations: age, heart failure, kidney damage and

more.

The presented case 2 describes the associated pathology recently

discovered: a form of heart failure (diffuse hypokinesia - weak contraction).

Consecutive, the decision of discontinuation of metformin is taken - long

medication in the patient's history. It is thus avoid the risk of lactic acidosis and

optimal glycemic control will be ensured with therapeutic alternatives that have

no precautions and contraindications associated with heart failure.


20

11.1.4. Conclusions

There were presented two clinical cases in which metformin therapy was

discontinued.

The measure was a precaution for the patient in question: to remove

manifest adverse effects associated with treatment (gastrointestinal side

effects), or to prevent a potentially serious side effect of metformin

(lactic acidosis).

Oral antidiabetic therapeutic alternatives were 2nd generation

sulfonylurea (gliclazide) - with better tolerability in the

gastrointestinal tract and acarbose - without triggering risk associated

lactic acidosis.

11.2. INFLUENCE OF METFORMIN TREATMENT ON SERUM

LEVELS OF VITAMIN B12

Metformin is incriminated in lowering serum levels of vitamin B12. In

specialist publications there are reported clinical studies and case reports where

the focus is this hypothesis (52, 146).

For this country there have been no studies to investigate the influence

of treatment with metformin on serum levels of vitamin B12.

11.2.3. Characterization of patients groups

Results and discussions

Characterization by age and sex

The total number of patients studied was 103, distribution between the

two analysis groups is unequal:

- 72 patients treated with metformin (Group 1),

- 31 patients who were not treated with metformin (Group 2).

Of the patients studied, females are the majority, with a percentage of

54.37%. Table 11.I describes groups 1 and 2 according to gender, with the average

age for each category.


21

Table 11.I. Characterization by age and sex

Sex Number of patients Percent (%) Average age (years)

Total patients

Female 56 54.37 61.20

Male 47 45.63 59.80

Total 103 100 60.57

Patients with metformin treatment (Group 1)

Female 43 59.72 63.14

Male 29 40.28 63.29

Total 72 100 63.18

Patients without metformin treatment (Group 2)

Female 13 41.94 54.07

Male 18 58.06 54.69

Total 31 100 54.30

Analysis of patients according to body weight, body mass index and

degree of obesity

Average values obtained for the patient's weight is 87.13 kg, with

standard deviation (SD) of 18.42. Body mass index has an average value of 32.25

kg/m2 (SD = 7.13).

Table 11.II. Body mass index and degree of obesity

Metformin

treatment Sex

No.

of cases

BMI (kg/m2) Obesity (degree)

>25 25-30 30-35 35-40 >40 NM* 1 2 3 NM

YES F 43 1 8 17 11 6 0 15 10 5 13

YES M 29 2 10 9 3 2 3 8 4 2 15

NO F 13 3 5 2 1 2 0 5 2 2 4

NO M 18 7 3 2 2 1 3 3 1 1 13

* Not mentioned.

Table 11.II summarizes the number of patients corresponding

distribution by gender, BMI ranges and diagnosis of obesity.

According to table 11.I, figure 11.2 highlights the differences between

the groups analyzed in terms of body mass index. Characteristic to diagnosis of

diabetes are frequent cases of obesity (BMI> 30 kg/m2). It appears in figure 11.2,

also a high number of female patients under treatment with metformin showing

high values for BMI.

This distribution of elevated BMI for patients with therapy with

metformin is justified by the fact that metformin helps weight reduction,

especially by reducing visceral fat, unlike other therapeutic classes of diabetes, for


22

which can be observed even a slight increase weight (sulfonylureas or

thiazolidinediones).

Fig. 11.2. Distribution of cases according to BMI

Metformin treatment

In this study, Group 1 is treated with metformin, duration of therapy is

between 1.25 and 19.25 years (mean 8.65 years) (Figure 11.3).

Dosage treatment with metformin dosages includes 1000 mg majority

(60 of 72 patients) administered 2 or 3 times a day. The dose of 3000 mg per day

is referred to in 35 patients (48.61%). Doses of 850 mg met only 12.5% of patients.

Fig. 11.3. The number and percentage of cases with metformin, depending on the

duration of treatment

Statistics on antidiabetic and associated drug therapy

0 5 10 15 20

under 25

25 to 30

30 to 35

35 to 40

over 40

Number of cases

BM

I (K

g/m

2)

Men (without metformin)

Women (without metformin)

Men (with metformin)

Women (with metformin)

5

7%

33

46%19

27%

14

20%

0-4 years 4-8 years 8-12 years over 12 years


23

Antidiabetic treatment

Table 11.III summarizes the number of cases associated with antidiabetic

medication. The highest values recorded for an oral therapy sulfonylureas and

insulin is the most frequent combination therapy for patients diagnosed with

diabetes, with or without metformin.

Table 11.III. Antidiabetic treatment

Metformin

treatment

Associated oral antidiabetic medication

Exenatide Insulin acarbose sitagliptin pioglitazone glimepiride gliclazide

YES 8 6 1 7 23 3 29

NO 1 0 0 0 0 0 28

Total 9 6 1 7 23 3 57

11.2.4. Analysis of the influence of metformin treatment on serum

vitamin B12

11.2.4.1. Data on vitamin B12 deficiency in patients treated with

metformin

We consider possible deficit and deficit the limit of vitamin B12 serum

levels below 300 pg/mL.


It is noted that a majority of patients with vitamin B12 deficiency have

metformin treatment.

Fig. 11.4 Average values for serum vitamin B12 to patients according to the presence

of metformin and duration of diabetes diagnosis

226,71

431,75

262,29

398,60

189,56

401,36

125,00

453,15

0,00 100,00 200,00 300,00 400,00 500,00

Deficit with

metformin

Normal with

metformin

Deficit with

metformin

Normal with

metformin

pg/mL

Mean vitamin B12 serum levels for

diagnosing diabetes duration of

more than 10 years

Mean vitamin B12 serum levels for

diagnosing diabetes duration of less

than 10 years


24

By comparing groups with B12 deficiency (using ANOVA) for DZ

diagnosis duration lower, respectively, more than 10 years, it was found that there

are significant differences between the mean vitamin B12 serum levels.

As figure 11.4, when comparing vitamin B12 average levels, there is a

downward trend in increasing duration of diabetes diagnostic for patients with

metformin.

In terms of percentage frequency of vitamin B12 deficiency cases for the

groups analyzed, there is a high rate for patients treated with metformin:

- 66.66% of patients with serum vitamin B12 levels less than 300 pg/mL

for patients treated with metformin,

- 32.25% of patients with serum vitamin B12 levels less than 300 pg/mL

for patients not treated with metformin.

11.2.4.4. Influence of treatment with metformin on serum levels of

vitamin B12, independent of the duration of treatment with metformin

The working hypothesis is considering the influence of treatment with

metformin on serum vitamin B12 levels, by analyzing vitamin serum levels for the

patients treated with metformin (Group 1/YES) compared with controls (Group

2/NO). Because the Group 1 shows a number of cases double the Group 2, it was

randomly divided the data set of Group 1 in two series. In this manner, statistical

analysis was applied.


The two series each have 31 data obtained, therefore it was possible to

apply the test, according to the method. Tables 11.XI and 11.XII centralize values

of the two series and the results of statistical processing.

Applying ANOVA for Set 1 we obtain function F (10.12) an amount 2.5

times higher than Fcritic (4) and the probability P (0.002326) for alpha equal to 0.05

is higher than 99%. So null hypothesis is invalidated and we can say that the

difference between serum vitamin B12 levels means for set 1 is statistically

significant (respectively 274.19 and 385.10 pg/mL).

Applying ANOVA for Set 2 we obtain F (16.98), an amount 4 times

higher than Fcritic (4) and the probability P (0.000117) for alpha equal to 0.05 is

higher than 99.9%. So null hypothesis is invalidated and we can say that the

difference between serum vitamin B12 levels means for set 2 is statistically

significant (respectively 251.94 and 385.10 pg/mL).

Patients treated with metformin in this study had serum vitamin B12

levels significantly lower than patients who did not received treatment with

metformin. Thus, for both series described study hypothesis is confirmed.


25

Table 11.XI. Comparison of serum level of vitamin B12 for set 1

Serum level of vitamin B12 (pg/mL)

Patients treated (YES) or untreated (NO) with

metformin

YES NO

190 459

294 531

30+8 564

256 279

212 257

205 296

372 341

125 125

187 302

573 751

238 261

196 308

322 212

424 585

349 416

125 731

125 386

174 295

183 447

207 359

191 353

281 384

501 407

571 294

356 482

430 317

226 527

125 466

125 322

427 221

202 260

Mean serum vitamin B12 levels

(pg/mL) 274,19 385,10

Anova: Single Factor

F P-value F crit

10,1167493 0,002326 4,001194


26

Table 11.XII. Comparison of serum level of vitamin B12 for set 2

Serum level of vitamin B12 (pg/mL)

Patients treated (YES) or untreated (NO) with

metformin

YES YES

181 459

326 531

258 564

125 279

164 257

357 296

125 341

125 125

177 302

300 751

517 261

201 308

270 212

430 585

283 416

193 731

173 386

279 295

242 447

274 359

212 353

164 384

125 407

541 294

125 482

335 317

205 527

214 466

283 322

315 221

291 260

Mean serum vitamin B12 levels

(pg/mL) 251,94 385,10

Anova: Single Factor

F P-value F crit

16,98481 0,000117 4,001194

The results of this investigation is in the same direction with other similar

studies reported in the literature. Thus, the published results confirmed the

influence of metformin on serum vitamin B12 levels. Specialists recommendations

include annual testing of vitamin B12 levels in patients treated with metformin (52,


27

154) or medication supplementation with calcium, involved in the absorption of

vitamin (155).

Although some studies illustrate resistance to vitamin B12 in patients

diagnosed with diabetes (156), published research confirm the direct role of

metformin in lowering serum vitamin B12 (from 146, 156–158).

11.3. CARDIOVASCULAR RISK ASSESSMENT IN PATIENTS WITH

TYPE 2 DIABETES

Cardiovascular risk in patients with type 2 diabetes in the study group

was identified using the UKPDS RE program (UK Prospective Diabetes Study

Risk Engine). UKPDS RE is a tool for calculating cardiovascular risk for use in

patients with type 2 diabetes. Based on data provided by clinical trials (UKPS-

United Kingdom Prospective Diabetes Study) in the last 20 years, the program

can estimate the probability for occurrence of coronary disease or stroke among

these patients (163, 164).


After applying inclusion and exclusion criteria, 21 cases were analyzed

to assess cardiovascular risk.

Fatal and non-fatal coronary heart disease (CHD) obtained the highest

values of risk estimated for the next 10 years, with an average of 19.81% (fig.

11.13).

Fig. 11.13. Cardiovascular risk in patients with type 2 diabetes

19,81%

14,59%

9,97%

1,50%

0,00%

5,00%

10,00%

15,00%

20,00%

25,00%

30,00%

35,00%

CHD F CHD Stroke F Stroke

Car

dio

vas

cula

r ri

sk (

%)


28

By comparing the values obtained, it appears that male patients show

higher cardiovascular risk compared to females for the 4 categories evaluated

(CHD, F CHD, Stroke, F Stroke) (fig. 11.14).

Fig. 11.14. Cardiovascular risk by sex of the patient

Analyzing the data obtained it shows that cardiovascular risk has a

tendency to increase with increasing patient age. Obtaining statistically significant

correlations are limited by the small number of patients analyzed, including

investigative parameters (glycosylated hemoglobin, cholesterol, blood pressure,

body mass index) or cardiovascular medication.

Chapter 12

GENERAL CONCLUSIONS. ORIGINAL CONTRIBUTIONS.

RESEARCH PERSPECTIVE

GENERAL CONCLUSIONS

The research conducted in this thesis led to the following conclusions:

Statistical studies of medication and associated pathology in patients

with type 2 diabetes were meant to highlight the incidence of diabetes related

pathology and drug classes prescribed:

CHD F CHD Stroke F Stroke

Female 14,73% 10,75% 8,67% 1,44%

Male 24,44% 18,08% 11,15% 1,55%

-5,00%

0,00%

5,00%

10,00%

15,00%

20,00%

25,00%

30,00%

35,00%

40,00%

Car

dio

vas

cula

r ri

sk (

%)

Female Male


29

The design was a descriptive retrospective study for 221 patients

hospitalized in the Center of Family Medicine Iaşi.

The results showed a high incidence of chronic complications of

type 2 diabetes and associated cardiovascular disease (hypertension

is present in 83.71% of cases), data which are consistent with reports

from special publications.

Antidiabetic medication most commonly prescribed is the biguanide

(76.01%) and sulfonylurea (31.67%). Data from this study support

the choice of oral antidiabetic drugs in terms of toxicology.

Associated cardiovascular medication present high prescribing rate,

first is angiotensin converting enzyme inhibitors (45.25%).

Metformin quantitative analysis by capillary electrophoresis with UV

detection provides a simple and effective method, with the possibility of

applications on biological samples.

Electrophoretic separation was carried out in phosphate buffer at

pH 4.0 at a voltage of 15 kV, and the detection was performed at

200 nm.

The method was validated with the establishment of validation

parameters. Concentration range for metformin hydrochloride was

5-1000 µg/mL.

It has been developed a method for extraction of metformin in

human serum samples for analysis by capillary electrophoresis with

UV detection with a recovery of 98.14%.

Simultaneous quantitative determination of metformin and glimepiride

by UPLC-QTOF/MS was performed for the first time for these compounds, being

a modern, specific and improved method analysis with qualitative assessments

and dosing applications in toxicology.

Compounds analyzed were separated on a C18 UPLC BEH column,

gradient elution with 0.05% formic acid and acetonitrile.

Detection was carried out using the QTOF/MS system on the basis

of accurate mass m/z.

Method was validated by checking the validation parameters.

By optimizing a method of extraction from human serum, the

ULPC-QTOF/MS method may be used for determination of

biological samples.

o The extraction recoveries obtained were 43.96% for

metformin and 102.83% for glimepiride.

o Internal standards were used to correct possible errors in

determination: propranolol for metformin, glibenclamide

for glimepiride.


30

o There were analyzed 15 serum samples from patients under

treatment with metformin; serum levels obtained where in

the range of 34.629 to 2346.857 µg/L, in accordance with

the literature.

The clinical study of patients hospitalized in Diabetes, Nutrition and

Metabolic Diseases of the Clinical Emergency County Hospital "St. Spiridon"

addressed several toxicological research directions.

There were presented two clinical cases in which metformin therapy

was discontinued as a precaution - improvement or prevention of

adverse effects due to metformin (gastrointestinal disturbance or

lactic acidosis).

It was evaluated the influence of metformin treatment on serum

levels of vitamin B12.

o They were enrolled in the study 103 patients, 72 of them

with metformin, and 31 were the control group.

o Frequency of vitamin B12 serum levels less than 300 pg/mL

was higher in patients treated with metformin, compared to

those treated with metformin.

o Metformin-treated patients had serum level of vitamin B12

significantly lower than patients who did not received

treatment with metformin.

o Serum vitamin B12 level is not influenced by the serum

concentration of metformin, treatment duration with

metformin or duration of diabetes.

o A slight decrease in serum vitamin B12 means depending

on the duration of treatment was observed in periods of

over 4 years of therapy with metformin.

Cardiovascular risk was determined for the next 10 years through

the program UKPDS RE.

o It was assessed the risk for fatal and non-fatal coronary

heart disease, fatal coronary heart disease, fatal and non-

fatal stroke and fatal stroke.

o The averages were higher for male patients, at all four risk

categories analyzed.

o A percentage of the highest risk were obtained for fatal and

non-fatal coronary heart disease, reaching a maximum of

46.70%.

ORIGINAL CONTRIBUTIONS

In the thesis "Chemical and clinical toxicological study of oral

antidiabetic drugs" were found following original contributions:


31

Statistical interpretation of pathology and medication profile for

patients diagnosed with type 2 diabetes, highlighting prescribing

oral hypoglycaemic agents at the local level.

Approach of modern analysis methods of oral antidiabetics, which

are improved tools for applications in toxicology.

The clinical study investigating the oral antidiabetic effects in

patients in the Emergency County Hospital "St. Spiridon", with the

analyze of current interest issues.

RESEARCH PERSPECTIVE

The results obtained in the thesis provides new directions for advanced

research:

Applications of developed methods for quantitative analysis of oral

antidiabetic drugs in chemical-toxicological studies or in the case of

drugs poisonings.

The analysis of correlations between concentrations of biological

samples for studied oral antidiabetics and the toxicological profile.

Study on toxicokinetic characterization for metformin and

glimepiride by analysing biological samples (blood, urine) at

different times after administration.

In vivo study on animal models regarding the evaluation of toxic

reactions for the approached compounds.

Development of clinical studies on monitoring and evaluation of

adverse reactions to oral antidiabetic drugs.


32

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34

List of published works

1. Papers published in ISI journals

1.1. 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)

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

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

Smoked Cigarettes in Romania


de impact 0,677)

1.3. 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


de impact 0,81)

1.4. 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


de impact 0,956)

2. Papers published in BDI indexed journals

2.1. 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)

2.2. 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


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


35

2.3. 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


7848)

2.4. 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), 2015: 119(2): 585-591 (ISSN 0048-

7848)

3. Papers presented at congresses and symposia

3.1. Anca-Monica Strugaru, Luminița Agoroaei, Elena Butnaru

File de istorie: Aretaeus din Cappadocia și diabetul zaharat

(oral communication – A XXII-a Reuniune Națională de Istoria Farmaciei,

4-6 aprilie 2013, Iași). In extenso: Pagini din istoria farmaciei, Editura “Gr.

T. Popa”, U.M.F. Iași, 2013, p. 159-162(ISSN 978-606-544-155-2)

3.2. 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)

3.3. Ioana-Cezara Grigoriu, Luminiţa Agoroaei, Anca-Monica Strugaru, Elena

Butnaru

Metode analitice pentru determinarea unor compuşi antihistaminici din

fluide biologice

Analytical Methods for Determination of Antihistamine Compounds in

Biological Fluids

(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)

3.4. 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

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

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


36

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

Bonaventura Orfila

(oral communication – 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)

3.5. 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)

3.6. 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)

3.7. 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)

3.8. Luminiţa Agoroaei, Anca-Monica Strugaru, Ioana-Cezara Grigoriu, Elena

Butnaru

Familia de farmacişti ieşeni Lochman




3.9. Luminiţa Agoroaei, Anca-Monica Strugaru, Ioana-Cezara Grigoriu, Elena

Butnaru

Mateo José Bonaventura Orfila – părintele toxicologiei moderne




3.10. Elena Butnaru, Ioana Cezara Grigoriu, Anca Monica Strugaru, Claudia

Călinescu, Luminiţa Agoroaei

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





37

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

Elena Butnaru

Istoria descoperirii histaminei și antihistaminicelor – H1




3.12. 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)

3.13. 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)

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

Agoroaei, Ioana-Cezara Grigoriu, Elena Butnaru

Influența metforminului asupra nivelului seric de vitamină B12

(oral communication – Zilele Medicamentului, Ediția a XXIV-a, 3-4

decembrie 2015, Iași)

3.15. 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)

3.16. Anca Monica Strugaru, Elena Butnaru, Julia Kazakova, Manuel

Callejon-Mochón, Rut Fernandez-Torres

Simultaneous quantitative determination of metformin and glimepiride in

human serum by ultra high performance liquid chromatography with mass

spectrometry/QTOF detection

(poster – XV Reunion del Grupo Regional Andaluz de la Sociedad Espanola

de Quimica Analitica, 30 iunie-1 iulie 2016, Almeria, Spania)

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

Elena Butnaru

Originile naturale ale metforminului


38

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)

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

Elena Butnaru

Profesorul Marțian Cotrău (1923-1998) și istoria farmaciei



Pharmakon, p. 27-33 (ISSN 2457-3027)

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

Cezara Grigoriu, Luminița Agoroaei

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



Pharmakon, p. 84-97 (ISSN 2457-3027)

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

Luminița Agoroaei

Cazuri celebre de intoxicații cu monoxid de carbon



Pharmakon, p. 176-180 (ISSN 2457-3027)

3.21. 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

(oral communication – Congresul Național de Farmacie din România, Ediția

a XVI-a, cu tema “Farmacia – centru al interdisciplinarității științelor vieții”

(28 septembrie-1 octombrie 2016, București)

phd thesis abstract chemical and clinical toxicological ... · pdf file- phd thesis abstract...

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