Development and Validation of Analytical Methods For the

Determination of Some Drugs Affecting the Circulatory System

A Thesis Presented by

Ahmed Mohammed Saleh Al-Ghani

B.Sc.Pharm. Sci. Sana’a University (2003)

M. Pharm. Sci. Sana’a University (2011)

Submitted For

The Degree of Doctor of Philosophy in Pharmaceutical Sciences

(Pharmaceutical Chemistry)

Supervised By

Prof. Dr. Asmaa Ahmed El-Zaher

Professor of Pharmaceutical Chemistry

Faculty of Pharmacy
Cairo University

Ass. Prof. Dr. Marianne Alphonse Mahrouse

Associate Professor of Pharmaceutical Chemistry

Faculty of Pharmacy
Cairo University

Faculty of Pharmacy
Cairo University
2018

1
 Abstract

Abstract

This thesis is concerned with studying analytical methods for the determination of some
drugs (Etamasylate, Cinnarizine and Piracetam) affecting the circulatory system. The thesis
consists of the following sections:
Section I: Introduction It includes:
I.A review of circulatory system (cardiovascular), vascular disorder drugs and its
classification.
II. Literature review about the reported methods for the quantitative determination of the
drugs.
Section II: Aim and basis of the work: this section includes the aim of this work and the
basis on which the proposed methods were chosen is clarified.
Section III: Experimental and Discussion
This section is divided into two parts:
Part I- Chromatographic Methods.
This part is further subdivided into two methods:
Part I-A. RP-HPLC Methods.
Part I-B. TLC-Densitometric Methods.
Part II- Spectroscopic methods.
This part is further subdivided into two methods:
Part II-A-. Derivative and Derivative ratio spectrophotometric methods.
Part II-B-Chemometric-Assisted Spectrophotometric Methods.
Section-IV: Summary.
Section-V: References.
Section-VI: Arabic summary.

This thesis also contain 72 tables and 45 figures.

1
 Introduction

Circulatory system

The circulatory system, also called the cardiovascular system, that consists of the
heart and two vascular systems, the pulmonary and systemic circulations . The heart pumps
blood from the right ventricle through the pulmonary circulation, in which gas exchange
occurs and the oxygenated blood then returns to the left side of the heart, where the left
ventricle pumps it to the systemic circulation which delivers blood to individual organs.
Arteries supply blood at high pressure whereas arterioles are smaller vessels with muscular
walls which allow direct control of flow through the capillary beds. Capillaries have thin
walls, comprising a single layer of endothelial cells, which allow exchange of substances
such as nutrients, hormones and waste products between blood and tissues. Veins return
blood from the capillary beds to the heart, and contain about 70% of the circulating blood
volume
The function of the cardiovascular system is to supply the needs of the body tissues
through transportation of nutrients to the cells, removing away the waste products,
transporting hormones from one part of the body to another, and, in general, maintain an
appropriate environment in all the tissue fluids of the body for optimal survival and function
of the cells [2]. The rate of blood flow through most tissues is controlled in response to tissue
need for nutrients. The heart and circulation in turn are controlled to provide the necessary
cardiac output and arterial pressure to cause the needed tissue blood flow.

Vascular disorders:
Cardiovascular disease is the most frequent cause of adult death in the western world;
in the UK one-third of men and one-quarter of women die as a result of ischemic heart
disease. There are predictions that cardiovascular diseases will soon become the leading
cause of death on all continents. Examples of these disorders include angina, arrhythmia,
hypertension, hyperlipidemia and fibrinolysis. Strategies for the treatment and prevention of
heart disease can be highly effective and have been subjected to rigorous evaluation in many
randomized controlled trials.

1
 Introduction

Figure : Simplified diagram of the human circulatory system in anterior view.

2
 Introduction

Classification of cardiovascular drugs:

Cardiovascular drugs are classified according to the type of disorder to:
1. Anti-anginal drugs:
They are directed mainly towards alleviating and preventing angina attacks by
alternating the oxygen supply/oxygen demand ratio to the cardiac muscle or dilating the
coronary vessels. They are classified into three classes each has different mechanism (organic
nitrates, calcium channel blockers and β adrenergic blocking agents.
Organic Nitrates: They act as pharmacological source of nitric oxide that is produced
naturally by vascular endothelial tissues and is important for relaxation of vascular smooth
muscle, inhibition of platelet aggregation (anti-thrombotic) and inhibition of leukocyte-
endothelial interaction (anti-inflammatory). Nitrate esters, such as nitroglycerin, lower
arterial blood pressure and, in turn, reduce the work of the left ventricle, resulting in
decreased oxygen requirements, allowing the coronary system to satisfy the oxygen demands
of myocardial tissue and relieve anginal pain.

Calcium Channel blockers: They inhibit vascular smooth muscle contraction by depriving
the cell from the calcium ions through decreasing of calcium influx as a result of blocking of
calcium channels. Classes of calcium channel blockers used for treatment of angina include
dihydropyridines, benzothiazepine derivatives and phenylalkylamine derivatives.
-Dihydropyridines: (as nifedipine and amlodipine) They have much less effect on the
cardiac tissues compared to benzothiazepine, but have higher specificity for the arteriolar
vascular bed.
-Benzothiazepines: (as diltiazem) They affect both the heart and the arteriolar bed.
-Phenylalkylamines: as verapamin.
β-adrenergic blocking agents: Their use as anti-anginal agents is limited to the treatment of
excretion-induced angina. Although they can be used alone, they are often used in
combination therapy with nitrates, calcium channel blockers, or both.

2. Anti-Arrhythmic drugs:
Classes of Anti-arrhythmic drugs;
This class of drugs is classified according to their mechanism of action, and their
effects on the cardiac action potential.
Class IA (Sodium channel blockers):

3
 Introduction

It slows the conduction by blocking fast sodium channels, inhibiting phase 0 of the action
potential so, prolongs duration of action potential. It has an intermediate rate of dissociation
from sodium channels. It acts on both atrial and ventricular tissues, e.g. Quinidine and
Procainamide.
Class IB (Sodium channel blockers):
It shorten phase 3 repolarization, so shorten the duration of action potential. It has rapid rate
of dissociation from sodium channels. It acts only on the ventricular tissues, e.g. Tocainide.
Class IC (Sodium channel blockers): It markedly' slows phase 0 depolarization that cause
the slowdown of the conduction. It has slow rate of dissociation from the sodium channels. It
acts only on the ventricular tissue, e.g. Flecainide.
Class II (β-adrenergic blocking agents): It slows phase 4 depolarization, it slows the firing
of SA node and conduction through AV node, prolonging repolarization. It blocks
sympathetic stimulation of β-adrenergic receptors affecting both SA node and AV node.

Class III (Potassium channel blockers): It prolongs phase repolarization, which causes
prolongation of the duration of action potential. Thus result in the prolongation of the
effective refractory period. It acts on both atrial and ventricular tissues.
Amiodarone: It was initially developed as an anti-anginal in 1961 , after few years it was
found that it has anti-arrhythmic effects. Its cardiac effects are not well characterized, but
clinical studies showed that it is a primarily a class III agent but act also as a class I, II and IV
anti-arrhythmic. In 1980 it was approved by FDA as anti-arrhythmic drug, but due to the
highly reported side effects FDA approved it only for life threatening arrhythmia.
Class IV (Calcium channel blocker): It slows phase 4 depolarization, slowing the firing of
SA node and conduction through AV node, prolonging the repolarization of AV node.
(Example: Verapamil and Diltiazem.

3.Anti-hypertensive drugs:
Selective classes of anti-hypertensive drugs:
I-Diuretics: Compounds that increase urine flow rate have been known for centimes. They
are classified into different classes including osmotic diuretics, loop diuretics, thiazide-like
diuretics, carbonic anhydrase inhibitors, potassium sparing diuretics and mineralocorticoid
receptor antagonists. Osmotic diuretics increase the intraluminal osmotic pressure causing
water to pass from the body to the tubule, causing a diuretic effect. Loop diuretics are
believed to act mainly on ascending loop of Henle, where they inhibit the luminal

4
 Introduction

Na+/K+/2C1-" symporter. They are characterized by rapid onset and short duration of action.
Thiazide-like diuretics are structurally diverse group of sulfonamide derivatives that don’t
contain benzothiadiazine rings, but have the same mechanism of action similar to that of
thiazide diuretics. Carbonic anhydrase inhibitors induce diuresis by inhibiting the formation
of carbonic acid within proximal and distal tubular cells to limit the number of hydrogen ions
available to promote sodium reabsorption. Potassium sparing diuretics block epithelial
sodium channel in principal cells of the late distal convoluted tubule and collecting duct.
Finally, mineralocorticoid receptor antagonists antagonize the effect of the aldosterone
causing decrease in reabsorption of sodium and chloride ions and decrease the excretion of
potassium ions.

II-Agents affecting the renin-angiotensin pathway:

A. Angiotensin-Converting Enzyme Inhibitors: (ACEI) they generally inhibit the

conversion of angiotensin I to angiotensin II, but they cause an increase in bradykinin due
to the decrease of angiotensin II, resulting in dry cough, e.g. Captopril and Prindopril.
B. Angiotensin II Receptor Blockers: (ARBs) they tend to block angiotensin receptor
preventing the binding of angiotensin II to its receptor, so they don’t cause any decrease
in the level of angiotensin II avoiding the side effect of the dry cough, e.g. Olmesartan.
C. Renin Inhibitors: they inhibit directly renin, therefore preventing the formation of
angiotensin I and angiotensin II. They don’t cause any increase in the level of renin in
plasma as a compensatory mechanism as in case of ACE inhibitors and ARBs,
e.g. Aliskiren.

4. Antifibrinolytic- haemostatic drugs:

Haemostatic agents act to conserve blood but have differing sites of action in the
complex pathways determining coagulation and fibrinolysis. Antifibrinolytics inhibit the
activation of plasminogen to plasmin, prevent the break-up of fibrin and maintain clot
stability. They are used to prevent excessive bleeding. Such as aminocaproic acid (α-
aminocaproic acid) and tranexamic acid are used as inhibitors of fibrinolysis.
Tranexamic acid is used to treat epistaxis, thrombolytic overdose, surgery (e.g.
prostatectomy and bladder surgery) and to cover the risk of haemorrhage over dental
extractions in haemophiliacs. It is also increasingly being used early in civilian and military
trauma.

5
 Introduction

Etamsylate (ETS) is a haemostatic drug. It works by increasing capillary endothelial

resistance and promoting platelet adhesion. It also inhibits biosynthesis and action of
those prostaglandins which cause platelet disaggregation, vasodilation and increased capillary
permeability.
It is noteworthy that antifibrinolytics are formulated alone or in combination with
each other or with nonsteroidal anti-inflammatory drugs (e.g. MFA) to alleviate pain
associated with menorrhagia and surgery.

The combination of etamsylate (ETS) and mefenamic acid (MFA) has demonstrated a
significant activity against painful menstruation.
Etamsylate is marketed alone (Dicynone® tablets, injections) or in admixture with
tranexamic acid (Theolate® tablets) or marketed in admixture with mefenamic acid (MFA)
( Sylate M® and Eklot FM® tablets) or with tranexamic acid and Mefenamic acid (No Blos
Forte® tablets).

5. Miscellaneous drugs used for vascular disorders:

Cinnarizine (CIN) is a derivative of piperazine that has antihistaminic (H blocker) , calcium
channel blocking and sedative activities. It is also known to promote cerebral blood flow,
and so is used to treat cerebral apoplexy, post-trauma cerebral symptoms, and cerebral
arteriosclerosis.

Piracetam (PRM) is a nootropic and psychopharmacological drug. It is a synthetic cyclic

derivative of GABA. It is described as a myoclonus and neuroprotective agent. The
mechanism of action depends on eliminating of calcium chloride those results in a decrease
of the rhythm rate and an increase of the contraction amplitude.
It is noteworthy that cinnarizine and piracetam are formulated alone or in combination
with each other. Combined preparation of cinnarizine and piracetam exhibits pronounced
antihypoxic effect. The effects of both these components are mutually enhanced with respect
to the antihypoxic and cerebrovascular resistance reducing effects. The preparation also
increases the cerebral blood flow. Cinnarizine could also be viewed as a nootropic drug
because of its vasorelaxating abilities due to calcium channel blockage, which happen mostly
in brain. It is also effectively combined with other nootropics, primarily piracetam; in such
combination each drug potentiate the other in boosting brain oxygen supply or with analgesic
drugs (e.g. paracetamol).

6
 Introduction

Cinnarizine is marketed alone (Stugeron® and Cinnarizine® tablets) or in admixture

with Piracetam (Cinaretam® capsules) to treatment and prevention of motion
sickness, prevention of migraine, treatment of symptoms of cerebrovascular origin and
treatment of symptoms of peripheral-circulatory disorders or marketed in admixture with
Domperidone (DOM) ( Vertigun® tablets) to treatment of motion sikness or with Paracetamol
(PCM) (Neurozine® tablets) for treatment of vertigo and brain disorders.

7
 Aim of the Work

Aim of the work

Cardiovascular drugs are proving to be very useful therapeutic agents used to treat
and alleviate cardiovascular disorders [121].
The wide use of cardiovascular drugs in medicine promoted development of simple,
accurate, sensitive and applicable methods for their determination in pure and dosage forms.
The aim of this work is to develop simple and accurate methods for the determination
of some of these drugs in their mixtures with analgesics, non-steroidal anti-inflammatory
drugs (NSAIDs), or in presence of some of their impurities and degradation products.
Drugs cited in this thesis are etamsylate, cinnarizine and piracetam in presence of
their co-formulated drugs mefenamic acid, paracetamol and domperidone.
HPLC and HPTLC are powerful analytical tools for the separation and quantitative
analysis of pharmaceutical products. They are incorporated in the plan of the work using
ultraviolet detection for the determination of some vascular disorder drugs in the presence of
their main impurities and related substances as in case of cinnarizine / paracetamol in their
binary mixture, cinnarizine/ domperidone/ paracetamol in their ternary mixture and
etamsylate/ mefenamic acid in their binary mixture in the presence of their main impurities
(Hydroquinone, Dimethylaniline) and analysis in pharmaceutical products.
UV Spectrophotometric methods such as first derivative, second derivative and first
derivative ratio absorbance spectrophotometry are involved in the plan of this work to
estimate some drugs in mixtures and related substances as in case of cinnarizine with
paracetamol in their binary mixture.
Reviewing literature, there was no published method using chemometric technique
for the determination of etamsylate with mefenamic acid binary mixture in the presence of
their degradation products or for cinnarizine with piracetam in their binary mixture. This
encouraged the application of multivariate calibrations (Chemometric techniques), such as
principal component regression (PCR) and partial least-squares (PLS) to resolve any spectral
overlap between this drugs in mixtures and in the presence of their degradation products.
The plan of the work comprises the utilization of the proposed methods for the
quantitative determination of the studied drugs in pure form, in laboratory prepared mixtures
and in their pharmaceutical formulations in addition to validation of these methods.
In addition, the present work comprises application of the proposed methods on drug
products, validation of those methods accordance with ICH guidelines and statistical
comparison with the reported or pharmacopoeial method.
8
 Summary

Summary

Development and Validation of Analytical Methods for the Determination of Some

Drugs Affecting the Circulatory System

This thesis is concerned with the development of simple and accurate methods for the
analysis of some drugs affecting the circulatory system, namely etamsylate, cinnarizine and
piracetam. The studied drugs were analyzed in pure forms, in laboratory prepared mixtures
and in their pharmaceutical formulations.
The thesis consists of the following sections:
Section –I: Introduction

This section includes an overview of circulatory system (cardiovascular system),

vascular disorder drugs and its classification followed by a detailed presentation of the
investigated drugs and different methods used in the literature for their estimation.
Section –II: Aim and Basis of the Work

In this section, the aim of this work and the basis on which the proposed methods
were chosen is clarified.
Section -III: Experimental and Discussion

This section was further divided into two parts:

Part I- Chromatographic Methods

This part was further divided methods:

I-A. RP-HPLC Methods:

I-A-I. RP-HPLC Method for the Simultaneous Determination of Etamsylate and

Mefenamic Acid in Presence of their Main Impurities in Pharmaceutical Dosage form.

In this method complete separation of ETS, MFA, HQ and DMA were

simultaneously separated and quantified on Inertsil ODS-3V C18 column using 40 mM
potassium dihydrogen phosphate buffer (pH 7 adjusted with 0.1M NaOH): acetonitrile
(45:55, v/v)as a mobile phase at a flow rate of 1ml/min at ambient temperature. Detection was
carried at 220nm.
The method was successfully applied for the determination of etamsylate and
mefenamic acid in presence of (hydroquinone and dimethylaniline) their main impurities in
laboratory prepared mixture with mean percentage recoveries of 100.87±0.692 for ETS and
99.73±0.983 for MFA.

9
 Summary

The method was successfully applied for the determination of etamsylate and
mefenamic acid in Sylate M®tablets with mean percentage recoveries of 99.38 ± 1.600 and
100.196 ± 0.369, respectively.
I-A-II. RP-HPLC Method for the Simultaneous Determination of Cinnarizine,
Domperidone and Paracetamol in Two Pharmaceutical Dosage Form.

In this method complete separation of CIN, DOM and PCM were simultaneously
separated and quantified on Inertsil ODS-3V C18 column using acetonitrile: methanol (55:45,
v/v) as a mobile phase at a flow rate of 1.0 ml/min at ambient temperature. Detection was
carried at 220.0 nm.
The method was successfully applied for the determination of cinnarizine,
domperidone and paracetamol in laboratory prepared mixture with mean percentage
recoveries of 99.52 ± 1.035 for CIN, 100.24 ± 0.931 for DOM and 101.04 ± 0.969 for PCM.
The method was successfully applied for the determination of cinnarizine and
domperidone in Vertigun® tablets with mean percentage recoveries of 99.73 ± 1.275 and
99.57 ± 0.959, respectively, and also was successfully applied for the determination of
cinnarizine and paracetamol in Cinnarizine® & Paracetamol® tablets with mean percentage
recoveries of 101.35 ± 1.087 and 99.16 ± 0.878,, respectively.

I-B TLC-Densitometric Methods:

I-B-I. TLC-Densitometric Methods for the Simultaneous Determination of Etamsylate

and Mefenamic Acid in Presence of their Main Impurities in Pharmaceutical Dosage
Form.

A TLC- densitometric method based on the separation of ETS and MFA in presence
of their main impurities hydroquinone (HQ) and 2,3-dimethylaniline (DMA) respectively,
was described. Dichloromethane: ethylacetate: methanol: triethylamine (6: 2: 2: 0.5, v/v/v/v)
was used as developing system and the chromatogram was scanned at 300 nm.
The proposed method was successfully applied for the determination of etamsylate
and mefenamic acid in presence of up to 40% of hydroquinone and mefenamic acid their
main impurities and degradation Products in laboratory prepared mixture with mean
percentage recoveries of 99.45 ± 0.832 for ETS and 100.02 ± 1.180 for MFA.
The method was successfully applied for the determination of etamsylate and
mefenamic acid in Sylate M®tablets with mean percentage recoveries of 99.13 ± 1.317 and
100.75 ± 1.575 for ETS and MFA, respectively.

10
 Summary

I-B-II. TLC-Densitometric Method for the Simultaneous Determination of Cinnarizine,

Domperidone and Paracetamol in Two Pharmaceutical Dosage Form.

The method was based on spotting a methanolic solution of CIN, DOM and PCM on
thin layer chromatographic plate of silica gel. Quantitation was achieved by recording the
area under the peaks obtained by scanning the chromatogram using a spectrodensitometer at
270 nm. The Rf value of CIN, DOM and PCM were found to be 0.75, 0.37 and 0.25
respectively, using toluene: ethylacetate: methanol: triethylamine (5: 4.3: 0.7: 0.5, v/v/v/v) as
developing system.
The proposed method was successfully applied for the determination of cinnarizine,
domperidone and paracetamol in laboratory prepared mixture with mean percentage
recoveries of 99.83 ± 0.968 for CIN, 100.14 ± 0.940 for DOM and 100.30 ± 1.179 for PCM.
The method was successfully applied for the determination of cinnarizine and
domperidone in Vertigun® tablets with mean percentage recoveries of 100.84 ± 0.977 and
99.61 ± 0.965, respectively, and also was successfully applied for the determination of
cinnarizine and paracetamol in Cinnarizine & Paracetamol® tablets with mean percentage
recoveries of 100.92 ± 1.142 and 100.11 ± 1.260, respectively.

Part II- Spectroscopic methods

This part was further divided methods:

II-A-1. Derivative Spectrophotometric Method Determination of Paracetamol and

Cinnarizine in Pharmaceutical Dosage Form.
The zero order spectrum scan of CIN and PCM shows severe overlap in the λmax
wavelength. So CIN and PCM cannot be directly determined. Hence, second derivative order
was obtained and zero crossing with PCM spectra was found at 287.5 nm where CIN
amplitudes could be measured for its quantitative determination. Linear relationship was
obtained over the concentration range of 4–24 µg/ml for CIN. First derivative order was
obtained and zero crossing with CIN spectra was found at 235.0 nm where PCM amplitudes
could be measured for its quantitative determination. Linear relationship was obtained over
the concentration range of 4–24 µg/ml for PCM.
The proposed method was successfully applied for the determination of CIN and
PCM in laboratory prepared mixture with mean percentage recoveries of 100.66 ± 0.746
and 100.48 ± 0.503 for CIN and PCM, respectively.

11
 Summary

The method was successfully applied for the determination of CIN and PCM in
Cinnarizine & Paracetamol® tablets with mean percentage recoveries of 100.84 ± 0.972 and
100.58 ± 0.533, respectively.

II-A-2. Derivative ratio Spectrophotometric Method Determination of Cinnarizine and

Paracetamol in Pharmaceutical Dosage Form.
The overlapped spectra of CIN and PCM were resolved by the 1DD technique. The
ratio spectra of CIN was obtained by dividing the absorption spectra of different
concentration of CIN by the spectrum of PCM ( 4 µg/ml divisor) and vice versa. Then, the
first derivative of ratio spectra was obtained. Linear relationship was obtained between the
peak amplitudes at 291 and 290 nm and the concentration over the concentration range (5-30
µg/ml for CIN and 2.5-15 µg/ml for PCM).
The proposed method was successfully applied for the determination of CIN and
PCM in laboratory prepared mixture with mean percentage recoveries of 100.52 ± 0.642 and
101.11 ± 0.777 for CIN and PCM, respectively.
The method was successfully applied for the determination of CIN and PCM in
Cinnarizine® & Paracetamol® tablets with mean percentage recoveries of 100.53 ± 1.187 and
100.96 ± 0.855, respectively.

Part II-B-Chemometric-Assisted Spectrophotometric Methods

II-B-1. Simultaneous Determination of Etamsylate and Mefenamic Acid in Presence of

Hydroquinone Using Chemometric-Assisted Spectrophotometric Methods.

In this section two different chemometric methods were applied for the simultaneous
determination of ETS and MFA in the presence of (HQ); degradation of etamsylate product
in mixture either in laboratory prepared mixtures or in pharmaceutical dosage form (Sylate
M® tablets). The chemometric methods applied were principal component regression (PCR)
and partial least squares (PLS).
II-B-2 Chemometric-Assisted Simultaneous Determination of Cinnarizine and
Piracetam in Binary Mixtures.

Different chemometric methods were applied for the determination of Cinnarizine

(CIN) and Piracetam (PRM) in binary mixtures either in lab prepared mixture or in
pharmaceutical dosage form (Cinaretam® capsules). The chemometric methods applied
were principal component regression (PCR) and partial least squares regression (PLS).

12
 Summary

Section-IV: Summary.
Section-V: References.
Section-VI: Arabic summary.

13
 Conclusion

The proposed chromatographic methods ( RP-HPLC and TLC-densitometric

methods) and spectrophotometric methods (derivative, ratio derivatives spectrophotometric
method and assisted chemometric methods) have the advantages of simplicity, precision,
accuracy and convenience for the simultaneous separation and quantification of ETS,
MFA, CIN, DOM, PCM and PRM, some in presence of their main impurities or in
combination with each other in laboratory prepared mixture and pharmaceutical
preparation. Hence, the proposed method can be used for the quality control of the cited
compounds.

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