Analytical Study of Some Pharmaceutical Compounds Sally

Analytical Study of Some Pharmaceutical Compounds

Thesis presented by Sally Saad El-Din Mahmoud El-Mosallamy

B. Pharm. Sci., 2009, M. Pharm. Sci. 2016 Assistant lecturer at Faculty of Pharmacy, Cairo University

Submitted for the fulfillment of the Degree of Doctor of Philosophy in Pharmaceutical Sciences "Analytical Chemistry" Under the Supervision of

Prof. Dr. Badr El-Dean Abd El-Haleam El-Zeany

Professor of Analytical Chemistry Faculty of Pharmacy, Cairo University

Assistant Prof. Dr. Nagiba Yehia Mohamed Hassan

Assistant Professor of Analytical Chemistry Faculty of Pharmacy, Cairo University

Prof. Dr. Hanan Abd El-Monem Merey

Professor of Analytical Chemistry Faculty of Pharmacy, Cairo University

Department of Analytical Chemistry Faculty of Pharmacy Cairo University 2019 Abstract

This thesis is concerned with the analysis of some pharmaceutical compounds in different mixtures. It comprises three parts, part I is about determination of acefylline piperazine and bromhexine hydrochloride in the presence of their degradation products and dosage form excipients (methyl and propylparabens). While part II is about the simultaneous analysis of a binary mixture of camylofin dihyrochloride and analgin in the presence of their degradation products. In addition to Part III which is concerned with the simultaneous analysis of a binary mixture of calcipotriol monohydrate and betamethasone dipropionate in the presences of dosage form excipients (butylated hydroxytoluene and alpha-tocopherol). Various analytical techniques were used for the simultaneous analysis of these mixtures. The applied techniques include spectrophotometry, chemmometry, thin layer chromatography (TLC), high performance liquid chromatography (HPLC) and electrochemistry. The proposed methods were simple, accurate, and reproducible, which would be feasible, and sensitive for the determination of the studied drugs. The proposed methods were validated according to ICH-guidelines.

Key Words: Spectrophotometry; chemmometry, thin layer chromatography; high performance liquid chromatography; electrochemistry; bromhexine hydrochloride, acefylline piperazine, camylofin dihydrochloride; analgin; calcipotriol monohydrate and betamethasone dipropionate.

Introduction

Drugs are co-formulated in order to improve medication compliance by reducing the pill burden of patients. Also, one of the advantages of combined drugs is the ability to compose combined profiles of pharmacokinetics, effects and adverse effects.

Quantitative analysis of pharmaceuticals is necessary throughout the various stages of drug development and manufacture. It is considered advantageous to explore accurate rapid methodologies that are low in cost and do not require hazardous solvents, prolonged sample pretreatment or extraction steps.

From this perspective, this thesis is concerned with the quantitative determination of pharmaceutical compounds in their mixtures in the presence of dosage form excipients. These mixtures are acefylline piperazine with bromhexine hydrochloride (for treatment of cough), analgin with camylofin dihydrate (for treatment of spasm pain) and calcipotriol monohydrate with betamethasone dipropionate (for treatment of psoraiasis)

The first studied mixture is acefylline piperazine with bromhexine hydrochloride. Acefylline piperazine (AC) is a stimulant drug of the xanthine chemical class. It acts as an adenosine receptor antagonist. Xanthines are drugs with complex actions that include, in varying degrees, relaxation of bronchial smooth muscle and relief of bronchospasm, stimulant effects on respiration, and anti-inflammatory effects. Theophylline and its derivatives have long been used for their bronchodilator properties in the management of asthma and chronic obstructive pulmonary disease. Bromhexine hydrochloride (BR) is a mucolytic used in the treatment of respiratory disorders associated with productive cough that associated with viscid or excessive mucus. In addition, BR has antioxidant properties. BR is intended to support the body's mechanisms for clearing mucus from the respiratory tract. In case of productive cough; cough suppressants are inappropriate, since the cough serves the purpose of clearing the airways.

 Expectorants have been used on the grounds that increasing the volume of secretions in the respiratory tract facilitates removal by ciliary action and coughing Commonly

used expectorants are ammonium salts.  Mucolytics have been shown to affect sputum viscosity and structure and patients have reported alleviation of their symptoms, but no consistent improvement in lung function has been found. One of the commonly used mucolytics is bromhexine,

 Hydrating agents liquefy mucus and also have a demulcent effect. Hydration may be achieved simply by inhaled warm moist air.

Cough may sometimes be associated with bronchospasm in patients with asthma.

 Bronchodilators such as beta 2 agonist or antimuscarinic alleviate cough associated with bronchospasm. However, they are not generally considered of benefit in other forms of cough, and hence are not recommended other than in asthma or selected patients with chronic obstructive pulmonary disease

Cough and cold preparations containing various combinations of cough suppressants and/or expectorants, with sympathomimetics, antihistamines, or analgesics are available. Some combinations, such as a cough suppressant and an expectorant, seem illogical and have little evidence to support their efficacy. As with many combinations, doses of individual drugs may be inadequate or inappropriate, and the large number of ingredients may expose the patient to unnecessary adverse effects. The second studied mixture is camylofin dihydrochloride and analgin. Analgin (ANA) is a popular analgetic, non-opioid drug, commonly used in human and veterinary medicine. In some cases, this agent is still incorrectly classified as a non-steroidal anti-inflammatory drug (NSAID). ANA is a pro-drug, which spontaneously breaks down after oral administration to structurally related pyrazolone compounds. Apart from its analgesic effect, the medication is an antipyretic and spasmolytic agent. The mechanism responsible for the analgesic effect is a complex one, and most probably rests on the inhibition of a central cyclooxygenase-3 and activation of the opioidergic system and cannabinoid system. ANA can block both PG-dependent and PG-independent pathways of fever induced by lipopolysaccharides (LPS), which suggests that this drug has a profile of antipyretic action distinctly different from that of NSAIDs. The mechanism responsible for the spasmolytic effect of ANA is associated with the inhibited release of intracellular Ca2+ as a result of the reduced synthesis of inositol phosphate. ANA is predominantly applied in the therapy of pain of different etiology, of spastic conditions, especially affecting the digestive tract, and of fever refractory to other treatments. Camylofin (CAM) is a smooth muscle relaxant with both anticholinergic action as well as direct smooth muscle action. Anticholinergic action is produced by inhibiting the binding of acetylcholine to muscarinic receptors, but the action is less pronounced. Direct smooth muscle relaxation is achieved by inhibiting phosphodiesterase type IV, which leads to increased cyclic AMP and eventually reduced cytosolic calcium. Thus camylofin has a

2 comprehensive action to relieve smooth muscle spasm. It is used to treat stomach ache in infants and children.

The third mixture is calcipotriol monohydrate and betamethasone dipropionate.

Calcipotriol monohydrate (CPM) is an analogue of vitamin D used in the treatment of psoriasis because they appear to induce differentiation and suppress proliferation of keratinocytes. Although vitamin D3 analogues affect epidermal growth, their immunosuppressive properties may be equally important for their antipsoriatic effect. Betamethasone dipropionate (BMD) is a synthetic glucocorticoid with metabolic, immunosuppressive and anti-inflammatory activities given orally, parenterally, by local injection, by inhalation, or applied topically in the management of various disorders in which corticosteroids are indicated. Topical corticosteroids are among the first-line treatments for psoriasis. Combination therapy using corticosteroids with other topical treatments, such as calcipotriol, may be used to reduce adverse effects and improve efficacy of both drugs. Both CPM and BMD are sensitive to oxidizing agents, so the antioxidants BHT and TOCO were co-formulated to preserve CPM and BMD. Therefore the aim of this work is to develop simple, accurate and precise different analytical methods for quantitative determination of CPM and BMD in the presence of TOCO and BHT.

 To develop different analytical methods that can be used for the determination of the studied drugs in their pure form and in their mixtures.  Study the stability of the studied drugs and their routes of degradation and to find methods for their analysis in the presence of their degradation products.  To validate the proposed methods according to the ICH guidelines.  To statistically compare the results obtained by the proposed methods to those obtained either by the reported or the official methods.  To study the applicability of the suggested methods in the analysis of the studied drugs in their pharmaceutical formulations.

 To explore the opportunities and the shortcomings offered by the ion selective electrodes in monitoring the degradation kinetics of BR.  To compare the in-line analyzer (ISE) and the off-line analyzer (HPLC) in monitoring the dissolution behavior of CAM and ANA

Literature review

The literature review reveals that different analytical methods were reported for the analysis of BR either alone or in combination with other drugs including spectrophotometry , HPLC, TLC, voltammetry and ion selective electrode. While only spectrophotometic and HPLC methods were reported for the determination of AC. For the studied mixture HPLc and chemometric (PLS and PCR models ) were reported for the determination of AC and BR besides the dosage form excipients (methyl and propyl parabens) and no stability indicating methods for determination of both drugs in the presence of their degradation products were reported.

By reviewing the literature in our hands we found that several methods were reported for determination of either CAM or ANA either alone or in combinations. These methods include chromatographic, spectrophotometric and potentiometric methods. This binary mixture was determined using HPLC, TLC and ion selective electrode. But no stability indicating methods were reported for the determination of both drugs in the presence of their degradation products and impurities.

While for the third studied mixture we found several HPLC methods were reported for the determination of CPM and BMD. But no methods were reported for determination of both drugs along with the dosage form excipients.

Various analytical techniques were used for the simultaneous analysis of these mixtures. The applied techniques include spectrophotometry, chemmometry, thin layer chromatography (TLC) high performance liquid chromatography (HPLC) and electrochemistry. Our scientific motivation was to develop simple, accurate, reproducible and rapid analytical methods, which would be feasible and sensitive for the determination of the studied drugs. The proposed methods were validated according to ICH-guidelines.

Summary

This thesis represents an analytical study on some pharmaceutical compounds present in mixtures; the selected mixtures were acefylline piperazine with bromhexine hydrochloride which were coformulated with methyl and propyl parabens, camylofine dihydrochloride with analgin and finally calipotriol monohydrate with betamethasone dipropionate which were co formulated with butylated hydroxytoluene and α-tocopherol. The aim of this thesis was to develop simple, accurate, precise and sensitive methods for the analysis of these drugs in their mixtures or in the presence of their degradation products. The thesis comprises three parts:

Part I: Determination of Acefylline Piperazine and Bromhexine Hydrochloride

This part includes four sections:

Section A: Introduction, Literature Review and Forced Degradation of Acefylline Piperazine and Bromhexine Hydrochloride

This section includes a general introduction about chemistry and physical properties of both acefylline piperazine and bromhexine hydrochloride followed by a summary of the different reported methods for their quantification. It also includes the degradation of acefylline piperazine in acidic and basic conditions and the oxidative degradation of bromhexine hydrochloride, then the degradation products were isolated and their structures were confirmed using IR and mass spectroscopy. Section B: Stability Indicating Chromatographic Methods for Simultaneous Determination of Acefylline Piperazine and Bromhexine Hydrochloride

This section includes two subsections:

Subsection B1: Stability Indicating TLC-densitometric Method for Simultaneous Determination of Acefylline Piperazine and Bromhexine Hydrochloride

In this section, TLC-densitometric technique was used for the determination of acefylline piperazine and bromhexine hydrochloride in the presence of acefylline acidic and basic stressed degradation products, bromhexine oxidative degradation product and impurity B, methyl and propyl parabens using butan-1-ol: acetic acid: water (7: 2: 2, by volume) as a developing solvent. Chromatogram was scanned at 245 nm for the two drugs and in the range of (2-15 μg/band) and (0.4-8 μg/band) for acefylline piperazine and bromhexine hydrochloride, respectively.

The proposed TLC-densitometric method was used for the determination of both drugs in pure powdered forms and in their pharmaceutical dosage form. Subsection B2: Stability Indicating HPLC Method for Simultaneous Determination of Acefylline Piperazine and Bromhexine Hydrochloride

In this section, an HPLC method was applied for the simultaneous determination of acefylline piperazine and bromhexine hydrochloride in the presence of their degradation ® products, bromhexine impurity and dosage form excipients using X-Select C18 as a stationary phase. The mobile phase consisted of acetonitrile and 0.1% orthophosphoric acid pH (2.3) and a gradient elution was used. The detection was carried out at 245 nm. The proposed method was used for the determination of both drugs in pure powdered forms and in their pharmaceutical dosage form. Section C: Stability-Indicating Potentiometric Ion-Selective Electrode for The Determination of Bromhexine Hydrochloride and Monitoring its Degradation Kinetic

This section included development and investigation of three sensitive and selective polyvinylchloride (PVC) matrix membrane electrodes for bromhexine hydrochloride determination. Sensor 1 was prepared using sodium tetraphenylborate (NaTPB) as an cation exchanger in PVC matrix and using dibutylsebacate (DBS) as a plasticizer without incorporation of ionophore; while 2-hydroxypropyl-β-cyclodextrin (2-HP β-CD) and 4-tert- butylcalix[8]arene (t-Bu-CX8) were used as ionophores in sensors 2 and 3. Ability of the proposed sensors for their selective determination of bromhexine hydrochloride in the presence of its oxidative degradation product or in the presence of co-formulated drug and excipients has been evaluated. Also this section was undertaken to further explore how potentiometry can be used for the reliable monitoring of the oxidation kinetics of bromhexine. In this work, sensor 1 was used to study the kinetics of the BR oxidation and calculate the rate constant and activation energy. Section D: Simultaneous Spectrophotometric Determination of Acefylline Piperazine and Bromhexine Hydrochloride in the Presence of Dosage Form Excipients

This section includes two subsections:

Subsection D1: Stability-Indicating Chemometric Method (PLS-1) for Determination of Acefylline Piperazine and Bromhexine Hydrochloride

In this section, partial least square (PLS-1) a multivariate calibration method was applied for the determination of acefylline piperazine and bromhexine hydrochloride in the presence of acefylline piperazine acidic and basic degradation products, bromhexine impurity B, methyl and propyl prabens. Twenty five different mixtures were prepared using five level seven factor experimental design. The calibration model was constructed using fifteen mixtures (calibration set), while the rest were used as an external validation set to assist the validity of the constructed model. During model development, mean centering was used as a preprocessing step and leave one out cross validation was applied. The proposed method was used for the determination of both drugs in laboratory prepared mixtures and in their pharmaceutical dosage form. Subsection D2: Different Approaches in Manipulating Ratio Spectra Applied for the Analysis of Acefylline Piperazine and Bromhexine Hydrochloride

In this section, four different methods manipulating ratio spectra were developed for the determination of acefylline piperazine and bromhexine hydrochloride in the presence of methyl and propyl parabens in pharmaceutical formulation and laboratory prepared mixtures without prior separation namely; dual wavelength in ratio spectra (DWRS), first derivative of double divisor ratio spectra (1DD-RS), derivative ratio spectra-zero crossing method (1DD) and ratio subtraction coupled with dual wavelength method (RS-DW) (Sequential spectrophotometry) . The proposed analytical methods were validated according to the ICH guidelines. Section E: Conclusion This section comprises a general conclusion for part I. The obtained results of all proposed methods in this part were statistically compared with those obtained by applying the reported method for acefylline piperazine and bromhexine hydrochloride and no significant difference were obtained. Part II: Determination of Camylofin Dihydrochloride and Analgin

This part includes three sections:

Section A: Introduction, Literature Review and Forced Degradation of Camylofin Dihydrochloride and Analgin

This section includes a general introduction about chemistry and physical properties of camylofin dihydrochloride and analgin followed by a summary of the different reported methods for their quantification. It also includes the procedure of acidic degradation of

7 camylofin and the acidic and oxidative degradation of analgin, then the degradation products were isolated and their structures were confirmed using IR and mass spectroscopy. Section B: Stability Indicating Chromatographic Methods for Simultaneous Determination of Camylofin Dihydrochloride and Analgin

This section includes two subsections:

Subsection B1: Stability Indicating TLC-densitometric Method for Simultaneous Determination of Camylofin Dihydrochloride and Analgin

In this section, TLC-densitometric technique was used for the determination of camylofin dihydrochloride and analgin in the presence of camylofin acidic degradation product, analgin oxidative and acidic degradation product and their impurities (4- aminoantipyrine and phenylglycine) using ethyl acetate: methanol: ammonia (7: 3: 0.4, by volume) as a developing solvent. Chromatogram was scanned at 220 nm for the two drugs and in the range of (0.6-11 μg/band) and (2-20 μg/band) for camylofin dihydrochloride and analgin, respectively. The proposed TLC-densitometric method was used for the determination of both drugs in pure powdered forms and in their pharmaceutical dosage form. Subsection B2: Stability Indicating HPLC Method for Simultaneous Determination of Camylofin Dihydrochloride and Analgin

In this section, an HPLC method was applied for the simultaneous determination of camylofin dihydrochloride and analgin in the presence of their degradation products, and impurities using Kinetex 2.6m C18 as a stationary phase. The mobile phase consisted of water (pH 3.5 adjusted with orthophosphoric acid): methanol: acetonitrile (70:20:10, by volume). The detection was carried out at 220 nm. The proposed method was used for the determination of both drugs in pure powdered forms and in their pharmaceutical dosage form. Section C: Potentiometric Ion-selective Electrode for Determination of Camylofin Dihydrochloride and Analgin and Monitoring Dissolution of Spasmopyralgin-M tablet

This section includes two subsections:

Subsection C1: Stability-Indicating Potentiometric Ion-selective Electrode for Determination of Camylofin dihydrochloride and Analgin

This section included development and investigation of three sensitive and selective polyvinylchloride matrix membrane electrodes for camylofin dihydrochloride and analgin determination. Sensor 1 was prepared using tetradodecyl ammonium bromide as an anaion exchanger in PVC matrix and using 2-nitrophenyloctyl ether as a plasticizer for determination of analgin. Sensor 2 and 3 were developed for determination of camylofin dihydrochloride, where potassium tetrakis 4-chlorophenyl borate was used as a cation exchanger and 2- nitrophenyloctyl ether as a plasticizer. To study the effect of ionophore incorporation in terms of sensitivity and selectivity; 2-hydroxypropyl-β-cyclodextrin and 4-tert-butylcalix[8]arene were incorborated as an ionophore in sensor 2 and 3. The ability of the proposed sensors for their selective determination of camylofin dihydrochloride and analgin in the presence of their degradation products has been evaluated. Subsection C2: A Comparative Study between Ion selective Electrode (in-line analyzer) and HPLC (off-line analyzer) for Green Real Time Monitoring of the Dissolution Behavior of Camylofin Dihydrochloride and Analgin

In this section, the two validated stability indicating methods stated in the previous sections; HPLC and ISE were used for monitoring of camylofin dihydrochloride and analgin dissolution from spsmopyralgin-M tablets. The novelty of the current study comes from performing a side by side comparison under similar experimental conditions and hence the advantages and shortcomings of each technique were directly highlighted with the aim of evaluating the sustainability of the analytical procedure. Section D: Conclusion This section comprises a general conclusion for part II. The obtained results of all proposed methods in this part were statistically compared with those obtained by applying the reported method for camylofin dihydrochloride and analgin, no significant difference were obtained. Part III: Determination of Calcipotriol Monohydrate and Betamethasone Dipropionate in the Presence of Dosage Form Excipients

This part includes three sections:

Section A: Introduction and Literature Review

This section includes a general introduction about chemistry and physical properties of calcipotriol monohydrate and betamethasone dipropionate followed by a summary of the different reported methods for their quantification.

Section B: Chromatographic Methods for Simultaneous Determination of Calcipotriol Monohydrate and Betamethasone Dipropionate

This section includes two subsections:

Subsection B1: Thin Layer Chromatography for Simultaneous Determination of Calcipotriol Monohydrate and Betamethasone Dipropionate

In this section, TLC-densitometric technique was used for the determination of calcipotriol monohydrate and betamethasone dipropionate in the presence of dosage form excipients named butylated hydroxytoluene and alpha tocopherol using chloroform: ethyl acetate: toluene (5: 5: 3, by volume) as a developing solvent. Chromatogram was scanned at 264 nm for the two drugs and in the range of (0.3-5 μg/band) and (1-15 μg/band) for calcipotriol monohydrate and betamethasone dipropionate, respectively. The proposed TLC-densitometric method was used for the determination of both drugs in pure powdered forms and in their pharmaceutical dosage form. Subsection B2: High Performance Liquid Chromatography for Simultaneous Determination of Calcipotriol Monohydrate and Betamethasone Dipropionate

In this section, an HPLC method was applied for the simultaneous determination of calcipotriol monohydrate and betamethasone dipropionate in the presence of their using ® OnyxMonolithic C18 as a stationary phase. The mobile phase consisted of solvent X (methanol) and solvent Y (water and acetic acid, 98:2, v:v) as mobile phase in gradient elution. The detection was carried out at 264 nm. The proposed method was used for the determination of both drugs in pure powdered forms and in their pharmaceutical dosage form. Section C: Multivariate Calibration Methods for Simultaneous Determination of Calcipotriol Monohydrate and Betamethasone Dipropionate

In this section, two multivariate calibration methods were applied, namely, principle component regression (PCR) and partial least square (PLS). Twenty five different mixtures were prepared using five level four factor experimental design containing different ratios of calcipotriol monohydrate, betamethasone dipropionate, butylated hydroxytoluene and alpha tocopherol. The calibration model was constructed using seventeen mixtures (calibration set), while the rest were used as an external validation set to assist the validity of the constructed model.

During model development, mean centering was used as a preprocessing step and leave one out cross validation was applied.The proposed methods were used for the determination of both drugs in laboratory prepared mixtures and in their pharmaceutical dosage form. Section D: Conclusion This section comprises a general conclusion for part III. The obtained results of all proposed methods in this part were statistically compared with those obtained by applying the official methods for calcipotriol monohydrate and betamethasone dipropionate, no significant difference were obtained.

Conclusions

 Acefylline piperazine (AC) is susceptible to acidic and basic stressed degradation, while bromhexine hydrochloride (BR) is susceptible to oxidation using hydrogen peroxide giving BR N-oxide.  Camylofin dihydrochloride (CAM) possess a hydrolysable ester linkage. CAM showed hydrolytic behavior under acidic accelerated conditions which affect both the stability and efficacy of the drug.  The obtained acidic degradation product was identified and the suggested pathway was confirmed by IR and Mass spectroscopy.  Analgin undergoes oxidative and acidic degradation and the obtained degradation products were identified and the suggested pathways were confirmed by IR and Mass spectroscopy.  As no stability indicating methods were reported for the studied mixtures, different validated stability indicating methodologies for the determinations of either CAM and ANA or AC and BR in the presence of their degradation products namely; TLC densitometric method, HPLC method, chemometrics and Ion-selective-electrode potentiometric method were investigated.  The results obtained for the analysis of the studied drugs in the pure powder form by the proposed methods were statistically compared with those obtained by applying the either reported or official methods and show no significant difference with respect to accuracy and precision.  The developed TLC method is sensitive. It has the advantages of short run time, large sample capacity and use of minimal volume of solvents.  Ion selective electrodes succeeded in monitoring either the degradation of BR or the dissolution of CAM and ANA also the proposed sensors are more sensitive than the reported ones.  The proposed sensors offer advantages of fast response and elimination of drug pretreatment or separation steps. They are also inexpensive and could compete with the many sophisticated and well-established methods currently available.  ISE (in–line analyzer) is superior over HPLC (off-line analyzer) being a greener alternative used in monitoring the dissolution behavior of spasmopyralgin-M® tablets. The HPLC-monitoring was relatively time-consuming due to multiple sampling and purification steps, which form non-continuous profiles.

 The suggested spectrophotometric methods are eco-friendly and simple as they neither needs sample preparation, buffer preparation, expensive solvents nor sophisticated liquid chromatographic instruments and special software or data preprocessing.  All the proposed methods are the first for the studied mixture. As no methods for determination of calcipotriol monohydrate (CPM) and betamethasone dipropionate (BMD) in the presence of dosage form excipients were reported.  The proposed HPLC method neither uses buffer nor acetonitrile so it is simple and eco- friendly in comparison to the reported ones, also it separate CPM and BMD at shorter retention time.  All the proposed methods are simple, accurate, precise and can be used in the routine analysis and quality control laboratories of the studied drugs in pure form and in their pharmaceutical formulation.