Development and Validation of Analytical Methods for the Determination of Some Dermatological Drugs Amaal Abdullah Al-Tahami

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Development and Validation of Analytical Methods for the Determination of Some Dermatological Drugs

A Thesis Presented by Amaal Abdullah Al-Tahami B. Pharm. Sci. (1993) Faculty of Pharmacy, Sana’a University

M. Pharm. Sci. (2001) Faculty of Pharmacy, Jordan University

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. Ehab Farouk El-Kady Associate Professor of Pharmaceutical Chemistry Faculty of Pharmacy Cairo University

Faculty of Pharmacy Cairo University 2018

Abstract:

This thesis is concerned with the development of simple and accurate methods for the determination of some dermatological drugs, namely; betamethasone dipropionate, clioquinol, clobetasol propionate, diflucortolone valerate, econazole nitrate, fusidic acid, hydrocortisone, metronidazole benzoate, miconazole nitrate, mometasone furoate, salicylic acid and triamcinolone acetonide either alone or in combination with other drugs.

Different methods have been introduced for determination of the analyzed drugs in their pure form, laboratory prepared mixture and in pharmaceutical dosage forms.

Introduction

Dermatosis is a term that refers to diseases of the integumentary system. This classification includes everything on the surface of the body: skin, nails, and hair. Any condition affecting the skin could be categorized a dermatosis. Skin has several layers, including the epidermis, the dermis, and the subcutaneous tissue Figure 1. A dermatosis may involve changes in any or all of these skin layers[1].

Figure 1: Skin layers Some of the most common dermatological disorders: Table 1: some of the most common dermatological disorders

Name Description [2] Causes [3]

Oil glands in skin cause pimples Acne Bacteria and hormone and scarring Alopecia Hair loss around patches Autoimmune disorders areata Impetigo Skin infection Bacteria Keratosis Crusty pre-cancerous growths Sun damage The most serious form of skin Melanoma Genetic susceptibility cancer basal

Nails curve around the fingertips Nail due to low oxygen levels in the Various types of lung disease clubbing blood Several causes such as The fingernails become loose and Onycholysis (Trauma, infection, and skin separate from the nail bed disease) Characterized by patches of Psoriasis Autoimmune disorders abnormal skin Fungal infection of skin that Tinea Fungus leaves round marks Vitiligo White of patches of skin Autoimmune disorders

Dermatological formulation Different dosage forms (e.g. creams, ointments, gels, pastes, suspensions, lotions, foams, sprays, aerosols, and solutions) containing special topical agents are directly applied to the malfunctioning skin to treat a specific disease or alleviate a condition. A unique aspect of dermatological pharmacology is the direct accessibility of the skin as a target organ for the diagnosis and treatment. Therapeutic agents can reach epidermal keratinocytes and immunocompetent cells in the epidermis and the underlying dermis that are involved in the pathogenesis of numerous cutaneous diseases. Topical agents can be applied directly to the skin but must penetrate into the tissue to achieve efficacy. Appropriate use of topical agents requires an appreciation of the factors that influence percutaneous absorption [4]. On the other hand, there are a lot of dermatological drugs used systemically involving several route of administration: oral, intramuscular (e.g., methotrexate, glucocorticoids), intravenous (e.g., immunoglobulin, alefacept) or subcutaneous (e.g., eflizumab, etanercept)[4].

Most common drugs for dermatological disorders: 1- Corticosteroids Corticosteroids have immunosuppressive and anti-inflammatory properties. They are used for treatment of psoriasis, eczema, contact dermatitis, and other skin conditions manifested by itching and inflammation [5]. Topical corticosteroids act by binding to a specific receptor in the cellular cytoplasm and modulating the transcription of multiple genes. This leads to the suppression of the production of inflammatory

substances such as prostaglandins and leukotrienes, and also inhibits the recruitment of inflammatory cells into the skin[6]. There are many topical steroids available, and they differ in potency and formulation Table 2. Table 2: Most commonly used corticosteroids for dermatological disorders:

Generic Name Chemical Structure

Alclometasone dipropionate

Amcinonide

Betamethasone dipropionate

Clobetasol propionate

Desonide

Diflucortolone valerate

Flucinolone acetonide

Fluticasone propionate

Halobetasol propionate

Hydrocortisone

Mometasone furoate

Triamcinolone acetonide

2- Antibacterial agents: Majority of skin infections are caused by Gram-positive bacteria, most commonly Staphylococcus aureus and group A β-hemolytic Streptococcus (GABHS). Some Gram-negative organisms and anaerobes may also cause pyogenic skin infections. Other organisms like Mycobacteria sp. and Treponema pallidum may also cause infections. Topical antibacterial agents may be useful in preventing infections in clean wounds, in the early treatment of infected dermatosis and wounds [5]. Each class of antibacterial drugs has a unique mode of action which is summarized in (inhibition of cell wall synthesis, inhibition of protein synthesis, inhibition of nucleic acid synthesis, alteration of cell membrane function or alteration of cell metabolism)[7]. Some topical anti-infectives contain corticosteroids in addition to antibiotics. There is no convincing evidence that topical corticosteroids inhibit the antibacterial effect of

antibiotics when the two are incorporated in the same preparation. In the treatment of secondarily infected dermatoses, which are usually colonized with streptococci, staphylococci, or both, combination therapy may prove superior to corticosteroid therapy alone. Antibiotic-corticosteroid combinations may be useful in treating diaper dermatitis and impetiginized eczema [8].

Table 3: Most commonly used antibacterial agents for dermatological disorders:

Generic Name Chemical Structure

Erythromycin

Clindamycin

Mupirocin

Fusidic acid

3-Antifungal agents:

The treatment of superficial fungal infections caused by dermatophytie fungi may be accomplished with topical antifungal agents Table 4. Topical antifungal corticosteroid fixed combinations have been introduced on the basis of providing more rapid symptomatic improvement than an antifungal agent alone. [4] Azole antifungal drugs inhibit the enzyme lanosterol 14 α-demethylase; the enzyme necessary to convert lanosterol to ergosterol. Depletion of ergosterol in fungal membrane disrupts the

structure and many functions of fungal membrane leading to inhibition of fungal growth [9]. Table 4: Most commonly used antifungal agents for dermatological disorders: Generic Name Chemical Structure

Butenafine

Clotrimazole

Econazole nitrate

HNO3

Fluconazole

Ketoconazole

Miconazole nitrate

HNO3

4- Agents for acne:

Acne, also known as acne vulgaris, is a long-term skin disease that occurs when hair follicles are clogged with dead skin cells and oil from the skin. It is characterized by blackheads or whiteheads, pimples, oily skin, and possible scarring. It primarily affects areas of the skin with a relatively high number of oil glands, including the face, upper part of the chest and back [10].

Many treatment options for acne are available, including lifestyle changes, medications, and medical procedures. Eating fewer simple carbohydrates such as sugar may help. Treatments applied directly to the affected skin, such as azelaic acid, benzoyl peroxide, and salicylic acid, are commonly used . Antibiotics and retinoids are available in formulations that are applied to the skin and taken by mouth for the treatment of acne

Table 5. Isotretinoin pills are usually reserved for severe acne due to greater potential side effects [10].

Table 5: Most commonly used agents of acne for dermatological disorders:

Generic Name Chemical Structure

Azelaic acid

Benzoyl peroxide

Isotretinoin

5-Agents for pigmentation disorders Agents for pigmentation disorders include hydroquinone and methoxsalen, which are used for the treatment of hyperpigmented skin condition and vitiligo, respectively [5].

Hydroquinone, monobenzone and mequinol (the monomethyl ether of hydroquinone) are used to reduce hyperpigmentation of the skin Table 6. Topical hydroquinone and mequinol usually result in temporary lightening, where a monobenzone causes irreversible depigmentation The mechanism of action of these compounds appears to involve inhibition of the enzyme tyrosinase, thus interfering with the bio-synthesis of melanin. In addition, monobenzone may be toxic to melanocytes, resulting in permanent loss of these cells. Some percutaneous absorption of these compounds takes place, because monobenzone may cause hypopigmentation at sites distant from the area of application. Both hydroquinone and monobenzone may cause local irritation. Allergic contact dermatitis to these compounds can occur. Prescription combinations of hydroquinone, fluocinolone acetonide, and retinoic acid (Tri-Luma®) and mequinol and retinoic acid (Solage®) are more effective than their individual components. Methoxsalen is a photoactive substance that stimulates melanocytes and is used as a repigmentation agent for patient with vitiligo [5].

Table 6: Most commonly used agents of pigmentation for dermatological disorders:

Generic Name Chemical Structure

Hydroquinone

Mequinol

Methoxsalen

6- Drugs for psoriasis: Psoriasis is a skin disease that presents with erythematous scaling plaques. It manifests with increased epidermal cell proliferation. It can be managed with topical treatment including retinoids, vitamin D analogues, keratolytic agents and corticosteroids[5] Table 7.

Retinoids are a class of chemical compounds that are vitamers of vitamin A or are chemically related to it. Retinoids have found use in medicine where they regulate epithelial cell growth [5].

Calcipotriol is synthetic derivative vitamin D. It is used in the treatment of plaque psoriasis [11].

Keratolytic topical agents such as coal tar and salicylic acid that break down keratin (by breaking intermolecular bonds and causing maceration); assist removal of tough, outer stratum corneum, and penetration of keratolytic or caustic medicaments to deeper tissues, are effective in localized Psoriasis, especially on the scalp. They improve corticosteroid penetration [5].

Table 7: Most commonly used agent for psoriasis:

Generic Name Chemical Structure

Vitamin A1

Calcipotriol

Salicylic acid

Aim of the Work:

Dermatological agents include many different classes of drugs that are used to treat dermatological disorders.

The aim of this work is to develop simple, accurate and precise methods for the determination of some dermatological drugs. The work involves the analysis of these drugs in their pure form or laboratory prepared mixtures and in commercially used pharmaceutical dosage forms using analytical techniques such as spectroscopy and chromatography.

Drugs cited in this thesis are corticosteroid [betamethasone dipropinoate (BD), clobetasole propionate (CP), diflucortolone valerate (DV), Hydrocortisone (HC), mometasone furoate (MF) and triamcinolone acetonide (TA)]; antibacterial agents [clioquinol (CQ), fusidic acid (FA)and metronidazole benzoate(MB)]; antifungal [econazole nitrate (EC nitrate), miconazole nitrate (MC nitrate); and Keratolytic [salicylic acid (SA)].

HPLC is a powerful analytical tool for the separation and analysis of pharmaceutical products. It is incorporated in the plan of the work using ultraviolet detection for the estimation of SA /MF and with SA/BD in their two binary mixtures as ternary mixture, for the estimation of EC nitrate, TA and BA in their ternary mixture and MB, MC nitrate, CP and FA in their quaternary mixture and analysis of their pharmaceutical products.

Since the forced degradation, study helps in prediction of drug stability. Forced degradation study of diflucortolone valerate under different stress conditions was proposed.

Chemometric techniques were suggested for resolving spectral overlap displayed by EC nitrate, TA and BA in their ternary mixture, CQ/ HC and CP/CC in their binary mixtures.

In addition, method validation of the proposed methods with statistical comparison between the obtained results and those of the reported methods were conducted.

Summary: The thesis consists of the following sections 1- Introduction I. A review about some of the most common forms of dermatosis and the most important drugs for dermatological disorders [topical corticosteroid, topical antibacterial drugs, topical antifungal drugs, drugs for acne, agents for pigmentation disorders and drugs for psoriasis] II. Literature review about the official and reported methods for the quantitative determination of the drugs under investigation. 2-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. 3-: Experimental and Discussion This section was further divided into two subsections: Part Ӏ: High Performance Liquid Chromatographic Methods with Ultraviolet Detection.

Part ӀӀ. Spectrophotometric Methods.

3- Statistical Analysis.

4- Summary.

5- References.

6- Arabic Summary.

Conclusion: The proposed methods can be used for simultaneous determination for analyt drugs in laboratory prepared mixtures and pharmaceutical dosage forms containing them without interference with each other and without the need for previous physical separation of the drug.

Part Ӏ.A. RP-HPLC-UV Detection Method for the Simultaneous Determination of Two Binary mixtures, Mometasone furoate/Salicylic acid and Betamethasone dipropionate /Salcylic acid as a Ternary Mixture.

A reversed phase high performance liquid chromatography (RP-HPLC) method for the simultaneous determination of two binary mixtures, Mometasone furoate (MF) and Betamethasone dipropionate (BD) with Salicylic acid (SA) in laboratory prepared mixtures and in pharmaceutical dosage form was developed and validated. The mobile phase used was methanol: 20 mM potassium dihydrogen phosphate buffer (pH 2.8±0.1 adjusted with o-phosphoric acid) (75:25, v/v). The method is based on HPLC separation of MF, BD and SA. Chromatographic separation was achieved on a MediterraneaTM Sea C18 (150mm x4.6 mm, 5 µm) column at room temperature. The flow rate was maintained at 1 ml/min and isocratic elution was applied throughout the analysis. UV detection was carried out at 254 nm.

Linear relationship was obtained for the three drugs over the concentration range of (0.4-20 μg/ml) for MF and BD, (1-200 μg/ml) for SA. The method was successfully applied for the determination of MF/SA and BD/SA, in their laboratory prepared mixtures with mean percentage recoveries of 99.59 ± 0.51 for MF and 101.40 ± 0.74 for SA and mean percentage recoveries of 99.20 ± 0.58 for BD and 100.77 ± 0.38 for SA.

The proposed method was also applied for the simultaneous determination of each mixture in their pharmaceutical dosage form (Elicasal® ointment and Diprosalic® ointment ) with mean percentage recoveries of 95.83 ± 0.89 and 99.46 ± 0.62, for MF and SA, respectively, and 97.54 ± 1.05 and 101.03 ±0.65, for BD and SA, respectively.

Part Ӏ.B. RP-HPLC-UV Detection Method for the Simultaneous Determination of Econazole Nitrate, Triamcinolone Acetonide and Benzoic Acid in Ternary Mixture

A simple reversed phase high performance liquid chromatography (RP-HPLC) method for simultaneous determination of econazole nitrate (EC nitrate), triamcinolone acetonide (TA) and benzoic acid (BA) in laboratory prepared mixtures and in pharmaceutical dosage forms was developed and validated. The mobile phase used was methanole: 50 mM potassium dihydrogen phosphate buffer (pH 2.6±0.1) (70:30, v/v). The method is based on HPLC separation of EC nitrate, TA and BA using a MediterraneaTM Sea C18 (4.6 x 150 mm, 5 μm) column at room temperature. The flow rate was maintained at 1 ml/min and isocratic elution was applied throughout the analysis. UV detection was carried out at 230 nm.

Linear relationship was obtained over the concentration range of (10-200 μg/ml) for EC nitrate, (1-20 μg/ml) for TA and (2-40 μg/ml) for BA. The method was successfully applied for the determination of EC nitrate, TA and BA in their laboratory prepared mixtures with mean percentage recoveries of 101.00 ± 0.57 for EC nitrate, 100.80 ± 0.86 for TA and100.80 ± 0.86 for BA. The proposed method was also applied for the simultaneous determination of each drug in their pharmaceutical dosage forms (Pevisone® cream) with mean percentage recoveries of 97.40 ± 0.70, 100.59 ± 0.91 and 100.99 ± 0.96, for EC nitrate, TA and BA, respectively.

Part. I.C. RP-HPLC-UV Detection Method for the Simultaneous Determination of Metronidazole Benzoate, Miconazole Nitrate, Clobetasole Propionate and Fusidic Acid in Quaternary Mixture

A reversed phase high performance liquid chromatography (RP-HPLC) method for the simultaneous determination of quaternary mixture, metronidazole benzoate (MB) miconazole nitrate (MC), clobetasole propionate (CP) and fusidic acid (FA) in laboratory prepared mixtures and in pharmaceutical dosage form was developed and validated. The mobile phase used was acetonitrile: 50 mM potassium dihydrogen phosphate buffer (pH 3.2 ±0.1) (65:35, v/v). The method is based on HPLC separation of MB, MC nitrate, CP and FA. Chromatographic separation was achieved on a MediterraneaTM Sea C18 (250mm x4.6 mm, 5 µm) column at room temperature. The flow rate was maintained at 1.2 ml/min

and isocratic elution was applied throughout the analysis. UV detection was carried out at 240 nm.

Linear relationship was obtained over the concentration range of (10-100 μg/ml) for MB, (20-200 μg/ml) for MC nitrate, (0.5-5 μg/ml) for CP and (20-200 μg/ml) for FA. The method was successfully applied for the determination of drugs in their laboratory prepared mixtures with mean percentage recoveries of 99.98 ± 0.46 for MB, 101.21 ± 0.45 for MC nitrate, 99.46 ± 0.58 for CP and 99.12 ± 0.30 for FA.

The proposed method was also applied for the determination of drugs in its pharmaceutical dosage form (Lobate®M) with mean percentage recoveries of 100.40 ± 0.38 for MB, 101.50 ± 0.30 for MC nitrate 99.58 ±0.64, for CP and 99.74 ±0.44, for FA.

Part I-D. Forced degradation study to develop and validate stability-indicating RP-LC method for the determination of Diflucortolone Valerate in bulk drug and in ointment.

This method was proposed to study the degradation behavior of Diflucortolone valerate (DV) under acidic, alkaline, oxidative, thermal, and photolysis conditions, the separation was carried out using Inertsil® ODS-3 column (150mm x 4.6mm, 5µm) and acetonitrile: water (70: 30 v/v) as a mobile phase, at a flow rate, 1ml/min. Isocratic elution was applied throughout the analysis. Detection was carried out at UV detection (238).

Linear relationship was obtained over the concentration range of (2 – 100 µg/ml). To determine the accuracy of the method, it was performed on pure samples of the intact drug, with mean percentage recovery 100.17 ±0.84. Satisfactory results were obtained on applying the method on Nerisone® ointment

Forced degradation study revealed that DV was found to be liable in alkaline medium. In addition, the drug was sensitive to oxidation process yet it was found to be stable towards thermal and photolysis conditions.

Part ӀI.A.1. Chemometric Methods for the Simultaneous Determination of Econazole Nitrate, Triamcinolone Acetonide and Benzoic Acid in Ternary Mixture

In this section, two different chemometric methods were applied for the simultaneous determination of EC nitrate, TA and BA in ternary mixtures either in

laboratory prepared mixtures or in pharmaceutical dosage forms (Pevisone® cream). The chemometric methods applied were Principal component regression (PCR) and Partial least squares (PLS).

Part ӀI.A.2. Chemometric Methods for the Simultaneous Determination of Clioquinol and Hydrocortisone in Binary Mixture

In this section, three different chemometric methods were applied for the simultaneous determination of CQ/HC in a binary mixture either in pure form or in pharmaceutical dosage form (Clioquinol-Hydrocortisone® cream) The chemometric methods applied were Classical least squares (CLS), Principal component regression (PCR) and Partial least squares (PLS).

Part. ӀI.A.3. Chemometric Methods for the Simultaneous Determination of Clobetasol Propinoate and Chlorocresol in Binary Mixture

In this section, three different chemometric methods were applied for the simultaneous determination of CP/CC in a binary mixtures either in pure form or in pharmaceutical dosage forms (Clop®-E cream)The chemometric methods applied were Classical least squares (CLS), Principal component regression (PCR) and Partial least squares (PLS).

Paret II .B.1. Simultaneous Determination of Econazole Nitrate, Triamcinolone Acetonide and Benzoic Acid in Ternary Mixture Using Artificial Neural Networks Technique

ANNs were applied for the simultaneous determination of EN nitrate, TA and BA in ternary mixture either in laboratory prepared mixtures or in pharmaceutical dosage form (Pevisone® cream).

Part ӀӀ.B.2. Simultaneous Determination of Clioquinol and Hydrocortisone in Binary Mixture Using Artificial Neural Networks Technique

In this section, ANNs were applied for the simultaneous determination of CQ/HC in a binary mixtures either in laboratory prepared mixtures or in pharmaceutical dosage forms (Clioquinol-Hydrocortisone® cream).

At the end of each section, the validation parameters according to ICH guidelines for analytical methods were summarized. In addition, a statistical comparison between the results of the proposed methods and that of the reported or pharmacopoeial methods was computed and no significant difference was found.