DEVELOPMENT AND VALIDATION OF ANALYTICAL METHODS FOR THE DETERMINATION OF SOME RELAXANTS

A Thesis Presented by Maged Mohammed Saleh Al-Ward B. Pharm. Sci. Faculty of Pharmacy, Damascus University 2011 Submitted in the Partial Fulfillment for The Degree of Master of 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 Elkady Assistant Professor of Pharmaceutical Chemistry Faculty of Pharmacy Cairo University

Faculty of Pharmacy Cairo University 2017

1

Introduction

(Key Words: , hydrochloride, , citrate, hydrochloride, degradation product, Liquid chromatographic, Spectrophotometric, Chemometric methods, Artificial Neural Network technique)

Skeletal Muscle Relaxants

Definition and uses

Stimulation of skeletal muscle originates from the brain, passing through the spinal cord to somatic neurons from which the impulses are transmitted by the neurotransmitter, (ACh), to the muscle [1]. Muscle spasm, which is a painful involuntary contraction of the muscle resulting from overexcitability, the term spasticity has been loosely applied to various disorders of motor control resulting from CNS disorders, can be relieved by administration of skeletal muscle relaxants (SMR) [2] [1].

Skeletal muscle relaxants (SMR) are agents used in the management of musculoskeletal and neuromuscular disorders, also to alleviate muscle spasm and spasticity [1]. SMR are commonly combined with such as paracetamol and non-steroidal anti-inflammatory drugs (NSAIDs) such as ketoprofen, ibuprofen and aspirin for their and anti-inflammatory actions[2].

Classification of SMR

SMR are classified according to their mode of action into two main groups, peripherally acting skeletal muscle relaxants (neuromuscular blocking agents) and centrally acting skeletal muscle relaxants[2].

1

Introduction

I -Peripherally acting muscle relaxants (neuromuscular blocking agents): Neuromuscular blocking agents are principally used as adjuncts in surgical anasthesia to obtain satisfactory muscle relaxation and also to reduce doses of the and are commonly used to paralyze patients for endotracheal intubation and to relax abdominal muscles before surgery.

Neuromuscular blocking agents are classified in many ways as they are classified according to their duration of action into long, intermediate and short acting[3] or classified according to mechanism of action into nondepolarizing drugs, depolarizing blocking agents, directly acting compounds and miscellaneous[2].

I -1. Nondepolarizing drugs (competitive neuromuscular blocking drugs): These agents compete with (Ach) on the nicotinic receptors preventing depolarization of the end plate by the neurotransmitter. represent prototypes of these drugs. Acetylcholinestrase inhibitors such as neostigmine and edrophonium chloride competitively inhibit these drugs [3]. Most commonly used members of this class are shown in table (1).

I -2. Depolarizing blocking agents: The initial action of depolarizing blocking agents, such as succinylcholine and is to depolarize the membrane by opening channels in the same manner as ACh. They persist for longer durations, primarily because of their resistance to acetylcholinestrase [2], except which has short duration because it is considered a combination of two acetylcholine molecules. Most commonly used members of this class are shown in table (2).

2

Introduction

Table 1 : Most commonly used nondepolarizing neuromuscular blockers: Drug structure Trade name Atracurium Tracrium® Besylate

Doxacurium Nuromax® chloride

Fazadinium Fazadon® bromide

Gallamine Flaxedil® triethiodide

Metocurine Metubin iodide iodide®

Mivacurium Mivacron® chloride

Pancuronium Pavulon® bromide

3

Introduction

Pipecuronium Arduan® bromide

Rocuronium Esmeron®

Tubocurarine Delacuratine® chloride

Table 2 : Most commonly used depolarizing neuromuscular blockers: Drug structure Trade name

H3C CH3 ® + + Decamethonium bromide - Syncurine H3C N (CH2)10 N CH3 . 2Br H C CH 3 3

Hexafluorenium bromide Mylaxen®

Suxamethonium chloride Anectine (Succinylcholine chloride) chloride®

I -3. Agents acting directly on skeletal muscles: and the cinchona alkaloids, including , exert a direct effect on skeletal muscle excitability. They are used to reduce muscle tone rather than to induce . Dantrolene reduces muscle tone by interfering

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Introduction with the release of and thereby blocks contraction of skeletal muscle [3]. It is used for the relief of spasticity associated with a variety of conditions[1]. Most commonly used members of this class are shown in table (3)

Table 3 : Most commonly used directly acting relaxants: Drug Structure Trade name Dantrolene Dantamacrin® Sodium (sodium salt)

Quinine sulfate Quinine sulfate®

I -4. Miscellaneous Blocking Agents: Botulinum toxins A and B, which inhibit the release of ACh at the motor nerve terminals, are examples of this group. Carbolonium bromide, which produces an initial depolarizing neuromuscular block followed by competitive block, is an another example [1].

II- Centrally acting relaxants:

In contrast to peripherally acting neuromuscular blocking drugs, the muscle relaxants acting on the central nervous system (CNS) are prescribed to outpatients for acute muscle spasms as well as for the treatment of chronic spasticity [3].

The mechanism of action of centrally acting relaxants may be due to their CNS-depressant activity [1]. Most commonly used members of this class are shown in table (4).

5

Introduction

Orphenadrine citrate is marketed alone (Norflex® tablets, injections) or in admixture with paracetamol (Orphenadrine plus ® tablets). Chlorzoxazone is marketed in admixture with paracetamol (Myolgin®& Relax® capsules) or with ketoprofen (Flexofan® capsules). Tizanidine hydrochloride is marketed alone (S.M.R. ® tablets) or with mefenamic acid (Meftal-MR® tablets). Methocarbamol is marketed alone (Robaxin®) or with aspirin (Robaxisal®). Cyclobenzaprine hydrochloride is marketed alone (Multi-Relax®).

Table 4 : Most commonly used centrally acting relaxants: Drug structure Trade name Lioresal®

Carisoprodole Somadril®

Chlorzoxazone Paraflex®

Cyclobenzaprine Flexeril® hydrochloride

.HCl Myloastan®

Idrocilamide Srilane®

6

Introduction

Mephenesin Tolseram®

Meprobamate Aneural®

Metaxalone Skelaxin®

Methocarbamol Robaxin®

Orphenadrine Norflex® (citrate salt)

Tizanidine Sirdalud® Hydrochloride

.HCl

7

Introduction

The Investigated drugs

Chlorzoxazone[2, 4, 5]

Structure[5]:

Chemical name:

5-chloro-2-benzoxazolol.

Molecular formula:

C7H4ClNO2.

Molecular weight: 169.57.

Properties:

Crystals from acetone. Sparingly soluble in water; soluble in methanol, and isopropanol. Freely soluble in aqueous solutions of alkali hydroxides and ammonia. The UV absorption spectrum in absolute methanol shows a maximum at 282 ± 2 nm and minimum at 248 ± 2nm.

Melting Point:

191-191.5 ºC.

Action and uses:

Centrally-acting skeletal .

8

Introduction

Cyclobenzaprine Hydrochloride[2, 4, 5]

Structure [5]:

Chemical name:

1-Propanamine, 3-(5H-dibenzo[a,d]cyclohepten-5-ylidene)-N,N- dimethyl-, hydrochloride.

Molecular formula:

C20H21N·HCl

Molecular weight: 311.85

Properties:

A white to off-white odourless, crystalline powder. Freely soluble in water, in , and in methyl alcohol; sparingly soluble in isopropyl alcohol; slightly soluble in and in dichloromethane; insoluble in hydrocarbons.

Melting Point:

217 -219ºC.

Action and uses:

Centrally acting skeletal muscle relaxant, related to the tricyclic antidepressants.

9

Introduction

Methocarbamol[2, 4, 5]

Structure [5]:

Chemical name:

1,2-Propanediol, 3-(2-methoxyphenoxy)-, 1-carbamate.

Molecular formula:

C11H15NO5

Molecular weight:

241.24.

Properties:

A white powder, odourless or having a slight characteristic odour, Soluble 1 in 40 of water at 20°; sparingly soluble in chloroform; soluble in alcohol only with heating; insoluble in n-hexane and in benzene. Store in airtight containers.

Melting Point:

90-94 ºC.

Action and uses:

Centrally-acting skeletal muscle relaxant.

10

Introduction

Orphenadrine Citrate[2, 4, 5]

Structure [5]:

.

Chemical name:

N,N-Dimethyl-2-[(2-methylphenyl)phenylmethoxy] ethanamine citrate.

Molecular formula:

C18H23NO. Citrate, C18H23NO. C6H8O7.

Molecular weight:

269.39 (orphenadrine base).

Properties:

White or almost white, crystalline powder. Soluble in water, alcohol, chloroform. Sparingly soluble in acetone, benzene. Practically insoluble in ether. Melting Point: 156-157 ºC. Action and uses: Centrally-acting skeletal muscle relaxant and antihistaminic.

11

Introduction

Tizanidine [2, 4, 5]

Structure[5]:

Chemical name:

5-Chloro-N-(4,5-dihydro-1H-imidazol-2-yl)-2,1,3-benzothiadiazol-4- amine.

Molecular formula:

C9H8ClN5S.

Molecular weight:

253.71.

Properties:

Crystals from methanol.

Melting Point:

221-223 ºC.

Action and uses:

Centrally-acting skeletal muscle relaxant.

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Aim of the Work

Aim of the Work

Skeletal muscle relaxants are proving to be very useful therapeutic agents used to alleviate muscle spasm, spasticity and as adjuncts in surgical [3].

The wide use of skeletal muscle relaxants in medicine promoted the 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 determination of some of these drugs in their mixtures with analgesics, non-steroidal anti-inflammatory drugs (NSAIDs) or in presence of some of their degradation products.

Drugs cited in this thesis are methocarbamol, cyclobenzaprine hydrochloride, tizanidine hydrochloride, orphenadrine citrate and chlorzoxazone. HPLC is a powerful analytical tool for the separation and quantitative analysis of pharmaceutical products. It is incorporated in the plan of the work using ultraviolet detection for the determination skeletal muscle relaxants in the presence of their degradation products and related substances as in case of methocarbamol /aspirin in mixture, orphenadrine citrate/ paracetamol in mixture or in the presence of degradation product only as in case of cyclobenzaprine hydrochloride in binary mixture, as well as and determination of tizanidine hydrochloride / mefenamic acid as binary mixture and analysis in pharmaceutical products.

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Aim of the Work

Multivariate calibrations, such as principal component regression (PCR) and partial least-squares (PLS) are involved in the plan of this work to resolve any spectral overlap between drugs in mixtures and in the presence of their degradation products and related substances as in case of chlorzoxazone with ketoprofen /ibuprofen in two different mixture and in case of orphenadrine citrate with paracetamol or in the presence of degradation product only as in case of cyclobenzaprine.

Artificial Neural Networks (ANNs) are computer programs, such as the radial basis function-artificial neural network (RBF-ANN) model is involved in the plan of this work to resolve any spectral overlap between orphenadrine citrate and paracetamol in the presence of orphenadrine degradation product.

The plan of the work comprises the utilization of the proposed methods for the quantitative analysis of the cited drugs in raw materials and in their pharmaceutical formulations.

In addition, the plan of the work comprises statistical analysis to evaluate the obtained results of the proposed methods in comparison with the reported methods.

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Summary

Summary

Development and Validation of Analytical Methods for the Determination of Some Skeletal Muscle Relaxants

This thesis is concerned with the development of simple and accurate methods for the analysis of some skeletal muscle relaxants, namely chlorzoxazone, cyclobenzaprine hydrochloride, methocarbamol, orphenadrine citrate and tizanidine hydrochloride. 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 skeletal muscle relaxants and structure-activity relationship and 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 are clarified. Section -III: Experimental and discussion

This section was further divided into two parts:

15

Summary

Part I- Chromatographic methods

I-A-Simultaneous determination of methocarbamol and aspirin in presence of their degradation products by HPLC Method.

In this method complete separation of methocarbamol (MET),aspirin (ASP), guaifenesin (GUF) and salicylic acid (SAL) were simultaneously separated and quantified on Inertsil ODS-3C18 column using 25 mM dihydrogen phosphate buffer (pH 6 adjusted with 0.1M NaOH): acetonitrile (65:35, 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 methocarbamol and aspirin in presence of guaifenesin and salicylic acid their degradation Products in laboratory prepared mixture with mean percentage recoveries of 99.90±0.99 for MET and 99.81±0.88 for ASP. The method was successfully applied for the determination of methocarbamol and aspirin in Robaxisal®tablets with mean percentage recoveries of 103.11 ± 1. 09 and 101.14 ± 1.08, respectively. Ӏ-B- RP-HPLC method for the determination of cyclobenzaprine hydrochloride in the presence of its degradation product.

In this method complete separation of cyclobenzaprine hydrochloride CYC and anthraquinone ANQ were simultaneously separated and quantified on Inertsil ODS-3 C18 (250 x 4.6 mm, 5 µm) column using 25mM potassium dihydrogen phosphate buffer (pH 6.0 adjusted with 0.1 M NaOH): acetonitrile (30:70, v/v) at a flow rate of 1.5 ml/min at ambient temperature. Detection was carried at 220 nm respectively.

The method was successfully applied for the determination cyclobenzaprine hydrochloride in the presence of anthraquinone in

16

Summary laboratory prepared mixture with mean percentage recoveries of 99.77±1.34 for CYC. The method was successfully applied for the determination of these drugs in their coformulated dosage forms.

Ӏ-C- Validated Liquid Chromatographic method for the simultaneous determination of tizanidine hydrochloride and mefenamic acid in dosage form.

In this method complete separation of tizanidine hydrochloride(TIZ) and mefenamic acid MEF were simultaneously separated and quantified on Inertsil ODS-3C18 column using 50 mM potassium dihydrogen phosphate buffer (pH 2.6 adjusted with 0.1M NaOH): acetonitrile (40:60, v/v)as a mobile phase at a flow rate of 1.0ml/minat ambient temperature. Detection was carried at 317nm. The method was successfully applied for the determination of tizanidine hydrochloride and mefenamic acid in laboratory prepared mixture with mean percentage recoveries of 99.90±1.39 for TIZ and 99.53±0.83 for MEF. The method was successfully applied for the determination of tizanidine hydrochloride and mefenamic acid in MEFTAL_MR® tablets with mean percentage recoveries of 100.85± 1.5 and 99.83 ± 1.25, respectively.

Ӏ-D- Validated LC method for the simultaneous determination of Orphenadrine citrate and paracetamol in presence of orphenadrine degradation product in mixture.

17

Summary

In this method complete separation of orphenadrine citrate (ORC), paracetamol (PAR) and Methylbenzhydrol (MBH) were simultaneously separated and quantified on Inertsil ODS-3C18 column using 50 mM potassium dihydrogen phosphate buffer (pH 7.2 adjusted with 0.1M NaOH): acetonitrile (30:70, v/v)as a mobile phase at a flow rate of 1.2ml/min at ambient temperature. Detection was carried at 220nm.

The method was successfully applied for the determination of orphenadrine citrate and paracetamol in presence the degradation product of orphenadrine (MBH) in laboratory prepared mixture with mean percentage recoveries of 100.25%±0.98 for ORC and 100.21%±1.09 for PAR.

The method was successfully applied for the determination of orphenadrine citrate and paracetamol in Orphenadrine plus ® tablets with mean percentage recoveries of 96.81± 1. 24 and 98.35 ± 1.02, respectively.

Part II- Chemometric Assisted Spectrophotometric methods. ӀӀ-A-1-Chemometric-Assisted Spectrophotometric methods for determination of cyclobenzaprine hydrochloride in the presence of its degradation product in mixture.

In this section two different chemometric methods were applied for the determination of Cyclobenzaprine Hydrochloride CYC in the presence of its degradation product (anthraquinone; ANQ) either in laboratory prepared mixturesor in pharmaceutical dosage form (Multi-

18

Summary

Relax® tablets). The chemometric methods applied were principal component regression (PCR) and partial least squares (PLS).

ӀӀ-A-2- Simultaneous determination of orphenadine citrate and paracetamol in presence of orphenadrine degradation product using Chemometric-assisted spectrophotometric methods.

In this section two different chemometric methods were applied for the simultaneous determination of ORC and PAR in the presence of (MBH); degradation product of orphenadrine in mixture either in laboratory prepared mixtures or in pharmaceutical dosage form (Orphenadrine plus ®tablets). The chemometric methods applied were principal component regression (PCR) and partial least squares (PLS).

ӀӀ-A-3- Chemometric-assisted Simultaneous determination of Chlorzoxazone and Ibuprofen / Ketoprofen in the presence of Chlorzoxazone degradation product in two ternary mixtures.

Different chemometric methods were applied for the determination Chlorzoxazone (CH) and Ibuprofen (IBU) or Ketoprofen (KET) in presence of Chlorzoxazone degradation product (2-amino-4- chlorophenol) ACP in two different ternary mixtures either in lab prepared mixture or in pharmaceutical dosage form (Myofen® capsules and Flexan® capsules). The chemometric methods applied were principal component regression (PCR) and partial least squares regression (PLS). Part ӀӀ-B-1- Simultaneous determination of orphenadine citrate and paracetamol in presence of orphenadrine degradation product using Artificial Neural Networks technique.

19

Summary

ANNs were applied to UV spectrophotometric data for the simultaneous determination of orphenadrine citrate (ORC) and paracetamol (PAR) in the presence of Methylbenzhydrol (MBH) the degradation product of Orphenadrine in binary mixture either in pure form or in pharmaceutical dosage form (Orphenadrine plus ®tablets).

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Conclusion

Conclusion

At the end of the present work, it can be concluded that rapid, specific and selective HPLC and spectrophotometric analytical methods were developed for quantitative determination of the proposed methods, beside, their wide range of applications and they can be used for routine analysis and quality control of the cited drugs as such or in combinations in many dosage forms.

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