Development and Validation of Analytical Methods for the Determination of Some Skeletal Muscle Relaxant Drugs A thesis presented by Marwa Alsayed Abd Elmoneim Omran Drug control specialist National Organization for Drug Control and Research Bachelor of Pharmaceutical Sciences Faculty of Pharmacy - Tanta University 2007 Submitted for the partial fulfillment for the degree of Master in Pharmaceutical Sciences (Pharmaceutical Chemistry) Supervised by Dr. Ehab Farouk Hassan Elkady Associate Professor of Pharmaceutical Chemistry Faculty of Pharmacy Cairo University Dr. Hoda Mostafa Mahmoud Elquadaby Associate Professor of Analytical Chemistry National Organization for Drug Control and Research Faculty of Pharmacy Cairo University 2018 English Abstract Development and Validation of Analytical Methods for the Determination of Some Skeletal Muscle Relaxant Drugs Different analytical methods are introduced through this thesis for the determination of some skeletal muscle relaxant drugs in bulk, dosage forms, as combination with co-administered analgesics, and in biological fluids. The cited skeletal muscle relaxant drugs are dantrolene sodium, mephenesin, chlorzoxazone and methocarbamol. The co-administered analgesics are paracetamol and diclofenac potassium. Voltammetric part includes the determination of dantrolene sodium and mephenesin, each in bulk and pharmaceutical dosage form at pencil graphite and glassy carbon electrodes and the application to human mother milk and urine for both drugs using differential pulse and square wave voltammetric methods. Chromatographic part includes simultaneous determination of methocarbamol or chlorzoxazone with paracetamol in bulk, laboratory-prepared synthetic binary mixtures and dosage forms using a high performance liquid chromatographic method. Spectrophotometric part includes simultaneous determination of methocarbamol with paracetamol or diclofenac potassium each in bulk, laboratory-prepared synthetic binary mixtures and dosage forms using ratio difference method. Statistical analysis was carried out for comparing results of the developed methods with those of reference methods exhibiting good values and revealing insignificant difference. Keywords: Voltammetry, Liquid chromatography, Spectrophotometry, Skeletal muscle relaxant drugs, Pharmaceutical preparations, Human biological fluids. Introduction Skeletal Muscle Relaxants I. Definition and uses Skeletal muscle relaxants (SMR) are drugs used to relieve muscle spasm which is a painful involuntary contraction of the muscle resulting from over-excitability. II. Classification of SMR SMR are classified into two main groups, peripherally acting muscle relaxants (neuromuscular blocking agents) and centrally acting muscle relaxants. II.1. Peripherally acting muscle relaxants (neuromuscular blocking agents) Neuromuscular blocking agents are principally used as adjuncts in surgical anesthesia to obtain satisfactory muscle relaxation and also to reduce doses of the anesthetic. Neuromuscular blocking agents are classified according to duration of action into long, intermediate, and short acting. Metocurine, d- tubocurarine, pancuronium and doxacurium are long acting, whereas vecuronium and atracurium are of intermediate action. Mivacurium is an example of short acting agents. Neuromuscular blocking agents are also classified according to mechanism of action into non-depolarizing drugs, depolarizing blocking agents and directly acting compounds. II.1.1 Non-depolarizing drugs (competitive neuromuscular blocking drugs) These agents compete with acetyl choline (Ach) on the nicotinic receptors preventing depolarization of the end-plate by the neurotransmitter. Curare alkaloids represent prototypes of these drugs, for example: Atracurium Besilate. II.1.2 Depolarizing blocking agents The initial action of depolarizing blocking agents, such as succinylcholine and decamethonium is to depolarize the membrane by opening channels in the same manner as acetyl choline. II.1.3. Agents acting directly on skeletal muscle Dantrolene and the cinchona alkaloids, including quinine, exert a direct effect on skeletal muscle excitability. They are used to reduce muscle tone rather than to induce paralysis. Dantrolene reduces muscle tone by interfering with the release of calcium ions and thereby blocks contraction of skeletal muscle. II.1.4 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. II.2. 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. The mechanism of action of centrally acting relaxants may be due to their CNS-depressant activity. It is noteworthy that SMR are formulated alone or in combination with analgesics (e.g. paracetamol) and non-steroidal anti-inflammatory drugs (e.g. ketoprofen and ibuprofen) to alleviate pain associated with muscle spasm. Chlorzoxazone is marketed in admixture with paracetamol (Myolgin®& Relax® capsules) or with ketoprofen (Flexofan® capsules). Mephenesin is marketed as tablets or topical preparation (Decontractyl®). Methocarbamol is marketed in admixture with paracetamol (Flexopro- Extra® tablets) or with diclofenac potassium (Flexilax®& Dimra® tablets). Investigated Drugs 1-Chlorzoxazone O OH CL N 2-Dantrolene sodium O O N N 3 1/2 H2O NNa O N 2 O 3- Mephenesin O OH OH 4- Methocarbamol O OH O O NH2 O Co-administered drugs: 1- Paracetamol 2- Diclofenac potassium 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 anesthesia. The wide use of skeletal muscle relaxants in medicine promoted the development of simple, accurate, sensitive and applicable methods for their determination in pure, dosage forms and biological fluids. The aim of this work is to develop simple and accurate methods for the determination of some of these drugs either alone or in their mixtures with some analgesics. Drugs cited in this thesis are dantrolene sodium, mephenesin, chlorzoxazone and methocarbamol. Voltammetry is one of the electroanalytical methods that introduce a rapid qualitative and quantitative analysis of the drugs in their pure form. It also provides a good way for the application to the biological fluids. In this work, it is incorporated for the determination of dantrolene sodium and mephenesin in their pure forms, pharmaceutical preparations, human mother milk and urine. HPLC is a powerful analytical tool for the separation and quantitative analysis of drugs in pure form and pharmaceutical products. It is incorporated in the plan of the work using ultraviolet detection for the determination of methocarbamol and chlorzoxazone in combination with paracetamol in pure form and combined binary pharmaceutical dosage forms of the skeletal muscle relaxant with paracetamol. Spectrophotometry is involved in this work by applying ratio difference (RD) method for the simultaneous determination of methocarbamol either with paracetamol or with diclofenac potassium in pure forms and combined binary pharmaceutical dosage forms. In addition, the plan of the work comprises statistical analysis to evaluate the obtained results of the proposed methods in comparison with the reference methods. English Summary and Conclusions Development and Validation of Analytical Methods For the Determination of Some Skeletal Muscle Relaxant Drugs The presented thesis contains different analytical methods for the determination of some skeletal muscle relaxant drugs with some analgesics in bulk, laboratory-prepared synthetic mixtures, pharmaceutical preparations and some biological fluids. Cited drugs are dantrolene sodium, mephenesin, chlorzoxazone and methocarbamol. The thesis consists of the following sections: Section 1: Introduction that contains: I. Clinical aspects about skeletal muscle spasm proceeding till drugs acting as skeletal muscle relaxants, their classifications, their chemical structures and mechanisms of action. II. Literature review about the official and reported methods for the determination of the drugs under investigation. Section 2: Experimental and Discussion Aim of the introduced work: in this part, the aim of the present work and proposed methods was generally headed and the aim of each method is further clarified with more elaboration. This section was further divided into three parts: Part I: Voltammetric methods: I.A. Comparative voltammetric behavior and determination of dantrolene sodium in pure form, pharmaceutical preparation, human mother milk and urine at disposable pencil graphite and glassy carbon electrodes Differential pulse and square wave voltammetric methods have been developed and successfully applied to the estimation of dantrolene sodium (DAN) in Britton-Robinson (0.04 M BR) buffer pH 9.0 as electrolyte at pencil graphite electrode (PGE) and glassy carbon electrode (GCE) in pure form, pharmaceutical preparation, human mother milk and urine samples. The optimum conditions at PGE are deposition time (sec) 10, deposition potential (V) -0.7; at GCE are deposition time (sec) 30 and deposition potential (V) 0.8. The presented method was successfully applied for the assay of drug in pure form