Analysis of Some Drugs Affecting the Respiratory System

Analysis of Some Drugs Affecting the Respiratory System A Thesis Presented By Nehal Fayek Farid Faragallah B. Pharm. Sci. (1996) M.Sc. in Analytical Chemistry (2003) For The Degree of Doctor of Philosophy in Pharmaceutical Sciences (Analytical Chemistry) Under the supervision of Prof. Dr. Mohamed Abdelkawy Mohamed Professor of Analytical Chemistry Faculty of Pharmacy Cairo University Prof. Dr. Nariman Abd El Ghany El Ragehy Professor of Analytical Chemistry Faculty of Pharmacy Cairo University Prof. Dr. Fadia Hussein Metwally Professor of Analytical Chemistry Faculty of Pharmacy Cairo University Dr. Maha Abd El Moneim Hegazy Professor of Analytical Chemistry Faculty of Pharmacy Cairo University (2012)

ACKNOWLEDGEMENT I would like to acknowledge with sincere thanks and appreciation Prof. Dr. Mohamed Abdelkawy Mohamed, Professor of Analytical Chemistry, Faculty of Pharmacy, Cairo University, to whom, I am indebted for suggesting the point of research, continuous guidance, encouragement and stimulant discussions without which this work would have never been finished. I wish to convey my deep appreciation and thanks to Prof. Dr. Nariman Abd El Ghany El Ragehy, Professor of Analytical Chemistry, Faculty of Pharmacy, Cairo University for her valuable advice, keen supervision and constructive comments. I am grateful to Prof. Dr. Fadia Hussein Metwally, Professor of Analytical Chemistry, Faculty of Pharmacy, Cairo University, for her assistance and guidance. Special thanks to Dr. Maha Abd EL Monein Hegazy, Lecturer of Analytical Chemistry, Faculty of Pharmacy, Cairo University for her continuous support, guidance, solving problems and teaching me new techniques especially in the practical part without which this work would have never been finished. I would like to express my appreciation to my parents, my husband and my two daughters, who provided the warm atmosphere for completing the thesis. I would like to extend my thanks to the staff members and my colleagues in Faculty of Pharmacy, Cairo University and Beni Suef University for their cooperation and encouragement. Nehal Fayek

Analysis of Some Drugs Affecting the Respiratory System Abstract Simultaneous determination of a mixture of Ambroxol HCl (AMB) and Guaifenesin (G) in presence of the oxidative degradate of AMB and guaicol, the main impurity of G by RP-HPLC, TLC-spectrodensitometry and multivariate calibration methods was done. Model update of multivariate calibration was used to determine AMB and G in syrup dosage form due to interfering additives. Simultaneous determination of a quaternary mixture of Oxomemazine, Sodium benzoate, Guaifenesin and Paracetamol by RP-HPLC, TLC-spectrodensitometry and multivariate calibration methods was also done. Spectrophotometric determination of Oxomemazine HCl, Guaifenesin and Carbocisteine in pure form and in tablets was also done. Simultaneous determination of a ternary mixture of Terbutaline sulfate, Guaifenesin and Bromhexine HCl by RP-HPLC, TLC-spectrodensitometry and multivariate calibration methods was done and the three methods were applied for the determination of the drugs in syrup dosage form. Key Words: Ambroxol HCl Guaifenesin Guaicol HPLC TLCspectrodensitometry PLS PCR Oxomemazine Paracetamol Sodium benzoate Carbocisteine Terbutaline sulfate Bromhexine HCl.

Contents: List of Figures List of Tables... List of Abbreviations. Preface. Summary.. Page xii xvi xxii xxv xxvi Part I General Introduction I.1. Pharmacology of drugs affecting respiratory system 2 I.1.1. Bronchodilators. 3 I.1.2. Corticosteroids 4 I.1.3. Cromolyn and nedocromil... 5 I.1.4. Leukotriene receptor antagonists 5 I.1.5. Antihistamines 5 I.1.6. Cough preparations. 7 I.1.7. Nasal Decongestants.. 8 1.1.8. Antibacterial agents for respiratory tract infections.. 8 I.2. Studied Drugs 9 Part II Simultaneous determination of Ambroxol Hydrochloride and Guaifenesin in presence of the oxidative degradation product of Ambroxol HCl and Guaicol the main impurity of Guaifenesin by RP-HPLC, TLC-spectrodensitometric and multivariate calibration methods. Section A Literature review of Ambroxol HCl and Guaifenesin II.A. Literature review for the determination of Ambroxol HCl (AMB) and i

Guaifenesin (G) 20 II. A.1. Literature review of Ambroxol HCl 20 II.A.1.1. Titrimetric methods. 20 II.A.1.2. Spectroscopic methods 20 II.A.1.3. Chromatographic methods.. 22 II.A.1.4. Electro-analytical methods.. 27 II.A.2. Literature review of Guaifenesin. 28 II.A.2.1. Pharmacopeial methods... 28 II.A.2.2. Spectrophotometric methods 28 II.A.2.3. Chemometric methods. 29 II.A.2.4. Chromatographic methods.. 31 II.A.2.5. Electro-analytical methods.. 39 II.A.2.6. X ray diffraction method 40 II.A.3. Simultaneous determination of Ambroxol HCl and Guaifenesin.. 40 II.A.3.1. High performance liquid chromatography 40 II.A.3.2. Capillary electrophoresis.. 41 Section B Simultaneous determination of Ambroxol HCl and Guaifenesin in presence of the oxidative degradation product of Ambroxol HCl and Guaicol, the main impurity of Guaifenesin by HPLC method. II.B.1. Introduction. 43 II.B.2. Experimental... 43 II.B.2.1. Instruments... 43 II.B.2.2. Samples... 44 II.B.2.3. Reagents and solvents 46 II.B.2.4. Standard solutions... 46 ii

II.B.2.5. Procedure... 47 II.B.2.5.1. Chromatographic conditions. 47 II.B.2.5.2. Method validation. 47 II.B.2.5.3. Assay of pharmaceutical dosage forms using the proposed HPLC method. 49 II.B.3. Results and discussion... 51 Section C Simultaneous determination of Ambroxol HCl and Guaifenesin in presence of the oxidative degradation product of Ambroxol HCl and Guaicol, the main impurity of Guaifenesin by TLC- Spectrodensitometric method. II.C.1. Introduction. 67 II.C.2. Experimental 67 II.C.2.1. Instruments.. 67 II.C.2.2. Samples.... 68 II.C.2.3. Reagents and solvents.. 68 II.C.2.4. Stock standard solutions... 68 II.C.2.5. Procedure... 69 II.C.2.5.1. Chromatographic conditions 69 II.C.2.5.2. Method validation... 69 II.C.2.5.3. Assay of pharmaceutical dosage forms 71 II.C.3. Results and discussion... 72 Section D Simultaneous determination of quaternary mixture of Ambroxol HCl, its oxidative degradation product, Guaifenesin and its main impurity (Guaicol) by Multivariate Calibration Methods. II.D.1. Introduction 85 II.D.2. Experimental.. 86 iii

II.D.2.1. Instruments. 86 II.D.2.2. Samples... 87 II.D.2.3. Reagents and solvents. 87 II.D.2.4. Solutions. 87 II.D.2.5. Procedure. 89 II.D.2.5.1. Spectral characteristics 89 II.D.2.5.2. Calibration and validation sets..... 89 II.D.2.5.3. Assay of pharmaceutical dosage forms 90 II.D.3. Results and discussion. 90 Part III Simultaneous determination of a quaternary mixture of Oxomemazine, Sodium benzoate, Guaifenesin and Paracetamol by RP-HPLC, TLC-spectrodensitometric and multivariate calibration methods. Section A Literature review of Oxomemazine, Sodium benzoate and Paracetamol III.A. Literature review for the determination of Oxomemazine, Sodium benzoate and Paracetamol.. 106 III.A.1. Literature review of Oxomemazine.. 106 III.A.1.1 Colorimetric methods... 106 III. A.1.2. Chromatographic Methods... 106 III.A.2. Literature review of Sodium benzoate. 107 III.A.2.1. Pharmacopeial methods 107 III.A.2.2. Titrimetric procedures.. 107 III.A.2.3. Spectrophotometric methods 108 III. A.2.4. Chemometric methods.. 108 III.A.2.5. Chromatographic methods 109 iv

III.A.2.6. Electro-analytical methods... 112 III.A.3. Literature review for Paracetamol.... 112 III.A.3.1. Pharmacopeial methods 112 III.A.3.2. Gravimetric method.. 113 IIII.A.3.3. Titrimetric method 113 III.A.3.4. Spectroscopic methods 114 III.A.3.5. Chemometric methods. 117 III.A.3.6. Flow injection analysis. 118 III.A.3.7. Chromatographic methods.. 119 III.A.3.8. Electro - analytical methods. 126 III.A.4. Simultaneous determination of a quaternary mixture of Guaifenesin, Paracetamol, Sodium benzoate and Oxomemazine. 127 Section B Simultaneous determination of a quaternary mixture of Oxomemazine, Sodium benzoate, Guaifenesin and Paracetamol by RP-HPLC method. III.B.1. Introduction.. 129 III.B.2. Experimental. 130 III.B.2.1. Instruments... 130 III.B.2.2. Samples. 130 III.B.2.3. Reagents and solvents.. 131 III.B.2.4. Standard solutions 132 III.B.2.5. Procedure.. 132 III.B.2.5.1. Chromatographic conditions. 132 III.B.2.5.2. Method validation... 133 III.B.2.5.3. Assay of pharmaceutical dosage forms using the proposed HPLC method.. 134 v

III.B.3. Results and discussion.. 135 Section C Simultaneous determination of a quaternary mixture of Oxomemazine, Paracetamol, Guaifenesin and Sodium benzoate by TLC-spectrodensitometric III.C. 1. Introduction.. 148 III.C. 2. Experimental. 148 III.C.2.1. Instruments... 148 III.C.2.2. Samples. 148 III.C.2.3. Reagents and solvents.. 149 III.C.2.4. Standard solutions. 149 III.C.2.5. Procedure. 149 III.C.2.5.1 Chromatographic conditions 149 III.C.2.5.2. Method validation... 150 III.C.2.5.3. Application of the proposed TLC-spectrodensitometric method for analysis of pharmaceutical dosage forms 152 III.C.3. Results and discussion. 152 Section D Simultaneous determination of a quaternary mixture of Oxomemazine, Sodium benzoate, Guaifenesin and Paracetamol by multivariate calibration methods. III.D.1. Introduction.. 167 III.D.2. Experimental 167 III.D.2.1. Instruments... 167 III.D.2.2. Samples. 167 III.D.2.3. Reagents and solvents... 168 III.C.2.4. Standard Solutions 168 III.D.2.5. Procedure.. 169 vi

III.D.2.5.1. Spectral characteristics. 169 III.D.2.5.2. Calibration and validation 169 III.D.2.5.3. Assay of dosage form... 170 III.D.3. Results and discussion.. 170 Part IV Spectrophotometric determination of Oxomemazine HCl, Guaiphenesin and Carbocisteine in pure form and in Ultrasolv tablets. Section A Literature review of Carbocisteine IV.A.1. Literature review for the determination of Carbocisteine... 184 IV.A.1.1. Pharmacopeial methods 184 IV.A.1.2. Colorimetric methods... 184 IV.A.1.3. Chromatographic methods... 185 IV.A.1.4. Electrochemical methods.. 188 Section B Spectrophotometric Determination of Guaiphenesin and Oxomemazine HCl in presence of Carbocisteine IV.B.1. Introduction. 190 IV.B.2. Experimental 190 IV.B.2.1. Instruments... 190 IV.B.2.2. Samples. 190 IV.B.2.3. Reagents and solvents.. 191 IV.B.2.4. Standard solutions 191 IV.B.2.5. Procedure.. 191 vii

IV.B.2.5.1. Spectral characteristics..... 191 IV.B.2.5.2. Method validation 192 IV.B.2.5.3. Assay of Ultrasolv tablets.. 193 IV.B.3. Results and discussion. 194 Section C Colorimetric determination of Carbocisteine using p-benzoquinone. IV.C.1. Introduction.. 207 IV.C.2. Experimental 207 IV.C.2.1. Instruments... 207 IV.C.2.2. Samples. 207 IV.C.2.3. Reagents and solvents.. 208 IV.C.2.4. Standard solutions 208 IV.C.2.5. Procedure.. 208 IV.C.2.5.1. Spectral characteristics. 208 IV.C.2.5.2. Linearity and range.. 209 IV.C.2.5.3. Assay of Ultrasolv tablets 209 IV.C.3. Results and discussion. 209 Part V Simultaneous determination of a ternary mixture of Terbutaline sulfate, Guaifenesin and Bromhexine HCl by RP-HPLC, TLC-spectrodensitometric and multivariate calibration methods. Section A Literature review of Terbutaline sulfate and Bromhexine HCl V.A.1. Literature review for the determination of Guaifenesin (G), Terbutaline 219 viii

sulfate (TS) and Bromhexine hydrochloride (BH) V.A.1. Literature review of Terbutaline sulfate. 219 V.A.1.1. Pharmacopeial methods. 219 V.A.1.2. Spectroscopic methods.. 220 V.A.1.3. Chromatographic methods. 221 V.A.1.4. Electro-analytical methods. 226 V.A.2. Literature review of Bromhexine hydrochloride 227 V.A.2.1. Pharmacopeial methods. 227 V.A.2.2. Titrimetric methods 227 V.A.2.3. Spectroscopic methods.. 228 V.A.2.4. Chemometric methods 229 V.A.2.5. Chromatographic methods.. 229 V.A.2.6. Electro-analytical methods.. 233 V.A.3. Simultaneous determination of a ternary mixture of Terbutaline sulfate, Bromhexine HCl and Guaifenesin. Section B 233 Simultaneous determination of a ternary mixture of Terbutaline sulfate, Guaifenesin and Bromhexine HCl by HPLC method V.B.1. Introduction. 236 V.B.2. Experimental... 236 V.B.2.1. Instruments.. 236 V. B.2.2. Samples... 236 V. B.2.3. Reagents and solvents. 237 V. B.2.4. Standard solutions 238 V. B.2.5. Procedure.. 238 ix

V.B.2.5.1. Chromatographic conditions. 238 V.B.5.5.2. Method validation... 238 V.B.5.5.3. Assay of pharmaceutical dosage form 240 V.B.3. Results and discussion 240 Section C Simultaneous determination of a ternary mixture of Terbutaline sulfate, Bromhexine HCl and Guaifenesin by TLC-spectrodensitometric method V. C.1. Introduction. 253 V.C.2. Experimental... 253 V.C.2.1. Instruments. 253 V.C.2.2. Samples.. 254 V.C.2.3. Reagents and solvents 254 V.C.2.4. Standard solutions... 254 V.C.2.5. Procedure 254 V.C. 2.5.1. Chromatographic conditions... 254 V.C. 2.5.2. Method validation... 255 V.C. 2.5.3. Assay of Pharmaceutical dosage form. 257 V.C.3. Results and discussion 257 Section D Simultaneous determination of a quaternary mixture of Terbutaline sulfate, Bromhexine HCl, Guaifenesin and Sodium benzoate by multivariate calibration methods V.D.1. Introduction. 271 V.D.2. Experimental... 271 V.D.2.1. Instruments 271 x

V.D.2.2. Samples.. 271 V.D.2.3. Reagents and solvents.. 272 V.D.2.4. Standard solutions... 272 V.D.2.5. Procedure 272 V.D.2.5.1. Spectral characteristic. 272 V.D.2.5.2. Calibration and validation.. 273 V.D.2.5.3. Assay of dosage form. 273 V.D.3. Results and Discussion.. 274 References. 285 xi

List of Figures: Figure No. Page 1 Suggested mechanism for the oxidation reaction of Ambroxol HCl by hydrogen peroxide 52 2 IR for Ambroxol HCl and Ambroxol HCl oxidative degradation product.. 53 3 Mass spectroscopy for Ambroxol HCl and Ambroxol HCl oxidative degradation product. 54 4 HPLC Chromatogram of Ambroxol HCl and Guaifenesin... 56 5 Linearity of Ambroxol HCl and Guaifenesin by the proposed HPLC method... 57 6 Thin layer chromatogram of laboratory prepared mixture containing Ambroxol HCl, Guaifenesin, Ambroxol HCl oxidative degradation product and Guaicol.. 74 7 TLC chromatogram of Ambroxol HCl 75 8 TLC chromatogram of Guaifenesin 75 9 Calibration curve of Ambroxol HCl and Guaifenesin by the proposed TLCspectrodensitometric method. 76 10 UV-Spectra of 40 µg ml -1 Ambroxol HCl, 20 µg ml -1 Ambroxol degradation product, 40 µg ml -1 Guaifenesin, and 20 µg ml -1 Guaicol..... 92 11 RMSEC plot of the cross validation of the training set as a function of the number of principal components used to construct PLS and PCR models for Ambroxol HCl.. 94 12 RMSEC plot of the cross validation of the training set as a function of the number of principal components used to construct PLS and PCR calibrations for Guaifenesin. 94 xii

13 RMSEC plot of the cross validation of the training set as a function of the number of principal components used to construct PLS and PCR calibrations for Ambroxol HCl oxidative degradation product... 95 14 RMSEC plot of the cross validation of the training set as a function of the number of principal components used to construct PLS and PCR calibrations for Guaicol. 95 15 HPLC chromatograms of (A) laboratory prepared mixture of Paracetamol, Guaifenesin, Sodium benzoate (t R = 6.05) and Oxomemazine and (B) Toplexil syrup 138 16 Linearity of Oxomemazine, Sodium benzoate, Guaifenesin and (D) Paracetamol by the proposed HPLC method 139 17 TLC Chromatogram of laboratory prepared mixture containing Oxomemazine, Paracetamol, Guaifenesin and Sodium benzoate. 154 18 TLC chromatogram of Oxomemazine.. 155 19 TLC chromatogram of Paracetamol. 155 20 TLC chromatogram of Guaifenesin 156 21 TLC chromatogram of Sodium benzoate. 156 22 Calibration curves of relative peak area at 270 nm to the corresponding concentration of Oxomemazine, Paracetamol, Guaifenesin and Sodium benzoate. 157 23 Zero order absorption spectra of Oxomemazine, Sodium benzoate, Guaifenesin and Paracetamol. 171 24 RMSEC plot of the cross validation of the training set as a function of the number of principal components used to construct PLS and PCR calibrations for Oxomemazine... 174 25 RMSEC plot of the cross validation of the training set as a function of the number of principal components used to construct PLS and PCR calibrations for Sodium benzoate.. 174 xiii

26 RMSEC plot of the cross validation of the training set as a function of the number of principal components used to construct PLS and PCR calibrations for Guaifenesin... 175 27 RMSEC plot of the cross validation of the training set as a function of the number of principal components used to construct PLS and PCR calibrations for Paracetamol... 175 28 Zero order absorption spectra of solutions of Oxomemazine HCl, Guaifenesin and Carbocisteine in 0.1N HCl... 195 29 First Derivative absorption spectra of Oxomemazine HCl, Guaifenesin and Carbocisteine in 0.1N HCl. 195 30 Zero order absorption spectra of Oxomemazine HCl in the range of 5 60 µg ml -1... 196 31 Linearity of Oxomemazine HCl in the range of 5 60 µg ml -1 by zero order absorption spectrophotometric method at 332.8 and 294.4nm.. 197 32 First derivative absorption spectra of Guaifenesin as solvent in the range of 10 100 µg ml -1 198 33 Linearity of Guaifenesin in the range of 10 100 µg ml -1 by first derivative absorption spectrophotometric method at 280.6 nm and 266.6nm. 198 34 Absorption spectra of Carbocisteine with PBQ, Oxomemazine HCl with PBQ, Guaifnesin with PBQ against a blank of PBQ in phosphate buffer and spectrum of 0.1 M PBQ in phosphate buffer ph 7 against a blank of distilled water.. 210 35 Mechanism of reaction between Carbocisteine and p-benzoquinone.. 211 36 Absorption spectra of Carbocisteine in the range of 30-150 µg ml -1 with 0.1 M PBQ in phosphate buffer ph 7 213 37 Linearity of Carbocisteine in the range of 30-150 µg ml -1 with 0.1 M PBQ in phosphate buffer ph 7 at 490 nm.. 213 38 HPLC Chromatogram on XTerra C18 column of a. Laboratory prepared mixture of Terbutaline sulfate, Guaifenesin and Bromhexine HCl and b. All Vent syrup 241 xiv