(MS.c. Degree) For: Nesma Mahmoud Mohammed Fahmy B.Pharm.Sci, Cairo University, 2009 Teaching Assistant - Pharmaceutical Chem. Department, Faculty of Pharmacy, Ahram Canadian University On “Analytical Study of Some Local Anaesthetic Drugs in Formulation”

Summary: The thesis consisted a medical introduction and two chapters: Chapter I: Simultaneous Determination of Ternary Mixture of Lidocaine Hydrochloride, Fluocortolone Pivalate, and Chlorquinaldol in Creams and Suppositories

This chapter was divided into five sections:

Section A: General introduction and literature review

This section provided a general introduction about Hemorroids, in addition to the chemistry, mode of action and literature review of the reported analyical methods of Lidocaine Hydrochloride (LH), Fluocortolone Pivalate (FCP), and Chlorquinaldol (CQ). A general consideration about the practical work was covered in the following sections.

Section B: Determination of the Ternary Mixture of Lidocaine Hydrochloride, Flucortolone Pivalate, and Chlorquinaldol, in Creams and suppositories by Spectrophotometric Methods Simultaneous determination of mixtures of LH, and FCP, in presence of CQ without prior separation steps was applied using either successive or progressive resolution techniques. According to the concentration of CQ the extent of overlapping changed so it could be eliminated from the mixture to get the binary mixture of LH and FCP using ratio subtraction method for partially overlapped spectra or constant value via amplitude difference followed by ratio subtraction or constant center followed by spectrum subtraction spectrum subtraction for severely overlapped spectra. Successive ratio subtraction was coupled with extended ratio subtraction ,constant multiplication, derivative subtraction coupled constant multiplication, and spectrum subtraction could be applied for the analysis of partially overlapped spectra. On the other hand severely overlapped spectra could be analyzed by constant center and the novel methods namely differential dual wavelength (D1 DWL) for CQ, ratio difference and differential derivative ratio (D1 DR) for FCP, while LH was determined by applying constant value via amplitude difference followed by successive ratio subtraction ,and either constant multiplication or constant center followed by spectrum subtraction and successive derivative subtraction. The spectra of the cited drugs could be resolved and their concentrations were determined progressively from the same ratio spectrum using amplitude modulation method. The specificity of the developed methods was investigated by analyzing laboratory prepared mixtures and were successfully applied for the analysis of pharmaceutical formulations containing the cited drugs with no interference from additives. The proposed methods were validated according to the ICH guidelines. The obtained results were statistically compared with those of the official or reported methods; using student t-test, F-test, and one way ANOVA, showing no significant difference with respect to accuracy and precision. Section C : Determination of the Ternary Mixture of Chlorquinaldol, Flucortolone Pivalate, and Lidocaine Hydrochloride in Creams and Suppositories by Chemometric- Assisted Spectrophotometric Methods The determination of LH, FCP, and CQ was developed using two chemometric-assisted spectrophotometric techniques, principal component regression (PCR) and partial least square (PLS).The first step in determination of the cited drugs by multivariate calibration methods involved constructing the calibration matrix for the ternary mixture. The calibration set was obtained by using the absorption spectra set of 15 mixtures of LH, FCP, and CQ with different ratios of each component listed, measured at 200 - 346 nm. In this study, the leave one out crossing validation was used, and the RMSECV values were used as diagnostic tests for examining the errors in the predicted concentrations. RMSECV values indicated both precision and accuracy of predictions. To validate the prediction ability of the suggested models, they were applied to predict the concentrations of LH, FCP, and CQ in the validation set of ten mixtures. The two models were applied for the analysis of LH, FCP, and CQ in pharmaceutical dosage form. Section D Determination of the Ternary Mixture of Lidocaine Hydrochloride, Fluocortolone Pivalate, and Chlorquinaldol by Chromatographic Methods

HPLC was carried out at ambient temperature on Whatman Partisil 5 C8 column. The mobile phase consisted of phosphate buffer [pH 3.6]: acetonitrile [40:60 v/v] with 0.5 % triethylamine and the final pH was adjusted to 3.3 ± 0.2 using o-phosphoric acid. The mobile phase was filtered using 0.45 µm Millipore membrane filter (Billerica, MA) and delivered at a rate of 1 mL/min. The injection volumes were 20 µL and the detection was done at 220 nm. All the analyses were done at ambient temperature 25˚C. The proposed methods were validated as per ICH guidelines for accuracy, specificity, precision, linearity, range, and LOD/LOQ; and the results were found to be within the acceptable limits. The proposed method was successfully applied for determination of laboratory prepared mixtures and commercial preparations. Section E : Statistical Comparison and Conclusion The statistical comparison of the results obtained by applying the proposed methods to pure samples of LH, FCP, and CQ versus the official or reported methods showed that the calculated t and F values were less than the theoretical ones indicating that there was no significant difference between the proposed and the official or reported methods with respect to accuracy and precision. One-way ANOVA was applied for the purpose of comparison of developed and official or reported methods, which showed that there was no significant difference between them for the determination of LH, FCP, and CQ regarding accuracy and precision.

Chapter II : Stability Indicating Methods for the Simultaneous Determination of Lidocaine Hydrochloride, Dexamethasone Acetate, Calcium Dobesilate, as well as its Degradation Product and/or Impurity Hydroquinone

This chapter was divided into five sections:

Section A : General introduction and literature review

This section provided a general introduction about the chemistry, mode of action and literature review of the reported analyical methods of Lidocaine Hydrochloride (LH), Dexamethasone Acetate (DA), Calcium Dobesilate (CD), as well as its degradation product and/or impurity Hydroquinone (HQ). A general consideration about the practical work was covered in the following sections.

Section B :Spectrophotometric Stability Indicating Methods for the Determination of the Ternary Mixture of Lidocaine Hydrochloride, Dexamethasone acetate, Calcium Dobesilate as well as the Degradation Product and/or Impurity Hydroquinone The proposed drugs were determined at their maxima 210 nm, 305 nm, 239 nm and 225 nm for LH, CD, DA and HQ respectively, by successive ratio subtraction coupled with constant multiplication method to obtain the zero order absorption spectra of each drug separately, while by applying successive derivative subtraction they were determined at their first derivative spectra at 210 nm, 320 nm, 256 nm or P225-252 and P220-233 for LH, CD ,DA, and HQ, respectively. In binary mixture lidocaine hydrochloride was determined using novel mathematical methods namely amplitude subtraction and amplitude factor, in addition to ratio subtraction coupled with first derivative. While dexamathasone acetate was assayed by first derivative method. Lidocaine and dexamethasone in ternary mixture could be determined by a novel resolution technique namely successive spectrophotometric resolution; including successive ratio subtraction coupled with either first derivative, or extended ratio subtraction for dexamethasone and ratio subtraction coupled with first derivative for lidocaine hydrochloride; and finally the calcium dobesilate could be determined by first derivative, derivative ratio and the novel modified amplitude subtraction methods.

Section C : Determination of the Quaternary Mixture of Lidocaine Hydrochloride, Calcium Dobesilate, Dexamethasone Acetate, and The Degradation Product and/or Impurity Hydroquinone in Ointments by Chemometric Methods Determination of the Ternary Mixture of LH, DA, CD as well as the Degradation Product and/or Impurity HQ was developed using two chemometric-assisted spectrophotometric techniques, principal component regression (PCR) and partial least square (PLS).The first step in determination of the cited drugs by multivariate calibration methods involved constructing the calibration matrix for the binary mixture. The calibration set was obtained by using the absorption spectra set of 15 mixtures of LH, DA, CD, and HQ with different ratios of each component listed, measured at 200 - 344 nm. In this study, the leave one out crossing validation was used, and the RMSECV values were used as diagnostic tests for examining the errors in the predicted concentrations. RMSECV values indicated both precision and accuracy of predictions. To validate the prediction ability of the suggested models, they were applied to predict the concentrations of LH, DA, CD, and HQ in the validation set of ten mixtures. The two models were applied for the analysis of LH, DA, CD, and HQ in pharmaceutical dosage form. Section D : Determination of the Quaternary Mixture of Lidocaine Hydrochloride, Calcium Dobesilate, Dexamethasone Acetate, and the Degradation Product and/or Impurity Hydroquinone in Ointments by Chromatographic Methods Two simple, accurate and precise chromatographic methods were developed for the determination of calcium dobesilate in the presence of its interfering substances as its degradation product and/or impurity hydroquinone in pharmaceutical dosage forms with lidocaine hydrochloride alone or in combination with dexamethasone acetate. The first method was TLC-spectrodensitometric one using benzene: methanol: ethyl acetate: ammonia: sodium lauryl sulphate (7: 2.1: 2.5: 0.1: 0.05 v/v/v/v/w) as a developing system and scanned at 220 nm.

Second one was an HPLC method where the mixture was separated on a C18 column with flow rate 1 mL/min and the mobile phase was phosphate buffer : acetonitrile (35:65 v/v) (adjusted to pH 3.4 with o-phosphoric acid), scanned at 220 nm. The robustness of the method was determined to assess the effect of small but deliberate variation of the chromatographic conditions on the determination of cited drugs in presence of interfering substances. Robustness was determined by changing the mobile phase flow rate to 0.5, 1,and 1.5 mL/min, pH to 3.5, 4, and 5, and the concentration of acetonitrile in the mobile phase to 60% and 80%. The proposed methods were checked using laboratory-prepared mixtures and were successfully applied for the analysis of pharmaceutical formulations containing the cited drugs and were validated via ICH guidelines. The proposed methods could be used for the routine analysis of the cited drugs in their pharmaceutical formulation in quality control laboratories. Section E : Statistical Comparison and Conclusion The statistical comparison of the results obtained by applying the proposed methods to pure samples of LH, DA, CD, and HQ versus the official methods showed that the calculated t and F values were less than the theoretical ones indicating that there was no significant difference between the proposed and the official methods with respect to accuracy and precision. One-way ANOVA was applied for the purpose of comparison of developed and official methods, which showed that there was no significant difference between them for the determination of LH, DA, CD, and HQ regarding accuracy and precision.

A general introduction in addition to appendices describing the common materials, apparatus, and reagents were given. The thesis comprises 77 figures, 43 tables, and 302 references from 1930-2015, and ends with an Arabic summary. Research plan 1- Literature review on the pharmacological action and the previously published methods for the determination of the investigated drugs: [Lidocaine combined with other local anaesthetics as Prilocaine or disinfectants as Cetrimide or Corticosteroids as Dexamethasone, Flucortolone Pivalate, Hydrocortisone in addition to Zinc Oxide or Carbonic anhydrase inhibitors as Ca Dobesylate or anti-microbials as Chlorquinaldol - Mepivacaine combined with nasal decongestants as Nordefrine –Pramoxine combined with Hydrocortisone - Prilocaine –] 2- Application of official methods to the analysis of these drugs. 3- Development of suitable methods adopting different techniques as spectrophotometric, electrochemical and chromatographic methods. 4- Separation techniques for the mentioned local anaesthetics from their co-administered drugs found in mixtures in the pharmaceutical market. 5- Application of the suggested methods to the analysis of the studied drugs in pure, pharmaceutical formulation or in biological fluids.

Under the Supervision of:

Ass. Prof. Dr. Hayam Mahmoud Lotfy Prof. Dr. Mostafa Abdel Aty Shehata

Associate Professor of Analytical Chemistry Professor of Analytical Chemistry Faculty of Pharmacy - Cairo University Faculty of Pharmacy - Cairo University

Assosciate Professor Dr. Shereen Mostafa Tawakkol Assosciate Professor of Analytical chemistry-Helwan University