( و قضى ربك ألا تعبدوا إلا إياه وبالوالدين إحسانا إما يبلغن عندك الكبر أحدهما أو كلاهما فلا تقل لهما أف ولا تنهرهما وقل لهما قولاً كريما * واخفض لهما.

( و قضى ربك ألا تعبدوا إلا إياه وبالوالدين إحسانا إما يبلغن عندك الكبر أحدهما أو كلاهما فلا تقل لهما أف ولا تنهرهما وقل لهما قولاً كريما * واخفض لهما جناح الذل من الرحمة وقل رب ارحمهما كما ربيانى صغيراً *)

Contribution for the analysis of certain drugs which treat cerebrovascular insufficiency

This thesis consists of six parts Part I: General introduction Part I: General introduction Part II: New spectrophotometric method for simultaneous determination of binary mixtures of nicergoline and cinnarizine and stability indicating for vincamine. Part II: New spectrophotometric method for simultaneous determination of binary mixtures of nicergoline and cinnarizine and stability indicating for vincamine. Part III: Simultaneous determination of nicergoline and cinnarizine. Part III: Simultaneous determination of nicergoline and cinnarizine. Part IV: Stability indicating methods for the determination of meclophenoxate hydrochloride. Part IV: Stability indicating methods for the determination of meclophenoxate hydrochloride. Part V: Stability indicating methods for the determination of vinpocetine. Part V: Stability indicating methods for the determination of vinpocetine. Part VI: Stability indicating methods for the determination of Pyritinol dihydrochloride. Part VI: Stability indicating methods for the determination of Pyritinol dihydrochloride.

Part I General introduction Types of cerebrovascular disease Types of cerebrovascular disease Cerebrovascular insufficiency Etiology and Pathophysiology Cerebrovascular insufficiency Etiology and Pathophysiology Complications of cerebrovascular insufficiency Complications of cerebrovascular insufficiency Mechanism of action of the selected drugs Mechanism of action of the selected drugs

Part II New spectrophotometric method for simultaneous determination of binary mixtures of nicergoline and cinnarizine and stability indicating for vincamine

This part includes a general introduction about the chemistry of nicergoline, cinnarizine and vincamine. This part includes a general introduction about the chemistry of nicergoline, cinnarizine and vincamine. Review article on the reported methods used for their quantitative determination. Review article on the reported methods used for their quantitative determination. This part is subdivided into two sections: This part is subdivided into two sections: Section(A): Determination of vincamine in presence of its acid degradation product by the ratio subtraction method Section(A): Determination of vincamine in presence of its acid degradation product by the ratio subtraction method Section(B): Determination of nicergoline and cinnarizine by the ratio subtraction and isosbestic point methods Section(B): Determination of nicergoline and cinnarizine by the ratio subtraction and isosbestic point methods

Section(A) Determination of vincamine in presence of its acid degradation product by the ratio subtraction method

Structure of Vincamine: Structure of Vincamine:

Theory of ratio subtraction method: The method depends on that, if you have a mixture of two drugs (X) and (Y) with overlapping spectra and the spectrum of (Y) is extended than (X), the determination of (X) can be done by dividing the spectrum of the mixture by a certain concentration of (Y) as a devisor (Y'). The division will give a new curve that represents.If we subtract this constant, then multiply the new curve obtained after subtraction by (Y') (the devisor), therefore we can obtain the original curve of (X). This can be summarized as follows: The constant can be determined directly from the curve by the straight line which is parallel to the wavelength axis in the region where (Y) is extended.

Figure (2): Absorption spectra of vincamine 20 µg ml -1 (———) degradation product 20 µg ml -1 (---------- ) and deg.product 16 µ g ml-1 (devisor) ( ——— ) using 0.1N hydrochloric acid as a solvent. 268nm

Figure (5): Division spectra of laboratory prepared mixtures of vincamine (X) and its degradation product (Y) using 16 µg ml-1 of degradation product (Y') as a divisor and 0.1 N HCl as a solvent. A (X+Y/Y’)

Figure (6): Division spectra of laboratory prepared mixtures of vincamine (X) and its degradation product (Y) using 16 µg ml-1 of degradation product (Y') as a divisor and 0.1 N HCl as a solvent after subtraction of the constant. A (X/Y')

Figure (7): The obtained absorption spectra of vincamine in lab.mixtures 8-32  g.ml-1 A (X/Y’*Y’ = X) Figure (7): The original absorption spectra of vincamine in cal.curve from 8-40  g.ml -1 268nm

Figure (4): Linearity of the absorbance of the zero order curve at 268.2 nm to the corresponding concentration of vincamine. Absorbance

Table (I): Determination of vincamine in laboratory prepared mixtures by the proposed method. Concentration (µg.ml -1 )Percentage %Ratio subtraction method Vincamine Degradation product Vincamine Degradation product Recovery % Vincamine 8322080 98.60 12283070 98.91 16244060 98.24 20 50 99.51 24166040 100.36 28127030 99.32 3288020 99.85 Mean99.25 S.D.0.732

Table (II): Determination of vincamine in oxybral capsules by the proposed method. Company method **Reported method *Ratio subtraction method Oxybral capsules claimed to contain 30 mg vincamine Batch number Found % ± S.D. ** 99.32 ± 0.95699.07 ± 0.46699.01 ± 0.762052831 A 97.56 ± 0.85784.11 ± 0.50184.63 ± 1.0250012261 A(expired 5/04) * Stability indicating spectrophotometric method. ** Spectrophotometric method

Table (III): Application of standard addition for the determination of vincamine by the proposed method. Ratio subtraction methodVincamine Batch number Recovery % of addedStandard added in mg 98.56 99.01 99.99 25 37.5 50 052831 A 99.98 ± 0.731Mean ± S.D.*

Table (IV): Statistical comparison for the results obtained by the proposed method and the reported method for the analysis of vincamine in pure powder form Table (IV): Statistical comparison for the results obtained by the proposed method and the reported method for the analysis of vincamine in pure powder form * Stability indicating spectrophotometric method. Reported method *Ratio subtraction methodItem 99.9099.72Mean 1.0410.917S.D. 1.0840.841Variance 69n 1.288 (3.69)F test 0.353 (2.160)Student’s t test The figures in parenthesis are the corresponding tabulated values at P=0.05

Section [B] Determination of nicergoline and cinnarizine by the ratio subtraction and isosbestic point methods

Structure of nicergoline: Structure of nicergoline: Structure of cinnarizine: Structure of cinnarizine:

Figure (9): Absorption spectra of Nicergoline 20 µg ml-1 (———) Cinnarizine 20 µg ml-1 (------------- ) and mixture of 10 µg ml-1 of each drug (……….) using methanol as a solvent. 235.8 nm 270.2 nm

Figure (10): Zero order absorption spectra of nicergoline 6- 36 μg ml-1

Figure (12): Linearity of the absorbance of the zero order curve at 270.2 nm to the corresponding concentration of nicergoline. Figure (13): Linearity of the absorbance of the zero order curve at 235.8 nm to the corresponding concentration of nicergoline Absorbance

A (X+Y/Y’) Figure (15): Division spectra of laboratory prepared mixtures of cinnarizine (X) and nicergoilne (Y) using 6 µg ml-1 of nicergoline (Y') as a divisor and methanol as a solvent. (Scale x 0.1)

Figure (16): Division spectra of cinnarizine (X) and nicergoilne (Y) using 6 µg ml-1 of nicergoline (Y') as a divisor and methanol as a solvent after subtraction of the constant.(scale x 0.1) A (X/Y')

Figure (17): The obtained absorption spectra of cinnarizine in lab.mixtures. A (X/Y‘*Y’ = X) Figure (17): The original absorption spectra of cinnarizine in cal.curve. 252nm

Figure (14): Linearity of the absorbance of the zero order curve at 252.0 nm to the corresponding concentration of cinnarizine. Absorbance

Table (V): Determination of nicergoline and cinnarizine in laboratory prepared mixtures by the proposed methods Ratio subtraction method Isosbestic point method RatioConcentration (µg ml -1 ) Recovery % Nicergoline : Cinnarizine cinnarizineNicergoline CinnarizineNicergoline 235 nm270 nm 100.97100.88100.51 2 : 313.59.0 101.45101.47101.05 2 : 3.510.56.0 100.4399.7799.54 2 : 414.07.0 101.0799.1798.96 2 : 4.513.56.0 99.4399.0798.68 2 : 515.06.0 100.00100.3199.75 2 : 5.516.56.0 100.1499.2799.31 2 : 618.06.0 99.9698.6699.03 2 : 6.519.56.0 100.48100.00100.25 2: 721.06.0 100.43  0.635 99.84  0.918 99.67  0.790 Mean  S.D.

Table (VI): Determination nicergoline and cinnarizine in cinibral tablets by the proposed method. Cinibral tablets claimed to contain 10 mg nicergoline & 25 mg cinnarizine Batch number Isosbestic point method Ratio subtraction method Reported method * Found % ± S.D. * for nicergoline Found % ± S.D. * for cinnarizine Found % ± S.D. * Nicergoline cinnarizine λ1λ2 39123 99.24 ± 0.877 98.61 ± 0.874 99.15±0.21299.32 ± 0.956 0.95698.37 ±0.892 40082 100.8 6 ± 0.619 99.86 ± 0.733 99.58 ± 0.301 0.30198.56± 0.857 0.85798.99±0.825 * HPLC method.

Table (VII): Application of standard addition for the determination of nicergoline and cinnarizine by the proposed method. Ratio subtraction method Isosbestic point method Standard added (mg)Batch number Recovery % of added cinnarizine Recovery % of added nicergoline cinnarizinenicergoline λ2λ1 98.18 98.93 98.06 99.66 100.83 101.05 99.26 100.03 100.32 25.00 37.50 50.00 10.00 15.00 20.00 39123 98.39  0.471 100.51  0.74 7 99.87  0.54 7 Mean  S.D.* * Average of four determinations.

Table (VIII): Statistical comparison for the results obtained by the proposed method and the reported method for the analysis of nicergoline and cinnarizine in pure powder form * HPLC method. Reported method*Ratio subtraction method Isosbestic point methodItem Cinnarizinenicergolinecinnarizinenicergoline 235 nm270 nm 99.2799.6499.9199.8399.58Mean 0.7140.9780.7031.0390.847S.D. 0.5090.9560.4941.0790.717Variance 66999n 1.030 (3.69) 1.128 (4.82) 1.333 (3.69) F test 1.717 (2.160) 0.354 (2.160) 0.126 (2.160) Student's t test The figures in parenthesis are the corresponding tabulated values at P=0.05

Table IX: Assay parameters and method validation Ratio subtraction method Isosbestic point methodRatio subtraction method Parameter CinnarizinenicergolineVincamine λ=235nmλ=270nm 6.0-22.06- 36 8.0-40.0 Range (µgml -1 ) 0.05780.0410.01190.0239Slope 0.01930.00349-0.00010.0017Intercept 99.9199.8399.5899.72Mean 0.7031.0390.8470.917S.D. 0.4941.0790.7170.840Variance 0.99970.99980.99990.9998Correlation coefficient (r) 0.732, 0.8970.942, 0.6410.620, 0.8050.788, 0.903* RSD% a 0.841, 0.8750.892, 1.2740.881, 1.1730.936, 0.984*RSD% b RSD %a, RSD %b the intra-day, inter-day respectively (n=5) relative standard deviation of concentrations (28, 32 µ g/ml) for vincamine, (6, 8 µ g/ml) for nicergoline and (14, 16 µ g/ml) for cinnarizine..

Part III Part III Simultaneous determination of nicergoline and cinnarizine in their binary mixture

This part is subdivided into four sections: This part is subdivided into four sections: Section [A]:Simultaneous determination of nicergoline and cinnarizine by the derivative spectrophotometry Section [A]:Simultaneous determination of nicergoline and cinnarizine by the derivative spectrophotometry Section [B]: Simultaneous determination of nicergoline and cinnarizine by densitometric methods Section [B]: Simultaneous determination of nicergoline and cinnarizine by densitometric methods Section [C]: Simultaneous determination of nicergoline and cinnarizine by high- performance liquid chromatography Section [C]: Simultaneous determination of nicergoline and cinnarizine by high- performance liquid chromatography Section [D]: Simultaneous determination of nicergoline and cinnarizine by chemometric methods Section [D]: Simultaneous determination of nicergoline and cinnarizine by chemometric methods

Section [A] Simultaneous determination of nicergoline and cinnarizine by the derivative spectrophotometry

Figure (67): Absorption spectra of Nicergoline 22 µg ml-1 (———) and Cinnarizine 12 µg ml-1 (---------- ) using methanol as a solvent.

dA/dλ Figure (68): First order spectra of Nicergoline 22 μg ml-1 (______) Cinnarizine 12 μg ml-1 (_ _ _ _ _ _) using methanol as a solvent. 307nm

dA/dλ Figure (69): First – derivative absorption spectra of 6 - 38 µg ml-1 nicergoline. 307nm

Figure (70): Linearity of the peak amplitude of the first derivative at 307.6 nm to the corresponding concentration of nicergoline.

A (cinnarizine/nicergoline) Figure (71): Zero order of ratio spectra of cinnarizine 6- 22 μg ml-1 using 10 µg ml-1 of nicergoline as a divisor.

dA(cinnarizine/nicergoline)/dλ Figure (72): First order of ratio spectra of cinnarizine 6- 22 μg ml-1 using 10 µg ml-1 of nicergoline as a divisor. 244nm

Figure (73): Linearity of the peak amplitude of the first derivative of the ratio spectra at 244.6 nm to the corresponding concentration of cinnarizine.

Table (LXIII): Simultaneous determination of nicergoline and cinnarizinee in laboratory prepared mixtures by the proposed methods. Concentration (µg ml -1 )RatioFirst derivative method Derivative ratio method NicergolinecinnarizineNicergoline : Cinnarizine Recovery % NicergolineCinnarizine 9.013.52 : 3 98.88100.74 6.010.52 : 3.5 99.25101.62 7.014.02 : 4 98.24100.89 6.013.52 : 4.5 99.35101.11 8.020.02 : 5 99.81 6.016.52 : 5.5 99.81100.09 7.021.02 : 6 99.3599.90 6.019.52 : 6.5 99.81100.06 6.021.02: 7 101.75100.63 Mean  S.D.99.58  0.961100.53  0.616

Table (LXIV): Determination of nicergoline and cinnarizine in cinibral tablets by the proposed methods. Cinibral tablets claimed to contain 10 mg nicergoline & 25 mg cinnarizine Batch number First derivative method Derivative ratio method Reported method * Found % ± S.D. * for nicergoline Found % ± S.D. * for cinnarizine Found % ± S.D. * for nicergoline Found % ± S.D. * for cinnarizine 3912399.54 ± 0.869100.25 ±0.39699.32 ± 0.95698.37 ± 0.892 4008298.86 ± 0.63999.46 ± 0.58998.56 ± 0.85798.99 ± 0.825 * HPLC method.

Table (LXV): Application of standard addition for the determination of nicergoline and cinnarizine by the proposed method. Batch numberStandard added (mg)First derivative method Derivative ratio method nicergolinecinnarizineRecovery % of added nicergoline Recovery % of added cinnarizine 3912310.00 15.00 20.00 25.00 37.50 50.00 99.62 98.54 98.14 98.18 98.93 98.06 Mean  S.D.*98.76  0.76598.39  0.471 * Average of four determinations.

Table (LXVI): Statistical comparison for the results obtained by the proposed method and the reported method for the analysis of nicergoline and cinnarizine in pure powder form * HPLC method. Reported method*Derivative ratio method First derivative method Item Cinnarizinenicergolinecinnarizinenicergoline 99.2799.6499.9599.77Mean 0.7140.9780.7520.767S.D. 0.5090.9560.5650.588Variance 6699n 1.110 (4.82)1.625 (3.69)F test 1.750 (2.160)0.288 (2.160)Student's t test The figures in parenthesis are the corresponding tabulated values at P=0.05

Section [B] Simultaneous determination of nicergoline and cinnarizine by densitometric methods

Figure (74): TLC chromatogram of nicergoline and cinnarizine A= nicergoline, R f = 0.505. B= cinnarizine, R f = 0.807. M= mixture of both drugs Developing system, chloroform : methanol : ethyl acetate(5: 3 :2 v/v/v)

Figure (75): Scanning profile of the TLC chromatogram of nicergoline at 287 nm. Figure (76): Scanning profile of the TLC chromatogram of cinnarizine at 252 nm. Distance (mm) Reflectance

Figure (77): Linearity of the area under the peak to the corresponding concentration of nicergoline Figure (78): Linearity of the area under the peak to the corresponding concentration of cinnarizine spot -1

Table (LXVII): Determination of nicergoline and cinnarizine in laboratory prepared mixtures by the proposed method. Densitometric methodRatio nicergo line : cinnari zine Concentration  g.spot -1 Volume taken from stock solns (ml) Recovery % for Cinnarizine Recovery % for nicergoline CinnarizinenicergolineCinnarizine B nicergoline 98.05 3: 2 12832 98.56 4: 2 16842 98.01 5: 2 20852 98.99 6: 2 24862 99.38 7:2 28872 100.02 3: 2 320.750.5 98.71 4: 2 4210.5 98.50 5: 2 521.250.5 98.64 6: 2 621.50.5 98.17 7: 2 721.750.5 98.80  0.708 98.59  0.594 Mean  S.D.

Table (LXVIII): Determination of nicergoline and cinnarizine in cinibral tablets by the proposed method. Cinibral tablets claimed to contain 5 mg Batch number Densitometric methodReported method * % Found for nicergoline ± S.D. * % Found for cinnarizine ± S.D. * % Found for nicergoline ± S.D. * % Found for cinnarizine ± S.D. * 39123100.19 ± 0.60599.74 ± 0.92799.32 ± 0.95698.37 ± 0.892 4008298.90 ± 1.12599.01 ± 1.40198.56 ± 0.85798.99 ± 0.825 * HPLC method.

Table (LXIX): Application of standard addition for the determination of nicergoline and cinnarizine by the proposed method. Batch numberStandard added (mg)Densitometric method nicergolinecinnarizineRecovery % of added nicergoline Recovery % of added cinnarizine 3912320.00 30.00 40.00 50.00 75.00 100.00 98.65 100.62 97.87 99.04 99.60 101.63 Mean  S.D.99.04  1.417100.09  1.362 * Average of four determinations.

Table (LXX): Statistical comparison for the results obtained by the proposed method and the reported method for the analysis of nicergoline and cinnarizine in pure powder form * HPLC method. Reported method*Densitometric methodItem cinnarizinenicergolinecinnarizinenicergoline 99.2799.6499.9599.93Mean 0.7140.9781.4181.128S.D. 0.5090.9562.0101.272Variance 6677n 3.949 (4.95)1.330 (4.95)F test 1.060 (2.201)0.490 (2.201)Student's t test The figures in parenthesis are the corresponding tabulated values at P=0.05

Section [C] Simultaneous determination of nicergoline and cinnarizine by high-performance liquid chromatography

Figure (79): Liquid chromatographic separation of nicergoline and cinnarizine using final assay conditions: Column: RP18 Mobile phase: methanol : acetonitrile : water (4: 4: 2 v/v/v). Flow rate: 1.5 ml min-1. Detection: 287 nm for nicergoline and 252 nm for cinnarizine. Retention time nicergoline: 2.15 min. Retention time cinnarizine: 4.72 min. Time (min) Detector response

Figure (80): Linearity of the relative peak area to the corresponding concentration of nicergoline. Figure (81): Linearity of the relative peak area to the corresponding concentration of cinnarizine.

Table (LXXI): Determination of nicergoline and cinnarizine in laboratory prepared mixtures by the proposed method. HPLC methodRatio nicergoline: cinnarizine Concentration  g.ml -1 Volume taken from stock solns (ml) cinnarizinenicergolineCinnarizinenicergolineCinnarizinenicergoline 101.0699.03 3: 2 30203.02.0 100.7898.74 4: 2 40204.02.0 100.1997.96 5: 2 25102.51.0 101.67100.01 6: 2 30103.01.0 101.9998.43 7:2 35103.51.0 101.13  0.714 98.83  0.767 Mean  S.D.

Table (LXXII): Parameters required for system suitability test of HPLC method ParameterObtained valueReference value Resolution (R)1.959R > 0.8 T ( tailing factor)nicergoline 1.208T = 1 for a typical symmetric peak cinnarizine 1.187  (relative retention time) 2.512> 1 K’ (column capacity)nicergoline 3.7821- 10 acceptable cinnarizine 9.504 no.of theoretical plates N (no.of theoretical plates)nicergoline 463.11Increases with efficiency of the separation cinnarizine 427.95 HETPnicergoline 0.05398The smaller the value, the higher the column efficiency cinnarizine 0.0584

Table (LXXIII): Determination of nicergoline and cinnarizine in cinibral tablets by the proposed method. Reported method *HPLC method Cinibral tablets claimed to contain 5 mg Batch number % Found for cinnarizine ± S.D. * % Found for nicergoline ± S.D. * % Found for cinnarizine ± S.D. * % Found for nicergoline ± S.D. * 98.37 ± 0.89299.32 ± 0.95699.51± 1.01498.04 ± 0.68339123 98.99 ± 0.82598.56 ± 0.85799.17 ± 0.96199.65 ± 0.71540082 * HPLC method.

Table (LXXIV): Application of standard addition for the determination of nicergoline and cinnarizine by the proposed method. Batch numberStandard added (mg)HPLC method nicergolinecinnarizineRecovery % of added nicergoline Recovery % of added cinnarizine 3912310.00 15.00 20.00 25.00 37.50 50.00 97.63 98.02 98.93 99.84 100.45 100.36 Mean  S.D.*98.19  0.667100.21  0329 * Average of four determinations.

Table (LXX): Statistical comparison for the results obtained by the proposed method and the reported method for the analysis of nicergoline and cinnarizine in pure powder form * HPLC method. The figures in parenthesis are the corresponding tabulated values at P=0.05 Reported method*HPLC methodItem cinnarizinenicergolinecinnarizinenicergoline 99.2799.6499.97100.32Mean 0.7140.9780.5501.664S.D. 0.5090.9560.3022.768Variance 6689n 1.685 (3.97)2.896 (4.82)F test 2.080 (2.179)1.384 (2.160)Student's t test

Table (LXXVI): Assay parameters and method validation for nicergoline and cinnarizine Parameter Derivative ratio spectrophotometric method Densitometric method HPLC method nicergolinecinnarizinenicergolinecinnarizinenicergolinecinnarizine Range (µg/ml) 6- 386- 228- 32 (µg/spot) 2- 8 (µg/spot) 10- 9010- 45 Slope0.02380.27060.21950.65170.05240.0504 Intercept0.00720.08540.15410.1104-0.0401-0.0031 Mean99.7799.9599.9399.95 100.32 99.97 S.D.0.7670.7521.1281.418 1.664 0.550 Variance0.5880.5651.2722.010 2.768 0.302 Coff. Of variation 0.7670.7521.1281.4181.6580.550 Correl. Coef.(r) 0.99980.99960.99970.9995 0.9998 * RSD% a 0.901, 0.8750.481, 0.5030.758, 0.9510.989, 1.4011.005, 1.1240.329, 0.684 *RSD % b 1.307, 1.1050.843, 0.7420.894, 1.1971.554, 1.2161.110, 1.3540.689, 0.921 * RSD%a, RSD%b: the intra-day, inter-day respectively (n=5) relative standard deviation of concentrations (20 and 30 µ g/ml nicergoline and 10 and 14 µ g/ml cinnarizine) for derivative ratio, (16 and 20 µ g/spot nicergoline and 4 and 6 µ g/spot cinnarizine) for densitometric method and (20 and 30 µ g/ml from both nicergoline and cinnarizine) for HPLC method.

Section [D] Simultaneous determination of nicergoline and cinnarizine by chemometric methods

Chemometrics is the application of mathematical and statistical methods to provide maximum chemical information through analysis of chemical data. Chemometrics is the application of mathematical and statistical methods to provide maximum chemical information through analysis of chemical data. In this section, three chemometric techniques were applied for simultaneous determination of nicergoline and cinnarizine In this section, three chemometric techniques were applied for simultaneous determination of nicergoline and cinnarizine Classical Least Squares (CLS) with non zero intercept, Classical Least Squares (CLS) with non zero intercept, principal component regression (PCR) principal component regression (PCR) partial least squares (PLS). partial least squares (PLS).

Table (LXXVII): The concentration of different mixtures of nicergoline and cinnarizine used in the training set Sample numberNicergoline (μg.ml –1 ) Cinnarizine (μg.ml –1 ) 17.215.0 27.213.5 36.618.0 46.613.5 56.018.0 66.016.5 76.012.0 85.416.5 94.816.5 104.815.0

Figure (82): RMSEC plot of the cross validation results of the training set as a function of the number of principal components used to construct the PCR calibration for nicergoline. Figure (83): RMSEC plot of the cross validation results of the training set as a function of the number of principal components used to construct the PLS calibration for nicergoline.

Figure (35): RMSEC plot of the cross validation results of the training set as a function of the number of principal components used to construct the PCR calibration for cinnarizine. Figure (36): RMSEC plot of the cross validation results of the training set as a function of the number of principal components used to construct the PLS calibration for cinnarizine.

Table (LXXVIII): Results of the analysis of the mixtures of the validation set of nicergoline & cinnarizine by the proposed methods. Cinnarizine Recovery %Nicergoline Recovery % Concentration  g.ml -1 Sample no. PLSPCRCLS non zero PLSPCRCLS non zero CinnarizineNicergoline 100.57100.56100.71100.10 100.5618.07.2 1 100.47 100.3899.72 99.5312.07.2 2 99.93 99.97100.29 100.4116.56.6 3 98.90 98.84100.46 100.2915.06.6 4 100.64 100.74100.60 100.9115.06.0 5 100.90 100.94100.55 100.7013.56.0 6 99.47 99.4598.62 98.5218.05.4 7 99.50 99.4499.42 99.0818.04.8 8 100.05 100.0699.97 100.00 Mean 0.7070.7060.7570.6850.6860.854 S.D

Figure (84): Predicted concentration versus actual concentration of nicergoline in the validation set using CLS method Figure (85): Predicted concentration versus actual concentration of cinnarizine in the validation set using CLS method

Figure (86): Predicted concentration versus actual concentration of nicergoline in the validation set using PCR method Figure (87): Predicted concentration versus actual concentration of cinnarizine in the validation set using PCR method

Figure (88) : Predicted concentration versus actual concentration of nicergoline in the validation set using PLS method Figure (89) : Predicted concentration versus actual concentration of cinnarizine in the validation set using PLS method

Figure (90): Concentration residuals versus actual concentration of nicergoline in the validation set using CLS method Figure (91): Concentration residuals versus actual concentration of cinnarizine in the validation set using CLS method

Figure (92): Concentration residuals versus actual concentration of nicergoline in the validation set using PCR method Figure (93): Concentration residuals versus actual concentration of cinnarizine in the validation set using PCR method

Figure (94): Concentration residuals versus actual concentration of nicergolinein the validation set using PLS method Figure (95): Concentration residuals versus actual concentration of cinnarizine in the validation set using PLS method

Table (LXXIX): RMSEP and Q2 values of the validation set analysis of cinnarizine hydrochloride by the proposed methods. ItemCLSPCRPLS nicergolinecinnarizinenicergolinecinnarizinenicergolinecinnarizine RMSEP0.04910.110270.03620.101390.036240.10143 Q2Q2 0.99680.99720.99790.99760.99790.9976

Table (LXXX): Quantitative determination of nicergoline and cinnarizine in cinibral tablets by the proposed methods Table (LXXX): Quantitative determination of nicergoline and cinnarizine in cinibral tablets by the proposed methods Bat ch no. CLSPCRPLS nicergolinecinnarizinenicergolinecinnarizinenicergolinecinnarizine Fou nd* Mean± S.D. Fou nd* Mean± S.D. Fou nd* Mean± S.D. Fou nd* Mean± S.D. Fou nd* Mean± S.D. Fou nd* Mean± S.D. 391 23 6.01 99.99  0.321 15.1 5 101.09  0.601 6.01 99.45  0. 524 15.1 0 99.98  0. 726 6.02 100.04  0.611 15.1 0 99.78  0. 820 400 48 6.04 99.54  0.710 15.0 8 100.23  0.265 6.07 100.13  0.522 15.1 3 101.02  0.754 6.06 100.00  0.540 15.1 4 100.58  0.738 * (μg.ml – 1).

Table (LXXXI): Results of the standard addition technique for the simultaneous determination of nicergoline and cinnarizine in cinibral tablets by the proposed methods Batch. no Standard added(mg)CLSPCRPLS nicergolinecinnarizine Recovery % of added nicergoline Recovery % of added cinnarizine Recovery % of added nicergoline Recovery % of added cinnarizine Recovery % of added nicergoline Recovery % of added cinnarizine 391230.25 0.375 0.50 0.25 0.375 0.50 99.21 99.98 100.17 98.11 98.46 100.36 100.72 100.99 100.52 99.20 100.47 100.58 102.13 101.69 101.15 100.73 100.75 99.87 Mean ± S.D. 99.78 ± 0.508 98.97 ± 1.210 100.74 ± 0.235 100.08 ± 0.766 101.65 ± 0.490 100.45 ± 0.502

Table (LXXXII): Statistical comparison for the results obtained by the proposed methods and the reported method for the analysis of nicergoline and cinnarizine in pure powder form. * HPLC method. Reported method*PLSPCRCLSItem cinniccinniccinniccinnic 99.2799.6499.92100.0199.91100.0199.99100.00Mean 0.7140.9780.9620.5660.9660.5670.9050.575S.D. 0.5090.9560.9250.3200.9330.3210.8190.330Variance 6610 n 1.817 (4.77) 2.987 (3.48) 1.833 (4.77) 2.978 (3.48) 1.609 (4.77) 2.896 (3.48) F test 1.428 (2.145) 0.968 (2.145) 1.401 (2.145) 0.968 (2.145) 1.656 (2.145) 0.936 (2.145) Student's t test The figures in parenthesis are the corresponding tabulated values at P=0.05

Part IV Stability indicating methods for the determination of meclophenoxate hydrochloride

This part includes a general introduction about the chemistry of meclophenoxate hydrochloride. This part includes a general introduction about the chemistry of meclophenoxate hydrochloride. Review article on the reported methods used for its quantitative determination. Review article on the reported methods used for its quantitative determination. This part is subdivided into three sections: This part is subdivided into three sections: Section(A): High-performance liquid chromatographic determination of meclophenoxate hydrochloride in presence of its acid degradation product Section(A): High-performance liquid chromatographic determination of meclophenoxate hydrochloride in presence of its acid degradation product Section(B): Kinetic study on the degradation of meclophenoxate hydrochloride Section(B): Kinetic study on the degradation of meclophenoxate hydrochloride Section (C): Determination of meclophenoxate hydrochloride in presence of its acid degradation product using ion selective electrodes Section (C): Determination of meclophenoxate hydrochloride in presence of its acid degradation product using ion selective electrodes

Section [A] High-performance liquid chromatographic determination of meclophenoxate hydrochloride in presence of its acid degradation product

-Structure of meclophenoxate 2 N NaOH Reflux 25 min. then neutralize with HCl -The proposed mechanism for preparing the degradation product:

Figure (19): Liquid chromatographic separation of meclophenoxate.HCl and its degradation product using final assay conditions: Column: RP18 Mobile phase: 0.01 M ammonium carbonate: acetonitrile (7:3 v/v). Flow rate: 1.0 ml min-1. Detection: 277 nm. Retention time meclophenoxate hydrochloride: 5.39 min. Retention time degradation product: 2.70 min. Time (min) Detector response

Figure (20): Linearity of the relative peak area to the corresponding concentration of meclophenoxate hydrochloride.

Table (X): Determination of meclophenoxate hydrochloride in laboratory prepared mixtures by the proposed method Concentration ( µ g.ml -1 ) Percentage % HPLCMethod Meclophenoxate. HCl Degradation product Meclophenoxate.HCl Recovery % Meclophenoxate. HCl 3505087.512.599.12 3001007525101.56 25015062.537.598.75 2002005050102.03 15025037.562.598.51 1003002575100.47 5035012.587.599.07 Mean99.94 S.D.1.319

Table (XI): Parameters required for system suitability test of HPLC method Parameter Obtained value Reference value Resolution (R) 3.676 R > 0.8 T ( tailing factor) Meclophenoxate.HCl 1.05 T = 1 for a typical symmetric peak Deg. product 1.0  (relative retention time) 2.224 > 1 K ’ (column capacity) Meclophenoxate.HCl 9.796 1- 10 acceptable Deg. product 2.404 N (no.of theoretical plates) Meclophenoxate.HCl 728.4 Increases with efficiency of the separation Deg. product 268.16 HETP Meclophenoxate.HCl 0.0343 The smaller the value, the higher the column efficiency Deg. product 0.093

Table (XII): Determination of meclophenoxate hydrochloride in lucidril tablets by the proposed method * Stability indicating HPLC method. Reported method *HPLC method Lucidril tablets claimed to contain 250 mg meclophenoxate.HCl Batch number Recovery % ± S.D.*Found % ± S.D.** 100.29 ± 1.411100.87 ± 1.1975GE0941 84.12 ± 1.37383.91 ± 1.002010156(expired 3/04)

Table (XIII): Application of standard addition for the determination of meclophenoxate hydrochloride by the proposed method HPLC methodStandard added (mg) Batch number Recovery % of addedFound of added (mg) Meclophenoxate. HCl 98.96 101.33 101.21 98.96 152.00 202.42 100.00 150.00 200.00 5GE0941 100.53 ± 1.277Mean ± S.D.* * Average of four determinations.

Table (XIV): Statistical comparison for the results obtained by the proposed method and the reported method for the analysis of meclophenoxate hydrochloride in pure powder form * Stability indicating HPLC method. Reported method *HPLC methodItem 99.3999.94Mean 1.1441.148S.D. 1.3081.317Variance 69n 1.006 (4.82)F test 0.910 (2.160)Student’s t test The figures in parenthesis are the corresponding tabulated values at P=0.05

Section [B] Kinetic study on degradation of meclophenoxate hydrochloride

Kinetic study on degradation of meclophenoxate hydrochloride includes: Study the kinetic order of the reaction Study the kinetic order of the reaction Study the effect of sodium hydroxide concentration on the reaction rate Study the effect of sodium hydroxide concentration on the reaction rate Study the effect of temperature on the reaction rate Study the effect of temperature on the reaction rate Calculate energy of activation (Ea): Calculate energy of activation (Ea): log = log =

Figure (22): First order plot of the hydrolysis of meclophenoxate hydrochloride (1000 mg %) with 2 N NaOH at 80 o C

Figure (23): First order plot of the hydrolysis of meclophenoxate hydrochloride (1000 mg %) with 2 N NaOH at different temperatures.

Figure (24): First order plot of the hydrolysis of meclophenoxate hydrochloride (1000 mg %) with 1.5 N NaOH at different temperatures.

Figure (25): First order plot of the hydrolysis of meclophenoxate hydrochloride (1000 mg %) with 1.0 N NaOH at different temperatures.

Figure (26): Arrhenius Plot for the hydrolysis of meclophenoxate hydrochloride (1000 mg %) with 1.0, 1.5, 2.0 N NaOH Ea" was found to be 12.331 kilo calories mol-1

Table (XVI): Kinetic data of meclophenoxate hydrochloride hydrolysis Normality of NaOH Temperature K in min -1 t 1/2 in min. 2.0 N NaOH 90 o C 80 o C 70 o C 60 o C 0.1250.0790.0490.0285.548.7714.1424.75 1.5 N NaOH 90 o C 80 o C 70 o C 60 o C 0.0930.0590.0380.0227.4511.7418.2331.50 1.0 N NaOH 90 o C 80 o C 70 o C 60 o C 0.0630.0400.0250.01411.0017.3227.7249.50

Section [C] Determination of meclophenoxate hydrochloride in presence of its acid degradation product using ion selective electrodes

Ion selective electrodes are electrodes containing membranes having a selective response for a particular ion. Ion selective electrodes are electrodes containing membranes having a selective response for a particular ion. Meclophenoxate hydrochloride (Cation) reacted with tetraphenylborate or reineckate (anionic ion exchangers) to form stable 1:1, water insoluble ion association complex. Meclophenoxate hydrochloride (Cation) reacted with tetraphenylborate or reineckate (anionic ion exchangers) to form stable 1:1, water insoluble ion association complex.  -CD-based sensors form inclusion complexes in the aqueous and in solid state with organic molecules.  -CD-based sensors form inclusion complexes in the aqueous and in solid state with organic molecules. Three membranes are studied in this part: Three membranes are studied in this part: a) meclo-TPB a) meclo-TPB b) meclo-RNC b) meclo-RNC c)  -CD-RNC c)  -CD-RNC

The electrode assembly Figure (27): PVC matrix membrane ion selective electrode: [1] Shielded cable. [2] Rubber sheath. [3] Quickfit cone. [4] Quickfit socket. [5] Mercury. [6] Calomel reference electrode. [7] Internal solution. [8] PVC tubing. [9] Sensor membrane.

Figure (28): Effect of pH on the response of meclo - TPB electrode

Figure (29): Effect of pH on the response of meclo - reineckate electrode

Figure (30): Effect of pH on the response of  CD- reineckate electrode.

Figure (31): Effect of temperature on the response of  CD- reineckate membrane electrode.

Figure (32): Profile of the potential in mV to the –log concentration of meclophenoxate hydrochloride with meclo- TPB, meclo – reineckate and  CD- reineckate.

Table (XVIII): Electrochemical response characteristics of the three investigated electrodes ParameterMeclo-TPBMeclo-RNC  -CD-RNC Slope (mV/decade)* -52.73-51.64-54.05 Intercept (mV) 222.03227.84268.6 Response time (seconds) 404030 Working pH range 4 – 7.5 5.5 - 7 4 – 7.5 Concentration range (M) 1 x 10 -5 - 1 x 10 -2 Stability (weeks) 333 Average recovery (%) 99.9299.96100.03 Standard deviation 1.0770.5020.763 Correlation coefficient 0.99950.99980.9996

Table (XIX) Potentiometric selectivity coefficients (K Plot primary ion, interferent) for the three proposed electrodes. Interferent Selectivity coefficient Meclo-TPBMeclo-RNC  CD-RNC NaCl 3.75 x 10 -2 3.79 x 10 -2 5.75 x 10 -3 KCl 3.01 x 10 -2 3.61 x 10 -2 6.07 x 10 -3 NH 4 Cl 2.05 x 10 -2 3.30 x 10 -2 5.12 x 10 -3 CaCl 2 2.67 x 10 -2 3.36 x 10 -2 5.32 x 10 -3 MgSO 4 3.51 x 10 -2 4.52 x 10 -2 7.17 x 10 -3 glucose 3.14 x 10 -2 3.05 x 10 -2 6.34 x 10 -3 lactose 3.15 x 10 -2 3.18 x 10 -2 6.38 x 10 -3 sucrose 2.92 x 10 -2 2.67 x 10 -2 5.87 x 10 -3 Urea 2.92 x 10 -2 3.18 x 10 -2 4.76 x 10 -3 L-phenyl alanine 2.88 x 10 -2 2.56 x 10 -2 4.33 x 10 -3 Deg.product 2.09 x 10 -2 2.19 x 10 -2 5.41 x 10 -3

Table (XX): Determination of meclophenoxate hydrochloride in laboratory prepared mixtures by the proposed method Concentration (M) Ratio* Recovery % of meclophenoxate. HCl Meclophenoxate HCl Degradation product Meclo- TPB Meclo- RNC  CD-RNC 1 x 10 -3 1 x 10 -4 10: 1 98.6397.5499.21 1 x 10 -3 5 x 10 -4 2: 1 99.0699.4199.23 1 x 10 -3 1: 1 99.58100.06100.53 1 x 10 -3 5 x 10 -3 1: 5 100.6998.73102.87

Table (XXI): Determination of meclophenoxate hydrochloride in lucidril tablets by the proposed method Lucidril tablets claimed to contain 250 mg Batch number Meclo-TPBMeclo-RNC  CD-RNC Reported method * % Found ± S.D.** Found % ± S.D.** 5GE0941 100.78  0.916 99.23  1.137 100.74  0.628 99.54 ± 1.232 010156(expired 3/04) 83.09  1.404 83.11  0.675 83.12  0.661 99.65 ± 0.951 * Stability indicating HPLC method.

Table (XXII): Determination of meclophenoxate hydrochloride in spiked human plasma by the proposed electrodes. Concentration(M)Meclo-TPBMeclo-RNC  CD-RNC Recovery % ± S.D.* 1 x 10 -3 101.77 ± 0.612 101.58 ± 0.663 101.03 ± 0.497 1 x 10 -4 102.14 ± 0.550 101.97 ± 0.601 101.24 ± 0.404 * Average of three determinations

Table (XXIII): Application of standard addition for the determination of meclophenoxate hydrochloride by the proposed method Batch numb er Standard added (mg)Meclo-TPBMeclo-RNC  CD-RNC Meclo. HCl FoundRecoveryFoundRecoveryFoundRecovery 5GE094114.6822.0229.3614.4121.6729.3198.1698.4199.8214.6521.6129.0099.7998.1398.7714.7022.3429.91100.13101.45101.87 Mean ± S.D.* 98.79 ± 0.895 98.89 ± 0.837 101.15 ± 0.907 * Average of three determinations

Table (XXIV): Statistical comparison for the results obtained by the proposed method and the reported method for the analysis of meclophenoxate hydrochloride in pure powder form * Stability indicating HPLC method. Reported method *  CD-RNC Meclo-RNCMeclo-TPBItem 99.39100.0399.9699.92Mean 1.1440.7630.9021.077S.D. 1.3080.5820.8131.159Variance 6444n 2.247 (9.01)1.608 (9.01)1.128 (9.01)F test 0.974 (2.306)0.833 (2.306)0.733 (2.306)Student’s t test The figures in parenthesis are the corresponding tabulated values at P=0.05

Table (XLIV): Assay parameters and method validation for meclophenoxate hydrochloride Suggested electrodesHPLC method Parameter  CD-RNC Meclo-RNCMeclo-TPBMeclophenoxate. HCl. 1 x 10 -5 - 1 x 10 -2 M 15-400Range (μg ml -1 ) 54.0551.6452.730.0095Slope 268.6227.84222.030.0229Intercept 100.0399.9699.9299.94Mean 0.7630.9021.0771.148S.D. 0.5820.8131.1591.317Variance 0.7620.9021.0771.149RSD% 0.99960.99980.99950.9997Correl. Coef.(r) 0.841, 0.8750.936, 0.9840.928, 0.9031.110, 1.236* RSD% a 1.004, 1.1731.378, 1.5211.132, 1.1971.428, 1.418*RSD % b * RSD%a, RSD%b the intra-day, inter-day respectively (n=5) relative standard deviation of concentrations (200 and 300 µ g ml- 1) and (10-3M and 10-4M) for ion selective electrodes method.

Part V Stability indicating methods for determination of vinpocetine

This part includes a general introduction about the chemistry of vinpocetine. This part includes a general introduction about the chemistry of vinpocetine. Review article on the reported methods used for its quantitative determination. Review article on the reported methods used for its quantitative determination. This part is subdivided into four sections: This part is subdivided into four sections: Section [A]: Determination of vinpocetine in presence of its acid degradation product by the derivative ratio spectrophotometry Section [A]: Determination of vinpocetine in presence of its acid degradation product by the derivative ratio spectrophotometry Section [B]: Densitometric determination of vinpocetine in presence of its acid degradation product Section [B]: Densitometric determination of vinpocetine in presence of its acid degradation product Section [C]: High-performance liquid chromatographic determination of vinpocetine in presence of its acid degradation product Section [C]: High-performance liquid chromatographic determination of vinpocetine in presence of its acid degradation product Section [D]: Chemometric determination of vinpocetine in presence of its acid degradation product Section [D]: Chemometric determination of vinpocetine in presence of its acid degradation product

Section [A] Determination of vinpocetine in presence of its acid degradation product by the derivative ratio spectrophotometry

Structure of vinpocetine Structure of vinpocetine

Figure (34): Absorption spectra of vinpocetine 12 µg ml-1 (———) and degradation product 10 µg ml-1 (---------- ) using 0.1N hydrochloric acid as a solvent.

Figure (35): First order spectra of vinpocetine 12 μg ml-1 (______) degradation product 10 μg ml-1 (_ _ _ _ _ _) using 0.1N hydrochloric acid as a solvent. dA/dλ

A (vinpocetine/deg.prod.) Figure (36): Zero order of ratio spectra of vinpocetine 4-32 μg ml-1 using 10 µg.ml-1 of degradation product as a divisor.

Figure (37): First order of ratio spectra of vinpocetine 4-32 μg ml-1 using 10 µg ml-1 of degradation product as a divisor. dA(vinpocetine/deg.product)/dλ 311nm

Figure (38): Linearity of the peak amplitude of the first derivative of the ratio spectra at 311 nm to the corresponding concentration of vinpocetine.

Table (XXV): Determination of vinpocetine in laboratory prepared mixtures by the proposed method. Concentration ( µ g.ml -1 ) Percentage (%) Derivative ratio method Vinpocetine Degradation product Vinpocetine Recovery % Vinpocetine 248752599.51 201262.537.5102.37 16165050101.08 122037.562.598.42 8242575101.05 42812.587.5100.91 Mean100.55 S.D.1.385

Table (XXVI): Determination of vinpocetine in vinporal tablets by the proposed method. Vinporal tablets claimed to contain 5 mg Batch number Derivative ratio method Reported method ** Found % ± S.D. * 710101 99.02 ± 0.933 99.32 ± 0.956 910101 98.98 ± 1.014 98.56 ± 0.857 * Spectrophotometric method.

Table (XXVII): Application of standard addition for the determination of vinpocetine in its pharmaceutical preparation by the proposed method. Batch number Standard added (mg) Derivative ratio method Vinpocetine Found of added (mg) Recovery % of added 71010125.0037.5050.0025.0638.6250.53100.24102.98101.06 Mean ± S.D.* 101.42 ± 1.406 * Average of four determinations.

Table (XXVIII): Statistical comparison for the results obtained by the proposed method and the reported method for the analysis of vinpocetine in pure powder form Table (XXVIII): Statistical comparison for the results obtained by the proposed method and the reported method for the analysis of vinpocetine in pure powder form * Spectrophotometric method. Reported method *Derivative ratio methodItem 99.73100.05Mean 1.0501.251S.D. 1.1021.565Variance 68n 1.420 (4.88)F test 0.505 (2.179)Student’s t test The figures in parenthesis are the corresponding tabulated values at P=0.05

Section [B] Densitometric determination of vinpocetine in presence of its acid degradation product

Figure (40): TLC chromatogram of vinpocetine and its degradation product. A= Vinpocetine R f = 0.80. B= Degradation product R f = 0.54 M= mixture of vinpocetine and its deg. product Developing system, methanol: chloroform: ethyl acetate (2:1:1 v/v/v).

Figure (41): Scanning profile of the TLC chromatogram of vinpocetine at 268 nm. Distance (mm) Reflectance

Figure (42): Linearity of the area under the peak to the corresponding concentration of vinpocetine

Table (XXIX): Determination of vinpocetine in laboratory prepared mixtures by the proposed method. Concentration ( µ g/spot) Percentage (%) DensitometricMethod Vinpocetine Degradation product Vinpocetine Recovery % Vinpocetine 919010101.23 82802098.09 73703097.82 646040100.68 555050101.09 464060100.15 373070102.03 Mean100.15 S.D.1.609

Table (XXX): Determination of vinpocetne in vinporal tablets by the proposed method Vinporal tablets claimed to contain 5 mg Batch number Densitometric method Reported method ** Found % ± S.D. * 710101 101.03 ± 1.027 99.32 ± 0.956 910101 100.31 ± 1.003 98.56 ± 0.857 * Spectrophotometric method.

Table (XXXI): Application of standard addition for the determination of vinpocetine in its pharmaceutical preparation by the proposed method. Batch number Standard added (mg) Densitometric method Vinpocetine Found of added (mg) Recovery % of added 71010125.0037.5050.0025.6938.0249.12102.76101.3898.24 Mean ± S.D.* 100.79 ± 2.316 * Average of four determinations.

Table (XXXII): Statistical comparison for the results obtained by the proposed method and the reported method for the analysis of vinpocetine in pure powder form. * Spectrophotometric method. Reported method *Densitometric methodItem 99.7399.76Mean 1.0501.644S.D. 1.1022.702Variance 68n 2.451 (4.88)F test 0.038 (2.179)Student’s t test The figures in parenthesis are the corresponding tabulated values at P=0.05

Section [C] High-performance liquid chromatographic determination of vinpocetine in presence of its acid degradation product

Figure (43): Liquid chromatographic separation of vinpocetine and its degradation product using final assay conditions: Column: RP18 Mobile phase: methanol: 0.01 M ammonium bicarbonate (60:40 v/v). Flow rate: 1.6 ml min-1. Detection: 268 nm. Retention time vinpocetine (II): 6.12 min. Retention time degradation product (I): 2.24 min. Time (min) Detector response

Figure (44): Linearity of the relative peak area to the corresponding concentration of vincamine.

Table (XXXIII): Determination of vinpocetine in laboratory prepared mixtures by the proposed method Concentration ( µ g ml -1 ) Percentage (%) HPLC method Vinpocetine Degradation product Vinpocetine Recovery % Vinpocetine 1648020100.39 1467030100.07 128604099.67 1010505099.32 8124060101.21 614307099.54 4162080100.82 2181090101.07 Mean100.26 S.D.0.724

Table (XXXIV): Parameters required for system suitability test of HPLC method Parameter Obtained value Reference value Resolution (R) 3.167 R > 0.8 T ( tailing factor) Vinpocetine 1.045 T = 1 for a typical symmetric peak Deg. product 1.166  (relative retention time) 3.365 > 1 K ’ (column capacity) Vinpocetine 9.200 1- 10 acceptable Deg. product 2.733 N ( no.of theoretical plates ) Vinpocetine 418.88 Increases with efficiency of the separation Deg. product 51.38 HETP Vinpocetine 0.05968 The smaller the value, the higher the column efficiency Deg. product 0.4865

Table (XXXV): Determination of vinpocetine in vinporal tablets by the proposed method Vinporal tablets claimed to contain 5 mg Batch number HPLC method Reported method * % Found ± S.D. Found % ± S.D. * 710101 99.28 ± 1.124 99.80 ± 0.905 910101 100.71 ± 1.105 100.31 ± 1.074 * Spectrophotometric method.

Table (XXXVI): Application of standard addition for the determination of vinpocetine in vinporal tablets by the proposed method Batch number Standard added (mg) HPLC method Vinpocetine Found % of added Recovery % of added 012261 A 10.0015.0020.0010.1214.8519.90101.2299.0699.51 Mean ± S.D.* 99.93 ± 1.139 * Average of four determinations.

Table (XXXVII): Statistical comparison for the results obtained by the proposed method and the reported method for the analysis of vinpocetine in pure powder form Table (XXXVII): Statistical comparison for the results obtained by the proposed method and the reported method for the analysis of vinpocetine in pure powder form * Spectrophotometric method. Reported method *HPLC methodItem 99.73100.00Mean 1.0501.219S.D. 1.1021.485Variance 68n 1.347 (4.88)F test 0.434 (2.179)Student’s t test The figures in parenthesis are the corresponding tabulated values at P=0.05

Table (XXXVIII): Assay parameters and method validation for vinpocetine Parameter Derivative ratio spectrophotometric method Densitometric method HPLC method VinpocetineVinpocetineVinpocetine Range ( µ g/ml) 4-32 1-15( µ g/spot) 2-16 Slope0.18560.40710.1005 Intercept0.01970.1530.0175 Mean100.0599.76100.00 S.D.1.2511.6441.219 Variance1.5652.7021.485 Coff. Of variation 1.2501.6471.219 Correl. Coef.(r) 0.99940.99950.9996 * RSD% a 0.205, 0.318 0.487, 0.339 0.294,0.463 *RSD % b 0.441, 0.409 0.629, 0.578 0.665,0.547 *RSD%a, RSD%b: the intra-day, inter-day respectively (n=5) relative standard deviation of concentrations (16 and 20 µ g/ml) for derivative ratio, (9 and 11 µ g/spot) for densitometric method and (8 and 12 µ g/ml) for HPLC method.

Section [D] Chemometric determination of vinpocetine in presence of its acid degradation product

Table (XXXIX): The concentration of different mixtures of vinpocetine and its degradation product used in the training set. Sample number Vinpocetine (μg.ml – 1 ) Deg. product (μg.ml – 1 ) 18.010.0 28.014.0 316.06.0 416.010.0 520.06.0 620.010.0 712.010.0 812.018.0 912.00.0 1016.00.0

Figure (45): RMSEC plot of the cross validation results of the training set as a function of the number of principal components used to construct the PCR calibration for vinpocetine. Figure (46): RMSEC plot of the cross validation results of the training set as a function of the number of principal components used to construct the PLS calibration for vinpocetine.

Table (XXXX): Results of the analysis of the mixtures of the validation set of vinpocetine and its degradation product by the proposed methods Table (XXXX): Results of the analysis of the mixtures of the validation set of vinpocetine and its degradation product by the proposed methods Sample no. Concentration (μg.ml – 1 ) Vinpocetine Recovery % Vinpocetine Deg. product CLSPCRPLS 14.014.0 102.19102.16102.1671 28.06.0 101.77102.26102.26 38.018.0 102.06102.14102.14 416.014.0 99.7399.5299.52 520.02.0 100.70100.39100.39 620.014.0 99.7299.6499.64 720.018.0 99.81100.03100.03 812.06.0 100.29100.46100.46 912.014.0 99.0999.0599.05 1020.00.0 100.62100.64100.64 Mean 100.60100.63100.63 S.D. 1.0841.1751.175

Figure (47): Predicted concentration versus actual concentration of vinpocetine in the validation set using CLS method

Figure (48): Predicted concentration versus actual concentration of vinpocetine in the validation set using PCR method

Figure (49): Predicted concentration versus actual concentration of vinpocetine in the validation set using PLS method

Figure (50): Concentration residuals versus actual concentration of vinpocetine in the validation set using CLS method

Figure (51): Concentration residuals versus actual concentration of vinpocetine in the validation set using PCR method

Figure (52): Concentration residuals versus actual concentration of vinpocetine in the validation set using PLS method

Table (XXXXI): RMSEP and Q2 values of the validation set analysis of vinpocetine by the proposed methods. ItemCLSPCRPLS RMSEP0.104820.109170.10917 Q2Q2Q2Q20.999660.9996320.999632

Table (XXXXII): Quantitative determination of vinpocetine in vinporal tablets by the proposed methods. Batch no CLSPCRPLS Found* (μg.ml – 1 ) Mean ± S.D. Found* (μg.ml – 1 ) Mean ± S.D. Found* (μg.ml – 1 ) Mean ± S.D. 71010120.12100.41±0.65219.6599.41±0.80819.6599.41±0.808 91010119.3699.03±0.78119.5499.65±0.84519.5499.65±0.845 * Spectrophotometric method.

Table (XXXXIII): Results of the standard addition technique for the determination of vinpocetine in vinporal tablets by the proposed methods Batch numb er Standard added (mg)CLSPCRPLS Vinpocetine Recovery % of added 71010125.0037.5050.00101.54100.98100.06100.96101.2099.03100.96101.2099.03 Mean ± S.D. 100.86 ± 0.747 100.39 ± 1.189

Table (XXXXIV): Statistical comparison for the results obtained by the proposed methods and the reported method for the analysis of vinpocetine in pure powder form. * Spectrophotometric method. Reported method*Proposed methodsItem PLSPCRCLS 99.73100.21 100.05Mean 1.0500.968 0.774S.D. 1.1020.937 0.599Variance 610 n 1.176 (3.48) 1.839 (3.48)F test 0.931(2.145) 0.702(2.145)Student’s t test The figures in parenthesis are the corresponding tabulated values at P=0.05

Part VI Stability indicating methods for the determination of Pyritinol dihydrochloride

This part includes a general introduction about the chemistry of pyritinol dihydrochloride. This part includes a general introduction about the chemistry of pyritinol dihydrochloride. Review article on the reported methods used for its quantitative determination. Review article on the reported methods used for its quantitative determination. This part is subdivided into three sections: This part is subdivided into three sections: Section [A]:Determination of pyritinol dihydrochloride in presence of its degradation product by the derivative ratio spectrophotometry Section [A]:Determination of pyritinol dihydrochloride in presence of its degradation product by the derivative ratio spectrophotometry Section [B]: High-performance liquid chromatographic determination of pyritinol dihydrochloride in presence of its degradation product Section [B]: High-performance liquid chromatographic determination of pyritinol dihydrochloride in presence of its degradation product Section [C]: Determination of pyritinol dihydrochloride in presence of its degradation product using ion selective electrodes Section [C]: Determination of pyritinol dihydrochloride in presence of its degradation product using ion selective electrodes

Structure of pyritinol.2HCl Structure of pyritinol.2HCl The proposed scheme for its degradation The proposed scheme for its degradation N N N CH 3 HO S -S CH 3 OHHO CH 3 HO H 2 O 2 30% v/v 1 hr at room temp 2 SO 3 H OH mol.wt 233

Section [A] Determination of pyritinol dihydrochloride in presence of its degradation product by the derivative ratio spectrophotometry

Figure (54): Absorption spectra of pyritinol dihydrochloride 20 µg ml-1 (———) and degradation product 20 µg ml-1 (---------- ) using distelled water as a solvent.

Figure (55): First order spectra of pyritinol dihydrochloride 20 µg ml-1 (———) and degradation product 20 µg ml-1 (---------- ) using distelled water as a solvent. dA/dλ

Figure (56): Zero order of ratio spectra of pyritinol dihydrochloride 10-26 μg ml-1 using 20 µg.ml-1 of degradation product as a divisor. A (pyritinol.2HCl/deg.prod.)

dA(pyritinol.2HCl/deg.product)/dλ Figure (57): First order of ratio spectra of pyritinol dihydrochloride 10-26 μg ml-1 using 20 µg ml-1 of degradation product as a divisor. 357nm

Figure (58): Linearity of the peak amplitude of the first derivative of the ratio spectra at 357.4 nm to the corresponding concentration of pyritinol dihydrochloride.

Table (XXXXV): Determination of pyritinol dihydrochloride in laboratory prepared mixtures by the proposed method. Concentration ( µ g.ml -1 ) Percentage (%) Derivative ratio method Pyritinol.2HCl Degradation product Pyritinol.2HCl Recovery % Pyritinol.2HCl 24292.317.6999.10 22484.6215.3899.75 18869.2430.76101.28 141253.8546.15100.15 101638.4761.5399.62 Mean99.98 S.D.0.817

Table (XXXXVI): Determination of pyritinol dihydrochloride in Encephabol tablets by the proposed method. * Stability indicating spectrophotometric method. Reported method *Derivative ratio method Encephabol tablets claimed to contain 200 mg pyritinol.2HCl Batch number Found % ± S.D. ** 99.36 ± 0.421100.04 ± 0.92113476 98.95 ± 0.55199.42 ± 0.77513168

Table (XXXXVII): Application of standard addition for the determination of pyritinol dihydrochloride by the proposed method. Derivative ratio methodStandard added (mg) Batch number Recovery % of addedFound of added (mg) Pyritinol dihydrochloride 101.52 98.89 100.61 25.38 37.08 50.30 25.00 37.50 50.00 13476 100.34 ±1.335Mean ± S.D.* * Average of four determinations.

Table (XXXXVIII): Statistical comparison for the results obtained by the proposed methods and the reported method for the analysis of pyritinol dihydrochloride in pure powder form. * Stability indicating spectrophotometric method. Reported method *Derivative ratio methodItem 99.9299.52Mean 1.1721.037S.D. 1.3751.075Variance 69n 1.279 (3.48)F test 0.695 (2.160)Student’s t test The figures in parenthesis are the corresponding tabulated values at P=0.05

Section [B] High-performance liquid chromatographic determination of pyritinol dihydrochloride in presence of its oxidative degradation product

Figure (60): Liquid chromatographic separation of pyritinol dihydrochloride and its degradation product using final assay conditions: Column: RP 8 Mobile phase:acetonitrile: solution pH 4 (58:42 v/v). Flow rate: 1.0 ml min-1. Detection: 296 nm. Retention time pyritinol dihydrochloride: 1.62 min. Retention time degradation product: 2.97 min. Time (min) Detector response

Figure (61): Linearity of the relative peak area to the corresponding concentration of pyritinol dihydrochloride.

Table (IL): Determination of pyritinol dihydrochloride in laboratory prepared mixtures by the proposed method Concentration ( µ g.ml -1 ) Percentage % HPLCMethod Pyritinol. 2HCl Deg. product Pyritinol. 2HCl Deg. product Recovery % Pyritinol. 2HCl 8020802099.01 6040604098.25 40604060101.74 2080208099.61 10901090100.85 Mean99.89 S.D.1.404

Table (L): Parameters required for system suitability test of HPLC method Parameter Obtained value Reference value Resolution (R) 1.59 R > 0.8 T ( tailing factor) Pyritinol dihydrochloride 1.23 T = 1 for a typical symmetric peak Deg. product 1.14  (relative retention time) 2.208 > 1 K ’ (column capacity) Pyritinol dihydrochloride 4.946 1- 10 acceptable Deg. product 2.240 N (no.of theoretical plates) Pyritinol dihydrochloride141.41 Increases with efficiency of the separation Deg. product 85.69 HETP Pyritinol dihydrochloride 0.176 The smaller the value, the higher the column efficiency Deg. product 0.291

Table (LI): Determination of pyritinol dihydrochloride in Encephabol tablets by the proposed method. Reported method *HPLC method Encephabol tablets claimed to contain 200 mg pyritinol.2HCl Batch number Found % ± S.D. ** 99.36 ± 0.421100.19 ± 0.91113476 98.95 ± 0.55199.22 ± 0.91213168 * Stability indicating spectrophotometric method.

Table (LII): Application of standard addition for the determination of pyritinol dihydrochloride by the proposed method. Batch number Standard added (mg) HPLC method Pyritinol dihydrochloride Found of added (mg) Recovery % of added 1347610.0015.0020.00 9.82 9.8214.9620.2298.2699.74101.12 Mean ± S.D.* 99.47 ±1.430 * Average of four determinations.

Table (LIII): Statistical comparison for the results obtained by the proposed methods and the reported method for the analysis of pyritinol dihydrochloride in pure powder form. * Stability indicating spectrophotometric method. Reported method *HPLC methodItem 99.9299.59Mean 1.1720.963S.D. 1.3750.927Variance 68n 1.483 (3.97)F test 0.579 (2.179)Student’s t test The figures in parenthesis are the corresponding tabulated values at P=0.05

Section [C] Determination of pyritinol dihydrochloride in presence of its degradation product using ion selective electrodes

Pyritinol dihydrochloride (Cation) reacted with tetraphenylborate or reineckate (anionic ion exchangers) to form stable 1:2, water insoluble ion association complex. Pyritinol dihydrochloride (Cation) reacted with tetraphenylborate or reineckate (anionic ion exchangers) to form stable 1:2, water insoluble ion association complex.  -CD-based sensors form ion seiving membranes.  -CD-based sensors form ion seiving membranes. Three membranes are studied in this part: Three membranes are studied in this part: a) Pyrit-TPB a) Pyrit-TPB b) Pyrit-RNC b) Pyrit-RNC c)  -CD-RNC c)  -CD-RNC

Figure (62): Effect of pH on the response of pyrit - TPB electrode

Figure (63): Effect of pH on the response of pyrit - reineckate electrode

Figure (64): Effect of pH on the response of  CD- reineckate electrode

Figure (65): Effect of temperature on the response of  CD- reineckate membrane electrode

Figure (66): Profile of the potential in mV to the –log concentration of pyritinol dihydrochloride with pyrit- TPB, pyrit – reineckate and  CD- renikate.

Table (LVI): Electrochemical response characteristics of the three investigated electrodes ParameterPyrit-TPB Pyrit- RNC  -CD- RNC Slope (mV/decade)* -30.600-31.100-32.891 Intercept (mV) 266.84286.89317.07 Response time (seconds) 404040 Working pH range 2.5- 4 Concentration range (M) 3.162 x 10 -6 - 3.162 x 10 -4 1 x 10 -6 - 3.162 x 10 -4 Stability (weeks) 444 Average recovery (%) 99.99100.0099.99 Standard deviation 0.8270.7750.680 Correlation coefficient 0.99940.99960.9990

Table (LVII) Potentiometric selectivity coefficients (K Plot primary ion, interferent) for the three proposed electrodes. Interferent Selectivity coefficient Pyrit-TPB Pyrit- RNC  CD- RNC NaCl 6.31 x 10 -3 2.22 x 10 -3 8.67 x 10 -4 KCl 5.97 x 10 -3 2.27 x 10 -3 8.88 x 10 -4 NH 4 Cl 6.02 x 10 -3 2.84 x 10 -3 8.97 x 10 -4 CaCl 2 5.28 x 10 -3 3.36 x 10 -3 9.24 x 10 -4 MgSO 4 6.07 x 10 -3 3.25 x 10 -3 9.61 x 10 -4 glucose 4.97 x 10 -3 3.77 x 10 -3 9.11 x 10 -4 lactose 5.04 x 10 -3 3.29 x 10 -3 8.45 x 10 -4 sucrose 5.23 x 10 -3 3.20 x 10 -3 8.27 x 10 -4 Urea 4.61 x 10 -3 1.99 x 10 -3 1.00 x 10 -3 L-phenyl alanine 5.22 x 10 -3 2.06 x 10 -3 9.85 x 10 -4 Deg.product 1.10 x 10 -1 1.02 x 10 -1 9.71 x 10 -2

Table (LVIII): Determination of pyritinol dihydrochloride in laboratory prepared mixtures by the proposed method Concentration (M) Ratio* Found % of pyritinol dihydrochloride Pyritinol.2HCl Degradation product Pyrit- TPB Pyrit- RNC  CD- RNC 1 x 10 -4 1 x 10 -5 10: 1 99.12100.1498.26 1 x 10 -4 5 x 10 -5 2: 1 98.65101.22100.26 1 x 10 -4 1: 1 108.27108.00107.01 1 x 10 -4 5 x 10 -4 1: 5 139.11136.56137.81

Table (LIX): Determination of Pyritinol diydrochloride in encephabol tablets by the proposed method Reported method *  CD-RNC Pyrit-RNCPyrit-TPB Encephabol tablets claimed to contain 200 mg Batch number Found % ± S.D.** % Found ± S.D.** 99.36 ± 0.421 100.32  0.413100.02  0.866100.25  0.923 13476 98.95 ± 0.55198.60 ± 0.74199.57 ± 1.30299.41 ± 1.36113168 * Stability indicating spectrophotometric method.

Table (LX): Determination of pyritinol dihydrochloride in spiked human plasma by the proposed electrodes. Concentration(M)Pyrit-TPBPyrit-RNC  CD-RNC Recovery % ± S.D.* 3.162 x 10 -4 100.92 ± 0.671 99.74 ±0.601 100.76 ± 0.341 1 x 10 -4 101.14 ± 0.770 100.41 ± 0.826 100.11 ± 0.475 * Average of three determinations.

Table (LXI): Application of standard addition for the determination of pyritinol dihydrochloride by the proposed method Batch numb er Standard added (mg)Pyrit-TPB Pyrit- RNC  CD- RNC Pyrit.2HCl FoundRecoveryFoundRecoveryFoundRecovery 1347623.0034.5046.0022.7134.6246.1198.73100.34100.2323.1134.2547.19100.4799.28102.5822.7134.1846.3498.7399.07100.76 Mean ± S.D.* 99.76 ± 0.789 100.77 ± 1.671 99.52 ± 1.087 * Average of three determinations.

Table (LXII): Statistical comparison for the results obtained by the proposed method and the reported method for the analysis of Pyritinol dihydrochloride in pure powder form * Stability indicating spectrophotometric method. Reported method **  CD-RNC Pyrit-RNCPyrit-TPBItem 99.9299.99100.0099.99Mean 1.1720.6800.7750.827S.D. 1.3750.4620.6000.683Variance 6655n 2.976 (5.05)2.291 (6.26)2.013 (6.26)F test 0.126 (2.228)0.130 (2.262)0.111 (2.262)Student’s t test The figures in parenthesis are the corresponding tabulated values at P=0.05

Table (LXXXII): Assay parameters and method validation for pyritinol dihydrochloride Suggested eledtrodeHPLC methodDerivative ratio method Paramete r  CD-RNC Pyrit-RNCPyrit-TPBPyritinol.2HCl 1 x 10 -6 - 3.162 x 10 -4 M 3.162 x 10 -6 - 3.162 x 10 -4 M 10-8010-26Range (μg ml -1 ) 32.89131.10030.6000.02470.119Slope 317.07286.89266.840.00330.0235Intercept 99.99100.0099.9999.5999.52Mean 0.6800.7750.8270.9631.037S.D. 0.4620.6000.6830.9271.075Variance 0.6800.6000.8270.9661.04RSD% 0.99900.99960.99940.99970.9991.(r) 0.857, 0.8160.821, 0.9730.954, 0.7781.401, 1.2770.962, 0.841* RSD% a 1.176, 1.2111.312, 1.2751.286, 1.3011.334, 1.3871.218, 1.300*RSD % b * RSD%a, RSD%b the intra-day, inter-day respectively (n=5) relative standard deviation of concentrations (18 and 20 µ g/ml) for derivative ratio method, (40 and 50 µ g ml-1) for HPLC method and (10-4M and 10-5M) for ion selective electrodes method.

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