Analytical study on certain drugs which treat brain agency and deterioration.

This thesis consists of four parts:- PartI:Stability indicating methods for the determination of Pyritinol dihydrochloride. Part II: Stability indicating colorimetric method for the determination of piracetam. Part III : Stability indicating colorimetric method for the determination of meclophenoxate hydrochloride. Part IV :Stability indicating methods for the determination of vincamine in presence of its degradation product.

Part I : Stability indicating methods for the determination of Pyritinol dihydrochloride.

This part includes a general introduction about the chemistry of pyritinol dihydrochloride,mode of action. A review on the reported methods used for pyritinol dihydrochloride quantitative determination.

Section(A): Determination of pyritinol dihydrochloride in presence of its precursor and its degradation product by derivative spectrophotometry.

The structure of pyritinol dihydrochloride:- How to obtain the precursor ? How to obtain the degradation product?

I.A.1First derivative spectrophotometric determination of pyritinol dihydrochloride in presence of its precursor and its degradation product.

Figure ( 1 ): Absorption spectra of Pyritinol dihydrochloride 20 µ g. ml -1 ( ——— ), precursor 14 µ g. ml -1 ( — — — — ) and degradation product 6 µ g. ml -1 ( ) Using 0.1N hydrochloric acid as a solvent

Figure ( 2 ): First – derivative absorption spectra of Pyritinol dihydrochloride 20 µ g. ml -1 ( ——— ), precursor 14 µ g. ml -1 ( — — — — ) and degradation product 6 µ g. ml -1 ( ). Using 0.1N hydrochloric acid as a solvent

Figure ( 3 ): First – derivative absorption spectra of 6-22 µ g. ml -1 Pyritinol dihydrochloride

Figure (4): Linearity of the peak amplitude of the first derivative at 284.4nm to the corresponding concentration of pyritinoldihydrochloride.

I.A.2.Derivative ratio zero crossing method for the determination of pyritinol dihydrochloride in presence of its precursor and its degradation product.

A ( pyritinol)/deg.prod.) Figure ( 5 ): Zero order of ratio spectra of 6-20 µ g. ml -1 pyritinol.2HCl / deg. Product (———) and 14 µ g. ml -1 precursor/ deg. Product ( ) using 6 µ g. ml -1 deg. product as a divisor.

dA(pyritinol.2HCl/ deg. prod.)/dλ Figure ( 5 ): First order of ratio spectra of 6-20 µ g. ml -1 pyritinol.2HCl / deg. Product (———) and 14 µ g. ml -1 precursor/ deg. Product ( ) using 6 µ g. ml -1 deg. product as a divisor.

Figure (7): Linearity of the peak amplitude of the first derivative of the ratio spectra at 316 nm to the corresponding concentration pyritinol dihydrochloride.

Table (I): determination of pyritinol dihydrochloride in laboratory prepared mixtures by the proposed procedures. Concentration ( µ g/ml)Percentage %First derivative spectrophotometry Derivative ratio- zero crossing Pyritinol.2HCl Precur sor Deg.produttPyritiol.2HCl PrecursorDeg.prod uct Recovery % Pyritinol.2HClPyritinol. 2HCl %5%10%98.23%98.56% %10% %100.23% %10%15%99.91%99.86% %10%20%99.05%99.98% %5%25%100.91%100.97% %10%30%102.07%101.95% %10%35%99.83%99.97% %10%40%100.39%100.71% %10%45%100.34%100.94% %10%50%100.12%99.98% Mean S.D

Table (II): Determination of pyritinol dihydrochloride in encephabol tablets by the proposed procedures. * Average of six determinations. **Spectrophotometric method Encephabol tablets claimed to contain 200 mg Batch number First derivative spectro photometry Derivative ratio-zero crossing Compendial method** % Found % Found Recovery % ± S.D. * ± ± ± 0.739

Table (III): Statistical comparison for the results obtained by the proposed method and the compendial method for the analysis of pyritinol dihydrochloride in pure powder form. The figures in parenthesis are the corresponding tabulated values at P=0.05. *Spectrophotometric method First derivative spectrophotometry Derivative ratio- zero crossing method. Compendial method* Pyritinol dihydrochloride Pyritinol dihydrochloride Mean S.D Variance N986 F test2.021 (4.82)1.24 (4.88) Student ’ s t test1.84 (2.160)1.42 (2.179)

Table (IV): Results of application of standard addition to the determination of pyritinol dihydrochloride by the proposed method. Batch number Standard added (mg.ml -1 ) First derivative spectro photometry Derivative ratio- zero crossing Pyritinol dihydrochloride Recovery % of added Recovery % of added Mean ± S.D ± ± Mean ± S.D ± ± Mean ± S.D ± ± 0.725

Section (B): Determination of pyritinol dihydrochloride in presence of its precursor and its degradation product by densitometric method.

Figure (12): TLC chromatogram of pyritinol dihydrochloride, its degradation product and its precursor. A= Pyritinol dihydrochloride. R f = 0.4 B= Degradation product. R f = 0.5 C= Precursor. R f = 0.66 Developing system: n-butanol : acetic acid : water (4:1:1 v/v/v).

Figure (14): Linearity of the area under the peak to the corresponding concentration of pyritinol dihydrochloride.

Table (V): Determination of pyritinol dihydrochloride in laboratory prepared mixtures by the proposed procedures. Concentration ( µ g/ml)Percentage %Densitometric method Pyritinol.2HClPrecursorDeg.productPyritinol.2HClPrecursorDeg.product Recovery % Pyritinol 2.HCl % % % % % % % Mean S.D. 0.54

Table (VI): Determination of pyritinol dihydrochloride in encephabol tablets by the proposed procedures. * Average of four determinations. **Spectrophotometric method. Encephabol tablets claimed to contain 200 mg Batch number Densitometric methodCompendial method** % Found Recovery % ± S.D. * ± ± ± 0.739

Table (VII): Statistical comparison for the results obtained by the proposed method and the compendial method for the analysis of pyritinol dihydrochloride in pure powder form. The figures in parenthesis are the corresponding tabulated values at P=0.05 (36). *Spectrophotometric method Densitometric method.Compendial method* Pyritinol dihydrochloride Mean S.D Variance N86 F test1.542(4.362) Student ’ s t test1.67(2.179)

Table (VIII): Results of application of standard addition to the determination of pyritinol dihydrochloride by the proposed method. Batch number Standard added (mg.ml -1 ) Densitometric method Pyritinol dihydrochloride Recovery % of added Mean ± S.D ± Mean ± S.D ± Mean ± S.D ± 0.886

Table (IX) : Assay parameters and method validation * RSD% a, RSD% b the intraday, interday respectively (n=5) relative standard deviation of concentrations ( µ g/ml) for first derivative and derivative methods, and concentrations ( 7-9 µ g/spot) for densitometric method. Parameter First Derivative spectrophotometery Derivative ratio – zero crossing Densitometric method Pyritinol dihydrochloride Range (µg.ml -1 ) (µg.spot -1 ) Slope Intercept Mean S.D Variance Coff. of variation Correl. Coef.(r) * RSD% a – – *RSD % b – – – 0.673

Section (C): Oxidation – reduction titrimetric method for the determination of pyritinol dihydrochloride in presence of its degradation product.

The proposed reaction mechanism : 3Br es 6 Br - (x10) R-S-S-R + 6H 2 O 2 RSO 3 H + 10 H es(x6) Add the the two equations 60 Br - 30 Br R-S-S-R + 36 H 2 O 12 RSO 3 H + 60 H + + Therfore 30Br 2 = 6 R-S-S-R SinceBrO Br - +6 H + 3 Br H 2 O Therfore 10 K BrO 3 / K Br = 30 Br 2 = 6 R-S-S-R 1M K BrO 3 / K Br = 6 R-S-S-R / 10 1 ml N/40 K BrO 3 / K Br = 6 R-S-S-R / (6x10x40x1000) = 441/(400x1000) = mg Where R-S-S-R =

Table (XI) : Determination of pyritinol dihydrochloride in laboratory prepared mixtures by the proposed method. Weight (mg)PercentageTitrimetric method Pyritinol dihydrochlorid e Degradation product Pyritinol dihydrochlorid e Degradation product Recovery % Pyritinol dihydrochloride 15575%25% % %75% Mean S.D

Table (XII): Determination of pyritinol dihydrochloride in encephabol tablets by the proposed procedures. * Average of six determinations. **Spectrophotometric method Encephabol tablets claimed to contain 200 mg Batch number Titrimetric methodCompendial method** % Found Recovery % ± S.D. * ± ± ± 0.739

Table (XIII): Statistical comparison for the results obtained by the proposed method and the compendial method for the analysis of pyritinol dihydrochloride in pure powder form. The figures in parenthesis are the corresponding tabulated values at P=0.05. *Spectrophotometric method Titrimetric methodCompendial method* Pyritinol dihydrochloride Mean S.D Variance N66 F test2.211 (5.05) Student ’ s t test1.115 (2.228)

Table (XIV): Results of application of standard addition to the determination of pyritinol dihydrochloride by the proposed method. Batch number Standard added (mg.ml -1 ) Titrimetric method Pyritinol dihydrochloride Recovery % of added Mean ± S.D ± Mean ± S.D ± Mean ± S.D ± 1.388

Table (XV) : Assay parameters and method validation * RSD% a, RSD% b the intraday, interday respectively (n=3) relative standard deviation of concentrations ( 5 – 10 mg). Parameter Titrimetric method Pyritinol dihydrochloride Range (mg)1 – 20 Mean S.D Variance2.452 Coff. of variation1.567 * RSD% a – *RSD % b – 0.562

Part II Stability indicating colorimetric method for the determination of piracetam.

- This part includes a general introduction about the chemistry and mode of action of piracetam. - A review on the reported methods for its quantitative determination. -Stability indicating colorimetric method for the determination of piracetam using ninhydrin reagent.

-Structure of piracetam. -The proposed reaction mechanism for preparing the degradation product

-The proposed reaction mechanism of piracetam with ninhydrin:

Figure (15): Absorption spectra of Piracetam in water 100 µ g. ml -1 ( …….) Ninhydrin 4 % ( _ _ _ _ _ ) Colored product 30 µ g. ml -1 ( _______ ).

Figure (20): Effect of volume (ml)of ninhydrin solution on the absorbance of the colored product.

Figure (21): Effect of pH on the absorbance of the colored product.

Figure (22): Effect of heating time on the absorbance of the colored product.

Figure (23): Effect of volume (ml) of pyridine on the absorbance of the colored product.

Figure (18): Absorption spectra of colored product ( piracetam with ninhydrin) μg. ml -1

Figure (19): Linearity of the absorbance of the colored product of piracetam with ninhydrin to the corresponding concentration of piracetam.

Concentration ( µ g/ml)Percentage %Ninhydrin Method Piracetam Degradatio n product Piracetam Degradatio n product Recovery % Piracetam 45590%10% %20% %30% %40% % %60% %70% %80% Mean S.D Table (XVI): Determination of piracetam in laboratory prepared mixtures by the proposed procedures.

Table (XVII): Determination of piracetam in nootropil and stimulan by the proposed procedures. * Average of six determinations. **Titrimetric method Commercial NameDosage formBatch number Ninhydrin methodCompendial method** % Found Recovery % ± S.D.* Nootropil Tablet Capsule Ampoule Syrup ± ± ± ± 1.54 Stimulan Tablet Capsule Ampoule Syrup ± ± ± ± 1.47

Table (XVIII): Statistical comparison for the results obtained by the proposed method and the compendial method for the analysis of piracetam in pure powder form. The figures in parenthesis are the corresponding tabulated values at P=0.05. *Titrimetric method Ninhydrin methodCompendial method* Piracetam Mean S.D Variance N96 F test1.309 (4.82) Student ’ s t test1.451 (2.160)

Table (XIX): Results of application of the standard addition to the determination of piracetam by the proposed method. Commercia l Name Batch numbe r Standard added mg.ml -1 Ninhydrin method Batch number Standard added mg.ml -1 Ninhydrin Method Piraceta m Recovery% of added Piraceta m Recovery % of added Nootropil tablet mean± S.D ± ampoule mean± S.D ± capusl e mean ± S.D ± syrup mean± S.D ± Stimulan1537 tablet mean ± S.D ± ampoule mean± S.D ± capsul e mean ± S.D ± syrup mean± S.D ± 0.530

Table (XX) : Assay parameters and method validation * RSD% a, RSD% b the intraday, interday respectively (n=5) relative standard deviation of concentrations (20-30 µ g/ml) for piracetam Parameter Ninhydrin method Piracetam Range ( µ g.ml -1 ) Slope Intercept Mean S.D Variance1.37 Coff. of variation1.173 Correl. Coef.(r) * RSD% a *RSD % b