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

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

2. 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.

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

4. 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.

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

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

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

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

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

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

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

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

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

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

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

16. Concentration (µg/ml) Percentage % First derivative spectrophotometry Derivative ratio- zero crossing Pyritinol .2HCl Precursor Deg.produtt Pyritiol.2HCl Precursor Deg.product Recovery % Recovery % Pyritinol.2HCl Pyritinol. 2HCl 17.00 1.00 2.00 85% 5% 10% 98.23% 98.56% 16.00 2.00 2.00 80% 10% 10% 100.77% 100.23% 15.00 2.00 3.00 75% 10% 15% 99.91% 99.86% 14.00 2.00 4.00 70% 10% 20% 99.05% 99.98% 13.00 1.00 5.00 65% 5% 25% 100.91% 100.97% 12.00 2.00 6.00 60% 10% 30% 102.07% 101.95% 11.00 2.00 7.00 55% 10% 35% 99.83% 99.97% 10.00 2.00 8.00 50% 10% 40% 100.39% 100.71% 9.00 2.00 9.00 45% 10% 45% 100.34% 100.94% 8.00 2.00 10.00 40% 10% 50% 100.12% 99.98% Mean 100.15 100.11 S.D. 0.615 0.572 Table (I): determination of pyritinol dihydrochloride in laboratory prepared mixtures by the proposed procedures.

17. Encephabol tablets claimed to contain 200 mg Batch number First derivative spectro photometry Derivative ratio-zero crossing Compendial method** % Found % Found Recovery % ± S.D.* 19578 99.27 99.43 99.36 ± 0.421 19357 99.61 99.85 99.72 ± 0.854 19722 99.93 100.41 99.91 ± 0.739 Table (II): Determination of pyritinol dihydrochloride in encephabol tablets by the proposed procedures. * Average of six determinations. **Spectrophotometric method

18. First derivative spectrophotometry Derivative ratio- zero crossing method. Compendial method* Pyritinol dihydrochloride Pyritinol dihydrochloride Pyritinol dihydrochloride Mean 100.364 99.922 99.061 S.D. 1.497 1.172 1.053 Variance 2.241 1.375 1.109 N 9 8 6 F test 2.021 (4.82) 1.24 (4.88) Student’s t test 1.84 (2.160) 1.42 (2.179) 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

19. Batch number Standard added (mg.ml-1) First derivative spectro photometry Derivative ratio-zero crossing Pyritinol dihydrochloride Recovery % of added Recovery % of added 19578 0.10 0.15 0.20 101.31 99.32 98.75 101.00 99.06 100.92 Mean ± S.D. 99.79 ± 1.344 100.33 ±1.097 19357 0.10 0.15 0.20 98.92 99.47 100.82 99.87 99.36 100.56 Mean ± S.D. 99.73 ± 0.977 99.93 ± 0.602 19722 0.10 0.15 0.20 98.75 98.75 100.82 99.38 100.26 98.82 Mean ± S.D. 99.44 ± 1.195 99.48± 0.725 Table (IV): Results of application of standard addition to the determination of pyritinol dihydrochloride by the proposed method.

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

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

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

24. Concentration (µg/ml) Percentage % Densitometric method Pyritinol.2HCl Precursor Deg.product Pyritinol.2HCl Precursor Deg.product Recovery % Pyritinol 2.HCl 900 50 100 90 5 10 99.98% 800 50 150 80 5 15 99.80% 700 100 200 70 10 20 100.06% 600 100 300 60 10 30 100.75% 500 100 400 50 10 40 98.96% 400 100 500 40 10 50 99.86% 300 100 600 30 10 60 99.51% Mean 99.84 S.D. 0.54 Table (V): Determination of pyritinol dihydrochloride in laboratory prepared mixtures by the proposed procedures.

25. Encephabol tablets claimed to contain 200 mg Batch number Densitometric method Compendial method** % Found Recovery % ± S.D.* 19578 99.63 99.36 ± 0.421 19357 99.22 99.72 ± 0.854 19722 100.15 99.91 ± 0.739 Table (VI): Determination of pyritinol dihydrochloride in encephabol tablets by the proposed procedures. * Average of four determinations. **Spectrophotometric method.

26. Densitometric method. Compendial method* Pyritinol dihydrochloride Pyritinol dihydrochloride Mean 99.908 99.061 S.D. 0.848 1.053 Variance 0.719 1.109 N 8 6 F test 1.542(4.362) Student’s t test 1.67(2.179) 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

27. Batch number Standard added (mg.ml-1) Densitometric method Pyritinol dihydrochloride Recovery % of added 19578 1.00 1.50 2.00 99.51 100.33 98.79 Mean ± S.D. 99.54± 0.770 19357 1.00 1.50 2.00 100.62 99.47 99.31 Mean ± S.D. 99.80 ± 0.714 19722 1.00 1.50 2.00 99.32 100.69 99.03 Mean ± S.D. 99.68 ± 0.886 Table (VIII): Results of application of standard addition to the determination of pyritinol dihydrochloride by the proposed method.

28. Parameter First Derivative spectrophotometery Derivative ratio – zero crossing Densitometric method Pyritinol dihydrochloride Pyritinol dihydrochloride Pyritinol dihydrochloride Range (µg.ml-1) 6-22 6-20 1-15 (µg.spot-1) Slope 0.0143 -0.4751 0.439 Intercept 0.0082 -0.071 0.0844 Mean 100.364 99.922 99.908 S.D. 1.497 1.172 0.848 Variance 2.241 1.375 0.719 Coff. of variation 1.491 1.173 0.848 Correl. Coef.(r) 0.9994 0.9995 0.9998 * RSD%a 0.128- 0.203 0.132 – 0.197 0.378 –0.521 *RSD %b 0.136 –0.184 0.141 –0.175 0.554 –0.673 Table (IX) : Assay parameters and method validation * RSD%a , RSD%b the intraday, interday respectively (n=5) relative standard deviation of concentrations ( 12- 14µg/ml) for first derivative and derivative methods, and concentrations ( 7-9µg/spot) for densitometric method.

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

30. Theproposed reaction mechanism : 3Br2 + 6 es 6 Br - (x10) R-S-S-R + 6H2O 2 RSO3H + 10 H + + 10 es(x6) Add the the two equations 30 Br2 + 6 R-S-S-R + 36 H2O 12 RSO3H + 60 H+ + 60Br- Therfore 30Br2 = 6 R-S-S-R Since BrO3- + 5Br - +6 H+ 3 Br2 + 3 H2O Therfore 10K BrO3 / K Br = 30 Br2 = 6 R-S-S-R 1M K BrO3 / K Br = 6 R-S-S-R / 10 1ml N/40 K BrO3 / K Br = 6 R-S-S-R / (6x10x40x1000) = 441/(400x1000) = 1.1025 mg Where R-S-S-R =

31. Weight (mg) Percentage Titrimetric method Pyritinol dihydrochloride Degradation product Pyritinol dihydrochloride Degradation product Recovery % Pyritinol dihydrochloride 15 5 75% 25% 101.04 10 10 50% 50% 100.12 5 15 25% 75% 101.79 Mean 101.13 S.D. 0.740 Table (XI) : Determination of pyritinol dihydrochloride in laboratory prepared mixtures by the proposed method .

32. Encephabol tablets claimed to contain 200 mg Batch number Titrimetric method Compendial method** % Found Recovery % ± S.D.* 19578 101.50 99.36 ± 0.421 19357 100.13 99.72 ± 0.854 19722 98.59 99.91 ± 0.739 Table (XII): Determination of pyritinol dihydrochloride in encephabol tablets by the proposed procedures. * Average of six determinations. **Spectrophotometric method

33. Titrimetric method Compendial method* Pyritinol dihydrochloride Pyritinol dihydrochloride Mean 99.928 99.061 S.D. 1.566 1.053 Variance 2.452 1.109 N 6 6 F test 2.211 (5.05) Student’s t test 1.115 (2.228) 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

34. Batch number Standard added (mg.ml-1) Titrimetric method Pyritinol dihydrochloride Recovery % of added 19578 1.00 1.50 2.00 99.32 100.40 98.23 Mean ± S.D. 99.31± 1.085 19357 1.00 1.50 2.00 101.56 98.79 100.61 Mean ± S.D. 100.32 ± 1.407 19722 1.00 1.50 2.00 99.13 101.50 99.06 Mean ± S.D. 99.89 ± 1.388 Table (XIV): Results of application of standard addition to the determination of pyritinol dihydrochloride by the proposed method.

35. Parameter Titrimetric method Pyritinol dihydrochloride Range (mg) 1 – 20 Mean 99.928 S.D. 1.566 Variance 2.452 Coff. of variation 1.567 * RSD%a 0.461 – 0.511 *RSD %b 0.673 – 0.562 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) .

36. Part IIStability indicating colorimetric method for the determination of piracetam.

37. - 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.

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

39. -The proposed reaction mechanism of piracetam with ninhydrin:

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

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

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

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

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

45. Figure (18): Absorption spectra of colored product ( piracetam with ninhydrin) 10-50 μg. ml-1

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

47. Concentration (µg/ml) Percentage % Ninhydrin Method Piracetam Degradation product Piracetam Degradation product Recovery % Piracetam 45 5 90% 10% 98.32 40 10 80% 20% 99.24 35 15 70% 30% 101.06 30 20 60% 40% 100.25 25 25 50% 50% 98.89 20 30 40% 60% 101.70 15 35 30% 70% 98.88 10 40 20% 80% 100.54 Mean 99.86 S.D. 1.201 Table (XVI): Determination of piracetam in laboratory prepared mixtures by the proposed procedures.

48. Commercial Name Dosage form Batch number Ninhydrin method Compendial method** % Found Recovery % ± S.D.* Nootropil Tablet Capsule Ampoule Syrup 001108 001108 501106 600114 99.61 99.93 98.95 98.45 98.63 ± 1.27 99.32 ± 0.93 98.21 ± 1.31 98.11 ± 1.54 Stimulan Tablet Capsule Ampoule Syrup 1537 1668 1594 1653 98.80 99.75 99.53 100.42 97.90 ± 0.98 98.95 ± 1.34 98.23 ± 1.27 98.62 ± 1.47 Table (XVII): Determination of piracetam in nootropil and stimulan by the proposed procedures. * Average of six determinations. **Titrimetric method

49. Ninhydrin method Compendial method* Piracetam Piracetam Mean 99.746 98.892 S.D. 1.171 1.023 Variance 1.371 1.046 N 9 6 F test 1.309 (4.82) Student’s t test 1.451 (2.160) 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

50. Commercial Name Batch number Standard added mg.ml -1 Ninhydrin method Batch number Standard added mg.ml -1 Ninhydrin Method Piracetam Recovery% of added Piracetam Recovery % of added Nootropil 001108 tablet mean± S.D. 0.50 0.75 1.00 99.32 98.23 100.40 99.31 ± 1.085 501106 ampoule mean± S.D. 0.50 0.75 1.00 100.14 100.50 100.16 100.26± 0.202 001108 capusle mean± S.D. 0.50 0.75 1.00 101.50 102.50 99.79 101.26 ±1.370 600114 syrup mean± S.D. 0.50 0.75 1.00 100.00 99.63 98.87 99.60± 0.576 Stimulan 1537 tablet mean± S.D. 0.50 0.75 1.00 99.13 99.93 98.70 98.25± 0.620 1594 ampoule mean± S.D. 0.50 0.75 1.00 99.21 99.74 101.21 100.05± 1.036 1668 capsule mean± S.D. 0.50 0.75 1.00 98.56 98.71 98.29 98.52± 0.210 1653 syrup mean± S.D. 0.50 0.75 1.00 99.68 98.91 99.93 99.50± 0.530 Table (XIX): Results of application of the standard addition to the determination of piracetam by the proposed method.