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?

N NCH3

HOS -S

CH3

OH

OH

2HCl.. H2O

HO

N N NCH3

HOS -S

CH3

OH

OHHO

CH3

HO

HO

Zn pd. /1NHCl

1hr2

SH.HCl

mol.wt 221

N N NCH3

HOS -S

CH3

.2HCl

OHHO

CH3

HO

HO

H2O2 30% v/v

leave 1hr.at room temp.

2SO3H.HCl

OH

mol.wt 269

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 µg. ml-1 ,)———(

precursor 14 µg. ml-1 (— — — — ) and degradation product 6 µg. ml-1 (--------- ) Using 0.1N hydrochloric acid as a solvent

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

 

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

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0 5 10 15 20 25

Concenteration g.ml-1

Pe

ak

am

plit

ud

e

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 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.

dA)pyritinol.2H

Cl/ 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.

-12

-10

-8

-6

-4

-2

0

0 5 10 15 20 25

Concenteration g.ml-1

Pea

k am

pli

tud

e

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 (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 (II): Determination of pyritinol dihydrochloride in encephabol tablets by the proposed procedures.

* Average of six determinations. **Spectrophotometric method

Encephabol tablets claimed to contain 200 mgBatch 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 (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 Pyritinoldihydrochloride

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 (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 19578 0.10

0.150.20

 

101.3199.3298.75

101.0099.06

100.92

Mean ± S.D.   99.79 ± 1.344 100.33 ±1.097

19357 0.100.150.20

 

98.9299.47

100.82

99.8799.36

100.56

Mean ± S.D.   99.73 ± 0.977 99.93 ± 0.602

19722 0.100.150.20

 

98.7598.75

100.82

99.38100.2698.82

Mean ± S.D.   99.44 ± 1.195 99.48± 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. 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).

Figure ) 13 (:

Scanning profile of TLCchromatogram of pyritinol

dihydrochloride at 300 nm.

0

1

2

3

4

5

6

7

8

0 5 10 15 20

Concentration g.spot-1

Inte

gra

ted

pe

ak

are

a

( x

10

-4)

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 (µg/ml) Percentage % Densitometric method

Pyritinol.2HCl Precursor Deg.product Pyritinol.2HCl Precursor Deg.productRecovery %

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 (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 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 (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 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 (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

 

19578 1.001.502.00

99.51100.3398.79

Mean ± S.D.   99.54± 0.770

19357 1.001.502.00

100.6299.4799.31

Mean ± S.D.   99.80 ± 0.714

19722 1.001.502.00

99.32100.6999.03

Mean ± S.D.   99.68 ± 0.886

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.

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

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

degradation product.

The proposed 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

60 Br -30 Br2 + 6 R-S-S-R + 36 H2O 12 RSO3H + 60 H+ +

Therfore 30Br2 = 6 R-S-S-R

Since BrO3 - + 5Br - +6 H+ 3 Br2 + 3 H2O

Therfore 10 K BrO3 / K Br = 30 Br2 = 6 R-S-S-R

1M K BrO3 / K Br = 6 R-S-S-R / 10

1 ml N/40 K BrO3 / K Br = 6 R-S-S-R / (6x10x40x1000)

= 441/(400x1000) = 1.1025 mg

Where R-S-S-R =

 

N NCH3

HOS -S

CH3

OH

OH

2HCl.. H2O

HO

Table (XI) : Determination of pyritinol dihydrochloride in laboratory prepared mixtures by the proposed method .

Weight (mg) Percentage Titrimetricmethod

Pyritinol dihydrochlorid

e

Degradation product

Pyritinol dihydrochlorid

e

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 (XII): Determination of pyritinol dihydrochloride in encephabol tablets by the proposed procedures.

* Average of six determinations. **Spectrophotometric method

Encephabol tablets claimed to contain 200 mgBatch 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 (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 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 (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

19578 1.001.502.00

99.32100.4098.23

Mean ± S.D.   99.31± 1.085

19357 1.001.502.00

101.5698.79

100.61

Mean ± S.D.   100.32 ± 1.407

19722 1.001.502.00

99.13101.5099.06

Mean ± S.D.   99.89 ± 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) .

ParameterTitrimetric 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

Part IIStability 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.   

N

O

O

NH2 

-The proposed reaction mechanism for preparing the degradation product  

N

O

O

NH21 N NaOH

reflux 1 hr

N

O

O

OH

+ NH3

 

-The proposed reaction mechanism of piracetam with ninhydrin: 

 

 N

O

O

NH2

O

OOH

OH N

O

O

N

O

O

+

+ 2 H2O

 

Figure (15): Absorption spectra of

Piracetam in water 100 µg. ml-1 ).……(

Ninhydrin 4( % _ _ _ _ _ )

Colored product 30 µg. ml-1 (_______.)

 

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0 2 4 6 8 10

Volume (ml) of ninhydrin solution

Ab

so

rba

nc

e

Figure (20): Effect of volume (ml)of ninhydrin solution on the

absorbance of the colored product.

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0 2 4 6 8

pH

Ab

so

rba

nc

e

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

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0 5 10 15 20 25 30

Heating time in min.

Ab

so

rba

nc

e

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

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0 0.5 1 1.5

Volume (ml) of pyridine

Ab

so

rba

nc

e 

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) 10-50 μg. ml-1

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0 20 40 60

Concentration g.ml-1

Ab

so

rba

nc

e

Figure (19): Linearity of the absorbance of the colored product of

piracetam with ninhydrin to the corresponding

concentration of piracetam.

Concentration (µg/ml) Percentage % NinhydrinMethod

Piracetam Degradation product

PiracetamDegradation 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.

Table (XVII): Determination of piracetam in nootropil and stimulan by the proposed procedures.

* Average of six determinations.

 **Titrimetric method

Commercial Name Dosage form Batch number

Ninhydrin method Compendial method**

 % Found

 Recovery % ± S.D.*

Nootropil

TabletCapsuleAmpoule

Syrup

001108001108501106600114

99.61 99.9398.95 98.45

98.63 ± 1.2799.32 ± 0.9398.21 ± 1.3198.11 ± 1.54

Stimulan

TabletCapsuleAmpoule

Syrup

1537166815941653

98.80 99.75 99.53

100.42

97.90 ± 0.9898.95 ± 1.3498.23 ± 1.2798.62 ± 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 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 (XIX): Results of application of the standard addition to the

determination of piracetam by the proposed method.

CommercialName

Batchnumbe

r

Standardadded

mg.ml -1

Ninhydrinmethod

Batchnumber

Standardadded

mg.ml -1

NinhydrinMethod

   Piraceta

m

Recovery%of added

 Piraceta

m

Recovery %of added

Nootropil

001108tablet

 mean± S.D.

0.500.751.00

99.3298.23100.4099.31 ± 1.085

501106ampoule

 mean±S.D.

0.500.751.00

100.14100.50100.16

100.26±0.202

001108capusl

e 

mean± S.D.

0.500.751.00

101.50102.5099.79101.26 ±1.370

600114syrup

 mean±S.D.

0.500.751.00

100.0099.6398.87

99.60±0.576

Stimulan 1537tablet

 mean± S.D.

0.500.751.00

99.1399.9398.70

98.25±0.620

1594ampoule

 mean±S.D.

0.500.751.00

99.2199.74

101.21100.05±1.036

  1668capsul

e 

mean± S.D.

0.500.751.00

98.5698.7198.29

98.52± 0.210

1653syrup

 mean±S.D.

0.500.751.00

99.6898.9199.93

99.50±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 µg/ml) for piracetam

Parameter Ninhydrin method

Piracetam

Range (µg.ml-1) 10-50

Slope 0.0185

Intercept 0.0246

Mean 99.746

S.D. 1. 1707

Variance 1.37

Coff. of variation 1.173

Correl. Coef.(r) 0.9993

* RSD%a 0.223-0.317

*RSD %b 0.421-0.584