Part IIIStability indicating colorimetric method for the determination

of meclophenoxate hydrochloride.

-This part includes a general introduction about the chemistry and mode of action of meclophenoxate hydrochloride.

-A review on the reported methods for its quantitative determination.

Stability indicating colorimetric method for the determination of meclophenoxate hydrochloride using ferric hydroxamate complex formation.

-Structure of meclophenoxate 

OO

Cl

O

NCH3

CH3

 

OO

Cl

O

NCH3

CH3

OOH

Cl

O

OHN

CH3

CH3

1 N NaOH

instantaneous

+

2 N NaOH

Reflux 25 min.

OO

Cl

O

NCH3

CH3

OOH

Cl

O

OHN

CH3

CH3

1 N NaOH

instantaneous

+

-The proposed mechanism for preparing the degradation product:    

 

The proposed reaction mechanism:

O

OO

Cl

O

NCH3

CH3

R OCOR\ + : N H2OH R - C - NHOH + R/ OH

Fe (R - C - NH-O)3 + 3 H+3 R - C - NHOH + Fe+3

where

R = R\ =

 

Figure ( 26 ): Absorption spectra of Meclophenoxate.HCL 100 µg. ml-1 (---------) and colored product 300 µg. ml-1 (———).

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0 1 2 3 4

Volume (ml) of hydroxyl amine hydrochloride solution.

Ab

so

rba

nc

e

Figure (30): Effect of volume (ml) of hydroxyl amine hydrochloride

solution on the absorbance of the ferric hydroxamate

complex with meclophenoxate hydrochloride.

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0 0.5 1 1.5

Volume (ml) of ferric chloride solution.

Ab

so

rba

nc

e

Figure (31): Effect of volume (ml) of ferric chloride solution on the

absorbance of the ferric hydroxamate complex with

meclophenoxate hydrochloride.

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0 0.5 1 1.5

Volume (ml) of 1 N sodium hydroxide.

Ab

so

rba

nc

e

Figure (32): Effect of volume (ml)of 1 N sodium hydroxide on the

absorbance of the ferric hydroxamate complex with

meclophenoxate hydrochloride.

 Figure (28): Absorption spectra of ferric hydroxamate complex of

meclophenoxate 100-400 μg. ml-1

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0 100 200 300 400 500

Concentration g.ml-1

Ab

so

rba

nc

e

Figure (29): Linearity of the absorbance of ferric hydroxamate

complex of meclophenoxate hydrochloride to the

corresponding concentration of meclophenoxate

hydrochloride.

Table (XXI): Determination of meclophenoxatehydrochloride in laboratory

prepared mixtures by the proposed procedures.

Concentration (µg/ml) Percentage % Ferric hydroxamateMethod

Meclophenoxate.HCl Degradation product

Meclophenoxate.HCl

Degradation product

Recovery %

Meclophenoxate.HCl

350 50 87.5% 12.5% 99.85%

300 100 75% 25% 102.50%

250 150 62.5% 37.5% 98.82%

200 200 50% 50% 99.99%

150 250 37.5% 62.5% 98.45%

100 300 25% 75% 100.40%

Mean       100.00

S.D.       1.431

Table (XXII): Determination of meclophenoxatehydrochloride in lucidril

tablets by the proposed procedures.

* Average of six determinations. **Spectrophotometric method

Lucidril tablets claimed to contain 250 mg

Batch number

Ferric hydroxamate method Compendial method**

 % Found

 Recovery % ± S.D.*

010132 100.87 99.54 ± 0.632

010156 99.39 99.65 ± 0.951

020512 99.01 100.01 ± 0.547

Table (XXIII): Statistical comparison for the results obtained by the proposed method

and the compendial method for the analysis of meclophenoxate hydrochloride in pure

powder form.

The figures in parenthesis are the corresponding tabulated values at P=0.05.*Spectrophotometric method

  Ferric hydroxamate method Compendial method*

  Meclophenoxate. HCl Meclophenoxate. HCl

Mean 101.478 101.233

S.D. 0.755 0.547

Variance 0.570 0.299

N 7 6

F test 1.906 (4.95)  

Student’s t test 0.658 (2.201)  

Table (XXIV): Results of application of standard addition to the determination of

meclophenoxate hydrochloride by the proposed method.

Batch number

Standard added(mg.ml-1)

Ferric hydroxamate method

Meclophenoxate. HClRecovery % of added

010132 1.001.502.00

99.06100.20101.06

Mean ± S.D.   100.10 ± 1.003

010156 1.001.502.00

98.3399.0799.50

Mean ± S.D.   98.96± 0.591

020512 1.001.502.00

98.5099.7599.18

Mean ± S.D.   99.14 ± 0.625

Table (XXV) : Assay parameters and method validation

* RSD%a , RSD%b the intraday, interday respectively (n=5) relative standard deviation of concentrations (200-300µg/ml) for meclophenoxate HCl.

Parameter Ferric hydroxamate method

Meclophenoxate. HCl.

Range (μg.ml-1) 100-400

Slope 0.0022

Intercept 0.0061

Mean 101.478

S.D. 0.755

Variance 0.57

Coff. of variation 0.744

Correl. Coef.(r) 0.9996

* RSD%a 0.167-0.179

*RSD %b 0.298-0.347

Part IVDifferent stability indicating methods for the determination

of vincamine in presence of its degradation product.

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

Section [A]Determination of vincamine in presence of its acid

degradation product by the derivative ratio spectrophotometry.

NN

O

HO

H3CO

H

CH3

NN

NN

O

HO

H3CO

H

CH3

2N HCl

reflux 7hrs

O

HO

HO

H

CH3

+CH3OH

m.p.251-252

-Structure of vincamine.  

-The proposed mechanism for degradation of vincamine   

 

  vincamine 20 µg. ml-1 (———) and its degradation product 20 µg. ml-1 (---------- ) Using 0.1N hydrochloric acid as a solvent.

Figure ( 37 ): Absorption spectra of

dA/dλ

 

Figure (38): First order spectra of Vincamine 20 μg.ml-1 (______) Degradation product 20 μg.ml-1 (_ _ _ _ _ _) Using 0.1N hydrochloric acid as a solvent.

A

(vincamine/deg.prod.)

Figure (39) : Zero order of the ratio spectra of vincamine 12-48 μg.ml-1

using 20 µg.ml-1 of deg. product as a divisor.

Figure (40): First order of the ratio spectra of vincamine 12-48 μg.ml-1

using 20 µg.ml-1 of deg. product as a divisor.

dA(vincam

ine/deg.product)/dλ

-2.5

-2

-1.5

-1

-0.5

0

0 20 40 60

Concentration g.ml-1

Pe

ak

am

plit

ud

e

Figure (41): Linearity of the peak amplitude of the first derivative of the

ratio spectra at 293.2 nm to the corresponding concentration

of vincamine.

Table (XXVI) : Determination of vincamine in laboratory prepared mixtures by the

proposed procedures.

Concentration (µg/ml) Percentage % Derivative ratio method

VincamineDegradation product

VincamineDegradation product

Recovery %

Vincamine

42 6 87.5 % 12.5 % 99.96

36 12 75 % 25 % 99.19

30 18 62.5 % 37.5 % 101.40

24 24 50 % 50 % 100.75

18 30 37.5 % 62.5 % 101.30

Mean       100.52

S.D.       0.937

Table (XXVII): Determination of vincamine in oxybral capsules by the proposed

procedures.

* Average of four determinations. **Spectrophotometric method

Oxybral capsules claimed to contain 30 mgBatch number

Derivative ratio method Compendial method**

 % Found

 Recovery % ± S.D.*

012261 A 99.02 99.32 ± 0.956

011345 A 98.98 98.56 ± 0.857

021554 A 99.52 99.21 ± 0.659

Table (XXVIII): Statistical comparison for the results obtained by the proposed method and the compendial method for the analysis of vincamine in pure powder form.

The figures in parenthesis are the corresponding tabulated values at P=0.05. *Spectrophotometric method

  Derivative ratio method Compendial method*

  Vincamine Vincamine

Mean 99.90 99.58

S.D. 1.041 1.011

Variance 1.084 1.022

N 10 6

F test 1.060 (4.77)  

Student’s t test 0.601 (2.145)  

Table (XXIX): Results of application of standard addition to the determination of vincamine by the proposed method.

Batch number

Standard added(mg.ml-1)

Derivative ratio method

Vincamine

 Recovery % of added

012261 A  

0.2500.3750.500

99.73101.50102.80

Mean ± S.D.   101.34 ± 1.541

011345 A 0.2500.3750.500

99.64100.0999.19

Mean ± S.D.   99.64 ± 0.450

021554A 0.2500.3750.500

98.1598.9899.23

Mean ± S.D.   98.78 ± 0.565

Section (B)Densitometric determination of vincamine in presence of its

acid degradation product

Scanning profile of the TLC chromatogram of vincamine at 281 nm.

Figure ( 44 ):

0

1

2

3

4

5

6

0 5 10 15 20

Concentration g.spot-1

Inte

gra

ted

pe

ak

are

a

( x

10

-4)

Figure (45): Linearity of the area under the peak to the

corresponding concentration of vincamine.

Table (XXX): Determination of vincamine in laboratory prepared mixtures by the

proposed procedures.

Concentration (µg/spot) Percentage % Densitometricmethod

Vincamine Degradation product

Vincamine Degradation product

Recovery %

Vincamine

9 1 90% 10% 98.18

8 2 80% 20% 99.40

7 3 70% 30% 100.04

6 4 60% 40% 99.13

5 5 50% 50% 100.01

4 6 40% 60% 101.36

3 7 30% 70% 99.00

Mean       99.70

S.D.       1.032

Table (XXXI): Determination of vincamine in oxybral capsules by the proposed

procedures.

* Average of four determinations. **Spectrophotometric method

Oxybral capsules claimed to contain 30

mgBatch number

Densitometric method Compendial method**

 % Found

 Recovery % ± S.D.*

012261 A 99.96 99.32 ± 0.956

011345 A 100.31 98.56 ± 0.857

021554 A 99.57 99.21 ± 0.659

Table (XXXII): Statistical comparison for the results obtained by the proposed method

and the compendial method for the analysis of vincamine in pure powder form.

The figures in parenthesis are the corresponding tabulated values at P=0.05.*Spectrophotometric method

  Densitometric method Compendial method*

  Vincamine Vincamine

Mean 100.09 99.58

S.D. 0.761 1.011

Variance 0.579 1.022

N 8 6

F test 1.765 (4.362)  

Student’s t test 1.08 (2.179)  

Table (XXXIII): Results of application of standard addition to the determination of

vincamine by the proposed method.

Batch number

Standard added(µg.ml-1)

Densitometric method

Vincamine Recovery % of added

012261 A  

1.001.502.00

97.9097.4099.80

Mean ± S.D.   98.36 ± 1.266

011345 A 1.001.502.00

99.6099.1099.59

Mean ± S.D.   99.43 ± 0.285

021554A 1.001.502.00

100.09100.68100.30

Mean ± S.D.   100.35 ± 0.299

Section ( C )Colorimetric determination of vincamine by using p-

chloranilic acid reagent ( ion pair complexation ).

  

O

OH

Cl

Cl

OOH

H2A H+

H+

H+

B

+ HA-

B +

Where P-CA (H2A) =

(basic drug) (colourless)

- The reaction between vincamine and p-chloraanilic acid can be presented as follows:-

Vincamine 20 µg. ml-1 (…….) p-Chloranilic acid ( _ _ _ _ _) Colored product 200 µg. ml-1 (_______).

Figure (46): Absorption spectra of

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0 1 2 3 4 5

Volume (ml) of p- chloranilic acid solution.

Ab

so

rba

nc

e

Figure (49): Effect of volume (ml)of p- chloranilic acid solution on the absorbance of the colored product with vincamine.

 

0

0.2

0.4

0.6

0.8

1

1.2

0 2 4 6 8 10

Mole fraction of vincamine

Ab

sorb

ance

Figure (50): Determination of the stoichiometry of the reaction of

vincamine with p- chloranilic acid by the continuous variation method.

p-chloranilic acid) 75 – 250 μg. ml-1

Figure (47): Absorption spectra of colored product ( vincamine with

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

0 100 200 300

Concentration g.ml-1

Ab

so

rba

nc

e

Figure (48): Linearity of the absorbance of the colored product of

vincamine with p-chloranilic acid to the corresponding

concentration of vincamine.

Table (XXXIV): Determination of vincamine in oxybral capsule by the proposed

procedures.

* Average of six determinations. **Spectrophotometric method

Oxybral capsules claimed to contain 30 mgBatch number

Colorimetric method Compendial method**

 % Found

 Recovery % ± S.D.*

012261A 97.00 99.32 ± 0.956

021554A 98.49 98.56 ± 0.857

011345A 100.45 99.21 ± 0.659

Table (XXXV): Statistical comparison for the results obtained by the proposed

method and the compendial method for the analysis of vincamine in pure powder form.

The figures in parenthesis are the corresponding tabulated values at P=0.05.*Spectrophotometric method

  Colorimetric method. Compendial method*

  vincamine vincamine

Mean 100.13 99.58

S.D. 1.096 1.011

Variance 1.201 1.022

N 8 6

F test 1.175 (4.88)  

Student’s t test 0.959 (2.179)  

Table (XXXVI): Results of application of standard addition to the determination of vincamine by the proposed method.

Batch number

Standard added(mg.ml -1)

Colorimetric method

Vincamine

 Recovery % of added

012261A 0.500.751.00

94.6097.2097.80

Mean ± S.D.   96.53 ± 1.700

021554A 0.500.751.00

96.5195.4498.02

Mean ± S.D.   96.65 ± 1.296

011345A 0.500.751.00

99.5298.8899.25

Mean ± S.D.   99.22 ± 0.321

Section (D) High performance liquid chromatographic determination of

vincamine in presence of its acid degradation product.

Final assay conditions of Liquid chromatographic separation of vincamine and its degradation product:

Column: RP18 Mobile phase:

acetonitrile: 0.01 M ammonium carbonate (70:30 v/v).

Flow rate: 1.6 ml. min-1. Detection:U.V.at 280

nm.

Rt vincamine: 6.61 min.

Rt degradation product: 3.67 min

0

1

2

3

4

5

6

7

8

0 5 10 15 20 25

Concentration of vincamine in g.ml-1

Pea

k ar

ea x

10-3

Figure (53): Linearity of the area under the peak to the corresponding

concentration of vincamine.

Table (XXXVII): Determination of vincamine in laboratory prepared mixtures by the

proposed procedures.

Concentration (µg.ml-1) Percentage % HPLCmethod

Vincamine Degradation product

Vincamine Degradation product

Recovery %

Vincamine

18 2 90% 10% 99.13

16 4 80% 20% 98.45

14 6 70% 30% 100.89

12 8 60% 40% 98.85

10 10 50% 50% 101.33

8 12 40% 60% 100.24

6 14 30% 70% 99.76

4 16 20% 80% 98.63

2 18 10% 90% 99.75

Mean       99.67

S.D.       1.007

Table (XXXVIII): Determination of vincamine in oxybral capsules by the

proposed procedures.

* Average of four determinations. **Spectrophotometric method

Oxybral capsules claimed to contain 30 mgBatch number

HPLC method Compendial method**

 % Found

 Recovery % ± S.D.*

012261 A 98.38 99.32 ± 0.956

011345 A 99.36 98.56 ± 0.857

021554 A 100.95 99.21 ± 0.659

Table (XXXIX): Statistical comparison for the results obtained by the proposed

method and the compendial method for the analysis of vincamine in pure powder form.

The figures in parenthesis are the corresponding tabulated values at P=0.05.*Spectrophotometric method

  HPLC method Compendial method*

  Vincamine Vincamine

Mean 100.16 99.58

S.D. 1.026 1.011

Variance 1.054 1.022

N 10 6

F test 1.031 (4.77)  

Student’s t test 1.099 (2.145)  

Table (XXXX): Results of application of standard addition to the determination of

vincamine by the proposed method.

Batch number

Standard added(mg.ml-1)

HPLC method

Vincamine

 Recovery % of added

012261 A 

 

0.100.150.20

99.9098.40

101.36

Mean ± S.D.   99.89 ± 1.480

011345 A 0.100.150.20

98.54101.5699.79

Mean ± S.D.   99.96 ± 1.517

021554A 0.100.150.20

97.96102.32100.87

Mean ± S.D.   100.38± 2.220

Table (XXXXI ) : Assay parameters and method validation

* RSD%a , RSD%b the intraday, interday respectively (n=5) relative standard deviation of concentrations (20-32 µg/ml) for derivative ratio, (9-11µg/spot) for densitometric method,( 150 – 175 µg/ml) for colourimetric method and (10-14 µg/ml) for HPLC method . 

ParameterDerivative ratio

spectrophotometric methodDensitometric

methodColorimetric

methodHPLC

methodVincamine Vincamine Vincamine Vincamine

Range (µg/ml)

12-48 3-17(µg/spot) 75 –250 2-20

Slope -0.04 0.324 0.0032 0.3643

Intercept -0.021 0.0958 -0.0106 -0.2196

Mean 99.90 100.09 100.13 100.16

S.D. 1.0413 0.761 1.096 1.026

Variance 1.084 0.579 1.201 1.054

Coff. of variation

1.042 0.760 1.094 1.024

Correl. Coef.(r)

0.9992 0.9998 0.9991 0.9994

* RSD%a 0.189-0.311 0.176-0.211 0.183 – 0.233 0.254-0.378

*RSD %b 0.312-0.275 0.286-0.301 0.357 – 0.316 0.421-0.573

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