Part III Stability 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       -The proposed mechanism for preparing the degradation product:   2 N NaOH Reflux 25 min.

The proposed reaction mechanism:  

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

Figure (30): Effect of volume (ml) of hydroxyl amine hydrochloride solution on the absorbance of the ferric hydroxamate complex with meclophenoxate hydrochloride.

Figure (31): Effect of volume (ml) of ferric chloride solution on the absorbance of the ferric hydroxamate complex with meclophenoxate hydrochloride.

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

Figure (29): Linearity of the absorbance of ferric hydroxamate complex of meclophenoxate hydrochloride to the corresponding concentration of meclophenoxate hydrochloride.

Concentration (µg/ml) Table (XXI): Determination of meclophenoxatehydrochloride in laboratory prepared mixtures by the proposed procedures. Concentration (µg/ml) Percentage % Ferric hydroxamate Method Meclophenoxate.HCl Degradation product Recovery % 350 50 87.5% 12.5% 99.85% 300 100 75% 25% 102.50% 250 150 62.5% 37.5% 98.82% 200 50% 99.99% 98.45% 100.40% Mean   100.00 S.D. 1.431

Table (XXII): Determination of meclophenoxatehydrochloride in lucidril tablets by the proposed procedures. 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 * Average of six determinations. **Spectrophotometric method

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

Ferric hydroxamate method 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. HCl Recovery % of added 010132 1.00 1.50 2.00 99.06 100.20 101.06 Mean ± S.D.   100.10 ± 1.003 010156 98.33 99.07 99.50 Mean ± S.D. 98.96± 0.591 020512 98.50 99.75 99.18 99.14 ± 0.625

Table (XXV) : Assay parameters and method validation 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 * RSD%a , RSD%b the intraday, interday respectively (n=5) relative standard deviation of concentrations (200-300µg/ml) for meclophenoxate HCl.

Part IV Different 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.

-Structure of vincamine.   -Structure of vincamine.   -The proposed mechanism for degradation of vincamine

Figure ( 37 ): Absorption spectra of   vincamine 20 µg. ml-1 (———) and its degradation product 20 µg. ml-1 (---------- ) Using 0.1N hydrochloric acid as a solvent.

dA/dλ Figure (38): First order spectra of   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.

First order of the ratio spectra of vincamine 12-48 μg.ml-1 dA(vincamine/deg.product)/dλ 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.

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 Vincamine Degradation product Recovery % 42 6 87.5 % 12.5 % 99.96 36 12 75 % 25 % 99.19 30 18 62.5 % 37.5 % 101.40 24 50 % 100.75 101.30 Mean   100.52 S.D. 0.937

Table (XXVII): Determination of vincamine in oxybral capsules by the proposed procedures. Oxybral capsules claimed to contain 30 mg Batch 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 * Average of four determinations. **Spectrophotometric method

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

Derivative ratio method 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 0.250 0.375 0.500 99.73 101.50 102.80 Mean ± S.D. 101.34 ± 1.541 011345 A 99.64 100.09 99.19 Mean ± S.D. 99.64 ± 0.450 021554A 98.15 98.98 99.23 98.78 ± 0.565

Section (B) Densitometric determination of vincamine in presence of its acid degradation product

Figure ( 44 ): Scanning profile of the TLC chromatogram of vincamine at 281 nm.

Figure (45): Linearity of the area under the peak to the corresponding concentration of vincamine.

Concentration (µg/spot) Table (XXX): Determination of vincamine in laboratory prepared mixtures by the proposed procedures. Concentration (µg/spot) Percentage % Densitometric method Vincamine Degradation product Recovery % 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 50% 100.01 101.36 99.00 Mean   99.70 S.D. 1.032  

Oxybral capsules claimed to contain 30 mg Table (XXXI): Determination of vincamine in oxybral capsules by the proposed procedures. Oxybral capsules claimed to contain 30 mg Batch 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 * Average of four determinations. **Spectrophotometric method

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

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   1.00 1.50 2.00 97.90 97.40 99.80 Mean ± S.D. 98.36 ± 1.266 011345 A 99.60 99.10 99.59 Mean ± S.D. 99.43 ± 0.285 021554A 100.09 100.68 100.30 100.35 ± 0.299

Section ( C ) Colorimetric determination of vincamine by using p-chloranilic acid reagent ( ion pair complexation ).

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

p-Chloranilic acid ( _ _ _ _ _) Figure (46): Absorption spectra of Vincamine 20 µg. ml-1 (…….) p-Chloranilic acid ( _ _ _ _ _) Colored product 200 µg. ml-1 (_______).

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

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 p-chloranilic acid) 75 – 250 μg. ml-1

Figure (48): Linearity of the absorbance of the colored product of vincamine with p-chloranilic acid to the corresponding concentration of vincamine.

Oxybral capsules claimed to contain 30 mg Table (XXXIV): Determination of vincamine in oxybral capsule by the proposed procedures. Oxybral capsules claimed to contain 30 mg Batch 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 * Average of six determinations. **Spectrophotometric method

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

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 0.50 0.75 1.00 94.60 97.20 97.80 Mean ± S.D. 96.53 ± 1.700 021554A 96.51 95.44 98.02 Mean ± S.D. 96.65 ± 1.296 011345A 99.52 98.88 99.25 99.22 ± 0.321

Section (D)  High performance liquid chromatographic determination of vincamine in presence of its acid degradation product.

Rt degradation product: 3.67 min 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

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

Concentration (µg.ml-1) Table (XXXVII): Determination of vincamine in laboratory prepared mixtures by the proposed procedures. Concentration (µg.ml-1) Percentage % HPLCmethod Vincamine Degradation product Recovery % 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 50% 101.33 100.24 99.76 98.63 99.75 Mean   99.67 S.D. 1.007

Oxybral capsules claimed to contain 30 mg Table (XXXVIII): Determination of vincamine in oxybral capsules by the proposed procedures. Oxybral capsules claimed to contain 30 mg Batch 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 * Average of four determinations. **Spectrophotometric method

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

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 0.10 0.15 0.20 99.90 98.40 101.36 Mean ± S.D. 99.89 ± 1.480 011345 A 98.54 101.56 99.79 Mean ± S.D. 99.96 ± 1.517 021554A 97.96 102.32 100.87 100.38± 2.220

Table (XXXXI ) : Assay parameters and method validation Derivative ratio spectrophotometric method Densitometric method Colorimetric method HPLC method 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 * 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 .  

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