INTRODUCTION:
Cinitapride1,2 (CNP) is a substituted benzamide gastroenteric prokinetic agent acting via complex, but synergistic effects on seratonergic 5-HT2 (inhibition) and 5-HT4 (stimulation) receptor and dopaminergic D2 (inhibition) receptors in the neuronal synapses of the myenteric plexi; it is used as an anti-ulcerative drug. Pantoprazole3is a gastric proton pump inhibitor. Cinitapride is chemically 4-amino –N-[1-(3-cyclohexen-1-ylmethyl)-4-piperidinyl]-2-ethoxy-5-nitrobenzamide. Pantoprazole is chemically 6-(difluoromethoxy-2-[(3,4-dimethoxy pyridine-2-yl) methyl sulfynyl] -1H-benzimidazole. Only first derivative4 and HPTLC5 method has been reported so far for the combination. The aim of this project is to develop novel, precise and accurate colorimetric methods for the estimation of CNP and PNP in combined oral dosage form, which has not been reported till date. In this study CNP has been diazotized and then complexed with a diketone and PNP has been oxidized using bromine and then indirectly determined from the amount of unreacted bromine by the well known ferric thiocyanate complex formation.
Structures:6
Cinitapride
Mol wt: 402.49
Pantoprazole sodium sesquihydrate
Mol wt: 432.4
MATERIALS AND METHODS:
Instrumentation
A Shimadzu model UV-1650 double beam UV-VIS spectrophotometer with a pair of 1cm matched quartz cells was used to measure absorbance.
Chemicals and reagents:
The capsule dosage form was obtained from the local market. All the chemicals used in the preparation of the reagents were of analytical grade. Distilled water was used for the preparation of the reagents. All the solutions were freshly prepared just before the analysis. The modes of preparation of various reagents used are given below:
For Cinitapride
Sodium nitrite solution(2%): 2gms of sodium nitrite dissolved in water and made up to volume with water.
Hydrochloric acid 0.5 mol L-1: 42.5gms of concentrated hydrochloric acid was made up to 1000 mL in water.
Acetyl acetone(AAC) (2%) solution: 2 mL of acetyl acetone was dissolved in 5mL of alcohol and made up to volume with water in a 100 mL volumetric flask.
Sodium hydroxide 1 mol L-1: 4gms of sodium hydroxide was dissolved and made up to volume (100 mL) with water.
For Pantoprazole
Potassium bromate – bromide mixture
Dissolved 100mg of potassium bromate and 1g of potassium bromide in 10 mL of water and made up to volume in a 100 ml volumetric flask. Appropriate dilution of the stock solution was made to obtain a concentration of 20µg/mL to be used in the determination.
Ferrous ammonium sulphate (400 µg/mL)
Dissolved 400mg of FAS in 50 ml of distilled water, added 1 mL of dilute sulphuric acid and made up to 100 mL in a volumetric flask. Further dilutions were made to obtain a concentration of 400µg/mL of FAS.
Ammonium thio cyanate (3 mol L-1)
Dissolved 23g of ammonium thiocyanate in water and made up to 100 mL in water.
Hydrochloric acid (5 mol L-1‑)
About 15.423mL of concentrated hydrochloric acid in water was made up to volume in 1000mL volumetric flask with distilled water.
Hydrochloric acid (1M)
About 85mL of concentrated hydrochloric acid was made up to volume in 1000 mL volumetric flask with distilled water.
Methods
Spectral characterization and linearity establishment
Cinitapride
A stock solution of CNP was prepared by dissolving accurately weighed quantity of standard CNP in distilled water and made up to volume with water to obtain a concentration of 1000 µg/ml. From the stock solution 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 ml were transferred into six 50ml volumetric flasks. The solutions were diazotized by the addition of 2mL of 2% sodium nitrite solution and 2mL of 1M hydrochloric acid at a temperature of about 0-5° C for 5 minutes. Then 2 ml of 2 % AAC was added to each reaction mixture followed by the addition of 1M sodium hydroxide, when a reddish orange coloured chromogen was formed. Finally the solutions were made up to volume with distilled water and shaken well. The absorbance of the reddish orange coloured chromogen was scanned between 350-800nm against distilled water blank. The chromogen gave maximum absorbance at 399nm.
Pantoprazole
A stock solution of PNP was prepared by dissolving accurately weighed quantity of standard PNP in 1M hydrochloric acid and made up to volume with the same hydrochloric acid to obtain a final stock concentration of 1000µg/mL. The stock solution was further diluted with distilled water to obtain a working standard solution of concentration 20 µg/mL. Transferred 0.5, 1, 1.5, 2, 2.5 and 3 mL of the working standard solution of PNP (20µg/mL) into six 25 ml volumetric flask. To each was added 2 mL of potassium bromate – bromide mixture (40µg/ml) using a burette and 2mL of hydrochloric acid (5M), stoppered immediately, shaken well and kept aside for 5 minutes. To the reaction mixtures, 2 ml of FAS was added(400 µg/ml), shaken well and again kept aside for fifteen minutes until the reaction is completed. This was followed by the addition of 1mL of 3 mol L-1 ammonium thiocyanate when a blood red coloured chromogen is obtained. Finally the solutions were made up to volume with distilled water. The absorbance of the reddish coloured chromogen was scanned between 350-800nm against reagent blank. The chromogen gave maximum absorbance at 399nm. The chromogen gave maximum absorbance at 477 nm
Analysis of the sample:
The capsule dosage form contains CNP and PNP as separate extended release and enteric coated tablets respectively. So the drugs are analysed as separate entities by the above visible spectrophotometric methods respectively.
Cinitapride
Twenty tablets of CNP from the capsules were accurately weighed and ground to fine powder. Tablet powder equivalent to 25mg of CNP was accuratey weighed and shaken well with water for 20 minutes, made up to volume with water to obtain a concentration of 250µg/mL. The solution was filtered through Whatmann filter paper No.41. First 10 ml of the filtrate was discarded, and then 6 ml of the filtrate was transferred to a 50 ml volumetric flask. The sample solution was diazotized, complexed with AAC and the absorbance was measured using the same procedure as that of standard CNP.
Pantoprazole
Twenty tablets of PNP from the capsules were accurately weighed and ground to fine powder. Tablet powder equivalent to 50mg of CNP was accuratey weighed and shaken well with 1M hydrochloric acid for 20 minutes and made up to volume with 1M hydrochloric acid to obtain a concentration of 1000µg/mL. The solution was filtered through Whatmann filter paper No.41. First 10mL of the filtrate was discarded and the filtrate was appropriately diluted to obtain a concentration of 20µg/mL with water. Transferred 1mL of the first dilution (20µg/mL) to a 25mL volumetric flask and the same procedure for standard PNP was followed. The absorbance of the resulting solutions was measured.
RESULTS AND DISCUSSION:
Cinitapride
The colorimetric method is based on the diazotization7 of the primary aromatic amino group in CNP. The diazotized CNP is then complexed with AAC in alkaline media. The diazotisation of drugs containing free amino group is a well known reaction. In this method the primary aromatic amino group in CNP is diazotized using sodium nitrite solution and hydrochloric acid. Diazonium compounds are unstable and would decompose at elevated temperature8, hence temperature was maintained below 5°C until the addition of sodium hydroxide. The amino group is converted into diazonium salt which on treatment with AAC forms a complex. The complex is formed only in an alkaline medium, provided by the addition of sodium hydroxide solution. The complex is reddish orange in colour for which the maximum absorbance was observed at 399nm.
Reaction scheme:
I. Diazotisation:
NaNO2/ HCl
1° amino group of CNP Diazonium salt of
CNP
0-5 ° C
II. Complexation:
NaOH
Diazonium salt of + Acetyl acetone
Complex of diazo
group-diketone CNP (Reddish orange chromogen)
Optimisation
Diazotization and complexation reaction condition:
The conditions for the diazotization are well established to be at
0-5°c. An increased temperature results incomplete diazotization and
decomposition of diazotized compound which could be assessed by the non linear
response of the diazotized mixture in UV spectrophotometry at 410nm.The volume
and strength of the sodium nitrite and hydrochloric acid used was optimized. It
was found that an increase or decrease from 1-2mL and 1-4% of sodium nitrite
brought about no change in response or very less absorbance. By trial and error
it was established that 2mL of 2% sodium nitrite solution, 2mL of 2% AAC were
found to give good linear relationship between absorbance and concentration.
Any change in the volume or strength of AAC showed deviation from the
linearity. Similarly the strength and volume of sodium hydroxide were also optimized.
It was observed that 1mL of 1M sodium hydroxide was optimal to ensure complete
complexation and good colour intensity. Finally the concentration of the
analyte was also studied. Lower the concentration (below 10µgm) lower was the
colour intensity as a result the absorbance was very less and did not show much
difference in the absorbance. Higher concentration (above 60µg/mL) showed
deviation form the linearity. Thus 10-60µg/mL of CNP obeyed Beer’s law9 and
was selected for the study. The correlation coefficient was found to be 0.996
as shown in fig 1.
Fig 1: Calibration chart of cinitapride.
Pantoprazole
The colorimetric estimation of PNP is based on the oxidation of PNP by the insitu liberation of bromine during the reaction between excess bromate-bromide and hydrochloric acid. The unreacted bromine left after oxidation of PNP is determined indirectly by the oxidation of FAS to ferric ammonium sulphate. This in turn is complexed with ammonium thiocyanate to form a blood red complex of ferric thio cyanate which is a very well known reaction. The complex shows λmax at 470 nm. When a known excess amount of bromate-bromide mixture is allowed to react with increasing amount of PNP, there occurs decrease in the amount of bromine for oxidation of FAS to ferric ammonium sulphate. Thus, the decrease in the concentration of the complex, which is observed from the decreasing absorbance, results in a negative slope in the calibration chart fig 2.
Fig 2: Calibration chart of pantoprazole.
Reaction scheme:
I. Oxidation
KBrO3 + 5KBr + 6HCl
6 KCl + 3Br2 + 3H2O
Br2 / HCl
Pantoprazole
Oxidised product of PNP
(O)
II. Complexation
FAS+Br2 unreacted Ferric
ammonim sulphate
Ferric ammonim sulphate
+
Ferric thio cyanate
Ammonium thio cyanate ( blood red chromogen)
The strength and volume of hydrochloric acid and bromated-bromide mixture was optimised. Any change in the volume of the reagent resulted in either insufficient liberation or large excess of bromine which did not give any good response. Thus 1ml of 5M hydrochloric acid and 2ml of bromate –bromide mixture and 2ml of 400µg/mL of FAS was selected for the completion of the redox reaction. Moreover, 1mL of 3 molL-1 ammonium thio cyanate was required for the complete complexation of oxidised ferric ammonium sulphate to ferric thio cyanate. The ferric thio cyanate complex is blood red in colour and was found to be stable for 3 hrs. It showed maximum absorbance at 470 nm. Higher concentration of the analyte, required very high concentration and large excess of the bromate-bromide mixture. The above developed method obeyed Beer’s law at the concentration of 0.4-2.4 µg/ml of PNP which showed a gradual decrease in the concentration of the bromine by the linear negative slope. The correlation coefficient was within the limit as 0.998. The optical parameters of both CNP and PNP are represented in table 1.
Accuracy and Precision (CNP&PNP)
Precision of the developed methods for the drugs have been ascertained by the reproducibility of the results, when repeated six times gave a percentage purity of 99.154 ±0.000062 for CNP and 98.834 ± 0.000137 for PNP.The drugs obeyed Beer’s law at the concentration range of 10-60 and 0.4-2.4 µg/ml for CNP & PNP respectively. The method precision was assessed from the results of six replicate analysis. The assay percentage was appreciable and shown in table 2.Accuracy of the developed method for the samples was determined by recovery studies. It was performed on spiked samples at three levels. To a known amount of the analyzed sample the standard drug was added and the total amount was analysed by the above proposed colorimetry method. The results of the recovery studies (table 3) show that there is no interference by the additives in the formulation and the proposed methods could be applied for the tablet formulation of both the drugs. All the essential parameters like assay percentage, correlation coefficient, LOD, LOQ, the results of precision and accuracy were within the limits and acceptable.
Table 1: Optical parameters of CNP and PNP by visible spectrophotometry
|
S.No |
Optical Parameter |
Cinitapride |
Pantoprazole |
|
1. |
Wavelength λmax |
399 |
477 nm |
|
2. |
Molar absorptivity |
6571.97 |
95581.60 mol L-1 |
|
3. |
Beer’s law limit µg/ml |
10 - 50 |
0.4 -2.4 |
|
4. |
Regression equation |
Y=0.015 + 0.011 |
Y=-0.12979 + 0.372 |
|
5. |
Slope |
0.015 |
-0.1297 |
|
6. |
Intercept |
0.011 |
0.372 |
|
7. |
Correlation coefficient |
0.998 |
0.998 |
|
8. |
Sandell’s sensitivity |
0.061 |
0.13 |
|
9. |
LOD |
5.18 |
18.9 |
|
10. |
LOQ |
15.68 |
57.4 |
CONCLUSION:
New visible spectrophotometric methods have been developed for the estimation of CNP and PNP. The methods were found to be simple, selective, sensitive, accurate and reproducible. Thus the above proposed methods could be applied for the quality control of CNP and PNP in pharmaceutical formulation in industry.
ACKNOWLEDGEMENT:
The authors are thankful M/S Zydus Cadila, Ankleshwar, Gujarat (India) and Knis Laboratories, Chennai, TN (India) for providing Cinitapride and pantoprazole as gift samples respectively. The authors are also thankful to the Department of Pharmaceutical Chemistry, College of Pharmacy, Madras Medical College for rendering full support to carry out the study.