Testing Procedure HPLC method is described for the determination of cinitapride hydrogen tartrate.
A drug was subjected to all stress conditions such as preparation reduction, oxidation acidic and alkaline medium. Chromatography, Standard stock solution preparation, Standard solution preparation, Test Preparation, Preparation of Diluent, Preparation of Buffer Preparation of Mobile Phase was recorded on an column using mixture of acetonitrile and phosphate buffer.
Description:
Take about 2.0 g of the test sample in a watch glass and observe visually over a black back ground.
Requirement: It should be a yellow crystalline powder.
Solubility:
Weigh about 0.1 g of the test sample and transfer into a clean and dry stoppered test tube . Add 10 mL of water and mix the contents .
Requirement: The solution should be clear without any suspended particles.
Identification by IR:
Standard preparation:
Weigh about 300 to 400 mg of KBr, previously dried at 200°C and cooled, into a mortar and grind to a fine powder.
Add about 1 to 2 mg of working standard, mix perfectly and grind to a uniform powder . Take a small quantity of the powder and prepare a thin semi-transparent disk.
Record the IR spectrum of the disk from 4000 cm·1 to 650 cm·1 taking air as reference .
Sample preparation:
Weigh about 300 to 400 mg of KBr, previously dried at 200°C and cooled, into a mortar and grind to a fine powder.
Add about 1 to 2 mg of test sample, mix perfectly and grind to a uniform powder .
Take a small quantity of the powder and prepare a thin semi-transparent disk .
Record the IR spectrum of the disk from 4000 cm-1 to 650 cm-1 taking air as reference .
Specification: The infrared absorption spectrum of the finely ground sample in KBr dispersion compressed into a disk should exhibit maxima only at the same wave numbers as that of similar preparation of working standard.
Loss on drying:
- Weigh about I to 2 g of the test sample and transfer it to a weighing bottle of weight (W1) with a stopper previously dried at I 05°C for 30 minutes.
- Note down the weight of the weighing bottle with the sample (Wz) .
- Distribute the sample evenly in the weighing bottle by sidewise shaking to a height of 5 mm .
- Place the weighing bottle uncovered at 105°C in an oven for 3 hours .
- After 3 hours, open the oven and immediately place the stopper on the weighing bottle .
- Place the weighing bottle in a desiccator to reduce to room temperature .
- Determine the weight of weighing bottle with the contents. Note down the weight (W3) and Calculate
Loss on drying with the following formula.
Loss on drying (%w/w) = (W2-W3) x 100 / (W2-W1)
Specification: Not more than 0.50 %w/w.
Residue on ignition:
- Weigh about 1 to 2 g of test sample and transfer to a platinum or silica crucible of weight (W1), previously ignited at 600 ± so0c for 30 minutes and cooled in a desiccator.
- Note down the weight of the crucible with the sample (W2).
- Moisten the sample with 1 mL of sulphuric acid.
- Heat gently at first, at a temperature as low as practicable until the sample is thoroughly charred.
- Cool the residue and moisten the residue with I mL of sulphuric acid and heat gently until white fumes are no longer evolved.
- Ignite the crucible at 600 ± so0c in a muffle furnace until the carbon is consumed.
- Cool in a desiccator, weigh the crucible (W3) and calculate the percentage of residue.
- Continue moistening the residue with sulphuric acid, heating and igniting as before until constant weight is attained.
Calculation:
- Weight of the crucible = W 1
- Weight of the crucible + test sample = W2
- Weight of the sample (W) = W2-W1
- Weight of the crucible + residue = W3
- Weight of residue = W3-W1
Residue on ignition (%w/w) = Weight of the residue x 100/Weight of the test sample = (W3-W1)x100/W
Note: If the result obtained is not meeting the specification, continue moistening the residue with sulphuric acid, heating and ignition until consecutive weighing do not differ by more than 0.50 mg per gram of test sample taken or until the percentage of residue complies with the specification.
- Weight of the crucible+ sample after ignition = Wn
- Weight of the sample after ignition =Wn-W1
- Residue on ignition (%w/w) = (Wn -W1)X 100 / W
- Specification: Not more than 0.10 %w/w.
- Quality Control Department.
Heavy metals:
Preparation of Lead standard solution (10 ppm):
Pipette out 10 mL of the Lead nitrate stock solution (Refer stock solution STP-“SS-001”) to a 100 mL volumetric flask.
- Dilute the above solution to 100 mL with water .
- Preparation of thioacetamide glycerin base solution:
- Mix 0.6 mL of thioacetamide solution (Refer stock solution STP “SS-012”) and 3 mL of glycerin
- base solution (Refer stock solution STP “SS-013”) and heat on a boiling water bath for 20 seconds.
- Use the mixture immediately .
pH 3.5 acetate buffer: (Refer stock solution STP “SS-044”) Standard preparation:
- Pipette out 2 mL of Lead standard solution to a suitable test tube .
- Dilute with 25 mL of water .
- Adjust the pH between 3.0 and 4.0 using in acetic acid (Refer stock solution STP- “SS-002”) or 6N ammonium hydroxide (Refer stock solution STP- “SS-005”).
(Using short range pH indicator paper as external indicator.)
- Dilute with water to 40 mL.
Test preparation:
- Weigh about 1 g of the test sample and transfer to a suitable crucible .
- Add sufficient sulphuric acid to wet the test sample .
- Ignite the mixture at a low temperature until it is thoroughly charred .
- Add 2 mL of Nitric acid and 5 drops of sulphuric acid to the charred mass and heat cautiously until white fumes are no longer evolved.
- Ignite the contents in a muffle furnace at 500 to 600°C until the carbon is completely burned off.
- Cool the mass and add 4 mL of 6N hydrochloric acid (Refer stock solution STP- “SS-006”) .
- Cover and digest over a steam bath for 15 minutes .
- Uncover, and slowly evaporate on a steam bath to dryness .
- Moisten the residue with 1 drop of hydrochloric acid .
- Add 10 mL of hot water, and digest for 2 minutes .
- Add 6N ammonium hydroxide drop wise until the solution is just alkaline to litmus paper .
- Dilute with water to 25 mL.
- Adjust pH of the above contents to between 3.0 and 4.0 with lN Acetic acid using short range pH indicator paper as external indicator.
- Filter the above solution if necessary; rinse the crucible and the filter with 10 mL water .
- Combine the filtrate and rinsing in a 50 mL color-comparison tube .
- TP Dilute the contents with water to 40 mL and mix thoroughly .
Procedure:
- To each of the tubes containing the standard preparation and the test preparation, add 2 mL of pH 3.5 Acetate Buffer.
- Add 1.2 mL of thioacetamide -glycerin base TS, and dilute with water to 50 mL
- Mix and allow to stand for 2 minutes .
- Compare the two solutions by viewing downward over a white surface .
Requirement:
The colour of the solution from the test preparation should not be darker than that of the solution from the standard preparation.
Chromatographic purity by HPLC:
- Chromatographic system:
- Column : Waters X Bridge C 18, 150mm x 4.6 mm; 3.5µm or Equivalent
- Detector wavelength : 268 nm
- Flow rate : 1.0mL/min
- Injection volume : 10 µL
- Run time : 75.00 min.
- Column oven temperature : 40°c
- Diluent : Methanol and Acetonitrile in the ratio of 50:50 v/v
Preparation of Diluent:
- Mix the Methanol and Acetonitrile in the ratio of 50:50 v/v.
Preparation of Buffer:
- Take 1.0 mL of Triethylamine into a beaker containing 1000 mL of water, mix and filter though 0.45µm
Preparation of Mobile Phase-A:
- Prepare a mixture of buffer and Acetonitrile in the ratio of 80:20 v/v and mix well and degas.
Preparation of Mobile Phase-B:
- Prepare a mixture of buffer and Acetonitrile in the ratio of 20:80 v/v and mix well and degas.
Gradient Program:
| Time(minutes) | Mobile phase-A(¾v/v) | Mobile phase-B(¾v/v) |
| 0.01 | 80 | 20 |
| 5.00 | 80 | 20 |
| 10.00 | 70 | 30 |
| 35.00 | 45 | 55 |
| 55.00 | 45 | 55 |
| 60.00 | 20 | 80 |
| 65.00 | 20 | 80 |
| 70.00 | 80 | 20 |
| 75.00 | 80 | 20 |
Standard stock solution preparation:
- Weigh about 25.0 mg of Cinitapride hydrogen tartrate standard into a 100 mL volumetric flask, dissolve and dilute to the volume with diluent.
- Transfer 5 .0 mL of above solution into a 100 mL volumetric flask and dilute to the volume with diluent.
Standard solution preparation :(0.10%)
- Transfer 4.0 mL of standard stock solution into a 100 mL volumetric flask and dilute to the volume with diluent.
Test Preparation: (Prepare in duplicate)
- Weigh accurately about 25.0 mg of test sample and transfer into a 5’0 mL volumetric flask, dissolve and dilute to volume with diluent.
Procedure:
- Equilibrate the column and system at 1.0 mL/min flow rate .
- Keep the data processor in area normalization mode .
- Inject diluent as blank into the system and record the chromatogram .
- Inject Standard solution for six times into the system and record the chromatograms.
- Inject each test solution into the system and record the chromatograms.
- Inject standard solution into the system as online standard and record the chromatogram. System
Suitability criteria:
- The %RSD for peak area responses of Cinitapride from replicate injections of standard solution should not be more than 10. 0
- The cumulative %RSD for peak area responses of Cinitapride from replicate injections of
- Standard solution and online standard should not be more than 10.0
- The Tailing factor of Cinitapride peak from Standard solution should not be more than 2.0
- The Theoretical plate count for Cinitapride peak from Standard solution should not be less than 5000.
- Determine the percentage of single maximum impurity and total impurities from the test chromatograms by using the following formula.
Calculation:
Single Maximum impurity:
= At/As x Cs/Ct x P1
Where
At: Area of single maximum impurity in test solution
As: Average area of Standard solution
Cs: Concentration of Standard solution (mg/mL)
Ct: Concentration of test solution (mg/mL)
P1: Assay of Cinitapride working standard (¾w/w on as is basis)
Total impurities:
= At/As x Cs/Ct x P1
Where
At: Area of total impurities in test solution
As: Average area of Standard solution
Cs: Concentration of Standard solution (mg/mL)
Ct: Concentration of test solution (mg/mL)
P1: Assay of Cinitapride working standard (¾w/w on as is basis)
Note : Calculate the content from each test solution separately and report the average result.
Specification:
Single maximum impurity : Not more than 0.5%
Total impurities : Not more than 1.5%
Assay on dried basis by Titrimetric: (Perform in duplicate)
Reagents:
- Anhydrous acetic acid.
- 0.1M Perchloric acid solution (Refer volumetric solution STP “VS-005”)
- Procedure:
- Weigh about 0.200g (C00) of sample into 100 mL glass beaker .
- Add 10 mL of anhydrous acetic acid and 40 mL of acetic anhydride mix thoroughly .
- Titrate the contents in the beaker against 0.1M Perchloric acid solution, determine end point (EP1) by potentiometrically.
- 1 mL of 0.1 M perchloric acid is equivalent to 402.49 mg .
- Perform the blank titration as above, without test sample and determine the blank value (C01) .
- Enter the normality of the perchloric acid (CO2), factor (C03 as 55.25), 100 as (C04).
- 100-Loss on drying (COs) and weight of the sample (COo) taken into the auto titrator.
Calculate the assay on dried basis as follows:
Assay (%w/w) = (EP1- C01) X C02X C03 X C04 / C0o X C05
Note: 1) Determine the assay on dried basis as an average of two trials.
2) The difference between two trials should not be more than 1.0 %.
3) Both the trial values should be with in the specification limit.
Specification: Between 98.0%w/w and 102.0%w/w.