It’s important to follow the official pharmacopeias such as United States Pharmacopeia (USP), European Pharmacopoeia (EP), and Japanese Pharmacopoeia (JP) for testing methods and acceptance criteria of How to Test Ivabradine HCl Raw Material Ivabradine HCl raw material.
Description:
Take about 1 g sample on butter paper and examine for color and appearance. The sample should be a white to off-white crystalline powder.
Solubility:
Freely soluble in Methanol, Chloroform, and Dimethyl Formamide:
Weigh accurately about 1.0 g of the test sample into a cleaned and dry test tube. Add 10 mL of Methanol and mix the content. The solution should be clear.
sample into a cleaned and dry test tube. Add 10 Weigh accurately about 1.0 g of the test mL of Chloroform and mix the content. The solution should be clear.
Weigh accurately about 1.0 g of the test sample into a cleaned and dry test tube. Add 10 mL of DMF and mix the content. The solution should be clear.
Slightly soluble in water:
Weigh accurately about 10 mg of the test sample into a cleaned and dry test tube. Add 10 mL of water and mix the content. The solution should be clear.
Identification:
A) By FTIR:
Weight accurately about 300mg of KBr, into a mortar and grind to fine powder. Add about 1 mg of test sample, mix perfectly and grind to a uniform powder. Fill the sample cups with powder and similarly do as Ivabradine HO Standard. Record the IR spectrum of the powder from 4000 cm-1 to 625 cm-1. Overlay IR spectrum of sample and standard How to Test Ivabradine HCl Raw Material.
B) By HPLC:
The retention time of the major peak in the chromatogram of the assay preparation corresponds to that of the standard preparation obtained as directed in the assay.
C) Test For Chloride:
Preparation of 0.1 N Silver Nitrate solution:
Weigh about 0.175 g of Silver nitrate and dissolve in 10 mL of water.
Procedure:
Weigh about 0.2 gm of sample into a 25 mL volumetric flask, containing 10 mL of Methanol. Dissolve and dilute to the volume with Methanol and mix. Take 4.0 mL of the above solution into a 10 mL test tube and add 2 drops of 0.1 N Silver nitrate solution. A white curdy precipitate appears. This precipitate is insoluble in Nitric acid but soluble in a slightly excess addition of 6 N Ammonium hydroxide solution.
Water Content (By KF):
Transfer 35 to 40 mL of methanol to the titration vessel, and titrate with K.F. reagent, standardized earlier, to the electrometric end point to consume any moisture that may be present. Quickly add about 300 mg of sample, mix and titrate with the reagent to the electrometric end point
Calculate water content as follows:
Calculations: Where,
Water Content = A X F X 100
W
Where,
A = mL of Karl Fischer reagent required to titrate the sample. F = Factor of KF reagent
W• Weight of sample taken in mg
The water content should be not more than 4.0%.
Specific Optical Rotation: (On anhydrous basis)
Weigh accurately about 0.250 g of the test sample and transfer into a 25 mL volumetric flask. Dissolve and dilute up to mark with dimethyl sulphoxide.
Procedure:
Fill with dimethyl sulphoxide and set in the sample compartment. Observe the reading. If any correction required, set zero. Empty the specific rotation tube, rinse with sample solution. Take sample solution in to the tube and set in the sample compartment. Give the sample name and concentration. Observe the reading.
Calculates Specific optical rotation on anhydrous basis by the following formula.
Observed rotation
SOR (on anhydrous basis)=———-——————–x 100
(100-Water)
pH:
Weigh accurately about 1.0 g of test sample into 20 mL volume of water in a beaker and mix. Determine the pH of the solution with suitable pH meter.
Residue on Ignition:
Determine on 1.0 g of sample.
Ignite a silica crucible at 600 ± 50°C for 30 min. allow to cool in a desiccators over silica gel and weigh (Wl).Transfer accurately about 1.0 g of sample in the crucible and weight (W2). Moisten the sample with about 1.0 ml of sulphuric acid and heat gently at a low temperature until the sample is thoroughly charred.
After cooling, moisten the residue again with about 1.0 ml of sulphuric acid, heat gently until white fumes are no longer evolved and ignite at 600 ± 50°C until the residue is completely incinerated. Allow the crucible to cool in desiccators over silica gel, weigh it again and calculate the weight of the residue (W3).
Note: If the residue on ignition so obtained exceeds the prescribed limit, continue the ignition, as previously, to constant weigh. Weighing of the residue do not differ by more than 0.5 mg or until the percentage of residue complies with the limit in the individual monograph.
W3-W1
Sulphated ash% = ——– x 100
W2-Wl
Where,
Wl: Weight of the crucible.
W2: Weight of the crucible with sample. W3: Weight of the crucible with ash
Limit: Not more than 0.10 %
Chromatographic Purity (By HPLC)
Reagents:
- Tetra butyl ammonium hydrogen sulphide
- Methanol (HPLC/ AR grade)
- Water (HPLC/Equivalent)
Chromatographic conditions:
Equipment: HPLC
Column : Zorbax SB CS, 2.50×4.6mm, 5 µm or equivalent
Flow rate : 1.0 ml/ min
Injection Volume : 20µl
Column Temperature: 40 °C
Wavelength : 285nm
Runtime : 40min
| Time (Minutes) | %Solvent-A | %Solvent-B |
| 0.01 | 90 | 10 |
| 10.00 | 75 | 25 |
| 15.00 | 60 | 40 |
| 30.00 | 50 | 50 |
| 35.00 | 90 | 10 |
| 40.00 | 90 | 10 |
Preparation of buffer solution:
1.0 g of Tetrabutylammonium hydrogen sulphide dissolve in 1000 mL of water. Filter and degas through 0.45µ filter.
Preparation of mobile phase:
Solvent-A
Use buffer solution as solvent – A
Solvent-B
Use Methanol as solvent – B
Preparation of diluent:
Mixture of buffer and methanol in the ratio of 50:50.
Preparation of standard solution:
Weigh accurately about 50.0 mg of Ivabradine HCl working standard in50 mL of volumetric flask dissolve and dilute to the mark with diluent. Take 0.3 mL of above solution into 100 mL volumetric flask dissolve and dilute to the mark with diluent.
Preparation of Test solution:
Weigh accurately about 50.0 mg of Ivabradine HCl in50 mL of volumetric flask dissolve and dilute to the mark with diluent.
Sequence of injections:
1. Blank (Diluent): 3 injection
2. Standard Solution: 1 injection
3. Sample solution: 1 injection
Procedure:
Examine the blank chromatogram for any peak due to diligent and disregard corresponding peaks observed in the chromatogram of sample solution. Report the percentage Maximum single impurity and total impurities of the test solution by area normalization method.
System suitability requirements:
1.The tailing factor of the principle peak is not more than 2.0 from standard solution. Sample information:
The retention time of lvabradine HCl is about 18.4 minutes.
Maximum single impurity: NMT 0.30%
Total Impurities: NMT1.0%
Assay (By HPLC):
Reagents:
- Tetra butyl ammonium hydrogen sulphide
- Methanol (HPLC/ AR grade)
- Water (HPLC/Equivalent)
Chromatographic conditions:
Equipment: HPLC
Column : Zorbax SB C8, 250×4.6nun, 5 µm or equivalent
Flow rate : 1.0 ml/ min
Injection Volume : 20µl
Column Temperature: 40 °C
Wavelength : 285nm
Runtime : 25min
Gradient Programe:
| Time (Minutes) | %Solvent-A | %Solvent-B |
| 0.01 | 90 | 10 |
| 10.00 | 75 | 25 |
| 15.00 | 60 | 40 |
| 20.00 | 50 | 50 |
| 25.00 | 90 | 10 |
Preparation of buffer solution:
1.0 g of Tetrabutylammonium hydrogen sulphide dissolve in 1000 mL of water. Filter and degas through 0.45µ filter.
Preparation of mobile phase:
Solvent-A
Use buffer solution as solvent -A
Solvent-B
Use Methanol as solvent -B
Preparation of diluent:
Mixture of buffer and methanol in the ratio of 50:50.
Preparation of standard solution:
Weigh accurately about 50.0 mg of Ivabradine HCI working standard in50 mL of volumetric flask, dissolve and dilute to the mark with diluent and mix. Dilute 2.0 mL of this solution to 10 mL with diluent.
Preparation of Test solution:
Weigh accurately about 50.0 mg of l’est sample into a 50 mL of volumetric flask, dissolve and dilute to the mark with diluent and mix. Dilute 2.0 mL of this solution to 10 mL with diluent.
Procedure:
Equilibrate the column for at list 30 minutes. Inject diluent as blank, standard solution (Five injections), test solution (duplicate) record the chromatogram.
Sequence of injections:
Blank (Diluent): 2 injections
Standard Solution: 5 injections
Sample solution: 2 injections
Standard Solution _BKT: 1 injections
System suitability requirements:
1. % RSD for area of principal peak from five replicate injections of standard solution should not be more than 2.0.
2. The tailing factor of Ivabradine HO peak from standard solution should not be more than2.0.
3. Cumulative % RSD for area of principal peak from initial five replicate injections of standard solution and online standard solution should not be more than 2.0.
Calculation:
At WS 2 50 10 P
%Assay=——x—–x—–x—–x—–x—– x 100
As 50 10 Wt 2 10
Where,
At = Area of sample peak in test solution.
As= Average area of sample peak in standard solution
Ws = Weight of working standard taken in standard solution Wt= Weight of the test sample taken in test solution
P = Potency of reference standard (% w /w, on as is basis)
% Assay
Calculate the Assay (on anhydrous basis) = ——————————x100
(100 – Water content)
Residual Solvent By GC:
Chromatographic Conditions:
Column Information:
Columns DB-624, 30 mt x 0.32 mm i.d., 1.8 µm film thickness capillary column
Injector temperature 180°C
Carrier gas Nitrogen
Column Flow 0.8 ml/min (Constant flow)
Split ratio 5:1
Parameters
Temperature Program 40°C (10 min. Hold) & rise to 150°C at a rate of 10°C/min. and then rise to 230°C at a rate of 40°C/ min and hold for 7.0 minutes.
Head Space Conditions:
Run time: 30 minutes
Detector temperature (FID): 260°C
Hydrogen: 40ml/min
Air: 400ml/min
Make up gas (Nitrogen): 30ml/min
| Parameters | Standard |
| Vial temperature | 80°C |
| Loop temperature | 90°c |
| Transfer line temperature | 100°c |
| Vial equilibration time | 30minutes |
| GC cycle time | 40minutes |
| Pressurize time | 0.2minutes |
| Loop equilibration time | 0.05 minutes |
| Loop fill time | 0.2minutes |
| Injection time | 1 minutes |
Diluent: 1-Methyl 2-Pyrolidinone (NMP)
Preparation of Blank solution:
Take 1.0 ml of diluent into a 20 mL of headspace sample vial immediately place septum and crimp the vial.
Preparation of standard solution:
Weight accurately about 500 mg of Ethanol and 500 mg of Isopropyl alcohol, 41 mg of Acetonitrile, 500 mg of Ethyl acetate, 88 mg of Dimethyl Formamide into a 100 mL volumetric flask containing diluent and dilute to the volume with diluent and mix.
Dilute 1.0 ml of this solution to 100 ml with diluent and mix.
Take 7 headspace vials, add 1 mL of standard solution into seven headspace vials and immediately place septum and crimp the vials.
Preparation of Test solution:
Weigh accurately about 100 mg of the test sample into a 20 mL of headspace sample vial containing about 1.0 mL of diluent and immediately septum and crimp the vial.
Procedure:
Equilibrate the column at 230°C for 30 minutes then cool to initial temperature. Program the headspace sample vial to maintain the vial at 80°C for 30 minutes. Inject blank solution into the system separately for two times and record the chromatograms.
Program the data processor to inhibit the peaks due to blank. Inject standard solution into the system separately for six times and record the chromatograms. Inject blank solution into the system and record the chromatogram, Inject test solution separately into the system and record the chromatograms. Take the average area of each solvent from
replicate standard injections. Consider blank corrected peak areas if any, in calculation. Calculate solvent content in ppm by the following formula.
System Suitability Acceptance Criteria:
1. % RSD calculated for the six injections of standard solution should not be more than 15.0.
2. Cumulative % RSD of areas of initial six standard injections and online standard injection should not be more than 15.0.
3. Resolution between the solvents in the standard solution should not be less than 1.0
Calculations:
Calculate the concentration of Methanol and Toluene in ppm using the following formula:
Sample area x Std. wt. of solvent x 1 xl
———————————————— X 106
Std. area x 100 x 100 x Sample. wt.
Limit:
i) Dimethyl Formamide: Not more than 880 ppm
ii) Ethyl Acetate : Not more than 5000 ppm
iii) Isopropyl Alcohol : Not more than 5000 ppm
iv) Acetonitrile : Not more than 410 ppm
v) Ethanol : Not more than 5000 ppm
Particle Size:
Equipment: Sympatec particle size analyser equipped with vacuum unit and air compressor. Mode: Dry Mode
Dry powder feeder : 4.0 mm Injector and vibry
Lens: R3
Slit height: 1.0 nm
Instrument parameters:
Pressure: 1.3 bar
Feeding rate: 50.0 %
Stop time : 10 seconds
Reference measurement: 6 second
Copp: 1.0 to 5.0 %
Time base : 1000ms
Type: OASISDRY
Procedure:
Take about 1.0 to 2.0 g of the test sample in to dry powder feeder. Measure the particle size of the test sample.