(Ph. Eur. 11.6 update)
Action and use
Bisphosphonate + Vitamin D3 analogue.
DEFINITION
Alendronic Acid and Colecalciferol Tablets contain Sodium Alendronate Trihydrate and Colecalciferol.
The tablets comply with the requirements stated under Tablets and with the following requirements.
Content of alendronic acid, C4H13NO7P2
92.5 to 105.0% of the stated amount.
Content of colecalciferol, C27H44O
90.0 to 105.0% of the stated amount.
IDENTIFICATION
A. Carry out the method for thin-layer chromatography, Appendix III A, using the following solutions.
(1) Mix, with the aid of ultrasound, a quantity of the powdered tablets containing the equivalent of 70 mg of alendronic acid with 50 mL of water for 30 minutes and filter.
(2) 0.2% w/v of sodium alendronate BPCRS in water.
CHROMATOGRAPHIC CONDITIONS
(a) Use as the coating cellulose.
(b) Use the mobile phase as described below.
(c) Apply 2 μL of each solution.
(d) Develop the plate to 15 cm.
(e) After removal of the plate, dry in a current of warm air, spray with ninhydrin solution, heat at 105° and examine in daylight.
MOBILE PHASE
1 volume of 13.5M ammonia, 8 volumes of 2.5% v/v trichloroacetic acid and 11 volumes of methanol.
CONFIRMATION
The principal spot in the chromatogram obtained with solution (1) is similar in size, position and colour to that in the chromatogram obtained with solution (2).
B. In the Assay for alendronic acid, the retention time of the principal peak in the chromatogram obtained with solution (1) is similar to that of the peak in the chromatogram obtained with solution (2).
C. Carry out the method for thin-layer chromatography, Appendix III A, using the following solutions. Prepare the solutions immediately before use.
Solution A A solution containing 1% w/v of squalane and 0.01% w/v of butylated hydroxytoluene in ethylene chloride.
(1) Mix, with the aid of ultrasound, a quantity of the powdered tablets containing 1 mg of Colecalciferol with 20 mL of methanol, centrifuge and filter. Evaporate the filtrate to dryness and dissolve the residue immediately in 0.4 mL of solution
(2) 0.25% w/v of colecalciferol BPCRS in solution A.
CHROMATOGRAPHIC CONDITIONS
(a) Use as the coating silica gel (Merck plates are suitable).
(b) Use the mobile phase as described below.
(c) Apply 20 μL of each solution.
(d) Develop the plate to 15 cm.
(e) After removal of the plate, dry in current of warm air, and spray with sulfuric acid.
MOBILE PHASE
0.01% w/v of butylated hydroxytoluene in a mixture of equal volumes of cyclohexane and peroxide-free ether.
CONFIRMATION
The principal spot in the chromatogram obtained with solution (1) is similar in size, position and colour to that in the chromatogram obtained with solution (2).
D. In the Assay for colecalciferol, the retention time of the principal peak in the chromatogram obtained with solution (1) is similar to that of the peak in the chromatogram obtained with solution (2).
TESTS
Dissolution
For alendronic acid
Comply with the dissolution test for tablets and capsules, Appendix XII B1.
TEST CONDITIONS
(a) Use Apparatus 2, rotating the paddle at 50 revolutions per minute.
(b) Use 900 mL of water, at a temperature of 37°, as the medium.
PROCEDURE
Carry out the method for liquid chromatography, Appendix III D, using the following solutions.
(1) After 15 minutes withdraw a sample of the medium and filter (Whatman GF/C is suitable). Use the filtered dissolution medium diluted, if necessary, to produce a solution expected to contain the equivalent of 0.0078% w/v of alendronic acid.
(2) 0.01% w/v of sodium alendronate BPCRS in water.
CHROMATOGRAPHIC CONDITIONS
(a) Use a stainless steel column (15 cm × 4.6 mm) packed with anion exchange resin (Waters IC PAK Anion HC is suitable).
(b) Use isocratic elution and the mobile phase described below.
(c) Use a flow rate of 2 mL per minute.
(d) Use a column temperature of 35°.
(e) Use a conductivity detector, maintained at 35°.
(f) Inject 100 μL of each solution.
MOBILE PHASE
0.59% w/v of succinic acid.
When the chromatograms are recorded under the prescribed conditions the retention time of the peak due to alendronic acid is about 7 minutes.
DETERMINATION OF CONTENT
Calculate the total content of alendronic acid, C4H13NO7P2 in the medium using the declared content of C4H12NNaO7P2 in sodium alendronate BPCRS and the peak heights in the chromatograms obtained. Each mg of C4H12NNaO7P2 is equivalent to 0.9189 mg of C4H12NNaO7P2
LIMITS
The amount of alendronic acid released is not less than 75% (Q) of the stated amount.
For colecalciferol
Comply with the dissolution test for tablets and capsules, Appendix XII B1.
TEST CONDITIONS
(a) Use Apparatus 2, rotating the paddle at 75 revolutions per minute.
(b) Use 500 mL of a mixture of 3% w/v of sodium lauryl sulfate and 0.9% w/v of sodium chloride, at a temperature of 37°, as the medium.
PROCEDURE
Carry out the method for liquid chromatography, Appendix III D, using the following solutions.
(1) After 15 minutes withdraw a sample of the medium and filter (Whatman GF/C is suitable). Use the filtered dissolution medium diluted with medium, if necessary, to produce a solution expected to contain 0.000014% w/v of colecalciferol.
(2) Dilute 1 volume of a solution containing 0.0014% w/v of colecalciferol BPCRS in methanol to 100 volumes with the dissolution medium.
(3) Heat an aliquot of solution (2) on a water bath at 50° for 2 hours (generation of pre-colecalciferol).
CHROMATOGRAPHIC CONDITIONS
(a) Use a stainless steel column (15 cm × 4.6 mm) packed with end-capped octadecylsilyl silica gel for chromatography
(4 μm) (Phenomenex Synergi-Hydro RP is suitable).
(b) Use isocratic elution and the mobile phase described below.
(c) Use a flow rate of 2 mL per minute.
(d) Use a column temperature of 30°.
(e) Use a detection wavelength of 269 nm.
(f) Inject 150 μL of each solution.
MOBILE PHASE
methanol (96%).
When the chromatograms are recorded under the prescribed conditions the retention times of pre-colecalciferol and colecalciferol are about 11 minutes and 12 minutes respectively.
SYSTEM SUITABILITY
The test is not valid unless, in the chromatogram obtained with solution (3), the resolution between the peaks due tocolecalciferol and pre-colecalciferol is at least 1.2.
DETERMINATION OF CONTENT
Use the sum of the peak areas of colecalciferol and pre-colecalciferol. Calculate the total content of colecalciferol, C27H44O, in the medium using the declared content of C27H44O, in colecalciferol BPCRS.
LIMITS
The amount of colecalciferol released is not less than 75% (Q) of the stated amount.
Related substances
For alendronic acid
Carry out the method for liquid chromatography, Appendix III D, using the following solutions. Derivatise each solution prior to analysis.
Solution A A solution containing 0.294% w/v of sodium citrate and 0.142% w/v of anhydrous disodium hydrogen orthophosphate, adjusted to pH 8.0 with orthophosphoric acid.
(1) Mix a quantity of the powdered tablets containing the equivalent of 30 mg of alendronic acid with a 2.94% w/v solution of sodium citrate, dilute to 50 mL with the same solvent and filter.
(2) 0.00012% w/v of 4-aminobutanoic acid in 2.94% w/v sodium citrate.
(3) Dilute 1 volume of solution (1) to 100 volumes with a 2.94% w/v solution of sodium citrate, further dilute 1 volume of this solution to 5 volumes with the same solvent.
(4) 0.06% w/v of sodium alendronate BPCRS and 0.01% w/v of 4-aminobutanoic acid(impurity A) in 2.94% w/v sodium citrate.
(5) 0.000065% w/v of sodium alendronate BPCRS in 2.94% w/v of sodium citrate.
Derivatisation procedure Transfer 5 mL of solutions (1) to (5) separately into screw-cap centrifuge tubes, add 5 mL of a 1.91% w/v solution of sodium tetrahydroborate, 10 mL of a 0.2% w/v solution of (9-fluorenyl)methyl chloroformate in acetonitrile, shake for 1 minute and allow to stand at room temperature for 30 minutes, add 20 mL of dichloromethane and shake vigorously for 1 minute; centrifuge and use the aqueous layer.
CHROMATOGRAPHIC CONDITIONS
(a) Use a stainless steel column (25 cm × 4.1 mm) packed with styrene-divinylbenzene copolymer (10 μm) (HamiltonPRP-1 is suitable).
(b) Use gradient elution and the mobile phase described below.
(c) Use a flow rate of 2 mL per minute.
(d) Use a column temperature of 45°.
(e) Use a detection wavelength of 266 nm.
(f) Inject 20 μL of each solution.
MOBILE PHASE
Mobile phase A 15 volumes of acetonitrile and 85 volumes of solution A.
Mobile phase B 30 volumes of solution A and 70 volumes of acetonitrile.
| Time (Minutes) | Mobile phase A (% v/v) | Mobile phase B (% v/v) | Comment |
| 0-15 | 100→50 | 0→50 | linear gradient |
| 15-25 | 50→0 | 50→100 | linear gradient |
| 25-27 | 0→100 | 100→0 | linear gradient |
| 27-32 | 100 | 0 | re-equilibration |
The retention time of the peaks due to alendronic acid and 4-aminobutanoic acid, as their derivatives, are about 4 minutes and about 9 minutes respectively.
SYSTEM SUITABILITY
The test is not valid unless, in the chromatogram obtained with solution (4), the resolution between the two principal peaks is at least 10.0.
LIMITS
In the chromatogram obtained with solution (1):
the area of any peak corresponding to 4-aminobutanoic acid is not greater than the area of the corresponding peak in the chromatogram obtained with solution (2) (0.2%);
the area of any other secondary peak is not greater than the area of the principal peak in the chromatogram obtained with solution (3) (0.2%);
the sum of all impurities is not more than 0.5%.
Disregard any peak with an area less than half the area of the principal peak in the chromatogram obtained with solution (3) (0.1%).
Related substances
For colecalciferol
Carry out the method for liquid chromatography, Appendix III D, using the following solutions protected from light.
(1) Mix, with the aid of ultrasound, a quantity of the powdered tablets containing 0.56 mg of Colecalciferol with 5 mL of water. Add 25 mL of ethanol, stir for 30 minutes, add sufficient methanol to produce 50 mL and centrifuge.
(2) Dilute 1 volume of solution (1) to 100 volumes with methanol.
(3) Heat 10 mL of a 0.00112% w/v solution of colecalciferol BPCRS in methanol in a capped vessel to 50° for two hours (generation of pre-colecalciferol).
(4) Dilute 1 volume of solution (2) to 10 volumes with methanol.
CHROMATOGRAPHIC CONDITIONS
(a) Use a stainless steel column (15 cm × 4.6 mm) packed with end-capped octadecylsilyl silica gel for chromatography (3 μm) (Supelco Discovery HS is suitable).
(b) Use isocratic elution and the mobile phase described below.
(c) Use a flow rate of 1 mL per minute.
(d) Use a column temperature of 35°.
(e) Use an auto-sampler temperature of 5°.
(f) Use a detection wavelength of 266 nm.
(g) Inject 20 μL of each solution.
(h) For solution (1), allow the chromatography to proceed for 2.5 times the retention time of colecalciferol
MOBILE PHASE
methanol (96%).
The retention time of the peaks due to pre-colecalciferol and colecalciferol are about 12 and 13 minutes respectively.
SYSTEM SUITABILITY
The test is not valid unless, in the chromatogram obtained with solution (3), the resolution between the two principal peaks is at least 1.0.
LIMITS
In the chromatogram obtained with solution (1):
the area of any secondary peak is not greater than the sum of the areas of the peaks due to pre-colecalciferol and colecalciferol in the chromatogram obtained with solution (2) (1%);
the sum of the areas of any secondary peaks is not greater than 2.5 times the sum of the areas of the peaks due to pre-colecalciferol and colecalciferol in the chromatogram obtained with solution (2) (2.5%).
Disregard any peak due to pre-colecalciferol and any peak with an area less than the sum of the areas of the peaks due to pre-colecalciferol and colecalciferol in the chromatogram obtained with solution (4) (0.1%).
Uniformity of content
For colecalciferol
For tablets containing less than 2 mg and/or less than 2% w/w of colecalciferol
Carry out the method for liquid chromatography, Appendix III D, using the following solutions.
(1) Disperse one whole tablet in 5 mL of water, add 25 mL of ethanol and stir for 30 minutes, add sufficient methanol to produce 0.00014% w/v of Colecalciferol and filter (Whatman GF/C is suitable).
2) 0.00014% w/v of colecalciferol BPCRS in methanol.
(3) Heat 10 mL solution of a 0.00112% w/v of colecalciferol BPCRS in methanol in a capped vessel to 50° for two hours (generation of pre-colecalciferol).
CHROMATOGRAPHIC CONDITIONS
The chromatographic conditions described under Related substances (for colecalciferol) may be used.
SYSTEM SUITABILITY
The test is not valid unless, in the chromatogram obtained with solution (3), the resolution between the two principal peaks is at least 1.0.
DETERMINATION OF CONTENT
Combine the peak areas of colecalciferol and pre-colecalciferol. Calculate the total content of colecalciferol, C27H44O, in the tablets using the declared content of C27H44O, in colecalciferol BPCRS.
ASSAY
For alendronic acid
Weigh and powder 20 tablets. Carry out the method for liquid chromatography, Appendix III D, using the following solutions.
(1) Mix a quantity of the powdered tablets containing the equivalent of 0.1 g of alendronic acid with 200 mL of water for 30 minutes with the aid of ultrasound and occasional shaking, add sufficient water to produce 250 mL and centrifuge.
(2) 0.052% w/v of sodium alendronate BPCRS in water.
(3) 0.02% w/v of sodium alendronate BPCRS and 0.01% w/v of sodium dihydrogen orthophosphate in water.
CHROMATOGRAPHIC CONDITIONS
(a) Use a stainless steel column (25 cm × 4.1 mm) packed with styrene-divinylbenzene copolymer for chromatography (10 μm) (Hamilton PRP-X100 is suitable).
(b) Use isocratic elution and the mobile phase described below.
(c) Use a flow rate of 1.6 mL per minute.
(d) Use an ambient column temperature.
(e) Use a detection wavelength of 240 nm.
(f) Inject 100 μL of each solution.
(g) The method is validated to measure the drop in absorbance due to alendronic acid and an inverted peak will be observed.
MOBILE PHASE
0.007M nitric acid.
When the chromatograms are recorded under the prescribed conditions the retention time of the inverted peak due to alendronic acid is about 3.5 minutes.
SYSTEM SUITABILITY
The test is not valid unless, in the chromatogram obtained with solution (3), the resolution between the two principal peaks is at least 2.0.
DETERMINATION OF CONTENT
Calculate the content of alendronic acid, C4H13NO7P2 in the tablets using the declared content of C4H12NNaO7P2 in sodium alendronate BPCRS and the peak depth in the chromatograms obtained. Each mg of C4H12NNaO7P2 is equivalent to 0.9189 mg of C4H13NO7P2
For colecalciferol
Use the average of the individual results obtained in the test for Uniformity of content.
LABELLING
The quantity of sodium alendronate is stated in terms of the equivalent amount of alendronic acid.
The quantity of Colecalciferol is expressed in terms of both mg and IU.
IMPURITIES
The impurities limited by the requirements of this monograph include those listed under Colecalciferol and impurities A and D listed under Sodium Alendronate Trihydrate.
