(Ph. Eur. 11.6 update)
Action and use
Nucleoside reverse transcriptase inhibitor; antiviral (HIV).
DEFINITION
Abacavir, Zidovudine and Lamivudine Tablets contain Abacavir Sulfate, Zidovudine and Lamivudine.
The tablets comply with the requirements stated under Tablets and with the following requirements.
Content of abacavir, C14H18N6O
95.0 to 105.0% of the stated amount.
Content of zidovudine, C10H13N5O4
95.0 to 105.0% of the stated amount.
Content of lamivudine, C8H11N3O3S
95.0 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) Shake a quantity of powdered tablets containing the equivalent of 0.2 g of abacavir with 50 mL of water, filter and use the filtrate.
(2) 0.2% w/v of zidovudine BPCRS in water.
(3) 0.23% w/v of abacavir sulfate BPCRS in water.
(4) 0.1% w/v of lamivudine BPCRS in water.
CHROMATOGRAPHIC CONDITIONS
(a) Use precoated silica gel F254 plates (Merck silica gel 60 F254 plates are suitable).
(b) Use the mobile phase described below.
(c) Apply 10 μL of each solution.
(d) Develop the plate to 12 cm.
(e) After removal of the plate, dry it in air and immediately examine under ultraviolet light (254 nm).
MOBILE PHASE
3 volumes of glacial acetic acid, 10 volumes of methanol and 90 volumes of dichloromethane.
SYSTEM SUITABILITY
The test is not valid unless the chromatogram obtained with solution (1) shows three clearly separated spots.
CONFIRMATION
The chromatogram obtained with solution (1) shows three spots corresponding in position, colour and size to the spots in the chromatograms obtained with solutions (2), (3) and (4).
B. In the Assay, the chromatogram obtained with solution (1) shows principal peaks with the same retention time as the principal peaks due to abacavir, zidovudine and lamivudine in the chromatograms obtained with solutions (2), (3) and (4) respectively.
TESTS
Dissolution
Comply with the dissolution test for tablets and capsules, Appendix XII B1.
TEST CONDITIONS
(a) Use Apparatus 2 and rotate the paddle at 75 revolutions per minute.
(b) Use 900 mL of 0.1M hydrochloric acid, 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 45 minutes withdraw a 10 mL sample of the medium and filter.
(2) Dissolve suitable quantities of abacavir sulfate BPCRS, zidovudine BPCRS and lamivudine BPCRS in solvent A, described under Related substances, to produce the same concentrations as that expected for solution (1).
(3) 0.075% w/v of zidovudine and lamivudine impurity standard BPCRS and 0.025% w/v of abacavir sulfate BPCRS in solvent A.
CHROMATOGRAPHIC CONDITIONS
The chromatographic conditions described under Related substances may be used.
DETERMINATION OF CONTENT
Calculate the total content of abacavir, C14H18N6O, zidovudine, C10H13N5O4, and lamivudine, C8H11N3O3S, in the medium using the declared content of C14H18N6O abacavir sulfate BPCRS, the declared content of C10H13N5O4 in zidovudine BPCRS and the declared content of C8H11N3O3S in lamivudine BPCRS.
LIMITS
The amounts of abacavir, zidovudine and lamivudine released are not less than 75% (Q) of the stated amounts.
Related substances
Carry out the method for liquid chromatography, Appendix III D, using the following solutions in solvent A.
SOLVENT A
Dissolve 1.9 g of ammonium acetate in 900 mL of water, adjust the pH to 3.9 with glacial acetic acid and dilute to 1000 mL.
(1) Shake a quantity of the powdered tablets containing the equivalent of 0.1g of abacavir in 60 mL with the aid of ultrasound for 30 minutes, dilute to 100 mL and filter.
(2) Dilute 1 volume of solution (1) to 100 volumes.
(3) Dilute 1 volume of solution (2) to 10 volumes.
(4) 0.002% w/v of thymine.
(5) 0.075% w/v of zidovudine and lamivudine impurity standard BPCRS and 0.025% w/v of abacavir sulfate BPCRS in solvent A.
CHROMATOGRAPHIC CONDITIONS
(a) Use a stainless steel column (25 cm × 4.6 mm) packed with octadecylsilyl silica gel for chromatography (5 μm) (YMC ODS-A is suitable).
(b) Use gradient elution and the mobile phase described below.
(c) Use a flow rate of 1 mL per minute.
(d) Use a column temperature of 30°.
(e) Use a detection wavelength of 270 nm.
(f) Inject 10 μL of each solution.
MOBILE PHASE
Mobile phase A 0.025M ammonium acetate, the pH adjusted to 3.9 with glacial acetic acid.
Mobile phase B methanol.
Mobile phase C acetonitrile.
| Time
(Minutes) |
Mobile phase A
(% v/v) |
Mobile phase B
(% v/v) |
Mobile phase C
(% v/v) |
Comment |
| 0-15 | 95 | 5 | 0 | isocratic |
| 15-30 | 95→70 | 5→30 | 0 | linear gradient |
| 30-38 | 70 | 30 | 0 | isocratic |
| 38-60 | 70→0 | 30→0 | 0→100 | change of solvent |
| 60-65 | 0 | 0 | 100 | washing column |
| 65-66 | 0→95 | 0→5 | 100→0 | change of solvent |
| 66-75 | 95 | 5 | 0 | re-equilibration |
SYSTEM SUITABILITY
The test is not valid unless:
the chromatogram obtained with solution (5) closely resembles the reference chromatogram supplied with zidovudine and lamivudine impurity standard BPCRS and the retention of abacavir relative to zidovudine is about 1.2;
the resolution between the peaks due to lamivudine impurity B and lamivudine is at least 2.0; the resolution between the peaks due to lamivudine and thymidine is at least 2.0;
the resolution between the peaks due to zidovudine and zidovudine impurity B is at least 4.0; the resolution between the peaks due to zidovudine and abacavir is at least 1.5.
LIMITS
Using the chromatogram obtained with solution (5) and the reference chromatogram supplied with zidovudine and lamivudine impurity standard BPCRS identify any peaks in solution (1) corresponding to the named lamivudine and zidovudine impurities.
In the chromatogram obtained with solution (1):
the area of any peak corresponding to thymine (zidovudine impurity C) is not greater than the area of the principal peak in the chromatogram obtained with solution (4) (2.0%);
the area of any peak corresponding to zidovudine impurity B is not greater than the area of the peak due to zidovudine in the chromatogram obtained with solution (2) (1.0%);
the area of any peak corresponding to zidovudine impurity G (retention relative to zidovudine about 1.4) is not greater than 0.5 times the area of the peak due to zidovudine in the chromatogram obtained with solution (2) (0.5%);
the area of any peak corresponding to zidovudine impurity 1 (eluting between lamivudine impurity G and zidovudine impurity C) is not greater than 0.4 times the area of the peak due to zidovudine in the chromatogram obtained with solution (2) (0.4%);
the area of any peak corresponding to lamivudine impurity A is not greater than 3 times the area of the peak due to lamivudine in the chromatogram obtained with solution (3) (0.3%);
the area of any peak corresponding to a named lamivudine impurity is not greater than 0.2 times the area of the peak due to lamivudine in the chromatogram obtained with solution (2) (0.2%);
the area of any peak corresponding to a named zidovudine impurity is not greater than 0.2 times the area of the peak due to zidovudine in the chromatogram obtained with solution (2) (0.2%);
the area of any other secondary peak is not greater than 0.2 times the area of the peak due to abacavir in the chromatogram obtained with solution (2) (0.2%);
the sum of the areas of all the named zidovudine impurities is not greater than 4 times the area of the peak due to zidovudine in the chromatogram obtained with solution (2) (4.0%);
the sum of the areas of all the named lamivudine impurities is not greater than the area of the peak due to lamivudine in the chromatogram obtained with solution (2) (1.0%);
the sum any other secondary peaks is not greater than the area of the peak due to abacavir in the chromatogram obtained with solution (2) (1.0%);
Disregard any peak with an area less than the area of the peak due to abacavir in the chromatogram obtained with solution (3) (0.1%).
ASSAY
Weigh and powder 20 tablets. Carry out the method for liquid chromatography, Appendix III D, using the following solutions dissolved in solvent A described under Related substances.
(1) Shake a quantity of the powdered tablets containing the equivalent of 0.1 g of abacavir with 50 mL of solvent A in a 100 mL amber volumetric flask for 30 minutes, dilute to 100 mL and filter. Dilute 1 volume to 5 volumes.
(2) 0.023% w/v of abacavir sulfate BPCRS
(3) 0.02% w/v of zidovudine BPCRS.
(4) 0.01% w/v of lamivudine BPCRS.
(5) 0.075% w/v of zidovudine and lamivudine impurity standard BPCRS and 0.025% w/v of abacavir sulfate BPCRS in solvent A.
CHROMATOGRAPHIC CONDITIONS
The chromatographic conditions described under Related substances may be used.
SYSTEM SUITABILITY
The test is not valid unless:
the chromatogram obtained with solution (5) closely resembles the reference chromatogram supplied with zidovudine and lamivudine impurity standard BPCRS and the retention of abacavir relative to Zidovudine is about 1.2;
the resolution between the peaks due to lamivudine impurity B and lamivudine is at least 2.0;
the resolution between the peaks due to lamivudine and thymidine is at least 2.0;
the resolution between the peaks due to zidovudine and zidovudine impurity B is at least 4.0;
the resolution between the peaks due to zidovudine and abacavir is at least 1.5.
DETERMINATION OF CONTENT
Using solutions (1) and (2), calculate the total content of C14H18N6O in the tablets from the chromatograms obtained using the declared content of C14H18N6O in abacavir sulfate BPCRS.
Using solutions (1) and (3), calculate the total content of C10H13N5O4 in the tablets from the chromatograms obtained using the declared content of C10H13N5O4 in zidovudine BPCRS.
Using solutions (1) and (4) calculate the total content C8H11N3O3S in the tablets from the chromatograms obtained using the declared content of C8H11N3O3S in lamivudine BPCRS.
IMPURITIES
The impurities limited by the requirements of this monograph include impurities, A, B, C, E, F, G, H and J listed under Lamivudine, impurities B, C, E and G listed under Zidovudine and the following:
1. 1-[(2R,4S,5S)-4-amino-5-(hydroxymethyl)oxolan-2-yl]-5-methylpyrimidin-2,4(1H,3H)-dione.
