Within the framework of the Procurement, Quality and Sourcing Project for HIV, Tuberculosis and Malaria (http://www.who.int/prequal), The International Pharmacopoeiais collaborating with manufacturers, independent analytical drug quality control laboratories, national and regional pharmacopoeial bodies, research, governments, and regulatory bodies to provide specifications and monographs for the following antiretroviral agents: abacavir, didanosine, efavirenz, indinavir, lamivudine, nelfinavir, nevirapine, ritonavir, saquinavir, stavudine, zidovudine. A draft for lamivudine is provided below for comment.
Lamivudinum - Lamivudine (first draft)
C8H11N3O3S
Relative molecular mass. 229.3
Chemical name. (-) 4-amino-1-[(2R,5S)-2-(hydroxymethyl)-1,3-oxathiolan-5-yl]pyrimidin-2(1H)-one; CAS Reg. NO. 134678-17-4.
Description. A white or almost white powder.
Solubility. Soluble in water; sparingly soluble in methanol R; insoluble in acetone R.
Category. Antiretroviral (nucleoside reverse transcriptase inhibitor).
Storage. Lamivudine should be kept in a well-closed container, protected from light.
Manufacturer. The production method is validated to demonstrate that the substance, if tested, would comply with a limit of not more than 0.3% for 2S, 5R lamivudine enantiomer using a suitable chiral chromatographic method.
[Note from WHO Secretariat: This statement could be included, if reference to enantiomeric purity is considered advisable based on the relative toxicity of the 2S, 5R enantiomer. Such a statement would avoid the need to include a chiral chromatographic test within the analytical requirements of the monograph. The status of statements under the heading Manufacture will be defined in the General Notices of The International Pharmacopoeia.]
* Refers to The International Pharmacopoeia
REQUIREMENTS
Lamivudine contains not less than 97.0% and not more than 103.0% of C8H11N3O3S, calculated with reference to the dried substance.
Identity test
Either tests A and B, or test C may be applied.
A. Carry out test A.1. or, where UV detection is not available, test A.2.
A.1. Carry out the test as described under ‘‘Thin-layer chromatography’’ (Vol. 1, p. 83*), using silica gel R6 as the coating substance and a mixture of 67 volumes of dichloromethane R, 20 volumes of acetonitrile R, 10 volumes of methanol R and 3 volumes of ammonia (~260 g/l) TS as the mobile phase. Apply separately to the plate 5 µl of each of 2 solutions in methanol containing (A) 5 mg of the test substance per ml and (B) 5 mg of lamivudine RS per ml. After removing the plate from the chromatographic chamber, allow it to dry exhaustively in air or in a current of cool air. Examine the chromatogram in ultraviolet light (254 nm).
The principal spot obtained with solution A corresponds in position, appearance, and intensity with that obtained with solution B.
A.2. Carry out the test as described under ‘‘Thin-layer chromatography’’ (Vol. 1, p. 83*), using silica gel R5 as the coating substance and a mixture of 67 volumes of dichloromethane R, 20 volumes of acetonitrile R, 10 volumes of methanol R and 3 volumes of ammonia (~260 g/l) TS as the mobile phase. Apply separately to the plate 5 µl of each of 2 solutions in methanol containing (A) 5 mg of the test substance per ml and (B) 5 mg of lamivudine RS per ml. After removing the plate from the chromatographic chamber, allow it to dry exhaustively in air or in a current of cool air. Spray with vanillin/ sulfuric acid TS1. Heat the plate for a few minutes at 120°C. Examine the chromatogram in daylight.
The principal spot obtained with solution A corresponds in position, appearance, and intensity with that obtained with solution B.
B. The absorption spectrum of the final solution prepared for the Assay, when observed between 210 nm and 300 nm, exhibits one maximum at about 280 nm; the specific absorbance (A 1%1cm) is between 577 to 637.
C. Carry out the examination as described under “Spectrophotometry in the infrared region” (Vol. 1, p. 40*). The infrared absorption spectrum is concordant with the spectrum obtained from lamivudine RS or with the reference spectrum of lamivudine.
* Refers to The International Pharmacopoeia
Specific optical rotation. Use a 10 mg/ml solution in methanol R and calculate with reference to the dried substance; [α]D25°C = -136° to -144°.
Heavy metals. Use 1.0 g for the preparation of the test solution as described under “Limit test for heavy metals”, procedure 1 (Vol. 1, p. 118*). Determine the heavy metals content according to method A (Vol. 1, p. 119*); not more than 10 mg/g.
* Refers to The International Pharmacopoeia
Sulfated ash. Not more than 2.0 mg/g.
Loss on Drying. Dry for 3 hours at 105°C; it loses not more than 5 mg/g.
Related substances
Carry out the test as described under “High-performance liquid chromatography” (Vol. 5, p. 257*), using a stainless steel column (25 cm x 4.6 mm) packed with octadecylsilyl silica gel for chromatography R (Stationary phase A) (5µm) (Waters Hypersil BDS is suitable). As the mobile phase, use a mixture of 5 volumes of methanol R and 95 volumes of 1.9 g/l solution of ammonium acetate R, buffer adjusted to pH 3.8 with glacial acetic acid R.
Prepare the following solutions. For solution (1) prepare 0.5 mg/ml solution of test substance in the mobile phase. For solution (2) dilute 1.0 ml of solution (1) to 100 ml with mobile phase and then dilute 1.0 ml of this solution to 10 ml. For solution (3) dissolve 25 mg of salicylic acid R in 100 ml of mobile phase. Then dilute 1.0 ml of this solution to 500 ml with the mobile phase.
Operate with a flow rate of 1.0 ml per minute. As a detector use an ultraviolet spectrophotometer set at a wavelength of about 277 nm.
Maintain the temperature of the column at 35°C using, for example, a water-bath.
Inject alternately 10 µl each of solutions (1), (2) and (3). Record the chromatograms for about 3 times the retention time of lamivudine in solution (2).
Measure the areas of the peak responses obtained in the chromatograms from solutions (1), (2) and (3) and calculate the content of the related substances as a percentage.
In the chromatogram obtained with solution (1), the area of the peak (at a relative retention time of about 0.4) is not greater than 3 times the area of the peak in the chromatogram obtained with solution (2) (0.3%). The area of the peak (at a relative retention time of about 0.9) is not greater than 2 times the area of the peak in the chromatogram obtained with solution (2) (0.2%). The area of the peak corresponding to salicylic acid is not greater than that of the corresponding peak in the chromatogram obtained with solution (3) (0.1%). The area of any other peak apart from the principal peak is not greater than the area of the peak in the chromatogram obtained with solution (2) (0.1%). The total area of all the peaks apart from the principal peak obtained in the chromatogram with solution (1) is not greater than 6 times the area of the peak obtained with solution (2) (0.6%). Disregard any peak with an area less than 0.5 times the area of the principal peak obtained with solution (2) (0.05%).
Assay: Weigh accurately about 25 mg of the test substance into a 200-ml volumetric flask. Add about 180 ml of water and dissolve by using an ultrasonic bath if necessary. Cool to room temperature and dilute to volume with water and mix.
Dilute 4 ml of this solution to 50 ml with 0.1M H2SO4 and mix. For the blank, use a solution prepared by mixing 4 ml of water with 50 ml of 0.1M H2SO4.
Measure the absorbance of a 1-cm layer of the final solution at a maximum about 280 nm against a solvent cell containing the blank. Calculate the content of C8H11N3O3S using the absorptivity value of 60.7 (A 1%1cm= 607).
Impurities
The following list of known and potential impurities that have been shown to be controlled by the tests in this monograph is given for information.
[Note from WHO Secretariat: Chemical structures will be included in the next version.]
A. cis-5-(4-amino-2-oxopyrimidin-1(2H)-yl)-1,3-oxathiolane-2-carboxylic acid and enantiomer
B. 4-amino-1-[trans-2-(hydroxymethyl)-1,3-oxathiolan-5yl]pyrimidin-2(1H)-one
C. salicylic acid
D. 4-amino-1-[(2S,5R)-2-(hydroxymethyl)-1,3-oxathiolan-5yl]pyrimidin-2(1H)-one
E. 4-aminopyrimidin-2(1H)-one
F. pyrimidine-2,4(1H,3H)-dione
G. 4-amino-1-[(2R,3S,5S)-2-(hydroxymethyl)-3-oxo-1,3λ4-oxathiolan-5-yl]pyrimidin-2(1H)-one
H. 4-amino-1-[(2R,3R,5S)-2-(hydroxymethyl)-3-oxo-1,3λ4-oxathiolan-5-yl]pyrimidin-2(1H)-one
I. 4-amino-1-[(2S,4S)-2-(hydroxymethyl)-1,3-dioxolan-4-yl]pyrimidin-2(1H)-one
J. 1-[(2R,5S)-2-(hydroxymethyl)-1,3-oxathiolan-5-yl]pyrimidin-2,4(1H,3H)-dione
Reagent
Salicylic acid R. 2-hydroxybenzoic acid; C7H6O3.
A commercially available reagent of suitable grade.
Storage. Keep protected from light.
Figure 1: HPLC chromatogram of lamivudine and its related impurities
Monographs for antiretrovirals
Loading...
