Leucovorin Calcium Tablets
» Leucovorin Calcium Tablets contain not less than 90.0 percent and not more than 110.0 percent of the labeled amount of leucovorin (C20H23N7O7).
Packaging and storage Preserve in well-closed containers, protected from light, at controlled room temperature.
USP Reference standards 11
USP 10-Formylfolic Acid RS
A: Transfer a quantity of finely powdered Tablets, equivalent to about 200 mg of leucovorin calcium, to a conical flask, add 10 mL of water, shake vigorously, sonicate for 10 minutes, and filter. Transfer the filtrate to a stoppered centrifuge tube, add about 125 mg of ammonium oxalate, shake vigorously, and centrifuge until a clear supernatant is obtained. Transfer the supernatant to another stoppered centrifuge tube, add 1 mL of methanol and 3 drops of hydrochloric acid, and shake vigorously. If the preparation is cloudy, add methanol until a clear solution is obtained, and filter if necessary to remove any undissolved material. Cool the preparation at 0 until a precipitate forms, and centrifuge for 1 to 2 minutes. [noteThe cooling and centrifuging steps may be repeated if necessary to increase the amount of precipitate collected. ] Decant the supernatant, add 2 mL of methanol to the tube, shake vigorously to dissolve the precipitate, and transfer the contents to a beaker. Evaporate under a current of air to dryness, and dry the residue at 50 for 30 minutes: the IR absorption spectrum of a potassium bromide dispersion of the residue so obtained exhibits maxima only at the same wavelengths as that of a similar preparation of USP Leucovorin Calcium RS.
B: The retention time of the leucovorin peak in the chromatogram of the Assay preparation corresponds to that of the Standard preparation, as obtained in the Assay.
Medium: water; 900 mL.
Apparatus 2: 50 rpm.
Time: 30 minutes.
Procedure Determine the amount of C20H21CaN7O7 dissolved from UV absorbances at the wavelength of maximum absorbance at about 284 nm using filtered portions of the solution under test, diluted with water if necessary, in comparison with a Standard solution having a known concentration of USP Leucovorin Calcium RS in the same Medium.
Tolerances Not less than 75% (Q) of the labeled amount of C20H21CaN7O7 is dissolved in 30 minutes.
Uniformity of dosage units 905: meet the requirements.
Procedure for content uniformity Using individual intact Tablets, prepare solutions in water having known concentrations of about 10 µg of leucovorin calcium per mL. Prepare a Standard solution of USP Leucovorin Calcium RS in water having a known concentration of about 10 µg per mL. Concomitantly determine the absorbances of the test solutions and the Standard solution in 1-cm cells at the wavelength of maximum absorbance at about 284 nm, with a suitable spectrophotometer, using water as the blank. Calculate the quantity, in mg, of C20H21CaN7O7 in each Tablet taken by the formula:
(T / D)C(AU / AS)in which T is the labeled quantity, in mg, of leucovorin calcium in the Tablet; D is the concentration, in µg per mL, of the solution from the Tablet, based on the labeled quantity per Tablet and the extent of dilution; C is the concentration, in µg per mL, of USP Leucovorin Calcium RS in the Standard solution; and AU and AS are the absorbances of the solution from the Tablet and the Standard solution, respectively.
Chromatographic purity Calculate the percentage of each peak, other than the leucovorin peak, in the chromatogram obtained from the Assay preparation by the formula:
100(ri / rt)in which ri is the response of each impurity peak, and rt is the sum of the responses of all of the peaks: not more than 2.5% of any individual impurity and not more than 4.0% of total impurities is found.
Diluting solvent Prepare a mixture of water and methanol (80:20).
Mobile phase Prepare a 0.005 M solution of tetrabutylammonium phosphate in Diluting solvent. Adjust the pH of this solution to 7.5 by the addition of 50% (w/v) sodium hydroxide solution, filter, and degas.
Standard preparation Dissolve accurately weighed quantities of USP Leucovorin Calcium RS and USP 10-Formylfolic Acid RS in water to obtain a solution having known concentrations of about 500 µg of USP Leucovorin Calcium RS and 10 µg of USP 10-Formylfolic Acid RS per mL.
Assay preparation Weigh and finely powder not fewer than 20 Tablets. Transfer an accurately weighed portion of the powder, equivalent to about 50 mg of leucovorin, to a 100-mL volumetric flask. Add about 50 mL of water, sonicate for 30 minutes, dilute with water to volume, mix, and filter.
Chromatographic system (see Chromatography 621)The liquid chromatograph is equipped with a 254-nm detector and a 4.6-mm × 15-cm column that contains packing L1. The flow rate is about 2.0 mL per minute. Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: the resolution, R, between leucovorin and 10-formylfolic acid is not less than 1.5; and the relative standard deviation for replicate injections, and retention times is not more than 2.0% for the leucovorin peak.
Procedure Separately inject equal volumes (about 20 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the peak area responses. The relative retention times are about 2.3 for 10-formylfolic acid and 1.0 for leucovorin. Calculate the quantity, in mg, of C20H23N7O7 in the portion of Tablets taken by the formula:
(473.45 / 511.50)(L / D)(C)(rU / rS)in which 473.45 and 511.50 are molecular weights of leucovorin and anhydrous leucovorin calcium, respectively; L is the labeled amount, in mg, of leucovorin in each Tablet; D is the concentration, in mg per mL, of leucovorin in the Assay preparation, based on the labeled quantity per Tablet and the extent of dilution; C is the concentration, in mg per mL, of anhydrous USP Leucovorin Calcium RS in the Standard preparation; and rU and rS are the peak area responses obtained from the Assay preparation and the Standard preparation, respectively.
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