(Official January 1, 2007)

Tetrabutylammonium hydroxide solution— Dissolve tetrabutylammonium hydroxide in methanol to obtain a solution containing 0.25 g per mL.

2 N Monobasic sodium phosphate solution— Dissolve monobasic sodium phosphate monohydrate in water to obtain a solution containing 276 mg per mL.

Mobile phase— Mix 15 mL of Tetrabutylammonium hydroxide solution with 835 mL of water. Add 125 mL of acetonitrile, adjust with 2 N Monobasic sodium phosphate solution to an apparent pH of 7.5 ± 0.1, mix, dilute with water to 1000 mL, and filter. Adjust the concentration of acetonitrile, if necessary (see System Suitability under Chromatography 621).

Diluting solution— Mix 15 mL of Tetrabutylammonium hydroxide solution with 900 mL of water and adjust with 2 N Monobasic sodium phosphate solution to a pH of 7.5 ± 0.1. Dilute with water to 1000 mL, and mix.

Standard preparation— Dissolve an accurately weighed quantity of USP Leucovorin Calcium RS in Diluting solution, and dilute quantitatively with Diluting solution to obtain a solution having a known concentration of about 175 µg of anhydrous USP Leucovorin Calcium RS per mL.

Assay preparation— Dissolve about 20 mg of Leucovorin Calcium, accurately weighed, in Diluting solution in a 100-mL volumetric flask. Dilute with Diluting solution to volume, and mix.

System suitability preparation— Dissolve folic acid in Diluting solution to obtain a solution containing about 175 µg per mL. Mix 1 part of this solution with 4 parts of the Standard preparation.

Chromatographic system (see Chromatography 621)—The liquid chromatograph is equipped with a 254-nm detector and a 4-mm × 30-cm column that contains packing L1. The flow rate is 1 to 2 mL per minute. Chromatograph the System suitability preparation, and record the peak responses as directed for Procedure: the relative retention times for leucovorin and folic acid are 1.0 and about 1.6, respectively; the resolution, R, between the leucovorin calcium and folic acid peaks is not less than 3.6; and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 15 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the responses for the major peaks appearing at corresponding retention times in the chromatograms. Calculate the quantity, in mg, of C20H21CaN7O7 in the portion of Leucovorin Calcium taken by the formula: in which C is the concentration, in µg per mL, of anhydrous USP Leucovorin Calcium RS in the Standard preparation; and rU and rS are the peak responses obtained from the Assay preparation and the Standard preparation, respectively.