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

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

Mobile phase— Mix 15mLof Tetrabutylammonium hydroxide solutionwith 835mLof water.Add 125mLof acetonitrile,adjust with 2N Monobasic sodium phosphate solutionto an apparent pHof 7.5±0.1,mix,dilute with water to 1000mL,and filter.Adjust the concentration of acetonitrile,if necessary (see System Suitabilityunder Chromatography á621ñ).

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

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

Assay preparation— Dissolve about 20mg of Leucovorin Calcium,accurately weighed,in Diluting solutionin a 100-mLvolumetric flask.Dilute with Diluting solutionto volume,and mix.

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

Chromatographic system (see Chromatography á621ñ)—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 1to 2mLper 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 1.0and 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 15µL)of the Standard preparationand the Assay preparationinto 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 C20H21CaN7O7in the portion of Leucovorin Calcium taken by the formula: in which Cis the concentration,in µg per mL,of anhydrous USP Leucovorin Calcium RSin the Standard preparation;and rUand rSare the peak responses obtained from the Assay preparationand the Standard preparation,respectively.