A degraded sample from an expired 10 mg/mL vial of LV calcium for injection USP (David Bull Laboratories; Vaudreuil, Quebec; lot 2024022, expiry December 1994) was analyzed by the HPLC method to separate LV from its degradation products. Chromatograms were inspected for the appearance of additional peaks, and the LV peak was compared to the LV peak obtained from HPLC analyses of a fresh sample of LV for changes in concentration, retention time, and peak shape.

Ultraviolet spectral purity (200-365 nm, 6-nm bandwidth, determined with a UV6000LP deuterium lamp, Thermo Separation Products) of the leading edge, middle, and tail of the LV peak in both the fresh and expired samples were compared. The expired LV was used to develop an HPLC method for separation of IR, LV, and their degradation products.

Accuracy and Reproducibility of the HPLC Assay

The accuracy and reproducibility of the HPLC method for simultaneous analysis of IR and LV was tested across 5 standard curves. Each sample containing both IR and LV standards was chromatographed in duplicate. Interday and intraday reproducibility were assessed using the coefficient of variation of the peak area for each compound determined in duplicate, and accuracy was determined on the basis of deviations from the known concentration.
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Because the standard curve had an upper limit of 0.4 mg/mL for both IR and LV, all samples required dilution of either 1:2, 1:4, or 1:10. To evaluate the accuracy and reproducibility of an experimental solution that may need to be diluted, 5 replicates of samples containing 0.45 mg/mL of IR and 3.6 mg/mL of LV were prepared and diluted, and the concentrations of IR and LV were measured. All experimental solutions assessed by HPLC had concentrations of IR and LV above 0.00625 mg/mL, the lower limit of the standard curve for both drugs.

HPLC Analyses of Solutions for Compatibility Study

On each study day, fresh standards of IR and LV were prepared and chromatographed separately to construct standard curves. A stock solution of IR was prepared by dissolving an accurately weighed quantity of approximately 10 mg of IR hydrochloride trihydrate powder (CPT-11, class A primary standard, Pharmacia Corporation; lot C, expiry September 1, 2004) in 25 mL of distilled water. This stock solution (0.40 mg/mL) was then diluted to prepare 7 concentrations of IR: 0.00625, 0.0125, 0.025, 0.050, 0.100, 0.250, and 0.400 mg/mL. Four-microlitre aliquots of each of these 7 standards and a blank were directly chromatographed in duplicate to allow construction of the standard curve.

A stock solution of LV was prepared by diluting various volumes of a 10 mg/mL solution (leucovorin calcium for injection USP, Novopharm, Toronto, Ontario; lot 0271202001, expiry December 2004) in 10 mL of distilled water to prepare 7 concentrations of LV: 0.00625, 0.0125, 0.025, 0.050, 0.100, 0.250, and 0.400 mg/mL. Four-microlitre aliquots of each of these 7 standards and a blank were used to construct a standard curve.
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IR and LV were quantified simultaneously each day using the newly developed HPLC method described above. The average peak area of 2 replicates from each sample of IR and LV was subjected to least-squares linear regression; the concentration of experimental solutions was interpolated from standard curves and recorded. Concentrations were recorded to the nearest 0.001 mg/mL.