Water-insoluble substances—
Dissolve 25 g in 100 mL of water, filter the solution through a tared filtering crucible, wash the filter with hot water, and dry at 100
to constant weight: the increase in weight is not more than 25 mg (0.1%).
Fumaric and maleic acids—
Mobile phase—
Prepare a suitable filtered and degassed 0.01 N sulfuric acid in water solution.
Standard preparation—
Using
Mobile phase as a solvent, prepare a solution containing accurately known concentrations of about 0.005 mg of
USP Fumaric Acid RS per mL and about 0.002 mg of
USP Maleic Acid RS per mL.
Test preparation—
Transfer about 100 mg, accurately weighed, of Malic Acid to a 100-mL volumetric flask, dissolve in and dilute with Mobile phase to volume, and mix.
Resolution solution—
Using
Mobile phase as a solvent, prepare a solution containing about 1 mg of Malic Acid per mL, about 10 µg of
USP Fumaric Acid RS per mL, and about 4 µg of
USP Maleic Acid RS per mL.
Chromatographic system
(see
Chromatography 
621
)—The liquid chromatograph is equipped with a 210-nm detector and a 6.5-mm × 30-cm column that contains packing L17. Maintain the temperature of the column at 37 ± 1
. The flow rate is about 0.6 mL per minute. Chromatograph the
Resolution solution, and record the peak responses: the resolution,
R, of the maleic acid and malic acid peaks is not less than 2.5, the resolution,
R, of the malic acid and fumaric acid peaks is not less than 7.0, and the relative standard deviation of the maleic acid peak for replicate injections is not more than 2.0%.
Procedure—
Separately inject equal volumes (about 20 µL) of the
Standard preparation and the
Test preparation into the chromatograph, record the chromatograms, and measure the peak responses. The relative retention times are about 0.6 for maleic acid, 1.0 for malic acid, and about 1.5 for fumaric acid. Calculate the quantities, in mg, of maleic acid and of fumaric acid in the portion of Malic Acid taken by the formula:
100C(rU / rS),
in which
C is the concentration, in mg per mL, of the corresponding reference standard in the
Standard preparation; and
rU and
rS are the responses of the corresponding peaks from the
Test preparation and the
Standard preparation, respectively. Not more than 1.0% of fumaric acid and not more than 0.05% of maleic acid are found.
Assay—
Transfer about 2 g of Malic Acid, accurately weighed, to a conical flask, dissolve in 40 mL of recently boiled and cooled water, add
phenolphthalein TS, and titrate with 1 N sodium hydroxide VS to the first appearance of a faint pink color that persists for not less than 30 seconds. Each mL of 1 N sodium hydroxide is equivalent to 67.04 mg of C
4H
6O
5.
;)
