Transfer about 500 mg of Choline Bitartrate, accurately weighed, to a centrifuge tube; add 2.0 mL of water; and swirl to dissolve. Add 0.5 mL of potassium chloride solution (7.5 in 25), centrifuge, and transfer 1.0 mL of the supernatant to a 24-mL screw-capped vial. Dry at 120
for 2 hours. Add 400 mg of 3,5-dinitrobenzoyl chloride and 10 mL of acetonitrile, and mix. Cap the vial, and heat at 55
for 2 hours. Cool to room temperature, add 5 mL of water, and allow to stand for 5 minutes. Quantitatively transfer this solution to a 50-mL volumetric flask, dilute with Mobile phase
to volume, and mix. Pipet 2.0 mL of the solution to a 25-mL volumetric flask, dilute with Mobile phase
to volume, and mix.
Separately inject equal volumes (about 20 µL) of the Standard solution
and the Test solution
into the chromatograph, record the chromatograms, and measure all the peak responses. Calculate the percentage of each impurity in the portion of Choline Bitartrate taken by the formula:
(253.25/139.62)62,500(C/W)(ri / rS),
in which 253.25 and 139.62 are the molecular weights of choline bitartrate and choline chloride, respectively; C
is the concentration of USP Choline Chloride RS
, in mg per mL, in the Standard solution; W
is the weight, in mg, of Choline Bitartrate taken to prepare the Test solution; ri
is the peak response for each impurity, other than that of the choline bitartrate derivative and 3,5-dinitrobenzoic acid; and rS
is the peak response for the choline chloride derivative in the Standard solution:
not more than 0.3% of any individual impurity is found; and not more than 2.0% of total impurities is found.