§ 436.322 - High-pressure liquid chromatographic assay for anthracycline antibiotics.

(a) Equipment. A suitable high-pressure liquid chromatograph, such as a Waters Associates Model 244 1 or equivalent equipped with:

(1) A low dead volume cell 8 to 20 microliters;

(2) A light path length of 1 centimeter;

(3) A suitable ultraviolet detection system operating at a wavelength of 254 nanometers;

(4) A suitable recorder of at least 25.4 centimeter deflection;

(5) A suitable integrator;

(6) A 30-centimeter column having an inside diameter of 4.6 millimeters and packed with a suitable reverse phase packing such as: Waters Associates, Micro-Bondapak C18.1

(b) Reagents. (1) Solvent mixture: Water: acetonitrile (69:31).

(2) Mobile phase: Water: acetonitrile (69:31) adjusted to pH 2 with phosphoric acid. Filter the mobile phase through a suitable glass fiber filter or equivalent that is capable of removing particulate contamination to 1 micron in diameter. Degas the mobile phase just prior to its introduction into the chromatograph pumping system.

(3) Internal standard solution: Prepare a 2.0-milligram-per-milliliter solution of 2-naphthalenesulfonic acid in the solvent mixture.

(c) Operating conditions. Perform the assay at ambient temperature with a typical flow rate of 1.5 milliliters per minute. Use a detector sensitivity setting that gives a peak height for the reference standard that is at least 50 percent of scale. The minimum between peaks must be no more than 2 millimeters above the initial baseline.

(d) Procedure. Use the standard and sample solutions prepared as directed in the individual monographs for the drug being tested. Use the equipment, reagents, and operating conditions listed in paragraphs (a), (b), and (c) of this section. Inject 5 microliters of the standard solution into the chromatograph. Allow an elution time sufficient to obtain satisfactory separation of expected components (ordinarily this time is 20 minutes). After separation of the standard solution has been completed, inject 5 microliters of the sample solution into the chromatograph and repeat the procedure described for the standard solution. The elution order is: Void volume, internal standard, doxorubicin, dihydrodaunomycin, daunomycin, adriamycinone, dihydrodaunomycinone, bromodaunomycin, daunomycinone, and bis-anhydrodaunomycinone.

(e) Calculations. Calculate the anthracycline content as directed in the individual monograph for the drug being tested.