§ 436.343 - High-pressure liquid chromatographic assay for cefuroxime.

(a) Equipment. A suitable high-pressure liquid chromatograph 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; and

(6) A 15-centimeter column having an inside diameter of 4.6 millimeters and packed with hexyl silane chemically bonded to porous silica or ceramic microparticles, 5 micrometers in diameter.

(b) Reagents—(1) Acetate buffer, pH 3.4. Place 50 milliliters of 0.1M sodium acetate into a 1,000-milliliter volumetric flask and dilute to volume with 0.1M acetic acid. Mix.

(2) Mobile phase. Mix 0.1M acetate buffer, pH 3.4:acetonitrile (10:1). 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 1.5 milligram per milliliter solution of orcinol monohydrate in water.

(c) Operating conditions. Perform the assay at ambient temperature with a typical flow rate of 2.0 milliliters per minute. Use a detector sensitivity setting that gives a peak height for the working standard that is at least 50 percent of scale.

(d) Preparation of working standard and sample solutions—(1) Preparation of working standard solution. Dissolve an accurately weighed portion of the cefuroxime working standard with sufficient distilled water to obtain a stock solution containing 1.0 milligram of cefuroxime per milliliter. Immediately transfer 5.0 milliliters of the stock solution to a 100-milliliter volumetric flask, add 20.0 milliliters of internal standard solution and dilute to 100 milliliters with distilled water and mix. Store the solution in a refrigerator and use within 6 hours.

(2) Preparation of sample solutions—(i) Product not packaged for dispensing (micrograms of cefuroxime per milligram). Dissolve an accurately weighed portion of the sample with sufficient distilled water to obtain a stock solution containing 1.0 milligram of cefuroxime per milliliter. Immediately transfer 5.0 milliliters of the stock solution to a 100-milliliter volumetric flask, add 20.0 milliliters of internal standard solution and dilute to 100 milliliters with distilled water and mix. Store the solution in a refrigerator and use within 6 hours. Using this sample solution, proceed as directed in paragraph (e) of this section.

(ii) Product packaged for dispensing. Determine both micrograms of cefuroxime per milligram of the sample and milligrams of cefuroxime per container. Use separate containers for preparation of each sample solution as described in paragraphs (d)(2)(ii) (a) and (b) of this section.

(a) Micrograms of cefuroxime per milligram. Dissolve an accurately weighed portion of the sample with sufficient distilled water to obtain a stock solution containing 1.0 milligram of cefuroxime per milliliter. Immediately transfer 5.0 milliliters of the stock solution to a 100-milliliter volumetric flask, add 20.0 milliliters of internal standard solution and dilute to 100 milliliters with distilled water and mix. Store the solution in a refrigerator and use within 6 hours. Using this sample solution, proceed as directed in paragraph (e) of this section.

(b) Milligrams of cefuroxime per container. Reconstitute the sample as directed in the labeling. Then using a suitable hypodermic needle and syringe, remove all of the withdrawable contents if it is represented as a single-dose container; or, if the labeling specifies the amount of potency in a given volume of the resultant preparation, remove an accurately measured representative portion from each container. Dilute the solution thus obtained with distilled water to obtain a stock solution of 1.0 milligram per milliliter. Immediately transfer 5.0 milliliters of the stock solution to a 100-milliliter volumetric flask, add 20.0 milliliters of internal standard solution and dilute to 100 milliliters with distilled water and mix. Store the solution in a refrigerator and use within 6 hours. Using this sample solution, proceed as directed in paragraph (e) of this section.

(e) Procedure. Using the equipment, reagents, and operating conditions as listed in paragraphs (a), (b), and (c) of this section, inject 10 microliters of the working standard solution into the chromatograph. Allow an elution time sufficient to obtain satisfactory separation of the expected components. After separation of the working standard solution has been completed, inject 10 microliters of the sample solution prepared as described in paragraph (d)(2)(i) of this section into the chromatograph and repeat the procedure described for the working standard solution. If the sample is packaged for dispensing, repeat the procedure for each sample solution prepared as described in paragraphs (d)(2)(ii)(a) and (d)(2)(ii)(b) of this section.

(f) Calculations. (1) Calculate the micrograms of cefuroxime per milligram of sample as follows:

ER01JA93.059

(2) Calculate the cefuroxime content of the vial as follows:

ER01JA93.060