§ 436.352 - High-performance liquid chromatographic assay for determining clavam-2-carboxylate content in clavulanate potassium.

(a) Apparatus. A suitable high-performance liquid chromatograph equipped with:

(1) A suitable detection system specified in the monograph for the drug being tested;

(2) A suitable recording device of at least 25-centimeter deflection;

(3) A suitable chromatographic data managing system; and

(4) An analytical column, approximately 30 centimeters in length, packed with a material as defined in the monograph for the drug being tested.

(b) Procedure. Perform the assay and calculate the drug content using the temperature, instrumental conditions, and calculations specified in the monograph for the drug being tested with a flow rate not to exceed 0.5 milliliter per minute. Use a detector sensitivity setting that gives a peak height for the working standard that is at least 50 percent of scale with typical chart speed of not less than 2.5 millimeters per minute. Use the apparatus described in paragraph (a) of this section; and the mobile phase and working standard and sample solutions described in the monograph for the drug being tested. Equilibrate and condition the column by passage of 10 to 15 void volumes of mobile phase followed by five replicate injections of the same volume (between 10 and 20 microliters) of the working standard solution. Allow an operating time sufficiently long to obtain satisfactory separation and elution of the expected components after each injection. The retention times for clavam-2-carboxylic acid and clavulanic acid are about 10 and 14 minutes, respectively, under these prescribed conditions. The sample solution should be injected at least in duplicate and an average should be taken. For each such series of samples injected, two injections of standard should be made, one before and one after the sample series, and an average should be taken. Record the peak responses and calculate the prescribed system suitability requirements as follows:

(c) System suitability test. Using the apparatus and procedure described in this section, test the chromatographic system for assay as follows:

(1) Tailing factor. Calculate the tailing factor (T), from distances measured along the horizontal line at 5 percent of the peak height above the baseline, as follows:

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(2) Efficiency of the column. Calculate the number of theoretical plates (n) of the column as follows:

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(3) Resolution factor. Calculate the resolution factor (R) as follows:

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(4) Coefficient of variation (Relative standard deviation). Calculate the coefficient of variation (SR

EC01AP94.019 If the complete operating system meets the system suitability requirements of the monograph for the drug being tested, proceed as described in paragraph (b) of this section, using the sample solution in lieu of the working standard solution.