§ 436.216 - High-performance liquid chromatographic assay.

(a) Equipment. 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, 3 to 30 centimeters long, packed with a material as defined in the monograph for the drug being tested; and if specified in that monograph, the inlet of this column may be connected to a guard column, 3 to 5 centimeters in length, packed with the same material of 40 to 60 micrometers particle size.

(b) Procedure. Perform the assay and calculate the drug content using the temperature, instrumental conditions, flow rate, and calculations specified in the monograph for the drug being tested. Use a detector sensitivity setting that gives a peak height for the working standard solution that is at least 50 percent of scale with typical chart speed of not less than 2.5 millimeters per minute. Use the equipment described in paragraph (a) of this section. Use the reagents, working standard solution, and sample solution 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 of the working standard solution. Allow an operating time sufficiently long to obtain satisfactory separation and elution of the expected components after each injection. Record the peak responses and calculate the prescribed system suitability requirements described for the system suitability test in paragraph (c) of this section.

(c) System suitability test. Select the system suitability requirements specified in the monograph for the drug being tested. Then, using the equipment and procedure described in this section, test the chromatographic system for assay as follows:

(1) Trailing factor or asymmetry factor. Calculate either the trailing factor (T), from distances measured along the horizontal line at 5 percent of the peak height above the baseline or the asymmetry factor (As) measured at a point 10 percent of the peak height from the baseline; whichever is required in the appropriate monograph, as follows:

ER01JA93.023 ER01JA93.024

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

ER01JA93.025 ER01JA93.026

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

ER01JA93.027

(4) Coefficient of variation (relative standard deviation). Calculate the coefficient of variation (SR in percent) as follows:

EC01AP94.005 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, except alternate injections of the working standard solution with injections of the sample solution.

(5) Capacity factor. Calculate the capacity factor (k), if required in the monograph as follows:

ER01JA93.028 ER01JA93.029