§ 436.358 - High-performance liquid chromatographic assay for pyridine.

(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, 15 to 25 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 pyridine 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 that is at least 25 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 for the system suitability test by five replicate injections of the same volume (between 10 and 20 microliters) of the system suitability test 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) Tailing factor. Calculate the tailing factor for the pyridine peak (T), from distances measured along the horizontal line at 5 percent of the peak height above the baseline, as follows:

ER01JA93.088

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

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(3) Coefficient of variation (relative standard deviation). Calculate the coefficient of variation for the pyridine peak (SR in percent ) as follows:

EC01AP94.025 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.