§ 436.304 - Clindamycin phosphate vapor phase chromatography.

(a) Equipment. Gas chromatograph equipped with an electronic integrator and with a flame ionization detector that has a sensitivity of at least 1 × 10− 1 0 amperes: Hewlett-Packard 7600 4 or equivalent.

(b) Reagents. (1) Trifluoroacetic anhydride.

(2) Intestinal alkaline phosphatase.

(3) pH 9.0 borate buffer: Transfer 3.1 grams of boric acid into a 1-liter volumetric flask containing 500 milliliters of water, mix, and add 21 milliliters of 1.0N sodium hydroxide and 10 milliliters of 0.1M magnesium chloride. Dilute to volume with water and mix well.

(4) Internal standard: Prepare a chloroform solution containing approximately 0.45 milligram hexacosane per milliliter.

(5) Anhydrous sodium carbonate.

(c) Typical conditions. (1) Column: 2 feet × 3 millimeters ID, glass, with 1 percent SE-30 on Diatoport S (80/100 mesh), or equivalent.

(2) Temperatures: Column, 180° C., detector, 215° C., injection port, ambient temperature.

(3) Carrier gas: Helium approximately 60 milliliters per minute.

(4) Detector: Hydrogen flame—hydrogen flow at 40 milliliters per minute. Air flow at 400 milliliters per minute.

(5) Sensitivity: 1 × 10− 9 amperes.

(d) Preparation of clindamycin phosphate sample solution. Accurately weigh approximately 12 milligrams of the clindamycin phosphate sample into a 50-milliliter glass-stoppered centrifuge tube. Pipet 25 milliliters of the pH 9.0 borate buffer into the centrifuge tube. Add 10 milliliters chloroform and shake vigorously for 15 minutes. Centrifuge the resulting mixture and pipet a 20-milliliter aliquot of the aqueous phase into a 35-milliliter centrifuge tube. Add a weighed amount of intestinal alkaline phosphatase equivalent to 50 units of activity 5 and allow the solution to stand until the enzyme has completely dissolved. Place the tube into a water bath at 37° C.±2° C. for 2.5 hours. After the 2.5-hour hydrolysis, allow the solution to cool and proceed as directed in paragraph (f) of this section.

(e) Preparation of the clindamycin hydrochloride standard solution. Accurately weigh approximately 9 milligrams of the clindamycin hydrochloride working standard into a 35-milliliter glass-stoppered centrifuge tube and dissolve in 20 milliliters of pH 9.0 borate buffer. Proceed as directed in paragraph (f) of this section.

(f) Procedure. Add 10 milliliters of the internal standard solution to each sample and standard solution. Shake the centrifuge tubes vigorously for 30 minutes and centrifuge. Remove the aqueous layer and discard. Shake the tubes again; mix in an ultrasonic mixer for 2 minutes, then centrifuge. No emulsion should be present at this stage. Remove the remaining aqueous layer by suction and transfer a 3-milliliter aliquot of the chloroform layer to a 1-dram tablet vial containing approximately 1 gram of anhydrous sodium sulfate. Swirl the vial to dry the chloroform and transfer a 1-milliliter aliquot to another 1-dram tablet vial. Using a 0.25-milliliter pipet, add 0.25 milliliter of trifluoracetic anhydride to each of the vials and place into a water bath at 45° C.±2° C. for 30 minutes. Remove the vials from the bath, add about 10 granules of anhydrous sodium carbonate to each vial, and allow to stand for approximately 30 minutes. Centrifuge the vials for approximately 10 minutes at 5,000 r.p.m. Inject 2 microliters of each of the resulting solutions into the gas chromatograph. Use the conditions and materials listed in paragraphs (a), (b), and (c) of this section. The elution order is: Epiclindamycin (if present), clindamycin B (if present), clindamycin, and internal standard. Calculate the clindamycin content as directed in paragraph (g) of this section.

(g) Calculations. Calculate the clindamycin content of the sample as follows:

EC01AP94.006