§ 436.302 - Clindamycin vapor phase chromatography.

(a) Equipment. Gas chromatograph equipped with a flame ionization detector: Barber-Colman 5,000 or equivalent.

(b) Reagents. (1) Pyridine, reagent grade, dried over sodium sulfate.

(2) Chloroform, reagent grade.

(3) Acetic anhydride, reagent grade, used as acteylating agent.

(4) Internal standard: Prepare a solution containing 3 milligrams of cholestane per milliliter in pyridine.

(c) Typical conditions. (1) Column: 4 feet × 4 millimeters ID, glass, with 1 percent SE-30 on Diatoport S (60/80 mesh), or equivalent.

(2) Temperatures: Column 200° C.; detector 215° C.; injection port, ambient temperature.

(3) Carrier gas: Helium approximately 120 milliliters per minute.

(4) Detector: Hydrogen flame—hydrogen at 120 pounds per square inch, air at 40 pounds per square inch.

(5) Sensitivity: 1,000; attenuation, 2 for clindamycin, 1 for internal standard: 2×10−8amperes.

(d) Preparation of clindamycin sample and working standard solutions. Accurately weigh approximately 15 milligrams of sample or working standard into a glass-stoppered conical 15-milliliter centrifuge tube. Add 1.0 milliliter of chloroform, 1.0 milliliter of internal standard solution, and 0.6 milliliter of acetic anhydride. Agitate the tubes to insure dissolution of the sample and complete mixing of the liquids. Proceed as directed in paragraph (e) of this section.

(e) Procedure. Cover the top of each centrifuge tube with a plastic cap. Punch a small hole in the top of each cap to allow vapor to escape. Place the tubes in a 100° C. drying oven for 2.5 hours. Remove the tubes from the oven and allow to cool. Take the plastic cap from each tube and replace with the glass stopper. Centrifuge 10-15 minutes at 2,000-2,500 r.p.m. to separate the white solid from the liquid in the tube. Inject 0.5 microliter of the clear liquid into the gas chromatograph. Use the conditions and materials listed in paragraphs (a), (b), and (c) of this section. The conditions should be adequate to maintain a stable baseline and provide at least 60 percent deflection of the recorder scale by the clindamycin peak. The resolution of the peaks should be complete. The elution order is: Internal standard, clindamycin, and epiclindamycin (if present). Calculate the clindamycin content as directed in paragraph (f) of this section.

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

ER01JA93.031