§ 436.307 - Spectinomycin vapor phase chromatography.

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

(b) Reagents. (1) Dimethylformamide, reagent grade, kept dry over anhydrous sodium sulfate.

(2) Internal standard: Prepare a solution containing 2 milligrams of triphenylantimony per milliliter in dry dimethylformamide.

(3) Silylating reagent: Hexamethyl-disilazane.

(c) Typical conditions. (1) Column: 4 feet by 4 millimeters ID, glass, with 5 percent SE-52 on Diatoport S (80/100 mesh), or equivalent.

(2) Temperatures: Column 215° C.; detector 270° C.; injection port 265° C.

(3) Carrier gas: Helium 93 milliliters per minute at 15 pounds per square inch.

(4) Detector: Hydrogen flame—hydrogen at 20 pounds per square inch, air at 40 pounds per square inch.

(5) Sensitivity: 1,000; attenuation, 10 for both spectinomycin and internal standard; 2 × 10− 6 amperes.

(d) Preparation of spectinomycin sample and working standard—(1) Working standard and bulk antibiotic solutions. (i) Accurately weigh approximately 30 milligrams of sample or working standard into separate glass-stoppered 25-milliliter Erlenmeyer flasks.

(ii) Add 10 milliliters of the internal standard solution and 1.0 milliliter of hexamethyldisilazane to each flask. Agitate the flasks to insure dissolution of the sample and working standard and complete mixing of the liquids. Shake the flasks intermittently for 1 hour. Proceed as directed in paragraph (e) of this section.

(2) Finished product solutions. Prepare the sample for assay as directed in the individual section for each antibiotic product to be tested.

(e) Procedure. Inject 2.5 microliters of each solution 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 base line and provide at least 60 percent deflection of the recorder scale by the spectinomycin peak. The resolution of the peaks should be complete. The internal standard will be eluted before spectinomycin. Calculate the spectinomycin content as directed in paragraph (f) of this section.

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

ER01JA93.035