where: m=moisture content of the sample. § 436.301 Thin layer chromatography identity test for carbenicillin indanyl. Using the sample solution prepared as described in the section for the antibiotic drug to be tested, proceed as described in paragraphs (a), (b), (c), and (d) of this section. Chromatography (a) Equipment-(1) tank. A rectangular tank, approximately 9 × 9 × 3.5 inches lined with Whatman's 3MM chromatographic paper (0.3 millimeters) or equivalent. (2) Iodine vapor chamber. A rectangular tank approximately 9 × 9 × 3.5 inches, with a suitable cover, containing iodine crystals. (3) Plates. Use 20 × 20 centimeters thin layer chromatography plates coated with silica gel G or equivalent to a thickness of 250 microns. (b) Reagents-(1) Extraction solvent. Mix ethyl acetate, acetone, pyridine, water, and acetic acid in volumetric proportions of 100:200:25:75:1.5 respectively. (2) Developing solvent. Mix ethyl acetate, acetone, pyridine, water, and acetic acid in volumetric proportions of 300:400:25:75:2 respectively. (3) Ferric chloride-potassium ferricyanide reagent. Immediately before use, mix 100 milliliters of a 1 percent ferric chloride solution in 1 percent hydrochloric acid with 100 milliliters of a 1 percent potassium ferricyanide solution and 75 milliliters of methanol. (c) Preparation of working standard solution. Weigh an amount of the carbenicillin indanyl working standard equivalent to approximately 10 milligrams of carbenicillin into a 50-milliliter Erlenmeyer flask. Dissolve the material in sufficient extraction solvent to make a solution containing 1 milligram carbenicillin per milliliter. (d) Procedure. Pour developing solvent into the bottom of the chromatography tank. Cover and seal the tank. Allow it to equilibrate for 1 hour. Prepare a plate as follows: On a line 2 centimeters from the base of the silica gel plate, and at intervals of 2 centimeters, spot 10 microliters of the standard solution and the sample solution. The plate should be air dried for 30 minutes. Place the plate into the chromatography tank. Allow the solvent front to travel about 15 centimeters from the starting line and then remove the plate from the tank. Heat the plate for 30 minutes at 80° C. in a circulating air oven and then allow the plate to cool to room temperature. Place the plate in the iodine vapor chamber for about 30 seconds, remove the plate and spray it with the ferric chloride-potassium ferricyanide reagent. Carbenicillin indanyl appears as a blue spot on a yellow-green background at an Rƒ of about 0.5. The test is satisfactory if the sample compares qualitatively with the standard. [39 FR 18944, May 30, 1974, as amended at 41 FR 18509, May 5, 1976] (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 x 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: 2×10-8 amperes. (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: Micrograms of clindamycin_R1 × W × ƒ per milligram where: RxW И Ru-Area of the clindamycin sample peak (at a retention time equal to that observed for the clindamycin standard)/ Area of internal standard peak; R=Area of the clindamycin standard peak/ Area of internal standard peak; W.-Weight of the clindamycin working standard in milligrams; Wu-Weight of the sample in milligrams; f=Potency of the clindamycin working standard in micrograms per milligram. § 436.303 Clindamycin content of clindamycin palmitate hydrochloride by vapor phase chromatography. (a) Equipment. Gas chromatograph equipped with a flame ionization detector: Hewlett-Packard 76064 or equivalent. (b) Reagents. (1) Acetic anhydride, reagent grade. 4 Available from: Hewlett Packard Co., P.O. Box 301, Loveland, CO 80537. (2) Pyridine, reagent grade. (4) Internal standard: Prepare a solution containing 5 milligrams of cholesteryl benzoate per milliliter in chloroform. (c) Typical conditions. (1) Column: 6 feet x 2 millimeters ID, glass, with 1 percent UC-W98 on Chromosorb WHP (80/100 mesh) or equivalent. (2) Temperatures: Column 275° C.; detector 290° C.; injection port 280° C. (3) Carrier gas: Helium approximately 60 milliliters per minute. (4) Detector: Hydrogen flame ionization-hydrogen at 12 pounds per square inch, air at 32 pounds per square inch. (5) Sensitivity: 1,000; attenuation, 16; 1 x 10-9 amperes. (d) Preparation of clindamycin palmitate hydrochloride sample and working standard solutions. Accurately weigh approximately 15 milligrams of both the sample and the working standard into separate glass-stoppered, conical 15-milliliter centrifuge tubes. Add 1.0 milliliter of internal standard solution, 1.0 milliliter of pyridine, and 0.5 milliliter of acetic anhydride to each tube. Agitate the tubes to insure dissolution 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 1 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 40 percent deflection of the recorder scale by the clindamycin palmitate peak. The resolution of the peaks should be complete. The internal standard will be eluted before the clindamycin palmitate. Calculate the clindamycin content as directed in paragraph (f) of this section. § 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 x 10-10 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 x 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 Helium approximately 60 milliliters per minute. 4 See footnote 4 to §436.303(a). (4) Detector: Hydrogen flame-hydrogen flow at 40 milliliters per minute. Air flow at 400 milliliters per minute. (5) Sensitivity: 1 x 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 35milliliter 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 1dram 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 5 Defined such that 50 units hydrolyzes at least 20 micromoles of a clindamycin phosphate authentic sample under the assay conditions described in this section. 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: Ru-Area of the clindamycin sample peak (at a retention time equal to that observed for the clindamycin standard)/ Area of internal standard peak; Ru-Area of the clindamycin standard peak/ Area of internal standard peak; W, Weight of the clindamycin working standard in milligrams; Wu-Weight of the sample in milligrams; f=Potency of the clindamycin working standard in micrograms per milligram. [39 FR 18944, May 30, 1974, as amended at 41 FR 24704, June 18, 1976] § 436.305 Thin layer chromatographic identity test for hetacillin. (a) Equipment-(1) Chromatography tank. A rectangular tank, approximately 9 × 9 × 3.5 inches with a glass solvent trough on the bottom. (2) Plates. Use 20 × 20 centimeter thin layer chromatography plates coated with Silica Gel G or equivalent to a thickness of 250 microns. (b) Developing solvent. Mix 650 milliliters acetone with 100 milliliters distilled water, 100 milliliters benzene, and 25 milliliters acetic acid. (c) Spray solution. Dissolve 300 milligrams of ninhydrin in 100 milliliters of ethanol. (d) Preparation of spotting solutions— (1) Sample solution. Use the sample solution prepared as described in the section for the particular product to be tested. (2) Reference solutions. Prepare a solution containing 10 milligrams of an authentic hetacillin sample per milliliter in a 4:1 solution of acetone and 0.1N hydrochloric acid, and a solution of ampicillin standard at 1 mg/ml in the same solvent. (e) Procedure. Spot a plate as follows: Apply approximately 10 microliters of the sample solution, 1 μ 1, of the reference hetacillin solution, and 1 μ 1, of the ampicillin reference solution on a line 1.5 centimeters from the base of the silica gel plate and at intervals of not less than 2.0 centimeters. Pour developing solvent into the glass trough in the bottom of the chromatography tank. After all spots are thoroughly dry, place the silica gel plate directly into the glass trough of the chromatography tank. Cover and seal the tank. Allow the solvent front to travel about 11.5 centimeters from the bottom of the plate, remove the plate from the tank, and allow to air dry. Apply the spray solution (do not saturate) and place immediately into an oven maintained at 90° C. Heat 15 minutes. (f) Evaluation. Measure the distance the solvent front traveled from the starting line and the distance the spots are from the starting line. Calculate the R value by dividing the latter by the former. The sample and standard should have spots of corresponding Rf values. [39 FR 18944, May 30, 1974, as amended at 45 FR 16472, Mar. 14, 1980] (3) Carrier gas: Helium at 15 pounds per square inch. (4) Detector: Hydrogen flame ionization-hydrogen at 20 pounds per square inch, air at 40 pounds per square inch. (5) Sensitivity: 100; attenuation 2; current 2 × 10-8 amperes. (d) Preparation of lincomycin sample and working standard solutions. Prepare the sample and working standard as follows: Weigh accurately an aliquot of about 40 milligrams into a 10-milliliter volumetric flask, add sufficient pyridine to dissolve, and make to mark. Transfer a 1-milliliter aliquot to a glass-stoppered conical centrifuge tube and proceed as directed in paragraph (e) of this section. (e) Procedure. Add 0.2 milliliter of the silylating reagent to each centrifuge tube and allow to stand at least 30 minutes. Then add exactly 1 milliliter of the internal standard, shake well, and centrifuge. Inject 5 microliters of the supernatant into the gas chromatograph. Use the typical conditions and materials listed in paragraphs (a), (b), and (c) of this section. The conditions should be adequate to provide at least 60 percent scale deflection with the lincomycin peak and to maintain a stable base line. The resolution of the peaks should be complete. The elution order is lincomycin B, lincomycin, and the internal standard. If necessary, adjust the current setting for the lincomycin B peak to give a satisfactory response relative to that of the lincomycin peak. Calculate the lincomycin content and lincomycin B content as directed in paragraph (f) of this section. R=Area of the lincomycin standard peak/ Wu-Weight of the sample in milligrams; Percent lincomycin where: B content B ×100 A+ B A=Area of lincomycin peak of the sample; B=Area of lincomycin B peak of the sample corrected for the attenuation adjustment. [39 FR 18944, May 30, 1974, as amended at 46 FR 3839, Jan. 16, 1981] § 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: Hexamethyldisilazane. (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 25milliliter 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 |