(d) Procedure. Pour the developing solvent into the glass trough on the bottom of the tank and onto the paper lining the walls of the 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 each of the standard solution and the sample solution. After all spots are thoroughly dry, place the silica gel plate directly into the glass trough. Cover and seal the tank. Allow the solvent front to travel about 15 centimeters from the starting line. Remove the plate from the tank and air dry. Expose the plate to iodine vapors for 40 minutes. Immediately circumscribe all spots using a suitable marker.

(e) Evaluation. Measure the distance the solvent front traveled from the starting line and the distance the spots are from the starting line. Calculate the Rf value by dividing the latter by the former. The sample and standard should have spots of corresponding Rf values and intensity.

[46 FR 61070, Dec. 15, 1981, as amended at 49 FR 2242, Jan. 19, 1984]

(a) Equipment. A high-pressure liquid chromatograph equipped with:

(1) A low dead volume cell 8 to 20 microliters;

(2) A light path length of 1 centimeter;

(3) A suitable ultraviolet detection system operating at a wavelength of 254 nanometers;

(4) A suitable recorder of at least 25.4-centimeter deflection;

(5) A suitable integrator;

(6) A 25-centimeter column having an inside diameter of 4.6 millimeters and packed with octadecyl silane chemically bonded to porous silica or ceramic microparticles, 5 to 10 micrometers in diameter (United States Pharmacopeia XX).

(b) Reagents. (1) 0.2M monobasic sodium phosphate: Dissolve 27.60 grams of monobasic sodium phosphate with sufficient water to make 1,000 milliliters.

(2) 10 percent tetrabutylammonium hydroxide in water.

(3) Ampicillin-piperacillin solution: Dissolve and dilute 25 milligrams of ampicillin and 5 milligrams of piperacillin monohydrate with sufficient mobile phase to obtain 100 milliliters, and mix.

(c) Mobile phase. Methanol:water:0.2M monobasic sodium phosphate:10 percent tetrabutylammonium hydroxide (450: 447: 100:3) adjusted to pH 5.5±0.02 with phosphoric acid. The concentration of reagents may be varied to obtain acceptable operation of the system. De-gas the mobile phase just prior to its introduction into the the chromatograph pumping system.

(d) Preparation of working standard and sample solutions—(1) Working standard solution. Place approximately 20 milligrams of the working standard, accurately weighed, into a 50-milliliter volumetric flask. Add 25 to 30 milliliters of mobile phase. Shake until dissolved. Dilute to volume with mobile phase.

(2) Sample solution—(i) Micrograms per milligram. Place approximately 20 milligrams of the sample, accurately weighed, into a 50-milliliter volumetric flask. Add 25 to 30 milliliters of mobile phase. Shake until dissolved. Dilute to volume with mobile phase.

(ii) Milligrams per vial. Reconstitute as directed in the labeling. Withdraw the total contents and dilute with mobile phase to a concentration of 0.4 milligram of piperacillin per milliliter.

(e) Procedure. Use the equipment, reagents, mobile phase, and working standard and sample solutions described in paragraphs (a), (b), (c), and (d) of this section and proceed as directed in paragraph (e) of this section.

(1) Systems suitability test. Chromatograph three replicate samples of ampicillin-piperacillin solution as directed in paragraph (e)(2) of this section. Allow an elution time sufficient to obtain satisfactory separation of expected components after each injection. Record the peak responses and calculate the resolution factor as described for system suitability tests in the United States Pharmacopeia XX General Chapter 621 for gas chromatography. The resolution factor between ampicillin and piperacillin is not

less than 15. If the resolution factor does not meet this limit, adjustments must be made to the system to obtain satisfactory operation before proceeding as described in paragraph (e)(2) of this section.

(2) Determination of the chromatogram. Operate the high-pressure liquid chromatograph at ambient temperature at a flow rate of one milliliter per minute. Use a detector sensitivity setting that gives a peak height for the reference standard that is at least 50 percent of scale. Purge the column with mobile phase until a steady baseline is estab

lished. Inject 10 microliters of the working standard solution into the chromatograph. Allow an elution time sufficient to obtain separation of the expected components. After separation of the working standard solution has been completed, inject 10 microliters of the sample solution into the chromatograph and repeat the procedure described for the working standard solution.

(f) Calculations-(1) Calculate the piperacillin content in micrograms per milligram as follows:

Micrograms of piperacillin per milligram of sample

where:

A=Area of the sample peak (at a retention time equal to that observed for the standard);

B=Area of the standard peak.

(2) Calculate the piperacillin content in grams per vial as follows:

where:

A=Area of the sample peak (at a retention time equal to that observed for the standard);

B=Area of the standard peak;

d=Dilution factor.

[47 FR 15768, Apr. 13, 1982; 47 FR 33493, Aug. 3, 1982]

§ 436.335 High-pressure

liquid chromatographic assay for chloramphenicol palmitate.

(a) Equipment. A suitable high-pressure liquid chromatograph equipped with:

(1) A low dead volume cell 8 to 20 microliters;

(2) A light path of 1 centimeter;

(3) A suitable ultraviolet detection system operating at a wavelength of 280 nanometers;

(4) A suitable recorder of at least 25.4-centimeter deflection;

(5) A suitable integrator; and

B×1,000 × 1,000

(6) A 30-centimeter column having an inside diameter of 4.0 millimeters and packed with octadecyl silane chemically bonded to porous silica or ceramic microparticles, 5 to 10 micrometers in diameter, U.S.P. XX. (b)

Mobile phase. Mix methanol:water:glacial acetic acid (170:30:1). Degas the mobile phase just prior to its introduction into the chromatograph pumping system.

(c) Operating conditions. Perform the assay at ambient temperature with a typical flow rate of 2.0 milliliters per minute. Use a detector sensitivity setting that gives a peak height for the reference standard that is at least 50 percent of scale. The minimum between peaks must be no more than 2 millimeters above the initial baseline.

(d) Preparation of sample and working standard solutions. Accurately weigh approximately 65 milligrams of sample

or chloramphenicol palmitate working standard each into a 50-milliliter volumetric flask. Add approximately 35 milliliters of methanol and 1 milliliter of glacial acetic acid. Place in an ultrasonic bath for 10 minutes and dilute to volume with methanol.

(e) Procedure. Using the equipment, mobile phase, and operating conditions listed in paragraphs (a), (b), and (c) of this section, inject 10 microliters of the working standard solution into the chromatograph. Allow an elution time sufficient to obtain satisfactory separation of expected components. After separation of the working standard solution has been completed, inject 10 microliters of the sample solution into the chromatograph and repeat the procedure described for the working standard solution.

(f) Calculations. Calculate the chloramphenicol content as follows:

§ 436.336 Thin layer chromatographic identity test for azlocillin.

(a) Equipment-(1) Chromatography tank. A rectangular tank, approximately 23 centimeters long, 23 centimeters high, and 9 centimeters wide, equipped with a glass solvent trough in the bottom and a tight-fitting cover for the top.

(2) Iodine vapor chamber. A rectangular tank approximately 23 centimeters long, 23 centimeters high, and 9 centimeters wide, with a suitable cover, containing iodine crystals.

(3) Plates. Use 20 x 20 centimeter thin layer chromatography plates coated with Silica Gel G or equivalent to a thickness of 250 microns.

(b) Reagents-(1) Buffer. Dissolve 9.078 grams of potassium phosphate, monobasic (KH2PO1) in sufficient dis

tilled water to make 1,000 milliliters (solution A). Dissolve 17.88 grams of sodium phosphate, dibasic, heptahydrate (Na2HPO4.7H2 O) in sufficient distilled water to make 1,000 milliliters (solution B). Place 12.1 milliliters of solution B into a 100-milliliter volumetric flask and dilute to volume with solution A.

(2) Developing solvent. Place 50 milliliters of n-butyl acetate, 9 milliliters of n-butanol, 25 milliliters of glacial acetic acid, and 15 milliliters of buffer into a separatory funnel. Shake well and allow the layers to separate. Discard the lower phase and use the upper phase as the developing solvent.

(c) Preparation of spotting solutions. Prepare solutions of the sample and working standard, each containing 20 milligrams of azlocillin per milliliter in distilled water.

(d) Procedure. Pour developing solvent into the glass trough on the bottom of the chromatography tank to a depth of about 1 centimeter. Use the chamber immediately. Prepare plate as follows: Apply spotting solutions on a line 2.5 centimeters from the base of the silica gel plate and at points 2.0 centimeters apart. Apply approximately 10 microliters of the working standard solution to points 1 and 3. When these spots are dry, apply approximately 10 microliters of sample solution to points 2 and 3. Place spotted plate in a desiccator until solvent has evaporated from spots. Place the plate into the glass trough at the bottom of the chromatography tank. Cover the tank. Allow the solvent to travel about 15 centimeters from the starting line. Remove the plate from the tank and allow to air dry. Warm the iodine vapor chamber to vaporize the iodine crystals and place the dry plate in the iodine vapor chamber until the spots are visible, usually about 10 minutes.

(e) Evaluation. Measure the distance the solvent front traveled from the starting line and the distance the spots are from the starting line. Calculate the Rf value by dividing the latter by the former. The azlocillin sample and the standard should have spots of corresponding Rf values (approximately 0.4), and standard and sample combined should appear as a single spot for

(a) Equipment. A suitable high-pressure liquid chromatograph equipped with:

(1) A low dead volume cell 8 to 20 microliters;

(2) A light path length of 8 millimeters;

(3) A suitable ultraviolet detection system operating at a wavelength of 254 nanometers;

(4) A suitable recorder that is compatible with the detector output;

(5) A suitable integrator (optional); and

(6) A 25-centimeter column having an inside diameter of 4.6 millimeters and packed with octadecyl silane chemically bonded to porous silica or ceramic microparticles, 10 micrometers in diameter, U.S.P. XX.

(b) Reagents. (1) 4 percent glacial acetic acid.

(2) 3.86 percent sodium acetate.

(c) Mobile phase. 4 percent glacial acetic acid:3.86 percent sodium acetate:methanol:distilled water (3:15:200:782). Filter the mobile phase through a suitable glass fiber filter or equivalent that is capable of removing particulate contamination to 1 micron in diameter. Degas the mobile phase prior to its introduction into the chromatograph pumping system. The distilled water:methanol ratio may be varied to obtain acceptable operation of the system.

(d) Operating conditions. Perform the assay at ambient temperature with a typical flow rate of 1.2 milliliters per minute. Use a detector sensitivity setting that gives a peak height for the cephradine in the cephradine working standard that is about 75 percent of full scale.

(e) Preparation of working standard and sample solutions—(1) Preparation of cephradine working standard solution. Place an accurately weighed portion of the cephradine working standard into a

suitably sized container. Add 5.0 milliliters of distilled water and place in an ultrasonic bath to facilitate dissolution. Dilute with a sufficient amount of mobile phase to obtain a solution containing 0.8 milligram of cephradine activity per milliliter.

(2) Preparation of cephalexin working standard solution. Dissolve an accurately weighed portion of the cephalexin working standard with mobile phase to obtain a solution containing 0.02 milligram of cephalexin activity per milliliter. Place in an untrasonic bath to facilitate dissolution.

(3) Preparation of sample solutions—(i) Product not packaged for dispensing (micrograms of cephradine per milligram). Dissolve an accurately weighed portion of the sample with mobile phase to obtain a solution containing 0.8 milligram per milliliter. Place in an ultrasonic bath to facilitate dissolution. Using this sample solution, proceed as directed in paragraph (f)(1) of this section.

(ii) Product packaged for dispensing. Determine both micrograms of cephradine per milligram of the sample and milligrams of cephradine per container. Use separate separate containers for preparation of each sample solution as described in paragraphs (e)(3)(ii) (a) and (b) of this section.

(a) Micrograms of cephradine per milligram. Dissolve an accurately weighed portion of the sample with mobile phase to obtain a solution containing 0.8 milligram per milliliter. Place in an ultrasonic bath to facilitate dissolution. Using this sample solution, proceed as directed in paragraph (f)(1) of this section.

(b) Milligrams of cephradine per container. Reconstitute the sample as directed in the labeling. Then, using a suitable hypodermic needle and syringe, remove all of the withdrawable contents if it is represented as a singledose container; or, if the labeling specifies the amount of potency in a given volume of the resultant preparation, remove an accurately measured representative portion from each container. Dilute the solution thus obtained with mobile phase to obtain a solution containing 0.8 milligram per milliliter. Using this sample solution,

proceed as directed in paragraph (f)(1) of this section.

an

(f) Procedure—(1) Cephradine content. Using the equipment, reagents, mobile phase, and operating conditions as listed in paragraphs (a), (b), (c), and (d) of this section, inject 10 microliters of the cephradine working standard solution into the chromatograph. Allow elution time sufficient to obtain satisfactory separation of the expected components. After separation of the working standard solution has been completed, inject 10 microliters of the sample solution prepared as described in paragraph (e)(3)(i) of this section into the chromatograph and repeat the procedure described for the working standard solution. The elution order is void volume, cephalexin, and cephradine. If the sample is packaged for dispensing, repeat the procedure for each sample solution prepared as described in paragraphs (e)(3)(ii) (a) and (b) of this section.

(2) Cephalexin content. Proceed as directed in paragraph (f)(1) of this section, except:

(i) Use a detector sensitivity setting that gives a peak height for the cephalexin in the cephalexin working standard that is about 75 percent of full scale; and

(ii) Use the cephalexin working standard in lieu of the cephradine working standard.

(g) Calculations. (1) Calculate the micrograms of cephradine per milligram of sample as follows:

Micrograms of cefoperazone per milligram milligram

A XP, x100

A, XCX(100 m)

A. Area of the cephalexin peak in the chromatogram of the sample (at a retention time equal to that observed for the standard);

A=Area of the cephalexin peak in the chromatogram of the cephalexin working standard;

W1-Milligrams of cephalexin per milliliter of cephalexin working standard solution; W=Milligrams of cephradine per milliliter of sample solution;

P-Micrograms of cephalexin per milligram of cephalexin working standard; and m=Percent moisture content of the sample. [49 FR 47483, Dec. 5, 1984]

(a) Equipment. A suitable high-pressure liquid chromatograph equipped with:

(1) A low dead volume cell 8 to 20 microliters;

(2) A light path length of 1 centimeter;

(3) A suitable ultraviolet detection system operating at a wavelength of 254 nanometers;

(4) A suitable recorder of at least 25.4 centimeter deflection;

(5) A suitable integrator;

(6) A 30-centimeter column having an inside diameter of 4.0 millimeters and