(a) Equipment. A suitable high pressure liquid chromatograph, such as a Waters Associates Model 2441 or equivalent 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 30-centimeter column having an inside diameter of 4.0 millimeters and packed with a suitable reverse phase packing such as: Waters Associates, Micro-Bondapak C18;1 and

(6) A suitable integrator.

(b) Reagents-(1) Mobile phase. Mix acetonitrile (high pressure liquid chromatography grade): water (40:60). Filter the mobile phase through a suitable glass fiber filter or equivalent which is capable of removing particulate contamination to 1 micron in diameter. De-gas the mobile phase just prior to its introduction into the chromatograph pumping system.

(2) Internal standard solution. Dissolve 0.33 milligram of benzanilide per milliliter in acetonitrile (high pressure liquid chromatography grade). Filter the solution through a suitable glass fiber filter or equivalent which is capable of removing particulate contamination to 1 micron in diameter.

(c) 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 ref

erence standard that is at least 50 percent of scale. The minimum between peaks must be no more than 2 millimeters above the baseline.

(d) Preparation of the working standard and sample solutions—(1) Working standard solution. Prepare a solution containing 1.0 milligram per milliliter of sulfisoxazole acetyl in the internal standard solution.

(2) Sample solution. Reconstitute the sample as directed in the labeling. Allow to stand for 1 hour. Shake gently and transfer 5.0 milliliters of the sample to a separatory funnel. Extract the suspension three times with 75-milliliter portions of chloroform. Collect the chloroform layers in a 250-milliliter volumetric flask. Dilute the flask to volume with chloroform and mix. Filter a portion of the solution through a suitable glass fiber filter or equivalent which is capable of removing particulate contamination to 1 micron in diameter. Transfer a 4.0-milliliter aliquot of the filtrate into a 25-milliliter glass-stoppered flask and evaporate to dryness under a stream of dry air. Dissolve the residue in 10.0 milliliters of the internal standard solution, stopper, and mix.

(e) Procedure. Using the equipment, reagents, and operating conditions listed in paragraphs (a), (b), and (c) of this section, inject 5 microliters of sample or working standard solution prepared as described in paragraph (d) of this section, into the chromatograph. Allow an elution time sufficient to obtain satisfactory separation of expected components. The elution order is void volume, sulfisoxazole acetyl and benzanilide.

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

(a) Equipment. A suitable high-pressure liquid chromatograph, such as a Waters Associates Model 2441 or equivalent 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 340 nanometers;

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

(5) A suitable integrator;

(6) A column approximately 25 centimeters in length having an inside diameter of approximately 4 millimeters and packed with a suitable reversephase packing such as: 10 micrometer silica gel particles bonded to octadecyl silane, Vydac 201 TP Reverse Phase 2 or equivalent.

(b) Reagents-(1) 0.001M Ammonium (ethylenedinitrilo) tetraacetate. Moisten 293 milligrams of (ethylenedinitrilo) tetraacetic acid with 1 milliliter of methanol and dissolve in 7 milliliters of concentrated ammonium hydroxide. Dilute to 900 milliliters with distilled water, adjust the pH to 6.6 with glacial acetic acid, and dilute to 1,000 milliliters with distilled water.

(2) Mobile phase. Mix 150 milliliters of tetrahydrofuran (high-pressure liquid chromatography grade) with 850 milliof ammonium (ethylenedinitrilo) tetraacetate. Filter

liters

0.001M

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 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 0.8 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. 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 an amount of sample or working standard equivalent to approximately 25 milligrams of meclocycline into a 50-milliliter volumetric flask. Dissolve and dilute to volume with methanol and mix. Transfer exactly 3.0 milliliters of this solution to a 25-milliliter volumetric flask, dilute to volume with mobile phase, and mix.

(e) Procedure. Using the equipment, reagents, and operating conditions listed in paragraphs (a), (b), and (c) of this section, inject 10 microliters of the sample or working standard solution prepared as described in paragraph (d) of this section into the chromatograph. Allow an elution time sufficient to obtain satisfactory separation of expected components. The elution order is void volume, oxytetracycline (if present), demeclocycline (if present), methacycline (if present), and meclocycline.

(f) Calculations. Calculate meclocycline content as follows:

the

(2) Plates. Use 20 × 20 centimeter thin layer chromatography plates coated with Silica Gel 60F 254 or equivalent to a thickness of 250 microns.

(b) Reagents-(1) Developing solvent. Mix methylene chloride, chloroform, and 95 percent ethyl alcohol in volumetric proportions of 100:10:10, respectively.

(2) Spray solution. Dissolve 1 gram of ninhydrin in 100 milliliters of n-butanol and add 1 milliliter of pyridine.

(c) Spotting solutions-(1) Preparation of working standard solution. Dissolve and dilute a weighed amount of the bacampicillin hydrochloride working standard with sufficient 95 percent ethyl alcohol to obtain a solution containing 2 milligrams per milliliter.

(2) Preparation of sample solution. Dissolve and dilute a weighed amount of the sample with sufficient 95 percent ethyl alcohol to obtain a solution containing 2 milligrams per milliliter. Proceed as described in paragraphs (d) and (e) of this section.

(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 one hour. Prepare a plate as follows: On a line 2.5 centimeters from the base of the thin layer chromatography plate and at intervals of 2.0 centimeters, spot 5 microliters of the working standard solution to positions 1 and 3. When these spots are dry, apply 5 microliters of the sample solution to points 2 and 3. After all the spots are thoroughly dry, place the plate into the trough in the bottom of the tank. Cover and tightly seal the tank, allow the solvent front to travel about 15 centimeters from the starting line (about 30 minutes) and then remove the plate from the tank. Air dry the plate. Visualize the spots by spraying with spray solution and heating in an oven at 100° C for approximately 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. Divide the latter by the former to calculate the R value. Bacampicillin appears as a purple spot at an R, value of approximately 0.52. The test is satisfactory if the R value of the sample compares with that of the working standard. The combined spot should appear as a single spot of corresponding R, value.

[46 FR 25602, May 8, 1981, as amended at 49 FR 2242, Jan. 19, 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; and

(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 micrometers to 10 micrometers in diameter, U.S.P. XX.

(b) Mobile phase. Mix acetonitrile (high-pressure liquid chromatography grade): water (60:40). 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 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 2.5 milliliters per minute. Use a detector sensitivity setting that gives a peak height for the working 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 working standard and sample solutions—(1) Preparation of

working standard solution. Prepare a solution containing 0.25 milligram per milliliter of dactinomycin in mobile phase.

(2) Preparation of sample solution. Prepare the sample solution as described in the individual monograph for the drug being tested.

(e) Procedure. Use the equipment, mobile phase, operating conditions, and working standard and sample solutions described in paragraphs (a), (b), (c), and (d) of this section, and proceed as directed in paragraph (e)(1) of this section.

(1) System suitability test. Equilibrate and condition the column by passage of about 10 to 15 void volumes of mobile phase followed by two or more replicate injections of 10 microliters each of the working standard solution. Allow an elution time sufficient to obtain satisfactory separation of expected components after each injection. Record the peak responses and, calculate the relative standard deviation as described for system suitability tests in the U.S.P. XX General Chapter 621 chromatography. Proceed as directed in paragraph (e)(2) of this section if the minimum performance requirement for the relative standard deviation is not more than 1.0 percent. If the minimum performance requirement is not met, adjustment 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. Inject 10 microliters of the working standard solution into the chromatograph. Allow an 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 into the chromatograph and repeat the procedure described for the working standard solution.

(f) Calculations. Calculate the dactinomycin content as described in the individual monograph for the drug being tested.

[49 FR 24017, June 11, 1984, as amended at 50 FR 5749, Feb. 12, 1985]

(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 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;

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

(6) A suitable integrator.

(b) Mobile phase. Mix 0.01M ammonium acetate:methanol (19:1). 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 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 0.5 milliliter per minute. Use a detector sensitivity setting that gives a peak height for the working standard that is at least 50 percent of scale.

(d) Preparation of working standard solution. Transfer the contents of an ampoule of working standard to a tared weighing bottle. Place the unstoppered weighing bottle in a desiccator containing a saturated aqueous solution of potassium carbonate to provide an atmosphere of 42 percent relative humidity. Allow the moisture content of the working standard to equilibrate for 16 hours. Determine the moisture content as described in § 436.201 of this chapter. Equilibrated standard material must be kept in a closed weighing bottle and used within 36 hours of equilibration. Dissolve approximately 50 milligrams of the working standard, accurately weighed and corrected for moisture, with sufficient distilled water to obtain a solution containing 0.5 milligram of

moxalactam per milliliter. Use the prepared solution immediately.

(e) Preparation of sample solution. Mix contents of vial thoroughly. Dissolve an accurately weighed portion of approximately 50 milligrams of sample with distilled water to obtain a concentration of 0.5 milligram per milliliter (estimated); also, 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 single dose 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. Further dilute an aliquot of this solution with distilled water to obtain a concentration of 0.5 milligram per milliliter (estimated). Use the prepared solution immediately.

(f) Procedure. Using the equipment, reagents, and operating conditions as listed in paragraphs (a), (b), and (c) of this section, inject 5 microliters of the working standard solution into the chromatograph. Allow an elution time sufficient to obtain satisfactory separation of the expected components. After separation of the working standard solution has been completed, inject 5 microliters of the sample solution into the chromatograph and repeat the procedure described for the working standard solution.

Ratio of R-isomer to S-isomer

[46 FR 61069, Dec. 15, 1981]

§ 436.333 Thin layer chromatographic identity test for moxalactam.

(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. Line the inside walls of the tank with Whatman #3MM chromatographic paper or equivalent.

Area of the R-isomer peak Area of the S-isomer peak

(2) Plates. Use a 20x20 centimeter thin layer chromotagraphy plate coated with silca gel G or equivalent to a thickness of 250 micrometers.

(b) Developing solvent. Mix ethyl acetate, glacial acetic acid, acetonitrile, and water in volumetric proportions of 42:14:14:18, respectively.

(c) Preparation of spotting solutions. Prepare solutions of the sample and working standard, each containing 10 milligrams per milliliter of moxalactam in distilled water.