A: Infrared Absorption 197K.

B: The retention time of the major peak in the chromatogram of the Assay preparation corresponds to that in the chromatogram of the Standard preparation, as obtained in the Assay.

Test solution: 10 mg per mL, in methanol.

Sulfuric acid solution— Carefully add 50 mL of sulfuric acid to 450 mL of water, and mix.

Standard solution— Prepare a solution in Sulfuric acid solution having a concentration of about 2.0 µg of boron per mL. Use of a commercially prepared boron ICP standard solution is recommended.

Test solution— Accurately weigh approximately 1.0 g of Zileuton into a 125-mL conical flask. Add 1 to 1.5 mL of sulfuric acid, and digest in a fume hood on a hot plate until charring begins. Add 2 mL of nitric acid to the cooled sample to aid digestion, and heat until brown fumes are not evolved. Cautiously add 30 percent hydrogen peroxide, dropwise, allowing the reaction to subside, and heating between drops. Add the first few drops very slowly with sufficient mixing in order to prevent a rapid reaction. Discontinue heating if foaming becomes excessive. When the reaction has abated, heat cautiously, rotating the flask occasionally to prevent the sample from caking on glass exposed to the heating unit. Maintain oxidizing conditions at all times during the digestion by adding small quantities of the 30 percent hydrogen peroxide, whenever the mixture turns brown or darkens. Approximately 1 to 2 mL of nitric acid can be added, if necessary, which will create a refluxing effect to wash down any particles adhering to the neck of the flask. Continue the digestion until the organic matter is destroyed, gradually raising the temperature of the hot plate until fumes of sulfur trioxide are copiously evolved and the solution becomes colorless or retains only a light straw color. Transfer the solution to a 25-mL volumetric flask using about 7 mL of water. Repeat the washing twice more, and combine the washings in the volumetric flask. Dilute with water to volume, and mix.

Procedure— The inductively coupled plasma-atomic emission spectrometer is set up with wavelength of 249.7 nm, RF power of 1.25 KW, argon torch flow of about 13 L per minute, argon nebulizer flow of about 1 L per minute, and argon auxillary flow of about 0.5 L per minute. Analyze the Standard solution and the Test solution, using Sulfuric acid solution as the blank. Calculate the quantity, in µg of boron per g, in the portion of Zileuton taken by the formula: where C is the concentration, in µg per mL, of boron in the Test solution determined from the instrument; and W is the weight, in g, of the Zileuton: not more than 10 µg per g is found.

Standard solution— Dissolve an accurately weighed quantity of pyridine, approximately 250 mg, in dimethyl sulfoxide, and dilute with dimethyl sulfoxide to 50 mL. Transfer 5 µL to a 100-mL sealed headspace vial.

Test solution, Chromatographic system, and Procedure— Proceed as directed for Test Solution, Chromatographic System, and Procedure under Organic Volatile Impurities, Method IV 467. Separately inject the Standard solution and the Test solution into the gas chromatograph, and record the peak responses. Calculate the quantity, in ppm, of pyridine in the portion of Zileuton taken by the formula: in which rU and rS are the peak responses for pyridine in the Test solution and the Standard solution, respectively; WS is the weight, in mg, of pyridine used to prepare the Standard solution; and WU is the weight, in mg, of Zileuton: not more than 100 ppm is found.

TEST 1—

TEST 2—

(Official January 1, 2007)

NOTE—The Standard preparation and the Assay preparation are to be refrigerated at or below 5 immediately after preparation and during analysis using a refrigerated autosampler. The solutions are stable at or below 5 for about 36 hours.

Buffer solution— Dissolve 7.7 g of ammonium acetate and 0.25 g of acetohydroxamic acid in about 900 mL of water in a 1000-mL volumetric flask, adjust with perchloric acid to a pH of 2.0, dilute with water to volume, and mix.

Mobile phase— Prepare a filtered and degassed mixture of Buffer solution and acetonitrile (72:28). Make adjustments if necessary (see System Suitability under Chromatography 621).

Internal standard preparation— Transfer about 30 mg of methylparaben, accurately weighed, to a 100-mL volumetric flask, dissolve in and dilute with acetonitrile to volume, and mix.

Standard stock preparation— Dissolve an accurately weighed quantity of USP Zileuton RS in acetonitrile to obtain a solution having a known concentration of about 1 mg per mL.

Standard preparation— Transfer 5.0 mL of the Standard stock preparation and 4.0 mL of the Internal standard preparation to a 50-mL volumetric flask, dilute with acetonitrile to volume, and mix.

Assay preparation— Transfer about 100 mg of Zileuton, accurately weighed, to a 100-mL volumetric flask, dissolve in and dilute with acetonitrile to volume, and mix. Transfer 5.0 mL of this solution and 4.0 mL of the Internal standard preparation to a 50-mL volumetric flask, dilute with acetonitrile to volume, and mix.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 260-nm detector and a 4.6-mm × 30-cm column that contains 10-µm packing L1. The flow rate is about 1.5 mL per minute. Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: the resolution, R, between zileuton and methylparaben is not less than 5.0; the tailing factor is not more than 1.3; and the relative standard deviation for replicate injections is not more than 0.6%.

Procedure— Separately inject equal volumes (about 20 µL) of the Assay preparation and the Standard preparation into the chromatograph, record the chromatograms, and measure the peak areas. Calculate the quantity, in mg, of C11H12N2O2S in the portion of Zileuton taken by the formula: in which C is the concentration, in mg per mL, of USP Zileuton RS in the Standard preparation; and RU and RS are the peak area ratios obtained from the Assay preparation and the Standard preparation, respectively.