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acid, and digest in a fume hood on a hot plate until charring

Zileuton 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
C11H12N2O2S 236.29 by adding small quantities of the 30 percent hydrogen
Urea, N-(1-benzo[b]thien-2-ylethyl)-N-hydroxy-, (±)-. peroxide, whenever the mixture turns brown or darkens.
(±)-1-(1-Benzo[b]thien-2-ylethyl)-1-hydroxyurea [111406- Approximately 1 to 2 mL of nitric acid can be added, if
87-2]. 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
» Zileuton contains not less than 98.5 percent and raising the temperature of the hot plate until fumes of sulfur
not more than 101.5 percent of C11H12N2O2S, trioxide are copiously evolved and the solution becomes
calculated on the anhydrous basis. colorless or retains only a light straw color. Transfer the
solution to a 25-mL volumetric flask using about 7 mL of water.
Packaging and storage—Preserve in tight, light-resistant Repeat the washing twice more, and combine the washings in
the volumetric flask. Dilute with water to volume, and mix.

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containers, and store at room temperature.
Procedure—The inductively coupled plasma-atomic
Change to read: 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
USP Reference standards á11ñ— minute, argon nebulizer flow of about 1 L per minute, and
USP Zileuton RS argon auxillary flow of about 0.5 L per minute. Analyze the
USP Zileuton Related Compound A RS
N-(1-Benzo-[b]thien-2-ylethyl)urea;
▲
Also known as 1-[1-(Benzo[b]thiophen-2-yl)ethyl]
ci 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:
urea.▲ (ERR 1-Apr-2021)
C11H12N2OS ▲220.29▲ (ERR 1-Apr-2021) 25C/W,
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USP Zileuton Related Compound B RS
▲
2-(Benzo[b]thien-2-oyl)benzo[b]thiophene; where C is the concentration, in µg per mL, of boron in the
Also known as Bis(benzo[b]thiophen-2-yl) Test solution determined from the instrument; and W is the
methanone.▲ (ERR 1-Apr-2021) weight, in g, of the Zileuton: not more than 10 µg per g is
C17H10OS2 ▲294.39▲ (ERR 1-Apr-2021) found.
USP Zileuton Related Compound C RS Limit of pyridine—
O

1-Benzo-[b]thien-2-ylethanone; Standard solution—Dissolve an accurately weighed quantity

▲
Also known as 1-(Benzo[b]thiophen-2-yl)ethan-1- of pyridine, approximately 250 mg, in dimethyl sulfoxide, and
one.▲ (ERR 1-Apr-2021) dilute with dimethyl sulfoxide to 50 mL. Transfer 5 µL to a
C10H8OS ▲176.23▲ (ERR 1-Apr-2021) 100-mL sealed headspace vial.
Test solution—Transfer about 100 mg of Zileuton,
Identification— accurately weighed, to a vial, add 5.0 mL of dimethyl sulfoxide
A: Spectroscopic Identification Tests á197ñ, Infrared and 1 g of anhydrous sodium sulfate, and seal with a septum
Spectroscopy: 197K. and crimp cap. Heat the sealed vial at 80° for 60 minutes.
B: The retention time of the major peak in the Chromatographic system (see Chromatography á621ñ)—
chromatogram of the Assay preparation corresponds to that in [NOTE—The use of a headspace apparatus is allowed.] The gas
the chromatogram of the Standard preparation, as obtained chromatograph is equipped with a flame-ionization
in the Assay. detector, a 0.53-mm × 30-m fused silica analytical column
Specific rotation á781Sñ: between –0.5° and +0.5°. coated with a 3.0-µm G43 stationary phase. The carrier gas is
Test solution: 10 mg per mL, in methanol. helium with a linear velocity of about 35 cm per second. The
Water Determination, Method I á921ñ: not more than 1.5%. injection port and detector temperatures are maintained at
Residue on ignition á281ñ: not more than 0.2%. 140° and 260°, respectively. The column temperature is
Specific surface area, Method I á846ñ—Outgas a portion of programmed according to the following steps. It is maintained
the test sample, about 100 mg, at 90° for 1 hour at ambient at 40° for 20 minutes, then increased rapidly to 240°, and
pressure using 0.001 mole fraction of krypton in helium as the maintained at 240° for 20 minutes. Inject the Standard
adsorbate gas: between 0.9 and 3.1. m2 per g. solution, and record the peak responses as directed for
Arsenic, Method II á211ñ: 2 µg per g. Procedure: the relative standard deviation for replicate
Limit of boron— injections is not more than 15%.
Sulfuric acid solution—Carefully add 50 mL of sulfuric acid Procedure—Using a heated gas-tight syringe separately
to 450 mL of water, and mix. inject equal volumes (about 1 mL) of the headspace of the
Standard solution—Prepare a solution in Sulfuric acid Standard solution and the Test solution into the gas
solution having a concentration of about 2.0 µg of boron per chromatograph, and record the peak responses. Calculate the
mL. Use of a commercially prepared boron ICP standard quantity, in ppm, of pyridine in the portion of Zileuton taken
solution is recommended. by the formula:
Test solution—Accurately weigh approximately 1.0 g of
Zileuton into a 125-mL conical flask. Add 1 to 1.5 mL of sulfuric 100(r U/r S)(W S/W U)

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in which r U and r S are the peak responses for pyridine in the acetonitrile to obtain a solution having a known concentration
Test solution and the Standard solution, respectively; W S is the of about 0.25 mg per mL. Transfer 5.0 mL of this solution to a
weight, in mg, of pyridine used to prepare the Standard 50-mL volumetric flask, dilute with acetonitrile to volume,
solution; and W U is the weight, in mg, of Zileuton: not more and mix.
than 100 ppm is found. System suitability solution—Dissolve an accurately weighed
Chromatographic purity— [NOTE—For Test 1 and Test 2, quantity of USP Zileuton Related Compound C RS in
the System suitability solution, the Standard solution, and the acetonitrile to obtain a solution having a known concentration
Test solution are to be refrigerated at or below 5° immediately of about 10 µg per mL. Transfer 5.0 mL of this solution and
after preparation and during analysis using a refrigerated 5.0 mL of the Standard stock solution to a 50-mL volumetric
autosampler. The solutions are stable at or below 5° for about flask, dilute with acetonitrile to volume, and mix.
36 hours.] Standard solution—Transfer 5.0 mL of the Standard stock
TEST 1— solution to a 50-mL volumetric flask, dilute with acetonitrile to
Buffer solution—Prepare as directed in the Assay. volume, and mix.
Mobile phase—Prepare a filtered and degassed mixture of Test solution—Proceed as directed for Test solution under Test
Buffer solution and acetonitrile (82:18). Make adjustments if 1.
necessary (see System Suitability under Chromatography Chromatographic system—Prepare as directed in the Assay.
á621ñ). Chromatograph the System suitability solution, and record the
System suitability solution—Dissolve accurately weighed peak responses as directed for Procedure: the resolution, R,
quantities of USP Zileuton RS and USP Zileuton Related between zileuton related compound B and zileuton related
Compound A RS in acetonitrile, and dilute quantitatively, and compound C is not less than 20. Chromatograph the Standard
stepwise if necessary, to obtain a solution having a known solution, and record the peak responses as directed for
concentration of about 5 µg of each USP Reference Standard Procedure: the relative standard deviation for replicate
injections is not more than 5.0%.

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per mL.
Standard solution—Dissolve an accurately weighed quantity of Procedure—Separately inject equal volumes (about 50 µL) of
USP Zileuton RS in acetonitrile to obtain a solution having a the Standard solution and the Test solution into the
known concentration of about 10 µg per mL. chromatograph, record the chromatograms, and measure the
Test solution—Transfer about 125 mg of Zileuton, accurately areas for the major peaks. Calculate the percentage of each
weighed, to a 50-mL volumetric flask, dissolve in and dilute impurity in the portion of Zileuton taken by the formula:
with acetonitrile to volume, and mix.
Chromatographic system—Prepare as directed in the Assay,
except to use a flow rate of 2.2 mL per minute.
ci 100(C S/C U)(r i/r S)

Chromatograph the System suitability solution, and record the in which C S is the concentration, in mg per mL, of USP
peak responses as directed for Procedure: the resolution, R, Zileuton Related Compound B RS in the Standard solution; C U
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between zileuton and zileuton related compound A is not less is the concentration, in mg per mL, of zileuton in the Test
than 1.5; and the relative standard deviation for replicate solution; r i is the peak response for each impurity obtained
injections is not more than 5.0%. from the Test solution; and r S is the peak response for zileuton
Procedure—Separately inject equal volumes (about 20 µL) of related compound B obtained from the Standard solution: not
the Standard solution and the Test solution into the more than 0.1% of any individual impurity is found; and not
chromatograph, and measure the areas for the major peaks. more than 0.7% of total impurities is found, the results for Test
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Calculate the percentage of each impurity in the portion of 1 and Test 2 being added.
Zileuton taken by the formula:
Assay—
100F(C S/C U)(r i/r S) [NOTE—The Standard preparation and the Assay
preparation are to be refrigerated at or below 5°
in which F is the relative response factor for each impurity, immediately after preparation and during analysis using a
which is 1.0 for any peak with a relative retention time of 0.5, refrigerated autosampler. The solutions are stable at or
0.7, 1.2, 1.6, 3.2, or 3.4, and is 1.2, 1.4, and 1.7 for peaks with below 5° for about 36 hours.]
relative retention times of 0.8, 2.1, and 2.8, respectively; C S is Buffer solution—Dissolve 7.7 g of ammonium acetate and
the concentration, in mg per mL, of USP Zileuton RS in the 0.25 g of acetohydroxamic acid in about 900 mL of water in a
Standard solution; C U is the concentration, in mg per mL, of 1000-mL volumetric flask, adjust with perchloric acid to a pH
of 2.0, dilute with water to volume, and mix.
zileuton in the Test solution; r i is the peak response for each
Mobile phase—Prepare a filtered and degassed mixture of
impurity obtained from the Test solution; and r S is the peak Buffer solution and acetonitrile (72:28). Make adjustments if
response for zileuton obtained from the Standard solution: not necessary (see System Suitability under Chromatography
more than 0.1% of any individual impurity with a relative á621ñ).
retention time of 0.8, 1.6, or 2.1 is found; not more than Internal standard preparation—Transfer about 30 mg of
0.10% of any individual impurity with a relative retention time methylparaben, accurately weighed, to a 100-mL volumetric
of 0.7, 3.2, or 3.4 is found; not more than 0.20% of any flask, dissolve in and dilute with acetonitrile to volume,
individual impurity with a relative retention time of 0.5 or 1.2 is and mix.
found; and not more than 0.07% of any individual impurity Standard stock preparation—Dissolve an accurately
with a relative retention time of 2.8 is found. weighed quantity of USP Zileuton RS in acetonitrile to obtain a
TEST 2— solution having a known concentration of about 1 mg per mL.
Perchloric acid solution—Dissolve 5.0 mL of perchloric acid in Standard preparation—Transfer 5.0 mL of the Standard
1000 mL of water. stock preparation and 4.0 mL of the Internal standard
Mobile phase—Prepare a filtered and degassed mixture of preparation to a 50-mL volumetric flask, dilute with acetonitrile
Perchloric acid solution and acetonitrile (1:1). Make to volume, and mix.
adjustments if necessary (see System Suitability under Assay preparation—Transfer about 100 mg of Zileuton,
Chromatography á621ñ). accurately weighed, to a 100-mL volumetric flask, dissolve in
Standard stock solution—Dissolve an accurately weighed and dilute with acetonitrile to volume, and mix. Transfer
quantity of USP Zileuton Related Compound B RS in

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5.0 mL of this solution and 4.0 mL of the Internal standard Procedure—Separately inject equal volumes (about 20 µL)
preparation to a 50-mL volumetric flask, dilute with acetonitrile of the Assay preparation and the Standard preparation into the
to volume, and mix. chromatograph, record the chromatograms, and measure the
Chromatographic system (see Chromatography á621ñ)—The peak areas. Calculate the quantity, in mg, of C 11 H 12 N 2 O 2
liquid chromatograph is equipped with a 260-nm detector S in the portion of Zileuton taken by the formula:
and a 4.6-mm × 30-cm column that contains 10-µm packing
L1. The flow rate is about 1.5 mL per minute. 1000C(R U/R S)
Chromatograph the Standard preparation, and record the peak
responses as directed for Procedure: the resolution, R, between in which C is the concentration, in mg per mL, of USP
zileuton and methylparaben is not less than 5.0; the tailing Zileuton RS in the Standard preparation; and R U and R S are the
factor is not more than 1.3; and the relative standard deviation peak area ratios obtained from the Assay preparation and the
for replicate injections is not more than 0.6%. Standard preparation, respectively.

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