Add the following:
Ezetimibe
(e zet' i mibe).
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C24H21F2NO3 409.43
2-Azetidinone, 1-(4-fluorophenyl)-3-[3-(4-fluorophenyl)-3-hydroxypropyl]-4-(4-hydroxyphenyl)-, [3R-[3(S*),4]]-;    
(3R,4S)-1-(p-Fluorophenyl)-3-[(3S)-3-(p-fluorophenyl)-3-hydroxypropyl]-4-(p-hydroxyphenyl)-2-azetidinone;    
(3R,4S)-1-(4-Fluorophenyl)-3-[(S)-3-(4-fluorophenyl)-3-hydroxypropyl]-4-(4-hydroxyphenyl)-azetidin-2-one     [163222-33-1].
DEFINITION
Ezetimibe contains NLT 98.0% and NMT 102.0% of ezetimibe (C24H21F2NO3), calculated on the anhydrous and solvent-free basis.
IDENTIFICATION
•  B. The retention time ratio of the major peak of the Sample solution to that of the ezetimibe peak from the System suitability solution in Organic Impurities, Procedure 2 is between 0.97 and 1.03.
ASSAY
•  Procedure
Solution A:  Water
Solution B:  Acetonitrile
Solution C:  Methanol
Mobile phase:  See Table 1.
Table 1
Time
(min) 		Solution A
(%) 		Solution B
(%) 		Solution C
(%)
0.0 63 27 10
37.0 63 27 10
60.0 40 50 10
70.0 40 50 10
80.0 10 80 10
90.0 10 80 10
90.1 63 27 10
100.0 63 27 10
Diluent:  Acetonitrile, methanol, and water (27:10:63). Add 1.0 mL of glacial acetic acid per L of the mixture.
Standard solution:  0.25 mg/mL of USP Ezetimibe RS prepared as follows. Dissolve a suitable amount of USP Ezetimibe RS in acetonitrile at about 1%–2% of the total volume in a suitable volumetric flask, and dilute with Diluent to volume.
Sample solution:  0.25 mg/mL of Ezetimibe prepared as follows. Dissolve a suitable amount of Ezetimibe in acetonitrile at about 1%–2% of the total volume in a suitable volumetric flask, and dilute with Diluent to volume.
Chromatographic system 
(See Chromatography 621, System Suitability.)
Mode:  LC
Detectors 
0–5 min:  UV 215 nm
5–100 min:  UV 248 nm
Column:  4.6-mm × 15-cm; 5-µm packing L43
Flow rate:  2 mL/min
Injection volume:  60 µL
System suitability 
Sample:  Standard solution
Suitability requirements 
Tailing factor:  NMT 1.5
Relative standard deviation:  NMT 0.73%
Analysis 
Samples:  Standard solution and Sample solution
Calculate the percentage of ezetimibe (C24H21F2NO3) in the portion of Ezetimibe taken:
Result = (rU/rS) × (CS/CU) × 100

rU== peak response of ezetimibe from the Sample solution
rS== peak response of ezetimibe from the Standard solution
CS== concentration of USP Ezetimibe RS in the Standard solution (mg/mL)
CU== concentration of Ezetimibe in the Sample solution (mg/mL)
Acceptance criteria:  98.0%–102.0% on the anhydrous and solvent-free basis
IMPURITIES
•  Residue on Ignition 281: NMT 0.2%
•  Organic Impurities, Procedure 1
Mobile phase, Diluent, Standard solution, Sample solution, and Chromatographic system:  Proceed as directed in the Assay.
System suitability solution:  0.25 mg/mL of USP Ezetimibe System Suitability Mixture RS prepared as follows. Dissolve a suitable amount of USP Ezetimibe System Suitability Mixture RS in acetonitrile at about 1%–2% of the total volume in a suitable volumetric flask, and dilute with Diluent to volume.
Sensitivity solution:  0.125 µg/mL of USP Ezetimibe RS in Diluent from the Standard solution
System suitability 
Samples:  Standard solution, System suitability solution, and Sensitivity solution
Suitability requirements 
Resolution:  NLT 1.5 between ezetimibe and o-fluorobenzene isomer, Standard solution
Tailing factor:  NMT 1.5, Standard solution
Relative standard deviation:  NMT 10%, Sensitivity solution
Analysis 
Sample:  Sample solution
Calculate the percentage of each impurity in the portion of Ezetimibe taken:
Result = (rU/rT) × (1/F) × 100

rU== peak response of each impurity from the Sample solution
rT== sum of all peak responses from the Sample solution
F== relative response factor from Table 2
Acceptance criteria:  See Table 2. Disregard peaks less than 0.05%.
Table 2
Name Relative
Retention
Time 		Relative
Response
Factor 		Acceptance
Criteria,
NMT (%)
Desfluoroaniline
analoga 		0.72 1.0 0.2
Ezetimibe
diastereomersb 		0.77 1.0 —
o-Fluorobenzene isomerc 0.89 1.0 0.2
Ezetimibe 1.0 — —
m-Flouroaniline
analogd 		1.13 1.0 0.2
Ezetimibe ketonee 1.42 1.5 0.1
Any unspecified
impurity 		— 1.0 0.10
Total achiral
impurities 		— — 0.6
a  (3R,4S)-3-[(S)-3-(4-Fluorophenyl)-3-hydroxypropyl]-4-(4-hydroxyphenyl)-1-phenylazetidin-2-one.
b  The two ezetimibe diastereomers, R,R,R- and S,S,S-, elute as one peak and are quantitated using Procedure 2.
c  (3R,4S)-1-(4-Fluorophenyl)-3-[(S)-3-(2-fluorophenyl)-3-hydroxypropyl]-4-(4-hydroxyphenyl)azetidin-2-one.
d  (3R,4S)-1-(3-Fluorophenyl)-3-[(S)-3-(4-fluorophenyl)-3-hydroxypropyl]-4-(4-hydroxyphenyl)azetidin-2-one.
e  (3R,4S)-1-(4-Fluorophenyl)-3-[3-(4-fluorophenyl)-3-oxopropyl]-4-(4-hydroxyphenyl)azetidin-2-one.
•  Organic Impurities, Procedure 2
Mobile phase:  Acetonitrile and water (450:550)
Diluent:  Acetonitrile with 0.1% glacial acetic acid (v/v)
System suitability solution:  0.4 mg/mL of USP Ezetimibe System Suitability Mixture RS in Diluent
Sensitivity solution:  0.2 µg/mL of USP Ezetimibe RS in Diluent
Sample solution:  0.4 mg/mL of Ezetimibe in Diluent
Chromatographic system 
(See Chromatography 621, System Suitability.)
Mode:  LC
Detector:  UV 248 nm
Column:  Two columns in series of 4.6-mm × 15-cm; 5-µm packing L##1
Column temperature:  5
Flow rate:  0.35 mL/min
Injection volume:  10 µL
Run time:  NLT 1.4 times the retention time of the ezetimibe peak
System suitability 
Samples:  System suitability solution and Sensitivity solution
Suitability requirements 
Resolution:  NLT 1.5 between ezetimibe and R,R,S-ezetimibe diastereomer peaks, System suitability solution
Tailing factor:  NMT 1.5 for the ezetimibe peak, System suitability solution
Relative standard deviation:  NMT 10%, Sensitivity solution
Analysis 
Sample:  Sample solution
Calculate the percentage of each enantiomeric or diastereomeric impurity in the portion of Ezetimibe taken:
Result = (rU/rT) × 100

rU== peak response of each impurity from the Sample solution
rT== sum of all peak responses from the Sample solution
Acceptance criteria:  See Table 3.
Table 3
Name Relative
Retention
Time 		Acceptance
Criteria,
NMT (%)
S,S,S-Ezetimibea 0.76 0.2
R,R,R-Ezetimibeb 0.82 0.1
Desfluoroaniline analogc 0.89 —
R,R,S-Ezetimibed 0.93 0.4
Ezetimibe 1.00 —
S,S,R-Ezetimibee 1.12 0.1
R,S,R-Ezetimibef 1.22 0.1
Total chiral impurities — 0.5
Total impuritiesg — 0.9
a  (3S,4S)-1-(4-Fluorophenyl)-3-[(S)-3-(4-fluorophenyl)-3-hydroxypropyl]-4-(4-hydroxyphenyl)azetidin-2-one.
b  (3R,4R)-1-(4-Fluorophenyl)-3-[(R)-3-(4-fluorophenyl)-3-hydroxypropyl]-4-(4-hydroxyphenyl)azetidin-2-one.
c  Desfluoroaniline analog impurity is quantitated using Procedure 1.
d  (3R,4S)-1-(4-Fluorophenyl)-3-[(R)-3-(4-fluorophenyl)-3-hydroxypropyl]-4-(4-hydroxyphenyl)azetidin-2-one.
e  (3S,4R)-1-(4-Fluorophenyl)-3-[(S)-3-(4-fluorophenyl)-3-hydroxypropyl]-4-(4-hydroxyphenyl)azetidin-2-one.
f  (3S,4R)-1-(4-Fluorophenyl)-3-[(R)-3-(4-fluorophenyl)-3-hydroxypropyl]-4-(4-hydroxyphenyl)azetidin-2-one.
g  Include all impurities from Procedure 1 and Procedure 2.
SPECIFIC TESTS
•  Optical Rotation, Specific Rotation 781S
Sample solution:  10 mg/mL of Ezetimibe previously dried for NLT 16 h over anhydrous calcium sulfate, in methanol
Temperature:  20
Acceptance criteria:  25.0 to 30.0
ADDITIONAL REQUIREMENTS
•  Packaging and Storage: Protect from moisture and store below 25.
•  USP Reference Standards 11
USP Ezetimibe RS Click to View Structure
USP Ezetimibe System Suitability Mixture RS
It contains ezetimibe, o-fluorobenzene isomer
(3R,4S)-1-(4-Fluorophenyl)-3-[(S)-3-(2-fluorophenyl)-3-hydroxypropyl]-4-(4-hydroxyphenyl)azetidin-2-one;
    C24H21F2NO3         409.43
and R,R,S-ezetimibe

((3R,4S)-1-(4-Fluorophenyl)-3-[(R)-3-(4-fluorophenyl)-3-hydroxypropyl]-4-(4-hydroxyphenyl)azetidin-2-one).
    C24H21F2NO3         409.43
[Note—It may also contain desfluoroaniline analog. ]
2S (USP38)
1   A suitable column is the Chiracel OD-RH brand containing 5-µm packing.
Auxiliary Information— Please check for your question in the FAQs before contacting USP.
Topic/Question Contact Expert Committee
Monograph Sujatha Ramakrishna, Ph.D., MBA
Senior Scientific Liaison
(301) 816-8349		 (SM22010) Monographs - Small Molecules 2
Reference Standards RS Technical Services
1-301-816-8129
rstech@usp.org
USP38–NF33 Supplement : No. 2 Page 8082
Pharmacopeial Forum: Volume No. 40(5)