Orlistat CAS 96829-58-2 API Weight Loss Drug Purity 98.0~101.5%

Orlistat
C29H53NO5 495.73
l-Leucine, N-formyl-, 1-[(3-hexyl-4-oxo-2-oxetanyl)methyl]dodecyl ester, [2S-[2(R*), 3]]-;
N-Formyl-l-leucine, ester with (3S,4S)-3-hexyl-4-[(2S)-2-hydroxytridecyl]-2-oxetanone [96829-58-2].
DEFINITION
Orlistat contains NLT 98.0% and NMT 101.5% of C29H53NO5, calculated on the anhydrous, solvent-free basis.
IDENTIFICATION
• A. Infrared Absorption <197M>
• B. The retention time of the major peak of the Sample solution corresponds to that of the Standard solution, as obtained in the Assay.
ASSAY
• Procedure
[Note-Avoid the use of plastic flasks for the preparation or containment of any solution in this analysis. ]
Mobile phase: Acetonitrile, phosphoric acid, and water (860: 0.05: 140)
Standard solution: 0.5 mg/mL of USP Orlistat RS in Mobile phase. Inject immediately after preparation or store at 5.
Sample solution: 0.5 mg/mL of Orlistat in Mobile phase. Inject immediately after preparation or store at 5.
Chromatographic system
(See Chromatography <621>, System Suitability.)
Mode: LC
Detector: UV 195
Column: 3.9-mm × 15-cm; 4-µm packing L1
Flow rate: 1.0 mL/min
Injection size: 20 µL
System suitability
Sample: Standard solution
Suitability requirements
Relative standard deviation: NMT 2.0%
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of orlistat (C29H53NO5) in the portion of Orlistat taken:
Result = (rU/rS) × (CS/CU) × 100
rU = peak response from the Sample solution
rS = peak response from the Standard solution
CS = concentration of USP Orlistat RS in the Standard solution (mg/mL)
CU = concentration of Orlistat in the Sample solution (mg/mL)
Acceptance criteria: 98.0%-101.5% on the anhydrous, solvent-free basis
IMPURITIES
Inorganic Impurities
• Residue on Ignition <281>: NMT 0.1%
• Heavy Metals, Method II <231>: 20 ppm
Organic Impurities
• Procedure 1: Limit of Orlistat Related Compound A
Standard solution: 0.1 mg/mL of USP Orlistat Related Compound A RS in acetone
Sample solution: 50 mg/mL of Orlistat in acetone
Chromatographic system
(See Chromatography <621>, Thin-Layer Chromatography.)
Mode: TLC
Adsorbent: 0.25-mm layer of chromatographic silica gel mixture
Application volume: 10 µL
Developing solvent system: Toluene and ethyl acetate (4:1)
Detection solution: Transfer 2.5 g of phosphomolybdic acid and 1 g of cerric sulfate into a 100-mL volumetric flask, dissolve in and dilute with methanol to volume.
Analysis
Samples: Standard solution and Sample solution
Remove the plate, and air-dry it thoroughly. Spray the dried plate with Detection solution, and place the plate in an oven at 120 for 30 min.
Acceptance criteria: Any secondary spot from the Sample solution corresponding to orlistat related compound A is not more intense than the corresponding spot from the Standard solution (0.2%).
• Procedure 2: Limit of Orlistat Related Compound B
Standard solution: 0.025 mg/mL of USP Orlistat Related Compound B RS in methylene chloride
Sample solution: 50 mg/mL of Orlistat in methylene chloride
Spiked sample solution: 50 mg/mL of Orlistat in Standard solution
Chromatographic system
(See Chromatography <621>, System Suitability.)
Mode: GC
Detector: Flame ionization
Column: 0.32-mm x 30-m fused silica, coated with a 0.25-µm G27 stationary phase
Column temperature: See the temperature program table below.
Initial Temperature () Temperature Ramp (/min) Final Temperature () Hold Time at Final Temperature (min)
50 4 170 -
170 30 300 30
Temperature
Injector: 270
Detector: 280
Carrier gas: Helium
Flow rate: 30 mL/min
Split ratio: 10:1
Injection size: 2 µL
System suitability
Sample: Standard solution
Suitability requirements
Relative standard deviation: NMT 10.0%
Analysis
Samples: Sample solution and Spiked sample solution
Calculate the percentage of orlistat related compound B in the portion of Orlistat taken:
Result = [rU/(rSP rU)] × (CS/CT) × 100
rU = peak response of orlistat related compound B from the Sample solution
rSP = peak response of orlistat related compound B from the Spiked sample solution
CS = concentration of USP Orlistat Related Compound B RS in the Standard solution (mg/mL)
CT = concentration of Orlistat in the Spiked sample solution (mg/mL)
Acceptance criteria: NMT 0.05% of orlistat related compound B is found.
• Procedure 3
[Note-Avoid the use of plastic flasks for the preparation or containment of any solution in this analysis. ]
Mobile phase, Standard solution, and Sample solution: Prepare as directed in the Assay.
System suitability solution: 10 µg/mL of USP Orlistat RS, 0.1 µg/mL of USP Orlistat Related Compound C RS, and 0.25 µg/mL of USP Orlistat Related Compound D RS in Mobile phase
Chromatographic system
(See Chromatography <621>, System Suitability.)
Proceed as directed in the Assay, except to chromatograph the System suitability solution.
System suitability
Sample: System suitability solution
Suitability requirements
Signal-to-noise ratio: NLT 3 for the orlistat related compound C and orlistat related compound D peaks
Relative standard deviation: NMT 10.0% for the orlistat peak
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of each impurity in the portion of Orlistat taken:
Result = (rU/rS) × (CS/CU) × (1/F) × 100
rU = peak response for each individual impurity from the Sample solution
rS = peak response of USP Orlistat RS from the Standard solution
CS = concentration of USP Orlistat RS in the Standard solution (mg/mL)
CU = concentration of Orlistat in the Sample solution (mg/mL)
F = relative response factor as given in Impurity Table 1
Acceptance criteria: See Impurity Table 1.
Impurity Table 1
Name Relative Retention Time Relative Response Factor Acceptance Criteria, NMT (%)
Formylleucinea 0.10 4.0 0.2
Orlistat related compound C 0.13 33 0.05
Orlistat open ring epimerb 0.44 1.0 0.2
Orlistat related compound D* 0.90 - Calculated in
Procedure 4
Orlistat open ring amidec* 0.90 - Calculated in
Procedure 4
Orlistat 1.00 - -
d-Leucine orlistatd 1.18 1.0 0.2
Individual unidentified impurity - 1.0 0.1
* Coelutes in this LC system, determined using Procedure 4.
a N-Formyl-l-leucine.
b (2S,3R,5S)-5-[(S)-2-Formylamino-4-methyl-pentanoyloxy]-2-hexyl-3-hydroxy-hexadecanoic acid.
c N-Formyl-l-leucine (S)-1-[(2S,3S)-2-hydroxy-3-[1-phenyl-R-ethylcarbomoyl]nonyl]-dodecyl ester.
d N-Formyl-d-leucine (S)-1-[[(2S,3S)-3-hexyl-4-oxo-2-oxetanyl]methyl]dodecyl ester or enantiomer.
• Procedure 4: Limit of Orlistat Related Compound D
Mobile phase: Methanol and water (83:17)
System suitability solution: 4 mg/mL of USP Orlistat RS and 2.4 µg/mL of USP Orlistat Related Compound D RS in acetonitrile, respectively
Standard solution: 5.0 mg/mL of USP Orlistat RS in acetonitrile
Sample solution: 5.0 mg/mL of Orlistat in acetonitrile
Chromatographic system
(See Chromatography 621, System Suitability.)
Mode: LC
Detector: 205 nm
Column: 4.0-mm × 25-cm; 5-µm packing L7
Flow rate: 0.6 mL/min
Injection size: 20 µL
System suitability
Sample: System suitability solution
Suitability requirements
Signal-to-noise ratio: NLT 3 for the orlistat related compound D peak
Relative standard deviation: NMT 10.0% for the orlistat peak
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of each impurity in the portion of Orlistat taken:
Result = (rU/rS) × (CS/CU) × (1/F) × 100
rU = peak response for each individual impurity from the Sample solution
rS = peak response for USP Orlistat RS from the Standard solution
CS = concentration of USP Orlistat RS in the Standard solution (µg/mL)
CU = concentration of Orlistat in the Sample solution (µg/mL)
F = relative response factor as obtained in Impurity Table 2
Acceptance criteria: See Impurity Table 2.
Impurity Table 2
Name Relative Retention Time Relative Response Factor Acceptance Criteria, NMT (%)
Orlistat related compound D 0.94 1.0 0.2
Orlistat 1.00 - -
Orlistat open ring amidea 1.25 4.3 0.1
a N-Formyl-l-leucine (S)-1-[(2S,3S)-2-hydroxy-3-[1-phenyl-R-ethylcarbomoyl]nonyl]-dodecyl ester.
• Procedure 5: Limit of Orlistat Related Compound E
Buffer: 0.4 N borate solution, adjusted to a pH of 10.2
Derivitizing agent: o-Phthaldehyde (OPA) solution. [Note-If unable to obtain commercially, the Derivitizing agent can be prepared as 1% each of 3-mercaptopropionic acid and o-phthaldialdehyde in 0.4 M borate buffer solution.]
Solution A: Transfer 4.1 g of sodium acetate trihydrate and 40 mg of ethylenediaminetetraacetic acid (EDTA) into a 1-L volumetric flask. Dissolve in 950 mL of water, and adjust with 0.1 N sodium hydroxide to a pH of 7.2. Dilute with water to volume, add 2.5 mL of tetrahydrofuran, and mix. Filter, and degas.
Solution B: Transfer 2.7 g of sodium acetate trihydrate and 40 mg of EDTA into a 1-L volumetric flask. Dissolve in 200 mL of water, and adjust with 0.1 N sodium hydroxide to a pH of 7.2. Add 800 mL of acetonitrile, filter, and degas.
Mobile phase: See the gradient table below.
Time (min) Solution A (%) Solution B (%)
0 96.7 3.3
20 60 40
24 0 100
38 0 100
38 96.7 3.3
45 96.7 3.3
Standard solution: Transfer a weighed quantity of about 0.2 mg of USP Orlistat Related compound E RS into a 20-mL head-space vial. Add 10 mL of 4 N sodium hydroxide, and close the vial. Heat the vial to 100 for 1 h, then allow to cool to room temperature. Transfer 2 mL of the resulting solution into a 50-mL volumetric flask, and dilute with water to volume. To 0.5 mL of this solution add 2.0 mL of Buffer and 0.5 mL of Derivatizing agent.
Sample solution: Proceed as directed for the Standard solution, but instead use 25 mg of Orlistat to replace the 0.2 mg of USP Orlistat Related Compound E RS.
Chromatographic system
(See Chromatography <621>, System Suitability.)
Mode: LC
Detector: Fluorescence 340 nm (excitation); 450 nm (emission)
Columns
Guard: 2.1-mm × 2-cm; 50-µm packing L1
Analytical: 2.1-mm × 20-cm; packing L1
Flow rate: 0.5 mL/min
Injection size: 20 µL
System suitability
Sample: Standard solution
Suitability requirements
Relative standard deviation: NMT 6.0% for the orlistat related compound E peak
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of this impurity in the portion of Orlistat taken:
Result = (rU/rS) × (CS/CU) × 100
rU = peak response for orlistat related compound E in the Sample solution
rS = peak response for USP Orlistat Related Compound E RS in the Standard solution
CS = concentration of USP Orlistat Related Compound E RS in the Standard solution (mg/mL)
CU = concentration of Orlistat in the Sample solution (mg/mL)
Acceptance criteria
Individual impurity: NMT 0.2% of orlistat related compound E is found.
Total impurities: NMT 1.0% of total impurities is found, the results for Procedures 1, 2, 3, 4, and 5 being added.
SPECIFIC TESTS
• Optical Rotation, Specific Rotation <781>
Sample solution: 30 mg/mL in dehydrated alcohol
Acceptance criteria: Between -48.0 and -51.0, at 20
• Water Determination, Method Ic <921>: NMT 0.2%
ADDITIONAL REQUIREMENTS
• Packaging and Storage: Preserve in well-closed containers between 2 and 8℃.
• USP Reference Standards <11>
USP Orlistat RS
USP Orlistat Related Compound A RS
USP Orlistat Related Compound B RS
USP Orlistat Related Compound C RS
USP Orlistat Related Compound D RS
USP Orlistat Related Compound E RS
USP35