Nateglinide
(na te glye' nide).
Click to View Image

C19H27NO3 317.42
d-Phenylalanine, N-[[trans-4-(1-methylethyl)cyclohexyl]carbonyl]-;    
()-N-[(trans-4-Isopropylcyclohexyl)carbonyl-d-phenylalanine     [105816-04-4].
DEFINITION
Nateglinide contains NLT 98.0% and NMT 102.0% of C19H27NO3, calculated on the dried basis.
IDENTIFICATION
•  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
Buffer:  8.5 g/L of anhydrous dibasic sodium phosphate in water. Adjust with phosphoric acid to a pH of 7.5.
Mobile phase:  Methanol and Buffer (1:1)
System suitability stock solution:  0.2 mg/mL each of USP Nateglinide Related Compound C RS and dl-phenylalanine in methanol. [Note—Sonicate, if necessary. ]
System suitability solution:  Transfer USP Nateglinide RS to a suitable volumetric flask, dissolve first in methanol, using 45% of the final volume, add System suitability stock solution equal to 5% of the final volume, and then dilute with Buffer to volume to obtain a solution containing about 1.0 mg/mL of nateglinide and about 0.01 mg/mL each of nateglinide related compound C and dl-phenylalanine.
Standard solution:  1.0 mg/mL of nateglinide prepared as follows: transfer USP Nateglinide RS to a suitable volumetric flask, dissolve first in methanol, using 50% of the final volume, and then dilute with Buffer to volume.
Sample solution:  Transfer about 100 mg of Nateglinide to a 100-mL volumetric flask, dissolve in 50 mL of methanol, and dilute with Buffer to volume.
Chromatographic system 
Mode:  LC
Detector:  UV 210 nm
Column:  6-mm × 15-cm; 6-µm packing L71 (see Chromatographic Reagents under Reagents, Indicators, and Solutions)
Column temperature:  30
Flow rate:  1 mL/min
Injection size:  20 µL
System suitability 
Samples:  System suitability solution and Standard solution
Suitability requirements 
Resolution:  NLT 0.9 between nateglinide related compound C and nateglinide, System suitability solution
Relative standard deviation:  NMT 1.0%, Standard solution
Analysis 
Samples:  Standard solution and Sample solution
Calculate the percentage of nateglinide (C19H27NO3) in the portion of Nateglinide 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 Nateglinide RS in the Standard solution (mg/mL)
CU== concentration of Nateglinide in the Sample solution (mg/mL)
Acceptance criteria:  98.0%–102.0% on the dried basis
IMPURITIES
•  Residue on Ignition 281: NMT 0.1%
•  Heavy Metals, Method II 231: NMT 10 ppm
•  Limit of Nateglinide Related Compound A and Other Impurities
Buffer:  7.8 g/L of monobasic sodium phosphate in water. Adjust with phosphoric acid to a pH of 2.5.
Mobile phase:  Acetonitrile and Buffer (7:13)
System suitability stock solution:  Dissolve USP Nateglinide Related Compound A RS in acetonitrile to obtain a solution containing about 0.6 mg/mL. Further dilute this solution with Mobile phase to obtain a solution containing about 0.12 mg/mL.
System suitability solution:  Transfer an amount of USP Nateglinide RS to a suitable volumetric flask, dissolve first in acetonitrile using 10% of the final volume, then add System suitability stock solution equal to 10% of the final volume, and dilute with Mobile phase to volume to obtain a solution containing about 6 mg/mL of nateglinide and about 0.012 mg/mL of nateglinide related compound A.
Standard solution:  Dissolve USP Nateglinide RS in acetonitrile to obtain a solution having a known concentration of about 0.3 mg/mL. Further dilute this solution with Mobile phase to obtain a solution having a known concentration of about 0.06 mg/mL.
Sample solution:  Transfer 60 mg of Nateglinide to a 10-mL volumetric flask, dissolve in a minimal amount of acetonitrile, and dilute with Mobile phase to volume.
Chromatographic system 
Mode:  LC
Detector:  UV 210 nm
Column:  3.9-mm × 5-cm; 5-µm packing L7
Column temperature:  40
Flow rate:  2 mL/min
Injection size:  100 µL
Run time:  5 times the retention time of nateglinide
System suitability 
Samples:  System suitability solution and Standard solution
Suitability requirements 
Resolution:  NLT 2.5 between nateglinide related compound A and nateglinide, System suitability solution
Relative standard deviation:  NMT 2.0%, Standard solution
Analysis 
Samples:  Standard solution and Sample solution
Calculate the percentage of each impurity in the portion of Nateglinide taken:
Result = (rU/rS) × (CS/CU) × (1/F) × 100
rU== peak response of each impurity from the Sample solution
rS== peak response of nateglinide from the Standard solution
CS== concentration of USP Nateglinide RS in the Standard solution (mg/mL)
CU== concentration of Nateglinide in the Sample solution (mg/mL)
F== relative response factor (see Table 1)
Acceptance criteria 
Individual impurities:  See Table 1.
Table 1
Name Relative
Retention
Time
Relative
Response
Factor
Acceptance
Criteria,
NMT (%)
Nateglinide related compound Aa 0.5 0.015 0.2
Ethyl analogb 0.6 1.0 0.1
Nateglinide 1.0
IPP impurityc 3.1 1.0 0.1
Ester impurityd 4.1 0.94 0.1
Any other individual impurity 1.0 0.1
a  trans-4-Isopropylcyclohexylcarboxylic acid.
b  N-(trans-4-Ethylcyclohexylcarbonyl)-d-phenylalanine.
c  N-(trans-4-Isopropylcyclohexylcarbonyl)-d-phenylalanine-d-phenylalanine.
d  N-(trans-4-isopropylcyclohexylcarbonyl)-d-phenylalanine-ethyl ester.
•  Limit of Nateglinide Related Compound B
Mobile phase:  0.77 g/L of ammonium acetate in methanol.
[Note—The following solutions are stable for up to 48 h when stored in a refrigerator. ]
System suitability solution:  10 mg/mL of USP Nateglinide RS and 0.02 mg/mL of USP Nateglinide Related Compound B RS in methanol
Standard solution:  0.02 mg/mL of USP Nateglinide Related Compound B RS in methanol. [Note—Nateglinide related compound B is N-(trans-4-isopropyl-cyclohexylcarbonyl)-l-phenylalanine. ]
Sample solution:  10 mg/mL of Nateglinide in methanol
Chromatographic system 
Mode:  LC
Detector:  UV 220 nm
Column:  4-mm × 25-cm or 4.6-mm × 25-cm; 5-µm packing L72 (see Chromatographic Reagents under Reagents, Indicators, and Solutions)
Column temperature:  40
Flow rate:  0.8 mL/min. [Note—The flow rate can be adjusted as needed to achieve a recommended retention time of nateglinide related compound B at about 25 min. ]
Injection size:  10 µL
System suitability 
[Note—The elution order is nateglinide related compound B, followed by the nateglinide peak. ]
Samples:  System suitability solution and Standard solution
Suitability requirements 
Resolution:  NLT 0.8 between nateglinide related compound B and nateglinide, System suitability solution
Relative standard deviation:  NMT 5%, Standard solution
Analysis 
Samples:  Standard solution and Sample solution
Calculate the percentage of nateglinide related compound B in the portion of Nateglinide taken:
Result = (rU/rS) × (CS/CU) × 100
rU== peak response of nateglinide related compound B from the Sample solution
rS== peak response of nateglinide related compound B from the Standard solution
CS== concentration of USP Nateglinide Related Compound B RS in the Standard solution (mg/mL)
CU== concentration of Nateglinide in the Sample solution (mg/mL)
Acceptance criteria:  NMT 0.2%
•  Limit of Nateglinide Related Compound C and Phenylalanine
Mobile phase, System suitability solution, Standard solution, Sample solution, and Chromatographic system:  Proceed as directed in the Assay.
Diluted standard solution:  Dilute the Standard solution with Mobile phase to obtain a solution having a known concentration of about 0.01 mg/mL of nateglinide.
Analysis 
Samples:  Sample solution and Diluted standard solution
Calculate the percentage of each specified impurity listed in Table 2 in the portion of Nateglinide taken:
Result = (rU/rS) × (CS/CU) × (1/F) × 100
rU== peak response of each impurity from the Sample solution
rS== peak response of nateglinide from the Diluted standard solution
CS== concentration of nateglinide in the Diluted standard solution (mg/mL)
CU== concentration of Nateglinide in the Sample solution (mg/mL)
F== relative response factor of each individual impurity (see Table 2)
Acceptance criteria 
Individual impurities:  See Table 2.
Table 2
Name Relative
Retention
Time
Relative
Response
Factor
Acceptance
Criteria,
NMT (%)
Phenylalanine 0.2 1.5 0.2
Nateglinide cis-isomera (related compound C) 0.9 0.97 0.2
Nateglinide 1.0
a  N-(cis-4-isopropylcyclohexylcarbonyl)-d-phenylalanine.
Total impurities:  The sum of all impurities found in the tests for Limit of Nateglinide Related Compound A and Other Impurities, Limit of Nateglinide Related Compound B, and Limit of Nateglinide Related Compound C and Phenylalanine is NMT 0.5%.
SPECIFIC TESTS
•  Loss on Drying 731: Dry a sample at 105 for 2 h: it loses NMT 0.5% of its weight.
ADDITIONAL REQUIREMENTS
•  Packaging and Storage: Preserve in tight containers. Store at room temperature.
•  USP Reference Standards 11
USP Nateglinide RS Click to View Structure
()-N-[(trans-4-Isopropylcyclohexyl)carbonyl-d-phenylalanine.
    C19H27NO3         317.42
USP Nateglinide Related Compound A RS
trans-4-Isopropylcyclohexylcarboxylic acid.
    C10H18O2        170.2
USP Nateglinide Related Compound B RS
N-(trans-4-Isopropylcyclohexylcarbonyl)-l-phenylalanine.
    C19H27NO3         317.4
USP Nateglinide Related Compound C RS
Nateglinide cis-isomer, N-(cis-4-isopropylcyclohexylcarbonyl)-d-phenylalanine.
    C19H27NO3        317.4
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Pharmacopeial Forum: Volume No. 34(6) Page 1463