L-4-Nitrophenylalanine Methyl Ester HCl CAS 17193-40-7 H-Phe(4-NO2)-OMe·HCl Assay >98.5% (HPLC) Factory

Method of Analysis of L-4-Nitrophenylalanine Methyl Ester HCl (H-Phe(4-NO2)-OMe·HCl) (CAS: 17193-40-7)
Description: Place about 2 g of sample in a clean and dry petri-dish and observe the appearance of sample. Reporting: Report the observation
Identificaiton: Compare the retention time of the major peak in the chromatogram of the test preparation with the retention time of the major peak in the chromatogram of system suitability praparation as obtained in chromatographic purity by HPLC.
Loss on Drying: Dry a glass-stoppered, shallow weighing bottle at 105±2℃ for 30 minutes. Allow it to cool to room temperature in a desciccator. Weigh accurately the weighing bottle with stopper and record the weight (W1). Transfer about 1 to 2g accurately weighed quantity of test substance into the weighing bottle. By gentle, sidewise shaking, distribute the test specimen as evenly as practicable to a depth of about 5 mm. Weigh the loaded weighing bottle and record the weight (W2). Place the loaded bottle in the drying oven, removing the stopper and leaving it also in the chamber. Dry the test sample at 105±2℃ for 3 hours. Upon opening the chamber, close the bottle promptly, and allowe it to come to room temperature in a desiccator. Weigh accurately the weighing bottle with stopper and recoed the weight (W3). Calculate the percentage loss on drying of the sample by using following formula:
Calculation Loss on drying (% w/w)= (W2-W3) ÷ (W2-W1) x 100
Enantiomer Content by HPLC:
Chemical & Reagents:
n-Hexane AR grade
Methanol HPLC grade
Isopropyl Alcohol HPLC grade
Diethylamine AR grade
Standards: L-4-Nitrophenylalanine Methyl Ester HCl standard; L-4-Nitrophenylalanine Methyl Ester HCl enantiomer standard
Mobile Phase: Prepare a homogeneous mixture of n-Hexane, Isopropyl Alcohol, Methanol and Diethylamine (65:20:15:0.1 v/v/v/v)
Blank Preparation: Transfer 2.5ml of Methanol in a 10-ml volumetric flask, dilute to volume with mobile phase and mix.
System suitability preparation:
Weigh accurately and transfer about 5 mg each of L-4-Nitrophenylalanine Methyl Ester HCl standard and L-4-Nitrophenylalanine Methyl Ester HCl enantiomer standard in a 10-ml volumetric flask, dissolve in about 2.5ml of methanol, dilute to volume with mobile phase and mix.
Test preparation:
Weigh accurately and transfer about 5 mg of sample in a 10-mol volumetric flask, dissolve in about 2.5ml of methanol, dilute to volume with mobile phase and mix.
Instrumental parameters:
Instrument: HPLC equipped with injector, Pump, UV detector and Recorder
Column: ChiralPak-IC (4.6mm x 250mm) 5 μ or equivalent
Flow rate: 1.0 ml/minute
Wavelength: 260nm
Injection volume: 10 μL
Column coven temp.: 25℃
Run time: 20 minutes
Procedure
Inject the solutions as mentioned in the following sequence. Record the chromatograms and confirm the criteria:
Description of Injections Number of Injections Criteria
Blank Preparation 1 No interfering peak should be observed at the retention time of L-4-Nitrophenylalanine Methyl Ester HCl or enantiomer.
System suitability preparation 1 Resolution between L-4-Nitrophenylalanine Methyl Ester HCl and enantiomer peak should be not less than 2.0
Test preparation 1 Use test chromatogram to determine enantiomer content in the sample.
Relative Retention Times:
L-4-Nitrophenylalanine Methyl Ester HCl 1.0
Enantiomer About 1.11
Retention time of L-4-Nitrophenylalanine Methyl Ester HCl is about 9.8 minutes.
Calculation: In the chromatogram obtained from test preparation, disregard all peaks expect L-4-Nitrophenylalanine Methyl Ester HCl and Enantiomer. Report the enantiomer content in the sample by area normalisation method.
Chromatogrpahic Purity and Impurities by HPLC: <In House>
Chemical & Reagents:
Diammonium phosphate AR grade
Phosphoric acid AR grade
Methanol HPLC grade
Purified Water
Standards
L-4-Nitrophenylalanine Methyl Ester HCl standard
4-Nitro-L-Phenylalanine monohydrate standard
Mobile Phase-A
Dissolve 6.6 g of Diammonium phosphate in 1000ml of purified water. Adjust the pH of this solution to 6.0 with Phosphoric acid. Filter and degas. Before measuring the pH of solution, calibrate the pH meter with standard buffer solution pH 4.01 AND 7.00.
Mobile Phase-B
Methanol HPLC grade
System Suitability Preparation
Weigh accurately and transfer about 25 mg each of L-4-Nitrophenylalanine Methyl Ester HCl standard and 4-Nitro-L-Phenylalanine monohydrate standard in a 50-ml volumetric flask, dissolve in and dilute to volume with methanol and mix.
Test Preparation:
Weigh accurately and transfer about 25 mg sample in a 50-ml volumetric flask, dissolve in and dilute to volume with methanol and mix.
Instrumental parameters:
Instrument: HPLC equipped with injector, Pump, UV detector and Recorder
Column: XTerra RP-18 (4.6mm x 150mm) 5 μ or equivalent
Flow rate: 1.0 ml/minute
Wavelength: 265nm
Injection volume: 20 μL
Column coven temp.: 25℃
Run time: 45 minutes
Gradient Program
Time (min) Mobile Phase-A % Mobile Phase-B %
0.01 90 10
15 60 40
35 40 60
40 35 65
41 90 10
45 90 10
Procedure:
Inject the solutions as mentioned in the following sequence. Record the chromatograms and confirm the criteria:
Blank Preparation 1 No interfering peak should be observed at the retention time of L-4-Nitrophenylalanine Methyl Ester HCl.
System suitability preparation 1 Tailing factor for L-4-Nitrophenylalanine Methyl Ester HCl peak should be not less than 2.0
Test preparation 1 Use test chromatogram to determine chromatographic purity and impurities.
Relative Retention Times:
L-4-Nitrophenylalanine Methyl Ester HCl 1.0 (retention time about 11.4 minutes)
4-Nitro-L-Phenylalanine About 0.4
Toluene About 2.3
Calculations:
In the chromatogram obtained from test preparation, after blank correction, disregard the peak due to toluene. Report the chromatographic purity, 4-Nitro-L-Phenylalanine content and total impurities in the sample by area normalisation method.