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Quantitative Analysis of N-Nitrosodimethylamine (NDMA), N-Nitroso-lephedrine and N-Nitrosopseudoephedrine in Pseudoephedrine Hydrochloride by High-Performance Liquid Chromatography-Tandem Mass Spectrometry
by: Dr. Balamurugan K | Assistant Vice president-Analytical R&D Malladi Drugs and Pharmaceuticals Ltd., India.
This analytical method describes a sensitive and robust procedure for the quantitation of N-Nitrosodimethylamine (NDMA), N-Nitroso-l-ephedrine, and N-Nitrosopseudoephedrine in Pseudoephedrine Hydrochloride using LC-MS/MS. The method was validated in accordance with United States Pharmacopeia (USP) General Chapters <1225> (Validation of Compendial Procedures) and <736> (Mass Spectrometry), the International Council for Harmonisation (ICH) M10 Guideline on Bioanalytical Method Validation, and the U.S. FDA Guidance for Industry: Control of Nitrosamine Impurities in Human Drugs.
LOQ (Limit of Quantitation): 0.04μg/mL with respect to 20 mg/mL sample concentration
Validated Range: 0.04μg/mL to 0.4μg/mL with respect to 20 mg/mL sample concentration.
Precision: %Relative standard deviation (RSD) of the 6 recoveries at 100% levels NDMA is 0.83%,
N-Nitroso-l-ephedrine is 0.50%, N-Nitroso pseudo ephedrine is 0.95%.
Accuracy: Mean % recoveries of NDMA, N-Nitroso-l-ephedrine, N-Nitroso pseudo ephedrine at
0.04μg/mL, 0.4μg/mL, and 0.6μg/mL are within 100.04 ~ 104.54%.
Instrument Conditions
Chromatographic Conditions
Column: Waters XBridge BEH C18 (150 mm × 4.6 mm, 2.5 μm)
Mobile Phase A: 0.1% Formic acid in water (1.0 mL formic acid in 1000 mL water),
filtered through a 0.45 μm membrane filter
Mobile Phase B: 0.1% Formic acid in methanol (1.0 mL formic acid in 1000 mL
methanol), filtered through a 0.45 μm membrane filter
Column Oven Temperature: 40 °C
Autosampler Temperature: 10 °C
Seal Wash/Needle Wash: Methanol:Water (1:1, v/v)
Flow Rate: 0.70 mL/min
Injection Volume: 30 μL
Detection Mode: Multiple Reaction Monitoring (MRM)
Total Run Time: 20 minutes
Gradient Program:
Mass Spectrometer Conditions
Interface: APCI (Atmospheric Pressure Chemical Ionization)
Interface Voltage: 4.5 kV
Interface Temperature: 370 °C
Heat Block Temperature: 220 °C
Desolvation Line (DL) Temperature: 220 °C
Drying Gas Flow: 3 L/min
Nebulizer Gas Flow: 3 L/min
Collision-Induced Dissociation (CID) Gas Pressure: 200 kPa
Ionization Mode: Positive
Detection Window:
o NDMA: 3.0 – 7.1 min
o NNL: 12.10 – 16.0 min
o NNP: 12.10 – 16.2 min
System Suitability Criteria
Inject 30 µL each of blank and standard solution (six replicates) using the defined chromatographic and MS parameters.
The system is considered suitable if the following conditions are met: The %RSD of peak areas for NDMA, NNL, and NNP across six injections is not more than 20.0%.
Typical retention times: NDMA: ~6.2 minutes NNL: ~13.0 minutes NNP: ~13.9 minutes
Results
Full detailed method, data and validation.
Quantitative Analysis of N-Nitrosodimethylamine (NDMA), N-Nitroso-l-ephedrine and N-Nitrosopseudoephedrine in Pseudoephedrine Hydrochloride by High-Performance Liquid Chromatography-Tandem Mass Spectrometry.pdf (1.8 MB)