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Quantitation of N-Nitrosodimethylamine (NDMA) and N-Nitrosophenylephrine (NNPE) in
Phenylephrine Hydrochloride (API) by LC-MS/MS
Author’s: Dr. Balamurugan K, Vice President-Analytical R&D
Elumalai Vinayagam, A. Suresh & R. Gunalan. Malladi Drugs and Pharmaceuticals Ltd.,
India.
Phenylephrine -Nitrosoamine.pdf (1.1 MB)
Abstract
We have developed and validated a sensitive LC-MS/MS procedure for the quantitation of
N-nitrosodimethylamine (NDMA) and N-Nitrosophenylephrine (NNPE) in Phenylephrine
Hydrochloride (API). Validation followed ICH Q2 (R2) and development principles from ICH Q14, aligned with USP <1469> (Nitrosamine Impurities) and FDA guidance on control of nitrosamine impurities. The method uses reversed-phase chromatography with APCI (+) MRM detection. The Limit of Quantitation (LOQ) is 0.018 μg/mL on solution basis, corresponding to ≈0.18 ppm at a 100 mg/mL test concentration. The validated analytical range is 0.018–0.18 μg/mL (≈0.18–1.8 ppm equivalent at 100 mg/mL). System precision at the 100% level showed %RSD ≤1.1% for both analytes; LOQ precision was ≤7.3% (NDMA) and 4.67(NNPE). Mean recoveries across LOQ, 100%, and 150% levels were 93–104%. Linearity was demonstrated over 10–150% of the target level with r² 0.98–0.99. Batch analyses of three API lots showed ND/BDL for both analytes, all well below calculated product-specific limits derived from acceptable intakes (AI). The method is suitable for routine QC and regulatory compliance for nitrosamines in Phenylephrine HCl.
Background
Nitrosamines are probable human carcinogens formed through reactions of secondary or
tertiary amines with nitrites under certain manufacturing or storage conditions. In recent
years, global regulatory agencies have issued stringent limits for nitrosamine impurities in
pharmaceuticals due to contamination events affecting several APIs. Phenylephrine Hydrochloride, a sympathomimetic amine commonly used as a nasal decongestant,
contains a secondary amine functional group susceptible to nitrosation, potentially forming NDMA, and NNPE. Given these risks, sensitive and robust analytical methods are
required to monitor these impurities and ensure compliance with regulatory limits of
NDMA-0.926 ppm and NNPE-0.965 ppm for each nitrosamine. MDD of Phenylephrine Hydrochloride is 103.6 mg.
1.0 Experimental Information
1.1 Objective
The objective of this study is to develop and validate a sensitive and robust LC-MS/MS
method for the quantitative determination of two nitrosamine impurities—NDMA
(N-Nitrosodimethylamine), and NNPE (N-Nitrosophenylephrine), in Phenylephrine
Hydrochloride(API). The method is designed to enable accurate detection of these impurities at trace levels in compliance with regulatory requirements.
1.2 Scope
The scope of this study is to evaluate the presence of nitrosamine impurities—NDMA,
and NNPE in Phenylephrine Hydrochloride (API) using a validated LC-MS/MS method. The method is intended for routine analysis to ensure that each impurity is controlled below the specified limit of NDMA-0.926 ppm and NNPE-0.965 ppm, in accordance with applicable regulatory guidelines for nitrosamine testing in pharmaceutical substances.
2.0 Instrumentation
The analysis was performed using a Shimadzu i-Series LC-2050C high-performance liquid
chromatography (HPLC) system coupled with a Shimadzu LCMS-8045 tandem mass
spectrometer equipped with an APCI interface. An autosampler was used for sample injection. Data acquisition, processing, and integration were carried out using Lab Solutions and Lab Solutions Insight software.
2.1 Sample Preparation
2.1.1 Sample Type Phenylephrine Hydrochloride (API)
2.1.2 Diluent Purified water
2.1.3 Sample Preparation Procedure Accurately weigh approximately 500.0 mg of Phenylephrine Hydrochloride into a 5.0 mL volumetric flask. Add diluent to volume and
mix well to ensure complete dissolution. Prepare two independent test solutions,
labelled T1 and T2
2.1.4 Standard Solution Preparation
Working Standards:
NDMA
NNPE
Standard stock solution-1: Accurately weigh approximately 37 mg of NDMA, and 38 mg of NNPE into a 100 mL volumetric flask. Dissolve and dilute to volume with methanol. Mix thoroughly.
Standard stock solution-2: Pipette 1.0 mL each of NDMA, and NNPE from Stock Solution-1 into separate 100.0 mL volumetric flasks. Dilute each to volume with diluent (water) and mix well.
Standard stock solution-3: Pipette 3.0 mL of each Standard Stock Solution-2 into a 50.0 mL
volumetric flask. Dilute to volume with diluent and mix.
Working Standard stock solution: Pipette 4.0 mL of Standard Stock Solution-3 into a
50.0 mL volumetric flask. Dilute to volume with diluent and mix well.
LOQ Solution: Pipette 1.0 mL of the Working Standard Solution into a 10.0 mL volumetric
flask. Dilute to volume with diluent and mix thoroughly.
LOQ Accuracy sample preparation: Weigh approximately 500 mg of Phenylephrine
Hydrochloride into a 5.0 mL volumetric flask. Add 0.5 mL of the Working Standard
Solution. Dilute to volume with diluent and mix well. 100 %Accuracy sample Preparation: Weigh approximately 500 mg of Phenylephrine Hydrochloride into a 5.0 mL volumetric flask. Add 0.4 mL of Standard Stock Solution-3. Dilute to volume with diluent and mix.
150 %Accuracy sample Preparation: Weigh approximately 500 mg of Phenylephrine
Hydrochloride into a 5.0 mL volumetric flask. Add 0.6 mL of Standard Stock Solution-3.
Dilute to volume with diluent and mix.
3.0 Instrument Conditions
3.1 Chromatographic Conditions
Column: COSMOSIL 3 C18 (150 mm × 4.6 mm, 3.0 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: 0.60 mL/min
Injection Volume: 20 μL
Detection Mode: Multiple Reaction
Monitoring (MRM)
Total Run Time: 20 minutes
Gradient Program:
3.5 System Suitability Criteria
Inject 20 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
and NNPE across six injections is not
more than 20.0%.
Typical retention times:
NDMA: ~5.3 minutes
NNPE : ~12.4 minutes
Phenylephrine: ~2.5 minutes
3.6 Sample Analysis Procedure
Inject 20 μL of each test sample (T1 and T2) into the LC-MS/MS system using the above
conditions. Record the mass chromatograms and measure the peak responses for NDMA
and NNPE.
4.0 Analysis and Results
4.1 Data Processing
Data acquisition, peak integration, and quantitative analysis were performed using
LabSolutions Insight software.
4.2 Calculation
The content of NDMA, and NNPE in the sample was calculated using the following formula:
Where,
TA = Peak area of respective impurity in test solution
SA = Average Peak area of respective impurity standard solution
SC = Concentration of respective impurity standard solution in mg/ml.
TC = Concentration of test solution in mg/ml.
P = Purity/potency of respective impurity standard
Report the average result of sample preparation T1 & T2.
Representative Data
NDMA Retention Time: 5.3 mins
NNPE Retention Time: 12.4 mins
4.3 Limit of Detection (LOD) and Limit of Quantification (LOQ):
The Limit of Quantification (LOQ) for NDMA, and NNPE was established based on signal-to-noise (S/N) ratio analysis. Six replicate injections of low-concentration standard solutions were analyzed to determine the minimum quantifiable amount with acceptable precision and accuracy. The Limit of Detection (LOD) was similarly determined based on the lowest concentration yielding a signal-to noise ratio ≥3:1.
The results are summarized in the table below:
4.4 Linearity
Linearity was evaluated by preparing five standard solutions for each analyte—NDMA, and NNPE across a concentration range of 18 ppb to 270 ppb, representing 10% to 150% of the target analyte level. All standards were prepared through serial dilution from a single stock solution to ensure consistency.
Linear regression analysis was performed using peak area (y-axis) versus concentration (x-axis),
excluding the origin. The resulting calibration curves demonstrated excellent linearity, with
Regression Coefficient (r²) of 0.99 for all two nitrosamine impurities. The y-intercepts were
minimal, indicating no significant deviation from the linear model.
The detailed results are presented in Table 4 to 5.
4.5 Accuracy
Accuracy of the method for the Test for NDMA and NNPE of Phenylephrine HCl, BP/EP was
demonstrated by analyzing data obtained from spiked solutions across the specified range of the analytical procedure. The percent recoveries of each individual sample and the average at each concentration level were determined and presented in Table 7 - 8. All acceptance criteria were met.
Method precision results are presented in Table 6. Results show that the method is precise for the determination of NDMA and NNPE in Table-6
5.0 Conclusion:
A highly sensitive and robust LC-MS/MS method was successfully developed and validated for the quantification of two nitrosamine impurities—NDMA, and NNPE in Phenylephrine Hydrochloride (API). The method meets all applicable validation criteria, including specificity, precision, accuracy, linearity, LOD, and LOQ, in accordance with USP, ICH, and FDA regulatory guidelines. Analytical results from batch analysis demonstrated that NDMA and NNPE not detected in any of the tested samples, well below the regulatory limit NDMA-0.926 ppm and NNPE-0.965 ppm. This validated method is suitable for routine quality control and regulatory compliance testing of nitrosamine impurities in Phenylephrine HCl and can serve as a reference framework for similar active pharmaceutical ingredients (APIs) requiring nitrosamine risk assessment.
References:
- European Medicines Agency (EMA). (2016, November 15). Guideline on the Chemistry of
Active Substances. Retrieved from https://www.ema.europa.eu/en/chemistry-activesubstances-chemistry-new-active-substances - International Council for Harmonisation (ICH). (2017, March 31). ICH Harmonised
Guideline M7(R1): Assessment and Control of DNA Reactive (Mutagenic) Impurities in
Pharmaceuticals to Limit Potential Carcinogenic Risk. Retrieved from
https://database.ich.org/sites/default/files/M7_R1_Guideline.pdf - U.S. Food and Drug Administration (FDA). (2021, February). Control of Nitrosamine
Impurities in Human Drugs: Guidance for Industry (Revision 1). Retrieved from
https://www.fda.gov/regulatory-information/search-fda-guidance-documents/controlnitrosamine-impurities-human-drugs - United States Pharmacopeia (USP). General Chapter <1469>: Nitrosamine Impurities.
Rockville, MD: The United States Pharmacopeial Convention. - United States Pharmacopeia (USP). General Chapter <1225>: Validation of Compendial
Procedures. Rockville, MD: The United States Pharmacopeial Convention. - United States Pharmacopeia (USP). General Chapter <736>: Mass Spectrometry.
Rockville, MD: The United States Pharmacopeial Convention.