Quantitation of 6 Nitrosamines in Losartan API by LC-MS/MS system as per the proposed USP General Chapter <1469>

Significance of the Topic

The presence of nitrosamine impurities in pharmaceutical ingredients poses significant health risks due to their mutagenic and carcinogenic properties. Regulatory bodies such as the US FDA and the United States Pharmacopeia have established stringent acceptable intake limits for key nitrosamines in drug substances to ensure patient safety. In response to recent findings of nitrosamines in losartan and other sartans, a robust analytical method is essential for reliable quantification at trace levels and compliance with the proposed USP general chapter 1469.

Objectives and Overview of the Study

This study aims to develop and validate an LC-MS/MS method for simultaneous quantitation of six nitrosamines (NDMA, NMBA, NDEA, NEIPA, NDIPA, NDBA) in losartan active pharmaceutical ingredient (API) following USP general chapter 1469 procedure 3. The work evaluates the performance of Shimadzu’s LCMS-8045 triple quadrupole system coupled with a UHPLC Nexera X S platform using Raptor ARC-18 and Shim-pack velox SP-C18 columns.

Methodology and Instrumentation Used

Sample Preparation:

• Weigh 80 mg of losartan API and transfer to a 2 mL centrifuge tube.
• Add 1188 µL of diluent (1% formic acid in water) and 12 µL of isotopically labeled internal standard mix.
• Vortex at 2,500 rpm for 5 minutes, centrifuge at 10,000 rpm for 10 minutes, then filter the supernatant through a 0.22 µm hydrophilic PTFE syringe filter.

Chromatographic Conditions:

• Column: Raptor ARC-18 or Shim-pack velox SP-C18, 150 mm × 3.0 mm, 2.7 µm.
• Mobile phases: A = 0.1% formic acid in water, B = 0.1% formic acid in methanol.
• Flow rate: 0.5 mL/min; column oven: 60 °C; injection volume: 20 µL.
• Gradient: 0–1.5 min (3% B), 1.5–4 min (3–50% B), 4–7 min (50–75% B), 7–8.1 min (75–85% B), 8.1–9.2 min (85–95% B), 9.2–12 min (95% B), re-equilibration to 3% B by 12.1 min; total run time 17 min.

MS/MS Detection:

• Instrument: Shimadzu LCMS-8045 with APCI interface.
• Interface temperature: 270 °C; desolvation line and heat block: 220 °C.
• Nebulizing and drying gas flows: 3 L/min each; polarity: positive ion mode.
• MRM transitions optimized for each analyte and internal standard.

Main Results and Discussion

Linearity and Sensitivity:

• Nine-point calibration curves (1.33–900 ppb for NDMA, up to 59.4 ppb for NDEA) yielded correlation coefficients (R²) > 0.997 for all six nitrosamines.
• Limits of quantitation (LOQ) ranged from 0.66 to 10.3 ppb, meeting USP requirements of ≤0.03 ppm for a maximum daily dose of 880 mg.

Precision and Accuracy:

• Repeatability at LOQ level showed RSD ≤ 25% (n=6).
• Recovery at LOQ for all nitrosamines fell within 70–130% acceptance criteria, confirming method accuracy in matrix.

Representative chromatograms demonstrated clear separation of nitrosamines from losartan peaks, with no interference due to sample matrix.

Benefits and Practical Applications of the Method

This LC-MS/MS protocol offers:

• High sensitivity and specificity for low-level nitrosamine detection.
• Rapid analysis with a total run time under 20 minutes.
• Robust performance using two equivalent C18 columns.
• Compliance with proposed USP general chapter 1469 guidelines, facilitating regulatory submissions and quality control in API manufacturing.

Future Trends and Opportunities for Application

As regulatory scrutiny of nitrosamines expands across pharmaceutical classes, this method can be adapted for:

• Analysis of additional nitrosamine compounds in other APIs and finished dosage forms.
• Integration with high-throughput automation and sample preparation workflows.
• Further miniaturization and coupling with advanced ionization sources to improve sensitivity.
• Monitoring nitrosamine formation during manufacturing and storage for risk mitigation.

Conclusion

A validated LC-MS/MS method on the Shimadzu LCMS-8045 system successfully quantified six nitrosamines in losartan API at trace levels. The approach exhibits excellent linearity, precision, and recovery, ensuring compliance with proposed USP general chapter 1469 limits and supporting rigorous quality assessment in pharmaceutical production.

References

• United States Pharmacopeia, General Chapter 1469: Determination of Nitrosamine Impurities in Drug Substances and Drug Products.
• US Food and Drug Administration Guidance on Nitrosamine Impurities in Human Drugs.