High-Throughput Analysis of Nitrosamines Using RapidFire Coupled to Ultivo Triple Quadrupole

Significance of the Topic

In pharmaceutical quality control and environmental monitoring, reliable and rapid detection of nitrosamine impurities is critical due to their potential carcinogenicity. Traditional liquid chromatography–mass spectrometry (LC–MS) methods, while sensitive, often require lengthy separations that limit throughput. The development of high-throughput screening techniques enables laboratories to process large sample sets efficiently, reducing backlog and supporting timely decision-making in drug safety, regulatory compliance, and industrial analytics.

Objectives and Study Overview

This work aimed to replicate a U.S. FDA rapid screening protocol for nitrosamine impurities and evaluate the feasibility of expanding the target panel. Specifically, the study focused on:

• Implementing online solid‐phase extraction (SPE) with an Agilent RapidFire system coupled to an Ultivo Triple Quadrupole MS.
• Quantifying a set of nitrosamine standards in under 15 seconds per sample injection.
• Assessing method precision, linearity, and potential for panel expansion.

Methodology and Instrumentation

The analytical workflow comprised five automated SPE cycles:

1. Aspiration of sample under vacuum (0.2 s).
2. Load and wash with aqueous buffer (3 s).
3. Optional additional wash step.
4. Elution with higher organic content to the mass spectrometer (3 s).
5. Re‐equilibration to initial aqueous conditions (0.5 s).

Key reagents:

• Buffer A: Water with 0.1% formic acid.
• Buffer B: Methanol with 0.1% formic acid.
• Graphitic carbon SPE cartridge (Type D G9206A).
• Nitrosamine standards and solvents (LC/MS grade).

Mass spectrometer settings on the Ultivo QqQ:

• Ionization: Positive APCI, 350 °C heater, 4 μA needle current.
• Drying gas: 6 L/min at 300 °C.
• Capillary voltage: 3,000 V; nebulizer gas at 55 psi.
• MRM transitions optimized for each nitrosamine.

Main Results and Discussion

Under optimized conditions, injections occurred at ~12.5 seconds each, enabling 72 analyses in under 15 minutes, including blanks. Triplicate calibrators yielded:

• Precision with coefficients of variation ranging from 4.5% to 9.0% across analytes.
• Linearity over concentration ranges of 1–100 ng/mL (e.g., NDBA, NPIP) and 5–100 ng/mL (e.g., NPyR, NMEA), with R² values between 0.997 and 0.999.

These performance metrics confirm that RapidFire SPE coupled with QqQ detection maintains analytical fidelity while drastically reducing cycle time compared to conventional LC–MS methods.

Benefits and Practical Applications

The described approach offers multiple advantages for analytical laboratories:

• High throughput—capacity for hundreds of samples per day.
• Reduced solvent and consumable consumption versus full chromatographic runs.
• Scalable panel expansion for emerging nitrosamine targets.
• Applicability to pharmaceutical API and formulation testing, environmental samples, and quality assurance workflows.

Future Trends and Potential Applications

Further developments may include:

• Integration of additional nitrosamine and related impurity targets in a single assay.
• Adaptation to other SPE cartridge chemistries for diverse sample matrices.
• Automation of data processing and real-time flagging of out‐of-specification results using advanced software or AI tools.
• Coupling with orthogonal detection techniques to confirm structural identity and enhance method robustness.

Conclusion

The RapidFire–Ultivo platform successfully replicated and extended an FDA nitrosamine screening method, delivering sub-15-second analysis times without compromising precision or linearity. This high-throughput workflow supports rapid decision-making in pharmaceutical quality control and can be adapted to broader analytical challenges requiring fast, reliable impurity quantitation.

Reference

1. FDA Updates and Press Announcements on Angiotensin II Receptor Blocker (ARB) Recalls. FDA, 2019.
2. Development and validation of a RapidFire-MS/MS method for screening of nitrosamine carcinogen impurities in ARB drugs. FDA, 2019.