A Sensitive and Cost-Effective LC–MS Method for Quantifying N-Nitroso-Atenolol in Drug Formulation

Importance of the Topic

Analytical monitoring of trace N-nitrosamines in pharmaceuticals is critical due to their potential mutagenic and carcinogenic effects even at low concentrations
Regulatory agencies such as FDA and EMA enforce strict acceptable intake limits for these impurities, driving the need for sensitive, reliable, and accessible analytical methods

Objectives and Study Overview

The study aimed to develop a sensitive, cost effective UHPLC–MS workflow using a single quadrupole detector to quantify N-nitroso-atenolol in atenolol drug substance and drug product
The target quantitation limit was set at 1.5 ppm, representing 10 % of the regulatory specification limit for CPCA Category IV NDSRIs

Methodology

Sample preparation involved diluting atenolol formulations to 0.5 mg/mL and spiking with N-nitroso-atenolol at defined levels
Calibration curves were constructed in both neat solvent (0.2–150 ng/mL) and matrix (0.5–150 ng/mL) using serial dilutions
An UHPLC gradient method separated atenolol (RT 3.45 min) from N-nitroso-atenolol (RT 6.72 min) to minimize matrix interference
Quantitation employed selected ion recording on a QDa II Mass Detector in positive ESI mode

Instrumentation

• UHPLC: ACQUITY Arc QSM-R with 2.7 µm CORTECS Premier T3 column at 45 °C
• Mass detection: ACQUITY QDa II single quadrupole mass detector with ESI+
• UV monitoring: Photo Diode Array detector at 225 nm
• Software: Empower CDS for data acquisition, processing, and reporting

Main Results and Discussion

Calibration in solvent showed excellent linearity (R2 = 0.999) down to 0.2 ng/mL and in matrix (R2 = 0.996) down to 0.5 ng/mL
Recoveries at the regulatory limit (15 ppm) ranged from 87–96 % in drug substance and 95–102 % in drug product, with RSD ≤ 1.9 %
At 1.5 ppm, recoveries were 71–96 % in substance and 84–95 % in product, with RSD ≤ 4.4 %
A divert valve and extension loop strategy enhanced robustness by protecting the mass detector from high concentration matrix components
Thermal and UV stress testing revealed stable N-nitroso-atenolol levels but increased N-formyl-atenolol, illustrating the method’s capability to detect related degradation products

Benefits and Practical Applications

• High sensitivity and specificity at sub-ppb levels using cost effective single quadrupole MS
• Clear chromatographic separation reduces matrix interference in complex formulations
• Streamlined regulated reporting with Empower CDS supports compliance with 21 CFR Part 11
• Applicable for routine quality control laboratories with limited access to high cost mass spectrometers

Future Trends and Applications

Expansion of single quadrupole workflows for other NDSRIs can democratize nitrosamine testing across diverse laboratories
Integration with automated sample preparation and data analytics will improve throughput and data integrity
Advancements in column technology and MS interfaces may further enhance sensitivity and resolution at lower cost

Conclusion

The developed UHPLC–MS method demonstrates that single quadrupole detection delivers reliable quantitation of N-nitroso-atenolol at regulatory levels
This accessible workflow supports robust routine testing, regulatory compliance, and patient safety by enabling trace monitoring of NDSRIs in pharmaceutical formulations

References

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