Quantitation of N-Nitroso Sitagliptin Impurity (NTTP) in Sitagliptin and Metformin Combination Drug Product Using the Agilent 6475 LC/TQ

Significance of the Topic

The control of nitrosamine impurities in pharmaceutical products is critical due to their potent carcinogenic potential. N-nitroso sitagliptin (NTTP) may arise during manufacturing or storage of sitagliptin-containing formulations. Regulatory bodies such as EMA and FDA have set strict intake limits (37 ng/day) based on a lifetime cancer risk model, necessitating highly sensitive analytical methods for routine monitoring.

Objectives and Study Overview

This work aimed to develop and validate an LC-MS/MS method for quantifying trace levels of NTTP in an extended-release sitagliptin-metformin tablet. The goals were to achieve sub-ng/mL detection limits, demonstrate linearity, accuracy, and precision, and establish suitability for both pre- and post-approval quality control.

Methodology

Multiple reaction monitoring (MRM) on an Agilent 6475 triple quadrupole MS coupled to an Agilent 1290 Infinity II LC system was used. Tablet samples were extracted in methanol by vortexing and shaking, followed by centrifugation and filtration. Calibration standards ranged from 0.04 to 1.3 ng/mL, and a specification-limit spike at 1.1 ng/mL (0.37 ppm relative to 3 mg/mL API). A diverter valve program directed primary sitagliptin and metformin peaks to waste, reducing background and enhancing sensitivity.

Instrumentation

• Agilent 1290 Infinity II LC (high-speed pump, multisampler, thermostat, VWD detector)
• Agilent 6475 triple quadrupole MS with APCI source in positive MRM mode
• Biphenyl analytical column (4.6 × 150 mm, 2.7 µm) at 50 °C

Key Results and Discussion

The method exhibited excellent linearity (R2 ≥ 0.994) over 0.04–1.3 ng/mL. Limits of detection and quantitation were 0.008 and 0.088 ng/mL, respectively, with signal-to-noise ratios exceeding regulatory thresholds. Accuracy (recovery) ranged between 80–120% for spiked samples, and precision (RSD) was below 5% at LLOQ, LOQ, and specification levels. Chromatographic selectivity was achieved using methanol-based mobile phases and a biphenyl column, leveraging π-π interactions. The diverter valve enhanced sensitivity by excluding API peaks from the MS.

Benefits and Practical Applications

This validated assay allows reliable sub-ng/mL quantitation of NTTP in pharmaceutical formulations. It supports compliance with EMA and FDA nitrosamine guidelines and can be deployed for pre-approval risk assessment and routine quality control in manufacturing laboratories, thereby preventing unacceptable impurity levels.

Future Trends and Potential Applications

Advances may include high-throughput automated workflows and adoption of high-resolution MS for comprehensive nitrosamine screening. Exploration of alternative stationary phases or ionization methods could extend applicability to other nitrosamine drug-substance-related impurities (NDSRIs) and dosage forms.

Conclusion

The presented LC-MS/MS method provides a sensitive, precise, and accurate approach for monitoring N-nitroso sitagliptin impurity in combination drug products. Its robust performance and alignment with current regulatory expectations make it suitable for implementation in both developmental and commercial QC settings.

References

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