Determination of Nitrosamine impurities in Pregabalin drug substance using Triple Quadrupole Liquid Chromatography Mass Spectrometry

Significance of the Topic

The detection of nitrosamine impurities in drug substances has become critical following global recalls of angiotensin receptor blocker medicines. Regulatory bodies such as the US FDA and EMA now require rigorous screening for nitrosamines, which are probable carcinogens formed as trace byproducts during synthesis. Extending this scrutiny to other pharmaceuticals like the anti-epileptic Pregabalin ensures patient safety and regulatory compliance.

Study Objectives and Overview

This work presents a targeted liquid chromatography tandem mass spectrometry method using a triple quadrupole instrument to quantify five nitrosamines in Pregabalin drug substance. The aim is to achieve low detection limits, high reproducibility, and chromatographic separation that prevents high concentration API interference.

Methodology and Instrumentation

Sample preparation was streamlined to a one-pot procedure:

• Weigh 100 mg Pregabalin into a centrifuge tube
• Add 5 mL of water–methanol diluent and vortex for two minutes
• Agitate the mixture at 450 rpm for forty minutes
• Centrifuge at 5000 rpm for ten minutes
• Filter the supernatant through a 0.2 µm nylon syringe filter into an LCMS vial and inject 20 µL

The instrumental setup combined a high-speed UHPLC system with a triple quadrupole mass spectrometer equipped with an APCI source in positive ion mode. Chromatographic separation employed a C18 column held at 40 °C with a gradient from 5 to 95 % methanol in aqueous formic acid at 0.5 mL/min. MS parameters such as gas temperature, flow, and capillary voltage were optimized for sensitivity and low background.

Results and Discussion

Atmospheric pressure chemical ionization in positive mode provided predominant [M+H]+ ions for all five nitrosamines. Multiple reaction monitoring transitions were fine-tuned to balance sensitivity and selectivity. Calibration curves spanning 0.1 to 100 ng/mL achieved correlation coefficients above 0.990. Seven replicates at 1 ng/mL showed peak area relative standard deviations below 2 %, demonstrating excellent reproducibility. Chromatographic divergence of the Pregabalin peak from nitrosamine peaks prevented API-induced contamination of the MS source.

Benefits and Practical Applications

This method meets or exceeds regulatory limit of quantitation requirements with robust performance at trace concentrations. Fast sample preparation, combined with sub-2 % RSD precision, makes it well suited for routine quality control of Pregabalin batches. The efficient chromatographic design minimizes carryover and instrument maintenance.

Future Trends and Potential Applications

Advancements may include high-throughput automation of sample handling, expansion of the target list to other nitrosamines or drug substances, and integration with high resolution mass spectrometry for confirmatory analysis. Ongoing method refinement will support evolving regulatory guidelines and broader pharmaceutical screening campaigns.

Conclusion

A triple quadrupole LC–MS/MS method with APCI source was successfully developed for the sensitive and reproducible determination of five nitrosamine impurities in Pregabalin drug substance. The procedure delivers rapid sample throughput, reliable quantitation, and effective chromatographic separation, making it ready for routine pharmaceutical quality control applications.