Quantitation of 8 Nitrosamines in Thiocolchicoside API by LCMS-8045 system

Importance of the Topic

Residual nitrosamines are classified as probable human carcinogens and have been detected in various drug products. Regulatory agencies impose stringent intake limits down to 26.5 ng/day, driving the need for highly sensitive analytical methods. Monitoring nitrosamine impurities in Thiocolchicoside API ensures patient safety and regulatory compliance.

Objectives and Study Overview

The study aimed to develop and validate a simple, robust, and high-throughput LC-MS/MS method for simultaneous quantitation of eight nitrosamines in Thiocolchicoside API without complex sample pretreatment. Eight target analytes include NDMA, NMBA, NDEA, NEIPA, NDIPA, NDPA, NMPA, and NDBA.

Methodology

Chromatographic separation was performed on a Shim-pack AQ C18 column (100 × 4.6 mm, 3 µm) using a gradient of 0.1% formic acid in water (A) and methanol (B) at 0.7 mL/min. A divert valve protected the MS by sending the API peak to waste. MRM transitions were optimized for each analyte with four isotope-labeled internal standards to ensure accurate quantitation. Sample preparation involved dilution of the API in aqueous diluent, spiking with internal standards, vortex mixing, filtration, and direct injection.

Instrumentation Used

Ultrahigh-performance liquid chromatography was performed on Nexera XS coupled to the Shimadzu LCMS-8045 triple quadrupole mass spectrometer equipped with an APCI interface. The instrument features ultra-fast scanning (30,000 u/sec), rapid polarity switching (5 msec), and high sensitivity. Column temperature was maintained at 40 °C, and interface temperatures were 350 °C (APCI), 200 °C (desolvation line), and 200 °C (heater block).

Main Results and Discussion

The method demonstrated linearity over 0.5 to 100 ppb for all eight nitrosamines with correlation coefficients exceeding 0.995. Limits of quantitation were established at 0.5 ppb (S/N ratio and RSD within acceptable criteria). Repeatability at LOQ (n=6) showed RSD below 15%. Recovery studies at 0.5, 1, and 3 ppb levels yielded recoveries between 70% and 130%, meeting regulatory acceptance criteria. Representative calibration curves and MRM chromatograms confirmed method robustness and selectivity.

Benefits and Practical Applications

• Simultaneous quantitation of eight nitrosamines in a single run
• No need for complex sample cleanup or derivatization
• High sensitivity and specificity with LOQ at sub-ppb levels
• Rapid analysis with a total run time of 25 minutes
• Protection of the MS from API matrix using a divert valve

Future Trends and Opportunities

Advancements may include extending the method to additional nitrosamine analogues, integrating high-resolution mass spectrometry for enhanced specificity, and automating sample preparation to support large-scale screening. On-line monitoring and coupling with process analytical technology could enable real-time quality control in manufacturing.

Conclusion

A single-run LC-MS/MS method on the Shimadzu LCMS-8045 system was successfully developed and validated for eight nitrosamines in Thiocolchicoside API. The approach offers high sensitivity, robustness, and compliance with regulatory nitrosamine limits, providing a practical solution for routine quality control and safety assessment.