Quantitation of N-Nitroso Hydrochlorothiazide in Losartan- Hydrochlorothiazide Tablets

Significance of the Topic

Losartan and hydrochlorothiazide combination therapy is widely prescribed for hypertension, but N-nitroso impurities pose safety concerns. Sensitive detection of N-nitroso HCTZ ensures drug safety and regulatory compliance.

Objectives and Study Overview

• Develop a sensitive and reproducible MRM LC/MS/MS method for N-nitroso HCTZ quantification in losartan-HCTZ tablets
• Establish method sensitivity, linearity, and reproducibility
• Apply the method for routine quality control and impurity monitoring

Methodology

Sample preparation involved tablet crushing, dilutions in methanol:water, and spiking placebo and tablet matrices at LOQ levels. Calibration standards ranged from 10 pg/mL to 10 ng/mL. Chromatography used an Agilent ZORBAX SB C18 column (4.6 × 150 mm, 5 µm) with a gradient of 1 mM ammonium fluoride in water (A) and acetonitrile (B) at 0.3 mL/min, injection volume 20 µL, and column temperature 40 °C.

Mass spectrometry operated in negative electrospray mode using MRM transition 325.0→293.9 m/z for N-nitroso HCTZ with optimized fragmentor and collision energy. Method validation covered linearity, LOD/LOQ, reproducibility, specificity, and recovery.

Used Instrumentation

• Agilent 1290 Infinity II LC system (high-speed pump, multisampler, multicolumn thermostat, diode array detector)
• Agilent 6495D Triple Quadrupole LC/MS/MS system with Jet Stream ion source

Key Results and Discussion

Chromatographic separation ensured clear resolution of N-nitroso impurity from losartan and HCTZ APIs, with a diverter valve diverting API elution to waste. The method achieved:

• LOD of 5 pg/mL and LOQ of 10 pg/mL with S/N of 121:1 at LOQ
• Linearity from 10 pg/mL to 10 ng/mL (R² = 0.9952)
• Spike recoveries of 114.35% in placebo and 93.85% in tablets
• %RSD of 2.7% for seven replicate injections at LOQ

Benefits and Practical Applications

• Ensures sensitive and reliable detection of nitrosamine impurities in combination drugs
• Supports compliance with regulatory limits and safety guidelines
• Facilitates routine quality control and impurity profiling

Future Trends and Potential Applications

• Extension of the method to other nitrosamine impurities and drug matrices
• Integration with high-resolution mass spectrometry for structural confirmation
• Automation for high-throughput screening in pharmaceutical QC labs
• Online and in-line monitoring for continuous manufacturing processes

Conclusion

The developed MRM LC/MS/MS method on the Agilent 6495D system offers high sensitivity, robustness, and reproducibility for quantifying N-nitroso HCTZ in losartan-HCTZ tablets, providing a valuable tool for pharmaceutical quality assurance and regulatory compliance.

Reference

• Mastovska K.; Zulkoski J.; Zweigenbaum J. Triggered MRM LC/MS/MS Method Development – Practical Considerations for MRM Optimization Using Agilent MassHunter Optimizer Software. Agilent Technologies, 2017.
• Sosienski T.; Covert K. Trace-Level Quantification of Potential Mutagenic Impurities in Pharmaceuticals. Agilent Technologies, 2019.
• Joseph P.; Banerjee S.; Vyas S. Determination of Genotoxic Nitrosamine Impurity in Bumetanide API and Tablets Using the Agilent 6470 Triple Quadrupole LC/MS. Agilent Technologies, 2020.