Trace-level Estimation of N-Nitroso Isavuconazonium Sulphate in Isavuconazonium Sulphate API Using Nominal Mass LC/MS

Importance of the topic

The control of N-nitroso impurities in pharmaceutical APIs is crucial due to their potential genotoxicity and regulatory limits at parts-per-billion levels. For isavuconazonium sulphate, accurate trace-level estimation of N-nitroso isavuconazonium sulphate ensures product safety and compliance. Moisture and pH sensitivity of this API pose analytical challenges that require a robust, sensitive approach.

Objectives and Study Overview

This study aims to develop and validate a liquid chromatography–tandem mass spectrometry method capable of quantifying N-nitroso isavuconazonium sulphate (NDSRI) in isavuconazonium sulphate API at sub-ppb concentrations. Key goals include improving sample stability, achieving low limits of detection and quantitation, and demonstrating ruggedness of preparation.

Methodology

• Sample preparation: Dilution in methanol–water (1:1) with 0.1% formic acid; stability assessed over three days.
• Chromatography: Poroshell HPH-C18 column (3.0×150 mm, 2.7 µm) with gradient from 75% aqueous (1 mM ammonium trifluoroacetate, 0.004% formic acid) to 98% acetonitrile over 20 minutes; flow rate 0.8 mL/min; injection volume 20 µL.

Used Instrumentation

• Agilent 6495D triple quadrupole mass spectrometer with AJS electrospray ionization source.
• MRM transitions: m/z 747.2→164.8 (quantifier), m/z 747.2→121.0 (qualifier).
• Source settings: gas flow 13 L/min, nebulizer 35 psi, capillary voltage 1600 V, gas temperature 280 °C, sheath gas temperature 325 °C.

Main Results and Discussion

• Limit of detection (LOD) of 1 ppt and limit of quantitation (LOQ) of 5 ppt at a test concentration of 1 mg/mL.
• Linearity over 1–1000 ppt with R²>0.998.
• Retention time difference of >2 minutes between API and impurity; no blank interferences observed.
• Interday precision (RSD 2.2% over 18 injections) and sample preparation ruggedness (RSD 2.9%–3.5% at 10 and 100 ppt levels).

Practical Benefits and Applications

This validated method delivers reliable quantification of N-nitroso impurity at ultra-trace levels in isavuconazonium sulphate API, supporting stringent quality control and regulatory compliance. Robust sample preparation and solution stability facilitate routine batch release testing.

Future Trends and Applications

• Integration of high-throughput solid-phase extraction to enhance sample cleanup and throughput.
• Adoption of high-resolution mass spectrometry for improved selectivity in complex matrices.
• Extension to formulated drug products, stability studies under stress conditions, and multi-nitrosamine screening.

Conclusion

A sensitive, precise LC-MS/MS method was established for trace quantitation of N-nitroso isavuconazonium sulphate in API. The approach meets stringent regulatory requirements, offering exceptional sensitivity, linearity, and reproducibility for quality assurance.

References

• Low-Level Quantitation of N-Nitroso Dabigatran Etexilate Impurity in Dabigatran Etexilate Mesylate API Using the Agilent 6495C. Agilent Application Note, 5994-7066EN.
• Highly Sensitive Quantification of Mutagenic NDSRI N-Nitroso Propranolol in Propranolol API and 40 mg Tablets Using LC/MS/MS. Agilent Application Note, 5994-5161EN.
• Quantitation of N-Nitroso Sitagliptin Impurity (NTTP) in Sitagliptin and Metformin Combination Drug Product Using the Agilent 6475 LC/TQ. Agilent Application Note, 5994-7161EN.
• Nitrosamine Impurities Application Guide. Agilent Application Guide, 5994-2393EN.
• Ultra-Fast Analysis of Nitrosamines Using SPE-QQQ. Agilent Application Note, 5994-3752EN.