Highly sensitive method for the determination of 12 nitrosamine impurities in multiple ARBs, the class of sartan drug formulations

Importance of the topic

Angiotensin II receptor blockers (ARBs) are widely used to manage hypertension, heart failure, and diabetic kidney disease. During their synthesis, potentially carcinogenic nitrosamine impurities can form, triggering global recalls when levels exceed regulatory limits. Sensitive and robust analytical methods are essential to ensure drug safety and compliance.

Objectives and article overview

This study aimed to develop and validate a single, highly sensitive LC–MS/MS method for simultaneous quantification of twelve nitrosamine impurities in three ARB drug substances—olmesartan, irbesartan, and valsartan. The goal was to achieve sub-nanogram detection limits, robust chromatographic separation from active pharmaceutical ingredients (APIs), and high throughput suitable for routine quality control.

Methodology and instrumentation

Standards for twelve nitrosamines were individually prepared at 100 µg/mL in methanol and diluted to generate linearity, LOQ (0.15 ng/mL), and LOD (0.0375–0.075 ng/mL) solutions. Drug substance samples (50 mg/mL) were extracted in 60/40 methanol–water, sonicated, centrifuged, and filtered through 0.2 µm PVDF membranes. Chromatographic separation employed a C18 reversed-phase column (4.6 × 250 mm, 5 µm) at 45 °C with a gradient of 0.1% formic acid in water and methanol, 0.5 mL/min flow, and 40 µL injection. A divert valve protected the MS from API overload. Detection used APCI in positive mode on a triple quadrupole mass spectrometer with selected reaction monitoring transitions tuned for each analyte.

Used instrumentation

• Vanquish Flex UHPLC system
• TSQ Altis Plus triple quadrupole mass spectrometer (APCI source)
• Chromeleon CDS v7.3.2 for data acquisition and reporting
• Acclaim 120 C18 column (4.6×250 mm, 5 µm)
• PEAK Scientific nitrogen generator, Chemyx infusion pump, Rheodyne 6-port valve
• PVDF syringe filters (0.2 µm) and glass screw-cap vials

Main results and discussion

System suitability tests yielded retention time RSD ≤0.3% and area RSD ≤5% for all nitrosamines. Linearity was excellent (R²>0.999) from 0.15 to 20 ng/mL. LOQ and LOD were below current regulatory thresholds. Recovery across low (0.15 ng/mL), mid (1.5 ng/mL), and high (20 ng/mL) levels ranged from 80% to 120% for all analytes in each ARB. A 41-hour sequence demonstrated method robustness with cumulative area RSD <8%. Chromatograms showed clear separation of twelve nitrosamines from API peaks.

Benefits and practical applications

• One validated method for twelve nitrosamine impurities in three APIs
• High sensitivity meeting or exceeding FDA and EMA guidelines
• Robust workflow for high-throughput screening in quality control
• Full compliance with ICH Q2(R2), FDA, and EMEA data integrity requirements

Future trends and possibilities

Emerging approaches may extend this method to other drug classes and unknown nitrosamine screening using high-resolution MS. Miniaturized sample preparation, automated online SPE, and AI-driven data evaluation will enhance throughput. Portable MS platforms could enable real-time monitoring of nitrosamine levels during manufacturing.

Conclusion

The developed LC–MS/MS assay provides a sensitive, reproducible, and regulatory-compliant solution for monitoring twelve nitrosamine impurities in multiple ARB drug substances. Its robustness, throughput, and simplicity make it ideal for routine pharmaceutical quality control.

Reference

• FDA. Angiotensin II receptor blockers (angiotensin II inhibitors). Drugs.com.
• European Medicines Agency. Angiotensin-II-receptor antagonists containing tetrazole group: referral opinion.
• FDA. Guidance for industry: Control of nitrosamine impurities in human drugs. Sept 2024.
• FDA. Search list of recalled angiotensin II receptor blockers including valsartan, losartan, irbesartan.
• FDA. Recommended acceptable intake limits for nitrosamine drug substance-related impurities.
• European Medicines Agency. QA Article 5(3) Implementation: Nitrosamines revision Oct 2023.
• European Medicines Agency. ICH Q2(R2) Validation of analytical procedures guideline.
• FDA. LC-HRMS method for six nitrosamine impurities in ARBs.