Highly Sensitive LC/MS/MS Method for the Simultaneous Quantification of Mutagenic Azido Impurity Analogues in Five Different Sartan APIs and Formulations

Importance of the Topic

Mutagenic azido impurities can form during the synthesis of sartan APIs bearing a tetrazole ring. Regulatory bodies worldwide have issued recalls and alerts due to the potential genotoxicity of impurities such as AZBT and AZBC. Adherence to ICH M7 and related guidelines demands highly sensitive and selective analytical methods to ensure patient safety and product quality.

Objectives and Study Overview

This application note describes the development and validation of two LC/MS/MS methods using an Agilent 6470 triple quadrupole for quantifying two azido impurities—5-(4'-(azidomethyl)-[1,1'-biphenyl]-2-yl)-1H-tetrazole (AZBT) and 4'-(azidomethyl)-[1,1'-biphenyl]-2-carbonitrile (AZBC)—in five sartan APIs and formulations (Losartan, Valsartan, Irbesartan, Candesartan, Olmesartan). Method 1 enables simultaneous quantification of AZBT and AZBC at a 2.5 mg/mL API level; Method 2 targets AZBT only at a 0.3 mg/mL API level.

Methodology and Instrumentation

• Sample Preparation
  • Crush tablets or weigh API, add organic diluent, vortex, sonicate or shake, centrifuge, filter, dilute.
  • Method 1: final API concentration 2.5 mg/mL; Method 2: 0.3 mg/mL.
• UHPLC System
  • Agilent 1290 Infinity II pump, multisampler, thermostat, wavelength detector.
  • Column: Poroshell 120 PFP, 3.0×100 mm, 2.7 µm.
  • Mobile phases: A = 0.1% formic acid in water; B = 0.1% formic acid in 95/5 acetonitrile/water; flow 0.4 mL/min with gradient elution.
• Mass Spectrometry
  • Agilent 6470 triple quadrupole with Jet Stream ESI source in positive mode.
  • MRM transitions AZBT: 278.2→235.1 (quantifier), 278.2→207.1 (qualifier); AZBC: 207.0→179.1, 207.0→151.0.
  • Gas temp 300 °C; gas flow 6 L/min; sheath gas 10 L/min; capillary 4 kV; dwell 50 ms.

Results and Discussion

• LOD/LOQ (Method 1): AZBT LOD 0.125 ng/mL (0.05 ppm), LOQ 0.5 ng/mL (0.2 ppm); AZBC LOD 0.625 ng/mL (0.25 ppm), LOQ 2.5 ng/mL (1 ppm). (Method 2): AZBT LOD 0.033 ng/mL (0.11 ppm), LOQ 0.1 ng/mL (0.33 ppm).
• Linearity: R2 ≥ 0.9955 (AZBT) and ≥ 0.9993 (AZBC) over 0.1–50 ng/mL.
• Recovery at 4 ppm: AZBT 89–107% in APIs; AZBC 80–103% (Candesartan formulation showed unsatisfactory AZBC recovery at LOQ).
• Reproducibility: RSD ≤ 5% at 4 ppm; ≤ 7.6% at LOQ.
• Efficient chromatographic separation of both impurities and all five sartan APIs; diverter valve programming protects the MS from high-concentration API elution.

Benefits and Practical Applications

• Trace-level sensitivity and selectivity for genotoxic azido impurities.
• Simultaneous dual-analyte detection accelerates routine QC workflows.
• Aligned with ICH M7 and regulatory agency requirements (US FDA, EMA, EDQM, MHRA, Health Canada, TGA).
• Adaptable to both API and finished dosage form testing.
• Integration of diverter valve minimizes instrument maintenance.

Future Trends and Potential Applications

• High-resolution MS to expand impurity panels including nitrosamines.
• Automated sample preparation and advanced data processing with machine learning.
• Harmonization of global analytical standards and impurity limits.
• Expansion to other API classes prone to mutagenic byproducts.

Conclusion

Two robust Agilent 6470 LC/MS/MS methods have been established for quantifying mutagenic azido impurities in five sartan APIs and formulations. Both methods achieve sub-ppm detection limits, strong linearity, high recoveries, and reproducible performance, supporting regulatory compliance and reliable quality control in pharmaceutical analysis.

References

• ICH M7(R1) Guideline on Assessment and Control of DNA Reactive (Mutagenic) Impurities.
• Health Canada recall alert for azide impurities in sartan drugs.
• TGA alert for azide impurity in sartan blood pressure medicines.
• UK MHRA recall of sartan batches due to azido impurities.
• EDQM risk alert for mutagenic azido impurities in sartan APIs.
• Swissmedic safety communication on monitoring sartan medicines.
• EDQM CEP general method for azido impurities in sartan substances.
• EDQM method parameters for AZBT determination by LC/MS.
• FDA method for azido compounds in sartan drug substances.