Liquid chromatography – high-resolution mass spectrometry method for determination of NDSRI in six beta-blocker drug substances

LC-HRMS method for determination of NDSRIs in six beta‑blocker drug substances — Application note summary

Importance of the topic

Nitrosamine drug substance‑related impurities (NDSRIs) are nitroso derivatives structurally related to active pharmaceutical ingredients (APIs). Their presence at unacceptable levels in drug substances or products has led to global regulatory concern and recalls. Reliable, selective and sensitive methods to detect and quantify NDSRIs at regulatory acceptable intake (AI) limits are therefore essential for risk assessment and quality control in pharmaceutical development and manufacturing.

Goals and study overview

• Develop a single, versatile LC‑HRMS workflow able to quantify NDSRIs associated with six beta‑blocker APIs: atenolol, bisoprolol fumarate, carvedilol, labetalol HCl, metoprolol fumarate and propranolol HCl.
• Achieve robust chromatographic separation between each API and its related NDSRI within a short runtime (20 min) while meeting regulatory expectations for sensitivity, accuracy and reproducibility.
• Demonstrate method performance relative to CPCA/AI guidance from regulatory agencies (FDA and EMEA) and ensure data integrity in acquisition and reporting.

Methodology and experimental design

• Analytical strategy: high‑resolution product‑ion LC‑HRMS quantitation with ion‑ratio confirmation using Thermo Scientific Orbitrap technology.
• Sample preparation: simple extraction of drug substance in 50:50 methanol:water diluent. Typical sample concentrations were 2 mg/mL for some APIs and 10 mg/mL for others; sonication/vortexing, centrifugation (12,000 rpm, 5 °C, 15 min) and 0.2 µm PVDF filtration prior to analysis.
• Calibration and validation design: LOD (triplicate), LOQ (six replicates), eight‑level linearity from 10% to 350% of the specification level (triplicates), reproducibility (six replicates at 100% level), recovery at 10%, 100% and 350% levels, and long‑run robustness with bracketing standards over a 21‑hour sequence.
• Data processing: extracted ion chromatograms (XIC) using mass precision to five decimal places; mass tolerances of 5 ppm (for NNCA, NNLA) and 10 ppm (others); ion‑ratio confirmation per EC decision criteria.

Instrumentation

• Liquid chromatography: Thermo Scientific Vanquish Flex UHPLC (Binary Pump F, Split Sampler FT, Column Compartment H, Vanquish Diode Array detector) with Thermo Scientific Hypersil GOLD C18 Selectivity column, 3 × 100 mm, 3 µm.
• Mass spectrometry: Thermo Scientific Orbitrap Exploris 120 high‑resolution mass spectrometer operating in positive H‑ESI mode, Orbitrap resolution 120,000, product‑ion MS/MS acquisition for selectivity and confirmation.
• Software: Chromeleon CDS for acquisition/processing/reporting and Mass Frontier 8.0 SR2 for fragmentation interpretation and product‑ion selection.
• Consumables and gases: Optima LC/MS grade solvents (water, methanol, formic acid), 0.2 µm PVDF filters, nitrogen generator.

Key method parameters (concise)

• Column temp 40 °C; flow 0.4 mL/min; mobile phases A = 0.1% formic acid in water, B = 0.1% formic acid in methanol; 20‑min gradient for separation and API/NDSRI resolution.
• Injection volumes varied by analyte (2–4 µL) to optimize sensitivity and peak shape.
• MS: positive polarity, product‑ion scans optimized per NDSRI (collision energies tuned), maximum injection time and scan range Auto, centroid data type.

Main results and discussion

• Sensitivity: LOQ set to 10% of the regulatory specification limit for each NDSRI; LOD ≈ 1/3 LOQ. Minimum observed S/N at LOD was excellent (≥102 across analytes).
• Linearity: coefficient of determination R2 > 0.99 for all six NDSRIs across 10%–350% calibration range.
• Precision and robustness: intra‑run reproducibility at specification level showed %RSD ≤ 3.2% for replicate injections; long‑run robustness including bracketing standards over 21 hours showed cumulative %RSD up to 6.4% (acceptable for routine QA/QC).
• Recovery: extraction recoveries ranged ~77%–107%, within the commonly accepted 70%–130% criteria for impurity assays.
• Selectivity: high‑resolution precursor and product‑ion scanning resolved isobaric interferences in several cases; ion‑ratio confirmation met target criteria for all analytes, supporting specificity of quantitation even when adduct formation was observed.
• Chromatography: Hypersil GOLD C18 Selectivity column and optimized gradient provided consistent retention time separation of each NDSRI from its parent API within the 20‑minute runtime.

Benefits and practical applications

• Single, transferable LC‑HRMS method applicable to six beta‑blocker APIs reduces method proliferation and supports streamlined workflows in pharmaceutical QC and stability testing.
• High resolution and accurate mass reduce false positives from isobaric interferences and improve confidence near regulatory limits (AI values determined by CPCA/FDA guidance).
• Chromeleon CDS and Mass Frontier integration enable traceable data handling, spectral interpretation and robust reporting suitable for regulated environments.
• Method is suitable for routine release testing, stability studies and investigational work during API or product development where nitrosamine risk assessment is required.

Future trends and potential uses

• Wider application: the demonstrated approach (LC‑HRMS with product‑ion confirmation and ion‑ratio checks) can be extended to NDSRIs of other API classes with appropriate method optimization of chromatography and MS transitions.
• Automation and higher throughput: further miniaturization of injection volumes, multiplexed sample preparation, or UHPLC column screening could shorten runtimes and increase sample throughput while retaining selectivity.
• Regulatory harmonization: as AI determination approaches (CPCA and guidance) evolve, adaptable HRMS workflows will be valuable for rapid implementation of revised limits and multi‑analyte surveillance.
• Software advances: improved automated fragment assignment and spectral libraries for common NDSRIs will speed method development and confirmation steps.

Conclusion

A robust, validated LC‑HRMS method using a Vanquish Flex UHPLC front‑end and Orbitrap Exploris 120 mass spectrometer was developed and demonstrated for the quantitation of six NDSRIs in their respective beta‑blocker drug substances. The method meets key validation criteria (sensitivity, linearity, precision, recovery, specificity) and provides a practical, single‑method solution for routine monitoring of these nitrosamine impurities in regulated pharmaceutical environments.

References

1. FDA guidance on recommended acceptable intake limits for nitrosamine drug substance related impurities (NDSRIs).
2. EMA guidance and updates on nitrosamine impurities including CPCA and acceptable intake advice.
3. ICH Q2(R2) — Validation of analytical procedures — scientific guideline.
4. European Commission Decision 2002/657/EC — analytical method confirmation criteria.
5. Methodological examples and LC‑HRMS nitrosamine workflows used by regulatory agencies and USP community resources.