Streamlining Analytical Method Development for NDSRIs and Mutagenic Impurities in Pharmaceutical Formulations Using Single-Quadrupole LC-MS

Importance of the topic

Control of nitrosamine drug substance-related impurities (NDSRIs) and mutagenic impurities is a regulatory and public-health priority in pharmaceutical quality control. NDSRIs arise from nitrosation of APIs or related impurities and can vary widely in structure and toxicity. Regulatory approaches such as the Carcinogenic Potency Categorization Approach (CPCA) require sensitive, compound-specific analytical methods to meet Acceptable Intake (AI) limits. The presented work demonstrates a streamlined workflow combining method-development software and a single-quadrupole LC‑MS to rapidly develop and validate LC conditions for simultaneous screening and quantification of an NDSRI (N‑nitrosoatenolol) and a mutagenic impurity (atenolol impurity D) in atenolol drug products.

Study objectives and overview

The study aimed to:

• Efficiently develop LC separation conditions (mobile phase, column, gradient) for atenolol, N‑nitrosoatenolol, and atenolol impurity D using LabSolutions MD;
• Assess the feasibility of quantitative analysis of these impurities in API and finished dosage forms using a single‑quadrupole LC‑MS (LCMS‑2050) with high sensitivity and precision;
• Demonstrate simultaneous screening for NDSRIs and mutagenic impurities at concentrations relevant to AI limits.

Methodology

Method development approach:

• LabSolutions MD was used to perform high‑throughput evaluation of column and mobile phase combinations via valve‑switching and to generate and rank gradient programs. The software also applied AI‑based retention predictions and gradient optimization to maximize resolution between closely eluting analytes.
• Comprehensive screening included two reversed‑phase C18 short columns and multiple aqueous modifiers (5 mM ammonium formate, 5 mM ammonium acetate, 0.1% formic acid, water) paired with methanol or acetonitrile as organic solvent.

Final chromatographic conditions (selected after optimization):

• Instrument: Nexera X3 LC coupled to LCMS‑2050 (single‑quadrupole, DUIS ionization);
• Column: Shim‑pack Velox SP‑C18, 50 × 2.1 mm, 2.7 µm; column temperature 40 °C;
• Mobile phases: A = 5 mM ammonium formate in water; B = methanol; flow 0.4 mL/min;
• Gradient: 20% B (0–2.00 min) → 99% B (5.00–7.00 min) → 20% B (7.01–10.00 min); injector divert to waste 0–1.5 min;
• Injection volume: 1 µL.

Mass spectrometry and detection:

• LCMS‑2050 operated in positive/negative DUIS with SIM monitoring for target masses: atenolol (m/z 267.17), N‑nitrosoatenolol (m/z 296.16), atenolol impurity D (m/z 244.07); overall scan range ±100–1000 m/z.
• Typical source/settings: nebulizing gas 3 L/min, drying gas 3 L/min, heating gas 7 L/min, DL 250 °C, desolvation 500 °C, interface voltage +0.5 kV / −2.0 kV.

Used instrumentation

• Shimadzu Nexera X3 UHPLC system.
• Shimadzu LCMS‑2050 single‑quadrupole mass spectrometer with DUIS ionization.
• Columns evaluated: Shim‑pack Velox SP‑C18 (selected), Shim‑pack Scepter C18‑120.
• Software: LabSolutions MD for method development, automatic gradient generation and AI‑based chromatogram prediction.

Main results and discussion

Method development outcomes:

• LabSolutions MD accelerated evaluation of column/mobile phase combinations and gradient permutations and enabled visual ranking by resolution and sensitivity. The optimal combination delivered improved separation between N‑nitrosoatenolol and atenolol impurity D, which initially co‑eluted closely.
• AI‑predicted chromatograms and gradient optimization produced a gradient that matched observed separations and enhanced resolution for the critical pair.

Analytical performance:

• Calibration and linearity: N‑nitrosoatenolol met the 80–120% accuracy criterion from 0.2–200 ng/mL; atenolol impurity D met it from 2–200 ng/mL. All calibration curves showed r² ≥ 0.999, indicating excellent linearity.
• Sensitivity: Good peak shapes and signal were obtained at the lowest calibration levels (0.2 ng/mL for N‑nitrosoatenolol, 2 ng/mL for impurity D) using SIM on a single‑quadrupole instrument.
• Spike recovery and precision — API sample: For N‑nitrosoatenolol spiked at 1.5 ppm (one‑tenth of AI) recovery = 106.6% (RSD 3.0%); at 15 ppm recovery = 100.4% (RSD 1.6%). Atenolol impurity D spiked at 15 ppm recovery = 96.3% (RSD 3.3%).
• Spike recovery and precision — drug product: For N‑nitrosoatenolol spiked at 1.5 ppm recovery = 106.4% (RSD 1.8%); at 15 ppm recovery = 110.0% (RSD 1.5%). Atenolol impurity D at 15 ppm recovery = 103.0% (RSD 1.9%). These results were achieved with a simple sample pretreatment (powder tablets, methanol extraction, centrifugation).
• Interferences: A nearby interfering peak (RT ≈ 3.25 min) produced signal at m/z 295 and 296; interpretation suggests this corresponds to the 13C isotope of protonated N‑formylatenolol (monoisotopic mass 294.16). Despite this interference, quantification of the target NDSRI remained reliable due to chromatographic resolution and SIM specificity.

Benefits and practical applications

• The combined use of LabSolutions MD and a single‑quadrupole LC‑MS enables rapid, resource‑efficient method development and validation for a variety of API‑related NDSRIs and mutagenic impurities without immediate recourse to high‑end HRMS instruments.
• Single‑quadrupole SIM offers sufficient sensitivity and linearity for routine screening and quantification of impurities at or below regulatory AI limits when coupled with optimized chromatography.
• Simple methanolic extraction of tablet formulations provided robust recoveries, supporting applicability in QC laboratories for initial screening and release testing workflows.

Future trends and potential applications

• Integration of method‑development software with AI retention and gradient prediction will further shorten development cycles and reduce experimental iterations for diverse NDSRIs.
• Broader application of targeted single‑quadrupole SIM workflows for routine surveillance of nitrosamines and mutagenic impurities across multiple APIs, with HRMS reserved for structure elucidation and confirmation of ambiguous signals.
• Automated sample‑preparation and higher‑throughput LC systems, combined with in‑line diversion strategies, will enable scalable screening in manufacturing and stability studies.
• Development of compound libraries and predicted mass/retention databases could facilitate multi‑analyte screening panels tailored to specific APIs and synthetic routes.

Conclusion

The case study demonstrates that LabSolutions MD guided method development paired with the LCMS‑2050 single‑quadrupole system can provide a fast, sensitive, and precise workflow for simultaneous analysis of NDSRIs and mutagenic impurities in both API and finished drug products. Optimized chromatography and targeted SIM detection achieved regulatory‑relevant sensitivity, excellent linearity, and reproducible recoveries with minimal sample preparation, supporting the practicality of single‑quadrupole LC‑MS for routine impurity screening in pharmaceutical quality control.

Reference

1. US Food and Drug Administration. Control of Nitrosamine Impurities in Human Drugs: Guidance for Industry, Revision 2, September 2024.
2. US Food and Drug Administration. Recommended Acceptable Intake Limits for Nitrosamine Drug Substance‑Related Impurities (NDSRIs) Guidance for Industry, August 2023.
3. US Food and Drug Administration. FDA Recommended AI Limits for Certain Hypothetical NDSRIs and Other Identified Nitrosamine Impurities, Updated 19 March 2026.
4. ICH M7(R2) Guideline. Assessment and Control of DNA Reactive (Mutagenic) Impurities in Pharmaceuticals to Limit Potential Carcinogenic Risk, Adopted 24 May 2022.