Quantitation of N-Nitroso-Desmethyl- Diltiazem, an NDSRI Impurity of Diltiazem Hydrochloride in Drug Product Using LCMS/ MS

Importance of the topic

The control of N-nitrosamine impurities in pharmaceutical products remains a high-priority safety issue because many nitrosamines are genotoxic and potentially carcinogenic. Nitrosamine drug substance related impurities (NDSRIs) such as N-nitroso-desmethyl-diltiazem (NDD) can form in final dosage forms derived from diltiazem hydrochloride; therefore robust, trace-level analytical methods are required to meet evolving regulatory expectations and to avoid batch-release complications.

Objectives and study overview

This application note reports the development and validation-style performance assessment of an ultra‑performance liquid chromatography tandem mass spectrometry (UPLC‑MS/MS) method for quantifying NDD in diltiazem tablets. The method was designed to achieve sensitivity well below regulatory thresholds (including an earlier conservative limit of 0.05 ppm with a 10% LOQ target of 0.005 ppm, and the more recent calculated threshold based on an AI of 100 ng/day of 0.185 ppm with a 10% target of 0.0185 ppm). Performance metrics evaluated include linearity, sensitivity, repeatability, spike recovery in the tablet matrix and chromatographic selectivity versus the API.

Methodology and analytical workflow

Sample preparation:

• Individual tablets (120 mg) were pulverized and extracted to yield a solution equivalent to 40 mg API/mL.
• Extracts were vortexed, centrifuged (3000 rcf), and filtered through 0.20 μm PVDF prior to analysis.
• Calibration solutions of authentic NDD standard covered 0.01–10 ng/mL (0.00025–0.25 ppm method equivalents).

Chromatography and mobile phases:

• Reversed‑phase separation on ACQUITY UPLC HSS T3 column (1.8 μm, 3.0 x 100 mm).
• Mobile phase A: 0.1% acetic acid in LC/MS water; Mobile phase B: LC/MS acetonitrile; gradient elution applied.
• Column temperature 45 °C, sample temperature 10 °C, injection volume 2.0 μL.

Mass spectrometry and data processing:

• Electrospray ionization in positive mode with MRM acquisition for resolved NDD isomers.
• Key MS settings: capillary 2.5 kV; source 150 °C; desolvation 500 °C; cone gas 50 L/hr; desolvation gas 1000 L/hr.
• MRM optimization, acquisition and quantitative processing were managed using waters_connect for Quantitation with integrated rule sets, impurity‑ppm calculations, and 21 CFR Part 11–capable audit features.

Used instrumentation

• ACQUITY UPLC H-Class Plus System (LC)
• ACQUITY UPLC PDA Detector
• Quaternary Solvent Manager and Sample Manager with Flow‑Through Needle
• Waters ACQUITY UPLC HSS T3 column, 1.8 μm, 3.0 x 100 mm
• Xevo TQ‑S micro Tandem Quadrupole Mass Spectrometer (ESI+)
• waters_connect for Quantitation software

Main results and discussion

Chromatographic selectivity:
The method achieved baseline separation between diltiazem API (tR ≈ 8.5 min) and two chromatographically resolved NDD isomers (tR ≈ 11.6 and 12.1 min), minimizing API-related matrix effects. The system fluidics allow diverting the high‑abundance API region to waste to protect MS linearity and sensitivity.

Linearity and sensitivity:
Calibration over 0.01–10 ng/mL showed excellent linearity (R² > 0.999) using 1/x weighting, covering at least three orders of magnitude. The method routinely detected NDD at or below the previously more stringent 10% LOQ target (0.005 ppm method equivalent) and provides sufficient margin relative to the current regulatory 10% target (0.0185 ppm equivalent for AI = 100 ng/day, assuming 540 mg MDD). Signal‑to‑noise for the lowest standard (0.01 ng/mL) was acceptable per automated RMS S/N calculation.

Repeatability and recovery in final dosage form:
Repeatability at the 10% threshold standard (0.2 ng/mL, 0.005 ppm equivalent) showed low area %RSDs (2.9% and 3.1% for the two isomers, n = 6). Spike recovery experiments in extracted tablet matrix across levels equivalent to 10–150% of the threshold (0.005–0.075 ppm method equivalents) returned mean recoveries between 90.7% and 106.6% with %RSD < 10% for all levels and isomers. These data indicate reliable quantitation in the presence of complex tablet matrix and endogenous impurity levels (pre‑existing NDD in the API were corrected for in recovery calculations).

Data handling and compliance features:
waters_connect MS Quan rules enabled automatic flagging of calibration and QC exceptions, impurity ppm calculations referenced to API concentration in the extract, and streamlined reporting. The platform supports data integrity via audit trails, access controls and electronic signatures, facilitating regulated workflows.

Benefits and practical applications

• High sensitivity and broad linear range enable monitoring of NDD at concentrations below regulatory 10% LOQ targets, supporting release and stability testing strategies.
• Chromatographic resolution of API and NDSRI isomers reduces risk of bias from matrix interferences and enables accurate isomer-specific reporting.
• Integrated software functionality simplifies method setup, MRM optimization, automated exception handling and impurity flagging—reducing manual review time in regulated QC environments.

Future trends and potential uses

Routine nitrosamine control will continue to evolve with updated acceptable intake values and read‑across strategies. Future work may include:

• Wider adoption of targeted LC‑MS/MS workflows for other NDSRIs across diverse APIs and formulations.
• Implementation of isotopically labelled internal standards to further improve accuracy and compensate for residual matrix effects.
• Automated sample‑preparation or on‑line extraction techniques to increase throughput and reduce variability in high‑volume QC labs.
• Expanded library building of RADAR/scan information to support rapid screening for unknown nitrosamines during method development.

Conclusion

The reported UPLC‑ESI‑MS/MS procedure reliably quantifies N‑nitroso‑desmethyl‑diltiazem in diltiazem tablets with sensitivity and precision that meets or exceeds relevant regulatory 10% LOQ targets, including historically stringent criteria. Chromatographic separation from the API, robust calibration metrics, satisfactory spike recoveries in final dosage form, and integrated compliant software tools make this approach suitable for regulatory control and routine QC monitoring of NDD in drug products.

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