Structural Analysis by In-Depth Impurity Search Using MetID Solution and High Accuracy MS/MS

Importance of the Topic

The detection and structural characterization of trace impurities in chemical and pharmaceutical products is essential for ensuring safety, efficacy, and regulatory compliance. Advanced software-assisted workflows combined with high-resolution MS/MS enable rapid, comprehensive profiling of unknown analogues, reducing manual interpretation time and improving confidence in impurity identification.

Objectives and Study Overview

This study demonstrates an automated impurity discovery and structural elucidation workflow applied to an alcoholic beverage adulterated with sildenafil-like compounds. The goals were to extract candidate impurities sharing fragmentation features with the drug, predict their structures, and confirm novel analogues using MetID Solution software and LCMS-IT-TOF instrumentation.

Methodology and Instrumentation

• Chromatography: Prominence HPLC with Phenomenex Gemini C18 column (2.0 × 150 mm, 5 µm).
• Mobile phases: 5 mM ammonium acetate in water (A) and acetonitrile (B); gradient from 5% B to 100% B over 35 min.
• Flow rate and injection: 0.2 mL/min, 1 µL injection, column at 40 °C.
• Mass spectrometry: LCMS-IT-TOF in positive ESI Auto MS/MS mode; probe +4.5 kV, nebulizing gas 1.5 L/min, CDL and block heater at 200 °C.
• Data processing: MetID Solution PLS analysis for impurity candidate extraction, followed by automated MS² fragmentation matching and neutral loss comparison.

Main Results and Discussion

The total ion chromatogram revealed multiple peaks, only one of which corresponded to the protonated sildenafil ion (m/z 475.2122). PLS scoring in MetID Solution highlighted substances sharing key MS/MS fragments and neutral losses with sildenafil. One candidate, P#212 (m/z 489.2276), matched the mass of known analogues (vardenafil, homosildenafil) but exhibited distinct fragmentation patterns. Analysis of overlapping neutral losses at 176 and 192 Da enabled pinpointing additional CH₂ insertions in the molecule, confirming P#212 as a novel sildenafil derivative differing from reported analogues in methylation sites.

Benefits and Practical Applications

• Streamlined impurity screening by automated extraction of structurally related compounds.
• Rapid fragment-based structure prediction reduces manual interpretation workload.
• Applicability to pharmaceutical QA/QC, metabolite profiling, and forensic analysis of adulterants.
• Flexibility to include user-defined transformations for targeted impurity searches.

Future Trends and Possibilities

• Integration of machine learning for predictive fragment assignment and impurity prioritization.
• High-throughput screening of complex mixtures using automated MSⁿ workflows.
• Extension to non-drug small molecules and environmental contaminant profiling.
• Real-time cloud-based data analytics for collaborative impurity database building.

Conclusion

The combined use of MetID Solution’s PLS-based impurity search and high-accuracy MS/MS enabled efficient discovery and structural elucidation of sildenafil analogues in a complex matrix. This approach significantly reduces manual analysis time and can be widely applied to impurity profiling in the pharmaceutical, food, and chemical industries.

Reference

• Shimadzu Corporation. Structural Analysis by In-Depth Impurity Search Using MetID Solution and High Accuracy MS/MS. Technical Report vol.16, C146-E118, January 2008.