Application Handbook Liquid Chromatography

Significance of the topic

The presence of low‐level impurities in active pharmaceutical ingredients (APIs) can compromise product safety and efficacy. Traditional one‐dimensional LC methods may not provide sufficient resolution or qualitative information on coeluting impurities. A rapid and reliable workflow for impurity separation, identification, and quantitation is essential for quality control, regulatory compliance, and root‐cause analysis in pharmaceutical development and manufacturing.

Objectives and study overview

This study demonstrates the use of Shimadzu’s Co‐Sense for Impurities System to analyze a trace impurity in rabeprazole sodium. The goals were to:

• Separate a minor impurity peak from the main API peak using two‐dimensional LC.
• Identify the chemical structure of the impurity by MS/MS.
• Establish a streamlined workflow for impurity source determination.

Methodology and instrumentation used

A two‐dimensional LC/MS workflow was set up with three columns and two pneumatic switching valves:

• ① First‐dimension separation on an ODS column with UV detection at 290 nm.
• ② Trap and exchange into volatile buffer on a second column, removing nonvolatile additives.
• ③ Second‐dimension re‐injection onto a microbore ODS column with MS detection (Shimadzu LCMS-8030, ESI positive).

Key operational parameters included phosphate buffer/methanol mobile phases in the first dimension and ammonium acetate buffers in the second dimension. Valve switching times were synchronized with the elution of the impurity peak.

Main results and discussion

Initial one‐dimensional LC‐UV of rabeprazole sodium revealed an impurity peak (0.06% of API area). With Co‐Sense, the target fraction was diverted off‐line, concentrated, and re‐separated, yielding three distinct impurity peaks by MS. Product‐ion scanning of the 508.2 m/z precursor ion enabled structural elucidation of one impurity as a benzimidazol‐2‐thiol derivative of rabeprazole. This approach overcame coelution challenges and provided MS/MS confirmation on a low‐level impurity.

Benefits and practical applications

• Efficient separation of trace impurities from major components and from each other.
• Structural identification via high‐sensitivity MS/MS without extensive sample prep.
• Fast method development and impurity source tracking in API batches.
• Compliance with ICH guidelines on impurity profiling and control.

Future trends and possibilities

Integration of this two‐dimensional LC/MS approach with high‐resolution MS, automated trapping protocols, and advanced data processing will enable untargeted impurity profiling across broader compound classes. Miniaturization and on‐line coupling could further reduce solvent consumption and analysis time, supporting real‐time batch release decisions.

Conclusion

Shimadzu’s Co-Sense for Impurities System successfully separated and identified a low‐level benzimidazol‐2‐thiol impurity in rabeprazole sodium. The two‐dimensional LC/MS workflow provides a robust platform for comprehensive impurity profiling in pharmaceutical development and QC.