Routine MS Detection for USP Chromatographic Methods

Importance of the Topic

Mass spectrometric analysis is an essential tool for the confirmation and characterization of pharmaceutical compounds and impurities. United States Pharmacopeia (USP) methods are widely adopted for routine assay and identification of APIs, yet their nonvolatile mobile phases preclude direct MS detection. A streamlined protocol that integrates MS without modifying established USP methods addresses critical needs in validation, impurity investigation, and quality control.

Objectives and Study Overview

This work presents a practical workflow for direct MS detection of peaks from unmodified USP HPLC methods. Using Irbesartan and its related compound A (RC-A) as test compounds, the study demonstrates a heart-cutting two-dimensional LC approach coupled with at-column dilution to transfer analyte fractions onto a second-dimension UPLC column and an ACQUITY QDa mass detector for identity confirmation and spectral acquisition.

Methodology and Instrumentation

A dual-pump ACQUITY UPLC system with 2D-LC technology was configured for heart-cutting. Key elements included:

• First-dimension USP method: XSelect HSS T3 4.6×250 mm, 5 μm column; isocratic 60:40 phosphoric acid buffer/acetonitrile at 1.0 mL/min; UV detection at 220 nm.
• Heart-cut transfer: timed valve events direct the target peak onto a second-dimension BEH C18 2.1×50 mm, 1.7 μm column while original USP flow is sent to waste.
• At-column dilution: simultaneous 0.2 mL/min 0.1% formic acid aqueous flow mixes with the directional fraction to reduce organic content from 40% to 20%, improving retention and peak shape.
• Mass detection: ACQUITY QDa operated in ESI+ mode; full scan 100–500 m/z alongside single-ion recording (SIR) at m/z 429.2 for Irbesartan and m/z 447.1 for RC-A.

Main Results and Discussion

The standard Irbesartan USP assay yielded a prominent API peak at 15.30 min and a minor unknown at 12.70 min by UV. Heart-cut transfers successfully isolated each peak for MS analysis without altering the original chromatographic method. SIR confirmed the API as Irbesartan (m/z 429.2) and the unknown as RC-A (m/z 447.1). Five consecutive transfers of RC-A showed excellent repeatability, with area %RSD of 4.2 and constant elution time. Comparative experiments demonstrated that at-column dilution was critical: without dilution, the transferred RC-A peak exhibited severe fronting, whereas with dilution it appeared symmetric and well retained.

Benefits and Practical Applications

• Direct MS confirmation of USP method peaks without fraction collection or mobile phase reformulation.
• Streamlined validation support through in-line specificity checks and impurity identification.
• Time and cost savings by avoiding full method redevelopment.
• Flexibility to transfer partial or entire peak volumes according to analyte abundance and analysis goals.

Future Trends and Opportunities

Advancements may include broader application to diverse USP monographs, integration with high-resolution mass spectrometry, and automated data processing with chemometric or AI-driven peak identification. Further miniaturization of dilution and trapping modules could enhance throughput and sensitivity for trace-level impurity profiling.

Conclusion

The described 2D-LC heart-cut protocol with at-column dilution and ACQUITY QDa detection provides a robust, reproducible solution for routine MS analysis of USP chromatographic peaks. It preserves original method selectivity while enabling rapid identity confirmation and impurity investigation in pharmaceutical quality control.

References

• USP Monograph Irbesartan, USP37-NF32, 3396, official August 1, 2014.