Mass Detection Coupled to a USP Method for Lidocaine and Prilocaine Cream Using Multi-dimensional Liquid Chromatography

Significance of the topic

The accurate detection and identification of impurities in pharmaceutical formulations is central to ensuring product safety and efficacy. Traditional USP methods frequently employ nonvolatile buffers that are incompatible with mass spectrometry, requiring time-consuming sample isolation and reanalysis steps. Integrating mass detection directly with unmodified USP monographs via multi-dimensional liquid chromatography significantly accelerates impurity characterization and reduces method revalidation burdens.

Objectives and overview

This study demonstrates a heart-cutting 2D LC approach combined with at-column dilution to couple mass detection with the USP assay for lidocaine and prilocaine cream. The workflow isolates a non-MS-compatible impurity peak from the first-dimension separation, refocuses and traps it, and transfers it under MS-friendly conditions to a second-dimension UPLC column for rapid mass spectral identification.

Methodology and instrumentation

First-dimension separation follows the USP monograph using a C18 column and phosphate buffer mobile phase with UV detection. A loop-and-trap interface captures a defined retention window (heart-cut), then an at-column dilution step lowers organic strength and focuses the analyte on a hydrophobic trap column. Finally, a second-dimension UPLC gradient with formic acid/acetonitrile elutes the trapped fraction to the mass detector.

Key results and discussion

An impurity peak at 3.68 min (RRT 0.37) was isolated and screened against a blank. Optimization of trap chemistry, dilution solvent pH, and 2D mobile phase identified the Oasis HLB trap with pH 10 aqueous loading and low-pH acetonitrile elution as optimal for maximum peak recovery. MS data confirmed a single [M+H]+ ion at m/z 108.0, corresponding to o-toluidine, a known related compound of prilocaine hydrolysis.

Benefits and practical applications

• Eliminates the need to modify and revalidate existing USP or proprietary LC methods before MS detection.
• Reduces sample preparation time and instrumentation requirements by performing impurity identification directly from assay runs.
• Provides unambiguous mass confirmation of unknown impurities to support formulation development and quality control.

Future trends and opportunities

Advances in multi-dimensional LC could expand compatibility to a wider range of compendial methods and tough sample matrices. Emerging trap chemistries and dynamic dilution schemes promise improved sensitivity for trace-level components. Integration with high-resolution and tandem MS platforms will further enhance structural elucidation capabilities. Automated event programming and real-time data analysis pipelines will streamline routine impurity profiling.

Conclusion

The described heart-cutting 2D LC with at-column dilution successfully couples mass detection to an unmodified USP method for lidocaine and prilocaine cream. The approach delivers rapid, accurate identification of an o-toluidine impurity without altering the compendial protocol, offering a straightforward path to MS-enhanced quality control.

Instrumentation used

• ACQUITY UPLC H-Class Quaternary Solvent Manager with UV detection (1D).
• Loop and trap interface with at-column dilution module.
• ACQUITY UPLC I-Class Binary Solvent Manager with ACQUITY QDa Mass Detector (2D).
• Empower 3 Chromatography Data Software for method control and data processing.

References

1. Sivakumar B., et al. Identification and Characterization of Process-Related Impurities of Trans-Resveratrol. Scientia Pharmaceutica. 2013;81:683–695.
2. USP Monograph for Lidocaine and Prilocaine Cream, USP40-NF35. United States Pharmacopeial Convention; December 1, 2016.
3. Mallet C.R., Murphy B.P. Multi-Dimensional Chromatography Compendium. Waters White Paper 720005339EN; 2015.
4. Mallet C.R., Botch-Jones S. Illicit Drug Analysis in Urine using 2D LC-MS/MS. Waters Application Note 720005533EN; 2015.
5. Waters Reversed-Phase Columns Selectivity Chart. Waters Corporation.