HPLC-CAD impurity profiling of carbocisteine using SCX-RP mixed-mode chromatography

Significance of the topic

Carbocisteine is a mucolytic agent used to manage acute and chronic respiratory conditions. Its impurity profiling is critical for patient safety and regulatory compliance, but the lack of chromophores and similar properties among amino acid derivatives complicates traditional detection methods. A direct, sensitive approach for non-volatile impurities is therefore highly valuable.

Study objectives and overview

This work sought to adapt and translate an existing HPLC method with charged aerosol detection (CAD) onto the latest Vanquish platform. The goal was to lower limits of quantitation (LOQs) for key impurities, notably cystine, and ensure compliance with ICH Q3A(R2) reporting thresholds of 0.03% per impurity.

Methodology and Instrumentation

An isocratic mobile phase of 18% acetonitrile and 10 mM trifluoroacetic acid was run at 1.3 mL/min on a SIELC Primesep 100 mixed-mode column (250 × 4.6 mm, 5 µm) at 20 °C over 20 minutes. Calibration standards covered 0.05–0.25% of assay concentration. CAD parameters were optimized to an evaporation temperature of 50 °C with a 10 s signal filter. Data acquisition and processing used Chromeleon CDS version 7.2.6.

Instrumentation

• Vanquish UHPLC system base
• Vanquish Charged Aerosol Detector H
• Vanquish Binary Pump Flex
• Vanquish Split Sampler
• Vanquish Column Compartment H
• Vanquish Diode Array Detector F

Main results and discussion

Calibration curves for five impurities exhibited linearity with R2 > 0.995. LOQs ranged from 0.01% to 0.02%, representing up to 80% improvement over the previous Corona CAD method. Cystine LOQ improved from 0.09% to 0.01%. Accuracy studies at 0.05%, 0.15%, and 0.25% spiking levels yielded recoveries between 90% and 120%. Intra- and interday precision showed RSDs below 10%. Analysis of eleven commercial batches revealed cystine in all samples, including those previously deemed non-detectable.

Benefits and practical applications

The enhanced sensitivity ensures impurity reporting below the ICH threshold of 0.03% for all analytes. Direct CAD detection eliminates derivatization, simplifying workflow and broadening the range of detectable impurities. This method supports robust quality control in pharmaceutical development and manufacturing.

Future trends and opportunities

• Coupling CAD with mass spectrometry for structural confirmation of impurities
• Development of environmentally friendly mobile phases to reduce solvent waste
• Automation and miniaturization of sample preparation protocols
• Extension of mixed-mode chromatography CAD methods to other amino acids and peptide impurities

Conclusion

Transferring the carbocisteine impurity profiling assay to the Vanquish CAD system achieved significantly lower LOQs and maintained robust accuracy and precision. The method meets regulatory requirements and offers a streamlined, derivatization-free approach for comprehensive impurity analysis.

Reference

1. Zheng JP et al Lancet 2008 371 2013-2018
2. Wahl O Holzgrabe U Talanta 2016 154 150-163
3. ICH Q3A(R2) guideline 2006
4. Wahl O Holzgrabe U J Pharm Biomed Anal 2014 95 1-10
5. Swartz M et al Charged Aerosol Detection for Liquid Chromatography Wiley 2017