Superior separation of nicotine and tobacco related alkaloids by utilizing the selectivity of hydrophilic interaction chromatography (HILIC)

Significance of the Topic

Polar tobacco alkaloids such as nicotine and its metabolites pose analytical challenges due to weak retention on conventional C18 phases and isobaric interferences in LC MS analysis

Study Objectives and Overview

This study aims to develop a fast sensitive and high efficiency HPLC MS MS method to separate six tobacco related alkaloids including nicotine nornicotine cotinine norcotinine trans 3 hydroxycotinine and anabasine using hydrophilic interaction chromatography on a core shell column

Methodology and Instrumentation

Instrumentation

• Vanquish Horizon UHPLC system with binary pump split sampler and column compartment
• TSQ Quantiva triple stage quadrupole mass spectrometer with HESI II source
• Accucore HILIC column 150 x 2.1 mm 2.6 µm core shell particles

Chromatographic Conditions

• Injection volume 2 µL
• Mobile phases 20 mM ammonium formate pH 3 and acetonitrile
• Gradient from 98 to 45 percent organic over 4 minutes total run 7 minutes at 0.75 mL/min

Mass Spectrometry Conditions

• Positive ion mode HESI II 3500 V vaporizer 450°C sheath gas 55 Arb aux gas 18 Arb
• Collision gas 1.5 mTorr ion transfer tube 365°C
• Optimized MRM transitions for each analyte

Main Results and Discussion

• Baseline separation of six isobaric compounds achieved on Accucore HILIC with resolution above 2 within 4 minutes
• Competitor HILIC column showed coelution of nicotine and anabasine resolution 1.26 insufficient for accurate quantitation
• Accucore HILIC delivered resolution 3.08 for anabasine minimizing isobaric interference
• High organic content enhanced ionization efficiency and assay sensitivity
• Laboratory background nicotine contamination identified as a challenge requiring dedicated clean areas

Benefits and Practical Applications

• Rapid analysis suitable for high throughput screening of tobacco related samples
• Improved retention and peak shape for polar analytes not retained on reversed phase
• Lower backpressure with core shell particles enabling robust UHPLC operation
• Enhanced MS signal sensitivity due to high organic solvent eluent

Future Trends and Potential Applications

Emerging uses of HILIC include profiling of polar metabolites in pharmaceutical environmental and food matrices

Advances in column chemistries and miniaturization will support faster separations with reduced solvent consumption

Integration with high resolution mass spectrometry and automated sample preparation will expand throughput and data quality

Conclusion

The Accucore HILIC core shell column provides superior separation of nicotine related alkaloids compared to conventional HILIC delivering accurate reliable and rapid quantitation in complex samples

References

1 Siegmund B Leitner E Pfannhauser W Determination of the Nicotine Content of Various Edible Nightshades and Their Products and Estimation of the Associated Dietary Nicotine Intake J Agric Food Chem 1999 47 8 3113 3120