SPE-LC-MS/MS Method for the Determination of Nicotine, Cotinine, and Trans-3-hydroxycotinine in Urine

Significance of the Topic

Accurate measurement of nicotine and its primary metabolites cotinine and trans-3-hydroxycotinine in urine is essential for clinical toxicology, smoking cessation monitoring, exposure assessment and pharmaceutical research. High-throughput, sensitive and reproducible methods enable laboratories to process large sample batches with confidence in data quality and consistency.

Objectives and Study Overview

This application note presents the development and validation of a solid phase extraction liquid chromatography tandem mass spectrometry method for quantifying nicotine, cotinine and trans-3-hydroxycotinine in human urine. The goals were to streamline sample cleanup, achieve rapid chromatographic separation and demonstrate robust analytical performance across a broad dynamic range.

Methodology

Sample pretreatment involved spiking blank urine with analyte standards and a deuterated internal standard, followed by centrifugation. Solid phase extraction was performed on SOLA CX cartridges under a conditioning, loading, washing and elution protocol. The dried extracts were reconstituted in aqueous methanol and injected onto a UHPLC system. Chromatographic separation used a C18 column with a methanol–ammonia gradient delivering complete elution within three minutes. Detection employed positive mode heated electrospray ionization and selective reaction monitoring transitions for each analyte and internal standard.

Used Instrumentation

• Sample concentrator and 96 well plate vacuum manifold for SPE processing
• Thermo Scientific Accela 600 pump coupled to a Syncronis C18 1.7 µm 50 x 2.1 mm analytical column with guard
• Thermo Scientific TSQ Vantage triple quadrupole mass spectrometer with HESI source
• Thermo Scientific LCQUAN software for data processing

Main Results and Discussion

Calibration curves were linear over 1–1000 ng/mL for nicotine and cotinine, and 10–1000 ng/mL for trans-3-hydroxycotinine with correlation coefficients above 0.999. Limits of quantitation ranged from 1 to 10 ng/mL. Intra- and inter-day precision at three QC levels showed relative standard deviations below 9.0 percent. Mean accuracy based on relative error was within ±15 percent (±20 percent at the LLOQ). Extraction recoveries exceeded 79 percent for all compounds. Chromatograms demonstrated sharp peaks and no significant matrix interferences.

Benefits and Practical Applications

• Rapid three-minute chromatographic cycle supports high sample throughput
• Minimal solvent and sample volume requirements reduce cost and waste
• High recovery and reproducibility ensure reliable quantitative data
• Applicable to clinical, forensic and pharmacokinetic studies

Future Trends and Potential Applications

Ongoing developments may include automation of SPE workflows, online SPE–LC–MS/MS integration, miniaturized and microflow chromatography for enhanced sensitivity, and extension to novel biomarkers in alternative matrices such as saliva or dried blood spots. Machine learning algorithms could further improve peak integration and quality control in large-scale studies.

Conclusion

The validated SPE-LC-MS/MS method combining SOLA CX extraction and Syncronis C18 UHPLC enables fast, sensitive and reproducible quantification of nicotine and its metabolites in urine. It meets rigorous accuracy, precision and recovery criteria, making it suitable for routine high-throughput bioanalytical applications.