Application Note 20709 SPE-LC-MS/MS Method for the Determination of Nicotine, Cotinine, and Trans-3-hydroxycotinine in Urine

Importance of the Topic

The quantification of nicotine and its major metabolites cotinine and trans-3-hydroxycotinine in urine is critical for assessing tobacco exposure, clinical toxicology, and pharmacokinetic studies. A rapid, reliable, and sensitive analytical method enables high-throughput screening in clinical laboratories and research settings, ensuring accurate biomarker assessment and supporting regulatory and compliance requirements.

Study Overview and Objectives

This work describes the development and validation of a solid-phase extraction (SPE) coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for nicotine, cotinine, and trans-3-hydroxycotinine in urine. The main objectives were to achieve:

• Fast sample preparation and chromatographic cycle time.
• High recovery and reproducibility across the calibration range.
• Sensitivity down to low nanogram-per-milliliter concentrations.

Methodology and Instrumentation

Sample Preparation:

• Matrix: Human urine spiked with analytes and cotinine-d3 internal standard.
• SPE cleanup: SOLA CX cartridges (10 mg/2 mL) conditioned with methanol and ammonium formate buffer, followed by sample loading, two wash steps (buffer and 1% formic acid in methanol), drying, and elution into 70:30 water/methanol.

Chromatography:

• Instrument: Thermo Scientific Accela 600 LC pump with Syncronis C18 1.7 µm, 50×2.1 mm column and guard column.
• Mobile phases: A = water + 0.1% ammonia; B = methanol + 0.1% ammonia.
• Gradient: 70:30 A/B to 0:100 A/B in 3 min, hold, then re-equilibrate by 6 min total cycle time.
• Flow rate: 0.2 mL/min; column temperature: 30 °C; injection volume: 10 µL.

Mass Spectrometry:

• Instrument: Thermo Scientific TSQ Vantage with HESI in positive mode.
• Key parameters: spray voltage 3,500 V; vaporizer 400 °C; capillary 380 °C; sheath and auxiliary gas; collision pressure 1.5 mTorr.
• SRM transitions: Nicotine m/z 163.1→130.1, cotinine 177.1→80.1, trans-3-hydroxycotinine 193.1→80.1, internal standard cotinine-d3 180.1→80.1.

Results and Discussion

Calibration and Sensitivity:

• Nicotine and cotinine calibration curves were linear from 1–1000 ng/mL (R² ≥ 0.9996).
• Trans-3-hydroxycotinine was linear from 10–1000 ng/mL (R² = 0.9991).
• Lower limits of quantitation (LLOQ): 1 ng/mL for nicotine and cotinine, 10 ng/mL for trans-3-hydroxycotinine.

Accuracy and Precision:

• Accuracy at low (15 ng/mL), mid (250 ng/mL), and high (600 ng/mL) QC levels was within ±15% (±20% at LLOQ).
• Intra-run precision (RSD) was below 9% for all analytes.

Recovery:

• Mean recovery across QC levels exceeded 79.3% for all compounds, demonstrating efficient SPE performance.

Benefits and Practical Applications

This method offers significant advantages for high-throughput bioanalytical and clinical research laboratories:

• Rapid 3-minute chromatographic runs increase sample throughput.
• Reduced solvent and sample volume requirements lower costs and environmental impact.
• High reproducibility and robust cleanup minimize matrix effects and failure rates.

Future Trends and Opportunities

Emerging directions to enhance tobacco biomarker analysis include:

• Automation and on-line SPE integration for further throughput gains.
• Miniaturized columns and microfluidic systems to reduce solvent use and improve sensitivity.
• Advanced sorbent materials for more selective extraction in complex matrices.
• Integration with high-resolution and ambient mass spectrometry for rapid screening in clinical and field settings.

Conclusion

The presented SPE-LC-MS/MS workflow employing SOLA CX cartridges and a Syncronis C18 UHPLC column delivers a fast, sensitive, and reproducible assay for urinary nicotine, cotinine, and trans-3-hydroxycotinine. The validated method meets stringent accuracy, precision, and recovery requirements, making it suitable for routine biomonitoring of tobacco exposure.

Reference

J. Jones, Thermo Fisher Scientific, Application Note 20709, 2013