Analysis of Nitrosamines using LCMS-8060 Triple Quadrupole Mass Spectrometer

Importance of Topic

Monitoring nitrosamines such as 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol NNAL in biological matrices is critical for assessing exposure to carcinogenic tobacco-specific compounds and other environmental sources. Accurate quantification at trace levels supports toxicological research, epidemiological studies, and quality control in pharmaceutical development.

Study Objectives and Overview

This work describes the development and validation of a sensitive LCMS-8060 method for quantifying NNAL in urine samples. The study evaluates method linearity, limits of detection and quantification, and performance across a wide dynamic range.

Instrumental Setup

• Shimadzu LCMS-8060 triple quadrupole mass spectrometer with electrospray ionization in positive mode
• Discovery HSF5 column 50 x 2 mm with 3 µm particle size
• Mobile phase A: 7 mM ammonium bicarbonate; mobile phase B: methanol

Methodology

• Chemical derivatization of NNAL and D3-labeled internal standard
• Multiple reaction monitoring transitions based on literature plus additional transitions for each analyte
• Six-minute gradient program per injection
• Injection volume of 0.1 µL for standards and samples
• Calibration curve constructed with 20 levels over 0.1 to 3000 pg/mL, fit using 1/C weighting

Key Results and Discussion

• Linear calibration from 100 pg/mL to 3000 pg/mL with r2=0.9942 and accuracy between 92% and 106% for levels C8–C20
• High concentration outliers (levels C1–C7 and C15) exceeded the calibrated range and were excluded from the linear fit
• Limit of quantification between 1 and 2 fg on column based on signal-to-noise ratios (S/N 23.5 at 10 fg; S/N 9.0 at 5 fg; S/N 2.5 at 1 fg; S/N 1.5 at 0.5 fg)
• Unknown samples Q1–Q5 showed three quantifiable results, one saturated response, and one below the quantification range
• Optimization of probe position and capillary protrusion improved sensitivity over previous acquisitions

Benefits and Practical Applications

• Femtogram-level sensitivity for trace nitrosamine detection in biological samples
• Short six-minute run time suitable for high-throughput workflows
• Robust quantification using internal standard normalization ensures reproducibility
• Applicable to exposure biomonitoring, toxicological studies, and regulatory compliance

Future Trends and Potential Applications

Further improvements in mass spectrometer source design and chromatography may lower detection limits by another order of magnitude. Integration with automation and high-resolution mass analyzers could broaden the range of detectable nitrosamines in complex matrices. Additional applications include environmental monitoring and drug safety assessment.

Conclusion

The validated LCMS-8060 method enables reliable quantification of NNAL at femtogram levels with excellent linearity and reproducibility in urine. This platform supports advanced bioanalytical studies of nitrosamine exposure and can be extended to other ultra-trace analytes.

References

No external literature references were cited in this application note.