Quantitation of N-nitrosamines in dietary supplement by using LC-MS/MS

Importance of the Topic

A growing number of consumers rely on protein powder supplements to meet daily nutritional requirements. However, these formulations can be contaminated with N-nitrosamines, potent genotoxic carcinogens formed from nitrites and secondary or tertiary amines. Regular monitoring of these impurities in dietary supplements is essential to ensure consumer safety and regulatory compliance.

Objectives and Study Overview

This study aimed to develop and validate a rapid, sensitive, and high-throughput liquid chromatography–tandem mass spectrometry (LC-MS/MS) method for the simultaneous quantification of four priority N-nitrosamines—NDMA, NEIPA, NDPA and NDIPA—in protein powder matrices.

Methodology and Instrumentation

• Sample Preparation: 0.4 g protein powder was spiked with N-nitrosamine standards (10 ppb), mixed with water and organic solvent, vortexed, salting-out extraction was performed, followed by centrifugation and filtration.
• Chromatographic Conditions: Shim-pack Scepter C8 column (3.0 × 150 mm, 5 µm); mobile phase A: 0.1% formic acid in water; B: 0.1% formic acid in methanol; gradient from 10% to 95% B; flow 0.5 mL/min; column at 40 °C.
• Mass Spectrometry: APCI interface; desolvation line 180 °C; heating block 200 °C; interface 300 °C; nebulizing gas 4 L/min; drying gas 5 L/min; MRM transitions optimized for each analyte.
• Calibration and Validation: Matrix-matched calibration from 0.1 to 5.0 ppb; linearity R² > 0.988; recovery and precision assessed at 10 ppb.

Used Instrumentation

• Shimadzu LCMS-8060NX triple quadrupole mass spectrometer
• Nexera X3 UHPLC system
• Shim-pack Scepter C8 column (3.0 × 150 mm, 5 µm)
• LC-MS grade water and methanol, formic acid

Main Results and Discussion

• Calibration curves exhibited excellent linearity (R² 0.989–0.995) over 0.1–5 ppb.
• Mean recoveries for all four N-nitrosamines ranged between 70% and 120%, meeting SANTE/12682/2019 guidelines.
• Instrument precision showed RSD values below 20% for reproducibility at the 10 ppb level.
• The method achieved low limits of quantitation suitable for trace-level monitoring in supplements.

Benefits and Practical Applications

This streamlined liquid–liquid extraction coupled with LC-MS/MS offers laboratories a robust protocol for routine quality control of dietary supplements. It enables simultaneous determination of multiple N-nitrosamines with minimal sample preparation and fast cycle times.

Future Trends and Opportunities

• Extension to other complex food and pharmaceutical matrices.
• Integration of high-resolution mass spectrometry for non-target screening.
• Automation of sample preparation to increase throughput.
• Alignment with evolving regulatory limits and global monitoring initiatives.

Conclusion

The validated LC-MS/MS method provides accurate, reproducible quantitation of NDMA, NEIPA, NDPA and NDIPA in protein powder supplements, complying with international guidelines. Its simplicity and sensitivity make it well suited for routine safety assessment in research, quality assurance and regulatory laboratories.

References

1. European Commission. Guidance document on analytical quality control and method validation procedures for pesticide residues and analysis in food and feed (SANTE/11312/2021).
2. World Health Organization. Information note on nitrosamine impurities (November 2019).
3. Harvard Health Publishing. The hidden dangers of protein powders. Harvard Health, 2020.