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

Significance of the Topic

Protein powders are widely consumed to meet daily nutritional requirements, but trace contaminants such as N-nitrosamines can pose significant health risks due to their mutagenic and carcinogenic properties. Reliable detection of these impurities in dietary supplements is essential for consumer safety and regulatory compliance.

Objectives and Study Overview

This work aimed to develop and validate a rapid, accurate, and high-throughput LC-MS/MS method for quantifying four key N-nitrosamines—NDMA, NEIPA, NDPA, and NDIPA—in protein powder formulations. The study focused on optimizing sample preparation, chromatographic separation, and mass spectrometric detection according to international guidelines.

Methodology and Instrumentation

The method comprised:

• Sample prep: 0.4 g of protein powder spiked at 10 ppb, slurry formation with water, addition of salts, vortex mixing, centrifugation, and filtration.
• Calibration: Matrix-matched standards prepared in extracted blank matrix, covering 0.1–5.0 ppb.
• Chromatography: Shim-pack Scepter C8 column (3.0 × 150 mm, 5 µm) with 0.1% formic acid in water/methanol gradient, flow rate 0.5 mL/min, column at 40 °C.
• Detection: Shimadzu LCMS-8060NX triple quadrupole with APCI interface (300 °C), optimized desolvation and block temperatures, and MRM transitions for each analyte.

Main Results and Discussion

The validated method demonstrated:

• Linearity: R² > 0.99 across 0.1–5.0 ppb for all analytes, with accuracy between 80% and 120%.
• Recovery: 70–120% average recoveries at 10 ppb spike level, meeting SANTE criteria.
• Precision: Repeatability with RSD < 20% for replicate injections.

These findings confirm the method’s robustness and suitability for trace-level nitrosamine analysis in complex protein matrices.

Benefits and Practical Applications

The protocol offers high sensitivity and selectivity for sub-ppb nitrosamine quantification, streamlined sample processing compatible with routine QA/QC laboratories, and compliance with food safety regulations.

Future Trends and Potential Applications

Prospective developments include:

• Extension to a wider range of supplement and food matrices prone to nitrosamine formation.
• Automation of sample preparation to increase throughput and reproducibility.
• Implementation of high-resolution MS for non-targeted screening of additional nitroso compounds.
• Standardization of methods to support global regulatory monitoring and risk assessment.

Conclusion

A validated LC-MS/MS approach employing simple extraction and APCI detection has been established for four N-nitrosamines in protein powders, achieving required performance criteria for sensitivity, accuracy, and precision. This method provides a reliable tool for ensuring the safety of dietary supplements.

References

1. SANTE/11312/2021: Guidance on analytical quality control and method validation procedures for pesticide residues in food and feed.
2. World Health Organization (2019): Medical alert on nitrosamine impurities.
3. Harvard Health Publishing: The hidden dangers of protein powders.