LC-MS/MS Method Development for Sulfite in Food and Beverage

Importance of the Topic

In the food and beverage industry, sulfites serve as key antioxidants and bleaching agents to preserve color, inhibit microbial growth, and prolong shelf life. However, their potential to trigger allergic reactions mandates accurate quantification and labeling. Regulatory bodies such as the U.S. FDA and the EU Commission set maximum thresholds of 10 mg/kg or 10 mg/L (as SO2 equivalents), above which disclosure is required. A rapid, reliable analytical approach supports both compliance and consumer safety.

Objectives and Overview

This study aimed to develop and validate a fast, sensitive UHPLC–MS/MS method for measuring free sulfite levels in diverse food matrices—specifically dried fruits and wines—using a formaldehyde derivatization strategy to convert unstable sulfite into a stable hydroxymethylsulfonate (HMS) adduct. By adapting elements of the latest FDA protocol, the goal was to achieve high throughput while maintaining regulatory compliance.

Methodology

Derivatization and Extraction:

• Sulfite in samples is stabilized by reacting with 0.2% formaldehyde (pH 4.5) to form HMS.
• Dried fruits are homogenized in formaldehyde solution, followed by shaking, sonication, centrifugation, and double extraction to generate a 50 mL combined extract.
• Wine samples are simply diluted 1:10 (w/v) with the formaldehyde solution.

SPE Cleanup and Adduct Conversion:

• C18 SPE cartridges remove lipophilic interferences: the first 2 mL of extract is discarded and the next 2 mL collected.
• Eluate is heated at 80 °C for 30 minutes to ensure complete HMS formation, then cooled to room temperature.

Chromatographic and Mass Spectrometric Conditions:

• Column: SeQuant ZIC HILIC, 150 × 2.1 mm, 5 μm particle size.
• Mobile phases: A = 10 mM ammonium acetate/90% acetonitrile; B = 10 mM ammonium acetate/50% acetonitrile.
• Gradient: B 30% (0–1 min) → 70% (3–5.5 min) → 100% (5.51–7.75 min) → 30% (8–12 min); flow rate 0.3 mL/min; column at 40 °C; injection 2 µL.
• MRM transitions (negative ESI): HMS (111 > 81, 13 V for quantification; 111 > 80, 27 V for reference); internal standard 34S-HMS (113 > 83, 13 V; 113 > 82, 27 V).
• MS parameters: DL 150 °C; interface 200 °C; heat block 500 °C; drying gas 10 L/min; nebulizing gas 2.5 L/min; heating gas 10 L/min.

Used Instrumentation

• UHPLC: Shimadzu Nexera X3 system.
• Mass Spectrometer: Shimadzu LCMS-8050 triple quadrupole.
• SPE manifold and InertSep C18 cartridges (500 mg/6 mL).

Main Results and Discussion

The method delivered a linear calibration up to 20 ppm HMS, permitting direct analysis without extensive dilution. The total run time per injection was halved from 24 to 12 minutes. Recovery experiments at spiking levels of 1, 5, and 10 mg SO2/kg yielded recoveries between 84% and 104% across raisin, mango, apricot, red wine, and white wine. Limit of quantification met regulatory needs, and matrix effects were effectively controlled by SPE cleanup.

Benefits and Practical Applications

• High throughput: 12 minute analysis increases sample capacity in QA/QC labs.
• Robust accuracy: recoveries within acceptable ranges ensure reliable compliance testing.
• Wide dynamic range: quantifies low-level residual sulfites and higher concentrations without repeated dilutions.
• Versatility: applicable to solid (dried fruit) and liquid (wine) matrices with minimal method modification.

Future Trends and Potential Applications

Emerging techniques may integrate high-resolution MS for improved selectivity and expand to other sulfite-containing commodities (e.g., seafood, vegetables). Automation of SPE cleanup or on-line derivatization could further streamline workflows. Coupling this approach with data analytics will support real-time monitoring of sulfite levels in industrial production.

Conclusion

A streamlined UHPLC–MS/MS method featuring formaldehyde derivatization to HMS offers rapid, accurate sulfite quantification in foods and beverages. The protocol meets regulatory thresholds, reduces sample preparation labor, and adapts to various matrices, making it a valuable tool for analytical laboratories.

Reference

• U.S. Food and Drug Administration. Code of Federal Regulations: Part 101.100(a)(4), Title 21. Office of the Federal Register, Washington, DC (2016).
• Carlos KS, de Jager LS. Determination of sulfite in food by liquid chromatography tandem mass spectrometry: Collaborative study. Journal of AOAC International. 100(6):1785–1794 (2017).
• U.S. Food and Drug Administration. Method number: C-004.03, Determination of Sulfites in Food using Liquid Chromatography–Tandem Mass Spectrometry (LC-MS/MS) (2021).