Highly Sensitive and Robust LC/MS/MS Method for Quantitative Analysis of Artifcial Sweeteners in Beverages

Importance of the Topic

Artificial sweeteners serve as low-calorie sugar alternatives in foods and beverages, addressing health concerns such as obesity and diabetes. Their widespread use demands accurate quantitation to ensure regulatory compliance and consumer safety. Additionally, these compounds are increasingly detected as emerging contaminants in surface and wastewater, highlighting the need for sensitive analytical methods.

Objectives and Study Overview

The primary aim was to develop a highly sensitive and robust LC/MS/MS method for quantifying ten artificial sweeteners in beverage matrices with minimal sample preparation. Key objectives included:

• Optimizing multiple reaction monitoring transitions for ten sweeteners.
• Evaluating ultra-small injection volumes (0.1–1 µL) to avoid extensive sample dilution or cleanup.
• Demonstrating method applicability in real beverage and mouthwash samples.

Methodology and Instrumentation

Standard stock solutions of ten high-purity sweeteners were prepared in methanol/water (50:50). Beverage and mouthwash samples were simply diluted with mobile phase prior to analysis. Instrumentation details:

• LC system: Shimadzu Nexera UHPLC with Synergi Polar-RP C18 column (100 × 2 mm, 2.5 µm), flow rate 0.25 mL/min.
• Mobile phases: A – water with 0.1% formic acid and 0.03% triethylamine; B – methanol with 0.1% formic acid and 0.03% trimethylamine. Gradient from 10% B (0–0.5 min) to 95% B (8–9 min) and re-equilibration to 10% B by 11 min.
• MS system: Shimadzu LCMS-8040 triple quadrupole with ESI source, positive/negative switching MRM mode; nebulizing gas 3 L/min, drying gas 15 L/min, block temperature 400 °C, interface temperature 250 °C.
• Injection volumes tested: 0.1, 0.5, 1, 5 and 10 µL.

Main Results and Discussion

Automated MRM optimization identified two transitions per compound (six in negative mode, four in positive). Chromatographic separation yielded sharp peaks between 2.0 and 8.2 min. Calibration curves exhibited excellent linearity across broad ranges (R² > 0.998). Using 0.1 µL injections, limits of quantitation reached sub-µg/L levels. Repeatability (RSD) for peak areas remained below 12%. Analysis of three soft drinks and one mouthwash revealed quantifiable levels of sweeteners such as aspartame, acesulfame K, saccharin, sucralose and glycyrrhizinate without significant matrix interferences.

Benefits and Practical Applications

• Direct injection of diluted samples streamlines workflow and increases throughput.
• Ultra-small injection volumes conserve sample and solvent while preserving sensitivity.
• High selectivity and robustness support routine quality control in beverage production.
• Method adaptability enables monitoring of artificial sweeteners in environmental waters.

Future Trends and Potential Applications

• Extension to wastewater and other food matrices with minimal sample preparation.
• Coupling with high-resolution MS for non-targeted screening of novel sweeteners.
• Automation of sample handling and injection to further improve throughput.
• Environmental fate studies leveraging the method’s high sensitivity.

Conclusion

A sensitive, robust LC/MS/MS method was established for ten artificial sweeteners in beverages using minimal sample pretreatment. The approach demonstrated excellent linearity, low detection limits and high repeatability. Employing ultra-small injection volumes enhances resource efficiency and throughput, facilitating regulatory compliance and environmental monitoring.

References

• EU directives 93/35/EC, 96/83/EC, 2003/115/EC, 2006/52/EC, 2009/163/EC; Wikipedia entry on sugar substitutes.
• Buchgraber P., Wasik A., Report EUR 22726 EN (2007).
• Large F.T., Scheurer M., Brauch H-J., Anal Bioanal Chem 403:2503-2518 (2012).
• Lim H-S. et al., Food Sci Biotechnol 22(S):233-240 (2013).