Automated Detection of Artificial Sweeteners

Significance of the Topic

Artificial sweeteners such as acesulfame-K, cyclamate, saccharin and sucralose are increasingly employed as sugar substitutes in food and beverage production. These compounds are minimally metabolized by humans and often pass through wastewater treatment plants untreated, making them persistent environmental pollutants and valuable indicators of water quality. Reliable, high-throughput analytical methods are therefore essential for monitoring these emerging contaminants at trace levels in groundwater and drinking water supplies.

Objectives and Overview

This application note describes the development and validation of an automated solid phase extraction (SPE) workflow using the FREESTYLE XANA robotic system, coupled with LC-MS/MS detection, for the quantification of four target artificial sweeteners in large-volume water samples. Key aims include reduction of manual labor, improvement of reproducibility, and achievement of detection limits suitable for environmental monitoring.

Methodology

Samples comprised 1 L of tap water spiked at 50 ng/L per analyte. Manual SPE on CHROMABOND HR-XAW cartridges (3 mL, 200 mg) was first established (conditioning with methanol and water, loading at 5–10 mL/min, washing, drying, elution with methanol/NH₃). The protocol was then fully automated on the FREESTYLE XANA, processing up to three samples in parallel:

• Conditioning: 3 mL MeOH, 3 mL water (10 mL/min)
• Loading: 1 L sample (8 mL/min)
• Washing: 4 mL water (8 mL/min)
• Drying: nitrogen, 1 min
• Elution: 3 mL MeOH, then 3 mL MeOH + 1 % NH₃ (2 mL/min)

Eluates were brought to 5 mL with methanol, diluted to 1 mL with water/acetonitrile (95/5 v/v) + 0.1 % formic acid, and injected into an LC-MS/MS system.

Used Instrumentation

• FREESTYLE XANA robotic platform with SPE and evaporation modules
• Agilent 1290 Infinity II UHPLC (G7120A pump, G7167B autosampler, G7116B column oven)
• NUCLEOSHELL RP 18plus column, 100×2 mm, 2.7 µm
• AB Sciex QTRAP 5500 with ESI in negative MRM mode

Main Results and Discussion

Recovery rates and reproducibility were determined (n=6):

• Acesulfame-K: 68 % ± 6 % RSD
• Cyclamate: 91 % ± 4 % RSD
• Saccharin: 93 % ± 3 % RSD
• Sucralose: 103 % ± 5 % RSD

MRM transitions and retention times provided clear, well-resolved peaks at 2.04–4.25 min. Automated SPE yielded performance comparable to manual extraction while enabling continuous, unattended operation.

Benefits and Practical Applications

• Significant reduction of hands-on time through full automation
• Parallel processing of up to three 1 L samples increases throughput 24/7
• Robust recoveries and low RSDs ensure reliable quantification at ng/L levels
• Applicable to routine environmental monitoring and QA/QC laboratories

Future Trends and Potential Applications

Emerging opportunities include integration of online SPE for real-time monitoring, expansion of the analyte panel to other micropollutants, and miniaturization of workflows to reduce solvent consumption. Advances in software algorithms and robotics will further streamline method customization and data handling for high-capacity laboratories.

Conclusion

The automated SPE-LC-MS/MS workflow on the FREESTYLE XANA platform provides a reliable, high-throughput solution for detecting artificial sweeteners in water at trace levels. The method’s robustness, unattended operation, and reproducibility make it well suited for large-scale environmental monitoring and regulatory compliance.