Analysis of Soft Drink Additives with No Interference from Aspartame Degradants Using Arc HPLC System with PDA Detection

Importance of the Topic

Quality control of natural and artificial additives in soft drinks is essential to ensure product safety and compliance with regulatory limits. Monitoring compounds such as caffeine, benzoate, sorbate, acesulfame K, saccharin and aspartame is challenged by the potential coelution of aspartame degradation products which can hamper accurate quantification.

Objectives and Overview

This study aimed to optimize an isocratic HPLC method on the Arc HPLC System using a pre-formulated beverage analysis kit to achieve a fast, robust 12-minute separation free of interference from aspartame degradants, while evaluating injector linearity and accuracy compared to volumetric pipettes.

Methodology and Used Instrumentation

Standards and Samples Preparation:

• Reconstituted Waters Beverage Analysis Kit standards to yield known concentrations of six additives.
• Soft drink samples degassed by ultrasonication and filtered through a 0.2 µm PVDF membrane.

Chromatographic Conditions:

• Column: XBridge BEH Phenyl XP, 2.5 µm, 4.6 × 100 mm.
• Mobile phase and wash solvents: ethanol-based pre-formulated reagents.
• Flow rate: 1.6 mL/min, isocratic, 35 °C, 12 min run time.
• Injection volume: 5 µL standard; adjusted to 2.5–4 µL for high-content samples.

Instrumentation:

• Arc HPLC System.
• 2998 Photodiode Array Detector (PDA) at 214 nm.
• Empower 3 Chromatography Data System.

Main Results and Discussion

Injection Performance:

• Peak area versus injection volume showed excellent linearity (R2>0.9999) over 0.2–20 µL.
• Relative injection errors were within ISO 8655-2 limits for volumetric pipettes for volumes ≥2.5 µL.

Aspartame Degradation Study:

• Heating standard solutions at 60 °C generated degradants identified as Asp-Phe and diketopiperazine (DKP) via PDA spectral library matching.
• Identified degradants were baseline-resolved from target additives under the optimized conditions.

Column Screening and Optimization:

• Compared 5 µm, 4.6×150 mm vs. 2.5 µm, 4.6×100 mm columns.
• Selected the 2.5 µm, 4.6×100 mm column at 1.6 mL/min for shortest runtime and resolution >2.0.

Soft Drink Sample Analysis:

• Measured caffeine, benzoate, sorbate, acesulfame K, saccharin and aspartame in three commercial colas.
• Caffeine content matched labels (98.5–100%), with repeatability (RSDr) <0.4% and intermediate precision (RSDr) <1.16%.
• Reduced injection volumes avoided manual dilutions while maintaining quantification accuracy.

Benefits and Practical Applications

• Rapid 12-minute isocratic analysis without interference from aspartame degradants.
• High injection accuracy enabling direct analysis of high-content samples with minimal dilution.
• Pre-formulated reagents simplify system setup and reduce hazardous waste.

Future Trends and Potential Applications

Advances in HPLC system design may further reduce run times, enhance automation and integrate high-resolution detectors such as mass spectrometers for broader screening. The adoption of inline sample preparation and artificial intelligence-driven data analysis will improve throughput and decision-making in beverage quality control.

Conclusion

An optimized 12-minute isocratic method on the Arc HPLC System with PDA detection and pre-formulated reagents enables accurate, interference-free determination of six common soft drink additives, improving productivity and data reliability in beverage manufacturing QC.

Reference

1. Van Vliet K et al. Nutrients 2020, 12, 1887.
2. ISO 8655-2:2002(E) Piston-operated volumetric apparatus – Part 2: Piston pipettes.
3. FDA Reviewer Guidance (1994) Validation of Chromatographic Methods.
4. Liu K et al. Waters Application Note 720007126EN, Jan 2021.
5. Waters Business Solution 720004489EN, Dec 2012.
6. Empower 3 Chromatography Data System documentation.