Measuring Total Sulfite (SO2) in Sucrose According to the USP43- NF38-6076 Method

Importance of the Topic

Pharmaceutical-grade sucrose must meet strict purity criteria to ensure product safety and performance. Sulfite impurities can affect stability, taste, and safety of oral formulations. Reliable quantification of total sulfite (SO₂) in sucrose supports regulatory compliance and maintains high quality standards in pharmaceutical manufacturing.

Study Objectives and Overview

This work evaluates the USP43-NF38-6076 enzymatic UV-Vis method for total sulfite determination in sucrose. It demonstrates how the Agilent Cary 3500 UV-Vis spectrophotometer, with in-cuvette stirring and multicell capability, streamlines sample analysis, improves data consistency, and reduces analysis time.

Methodology

An enzymatic cascade converts sulfite to sulfate and hydrogen peroxide (via sulfite oxidase), followed by NADH oxidation (via NADH peroxidase). The consumption of NADH is tracked by measuring absorbance decrease at 340 nm. Key steps:

• Sample preparation: Pharmaceutical and commercial sucrose solutions (400 mg/mL) and a reference spiked with 80 ppm SO₂ standard.
• Calibration: Seven sulfite standards (0–407 ppm) prepared in citric acid buffer.
• Reaction setup: Cuvettes loaded with sample or standard, buffer, NADH, enzymatic reagents, and water to 3.340 mL total volume; stirred at 500 rpm.
• Measurement: Initial absorbance (A₁) recorded after 4 min stirring; sulfite oxidase added; final absorbance (A₂) after 45 min. ∆A=(A₁–A₂) corrected by blank yields sulfite concentration using Beer–Lambert law.

Instrumentation

• Agilent Cary 3500 Multicell UV-Vis spectrophotometer with eight-cell sample holder.
• Agilent OpenLab software for data acquisition, kinetic monitoring, and compliance with 21 CFR Part 11/Annex 11.
• Megazyme total sulfite enzymatic kit (sulfite oxidase, NADH peroxidase, NADH, buffer).
• Quartz cuvettes (10 mm pathlength) with magnetic stirring in-cuvette.

Main Results and Discussion

• Calibration curve across 0–407 ppm SO₂ showed linear ∆A response at 340 nm.
• Sucrose sample analysis yielded ∆A of 0.236 and 0.242, corresponding to 2.37 ppm and 4.47 ppm total sulfite, both below half the reference limit.
• Simultaneous monitoring of seven standards reduced analysis time by over five hours compared to sequential measurements.
• In-cuvette stirring ensured homogeneity and minimized contamination risks from manual mixing.

Benefits and Practical Applications of the Method

• High sample throughput through multicell, simultaneous measurements.
• Improved accuracy by eliminating variability from separate runs.
• Automated stirring reduces sample handling and potential errors.
• Compliance readiness via integrated software audit trails.
• Suitable for routine quality control of pharmaceutical excipients.

Future Trends and Applications

• Integration of automated sample dispensers for fully automated workflows.
• Extension to other excipients and complex matrices (e.g., syrups, syrups with active APIs).
• Implementation of diode-array kinetic scanning for simultaneous multi-wavelength monitoring.
• Enhanced compliance features for emerging regulatory frameworks.
• Use of chemometric methods to refine detection limits and improve selectivity.

Conclusion

The Cary 3500 UV-Vis spectrophotometer, combined with the USP enzymatic assay, delivers reliable, high-throughput total sulfite analysis in sucrose. Its multicell design, in-cuvette stirring, and integrated software significantly reduce hands-on time, enhance reproducibility, and support regulatory compliance, making it a robust solution for pharmaceutical quality control.

Reference

1. Nováková L. et al. Pharmaceutical Analysis: Overview. Encyclopedia of Analytical Science, Elsevier, 2019; pp. 200–218.
2. United States Pharmacopeia. USP43-NF38-6076. Official Monograph for Total Sulfite Determination in Sucrose.