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

Significance of the Topic

Sulfite impurities in pharmaceutical sucrose can affect product stability, safety and efficacy. Accurate quantification according to USP standards ensures excipient quality, protects patient health and maintains regulatory compliance.

Objectives and Study Overview

This study evaluates the Agilent Cary 3500 UV-Vis spectrophotometer for measuring total sulfite (SO₂) in sucrose following the USP43-NF38-6076 enzymatic method. It demonstrates improvements in throughput, accuracy and data integrity.

Methodology and Instrumentation

The USP method uses a two-step enzymatic reaction:

• Sulfite oxidase converts sulfite to sulfate and hydrogen peroxide.
• NADH peroxidase reduces hydrogen peroxide while oxidizing NADH to NAD⁺.

Total sulfite is proportional to NADH consumption, monitored by measuring absorbance decrease at 340 nm before and after reaction. Sample and standard preparations include:

• Sucrose solutions (400 mg/mL) from reference and commercial sources.
• Sulfite standard solutions (0–407 ppm SO₂) in citric acid buffer.
• Enzymatic reagents: buffer, NADH, NADH peroxidase and sulfite oxidase from Megazyme kit.

Used Instrumentation

An Agilent Cary 3500 Multicell UV-Vis spectrophotometer with software-controlled in-cuvette stirring was employed. Key settings:

• Wavelength scan 300–400 nm, bandwidth 2 nm.
• Discrete 340 nm kinetic measurement, data interval 0.2 s, stir rate 500 rpm.
• Simultaneous monitoring of eight cuvettes using OpenLab software for data integrity and 21 CFR Part 11 compliance.

Main Results and Discussion

The multicell setup measured seven sulfite standards in parallel, reducing analysis time by over five hours compared with single-cell runs. Calibration produced linear response across 0–407 ppm SO₂. Measured ∆Abs for commercial and reference sucrose samples were below half the reference ∆Abs and within USP limits, confirming low sulfite content (2.4 ppm and 4.5 ppm).

Benefits and Practical Applications

The Cary 3500’s simultaneous multicell measurement and integrated stirring:

• Increases sample throughput and consistency.
• Minimizes operator error and contamination risk.
• Ensures homogenous mixing without manual intervention.
• Supports regulatory compliance via OpenLab audit trails.

Future Trends and Possibilities

Advances may include integration of automated sample handling, expanded multi-wavelength kinetics for complex reactions, and AI-driven spectral analysis. Coupling UV-Vis enzymatic assays with microplate platforms could further improve throughput in quality control laboratories.

Conclusion

The Agilent Cary 3500 UV-Vis spectrophotometer efficiently determines total sulfite in sucrose per USP43-NF38-6076. Simultaneous multicell analysis and automated stirring deliver high accuracy, reduced analysis time and robust data integrity, enhancing routine quality control workflows.

References

1. Lucie Nováková et al., Pharmaceutical Analysis, Encyclopedia of Analytical Science, Third Edition, Academic Press, 2019.
2. United States Pharmacopeia USP43-NF38-6076.