Analysis of Cyanocobalamin tablets, assay procedure 2 as per USP41-NF36 monograph,using Shimadzu Nexera X2 Fast LC system

Importance of the Topic

Cyanocobalamin, a synthetic form of vitamin B12, is essential for treating and preventing B12 deficiencies. Reliable quantification in tablet formulations ensures patient safety, dosage accuracy, and regulatory compliance. Fast and sensitive UHPLC methods address the pharmaceutical industry’s need for high-throughput quality control.

Objectives and Study Overview

This study demonstrates the assay of cyanocobalamin tablets (1000 µg) following USP41–NF36 Monograph 1114, Procedure 2, using the Shimadzu Nexera X2 Fast LC system. The aim is to verify system suitability, method precision, and compliance with USP criteria for tablet analysis.

Methodology and Instrumentation

The procedure follows USP monograph guidelines:

• Buffer: 470.7 mg hexane sulfonic acid sodium salt in water with phosphoric acid, pH adjusted to 3.5 using 50% KOH.
• Mobile phases: A – buffer; B – acetonitrile.
• Gradient: 0.0–0.5 min (1% B), 0.5–1.2 min (1→2.3% B), 1.2–1.4 min (2.3→5% B), 1.4–2.5 min (5→7% B), 2.5–5.0 min (7→18% B), 5.0–5.5 min (18→25% B), 5.5–6.5 min (25% B), 6.5–7.0 min (25→1% B), 7.0–8.0 min (1% B).
• Flow rate: 0.5 mL/min; column temperature: 35 °C; injection volume: 15 µL; run time: 8 min; detection at 361 nm (UV).

Standard (1 µg/mL) and sample solutions were prepared by tablet powder sonication and filtration through 0.22 µm membranes.

Used Instrumentation

• Shimadzu Nexera X2 Fast LC system (up to 130 MPa).
• UHPLC column: 2.1 mm × 10 cm, 1.7 µm L1 packing.
• Autosampler, column oven and mixer modules for high-speed, low carryover performance.
• Consumables: amber screw‐thread vials, PTFE/silicone septa, HPLC filters, high‐pressure PEEK fittings.

Key Results and Discussion

The assay yielded a retention time of 6.38 min. System suitability met USP criteria: tailing factor 1.43–1.51 (<2.0), theoretical plates ~80 833, %RSD retention time 0.09–0.19% and %RSD area 0.26–0.30% across six replicates. Overlay chromatograms confirmed excellent reproducibility and resolution.

Benefits and Practical Applications

• High throughput: complete analysis in 8 min suits large-scale QC operations.
• Superior precision and sensitivity ensure reliable dosage verification.
• Modular design allows expansion to high-temperature, multidimensional or green LC applications.

Future Trends and Applications

Emerging UHPLC technologies will further cut analysis time and solvent use, advancing sustainable ‘green’ methods. Integration with automated sample preparation and multidimensional separations will improve detection of trace impurities and complex vitamers. Coupling with mass spectrometry will enhance structural characterization and impurity profiling.

Conclusion

The Shimadzu Nexera X2 Fast LC system successfully complies with USP41 monograph requirements for cyanocobalamin tablet assay, offering high resolution, precision and rapid throughput for pharmaceutical quality control.

References

• USP Monograph: Cyanocobalamin Tablets, USP41–NF36, Monograph 1114, Procedure 2.
• USP General Chapter 621: Chromatography, First Supplement to USP41–NF36.