High Speed Analysis of Cyanocobalamin in compliance with Chapter 621 of USP40

Significance of the Topic

The analysis of cyanocobalamin, a vital B12 vitamin, is a critical quality control test in pharmaceutical production. Traditional USP monograph HPLC methods use 5 µm columns with long run times, high solvent consumption, and operational costs. Advances in ultra-high-performance liquid chromatography (UHPLC) and core-shell column packing enable significant productivity gains and cost reduction while maintaining regulatory compliance.

Objectives and Study Overview

This study aimed to demonstrate the transfer and optimization of the USP monograph method for cyanocobalamin analysis in compliance with USP General Chapter 621. The goal was to shorten analysis time, lower solvent use, and maintain chromatographic performance by switching from a conventional 5 µm, 4.6 × 150 mm column to a 2.7 µm, 4.6 × 100 mm core-shell column using Shimadzu instrumentation.

Methodology

The original isocratic method employed a 5 µm ODS column (4.6 × 150 mm), mobile phase water/methanol (13:7, v/v), flow rate 0.50 mL/min, injection volume 100 µL, column temperature 25 °C, and UV detection at 361 nm. According to Chapter 621, permissible adjustments include:

• Column length (L) to particle size (dp) ratio alteration: –25 % to +50 %.
• Particle size and column internal diameter modifications maintaining linear velocity.
• Flow rate changes as long as precision and system suitability are preserved.

The L/dp ratio was kept constant by reducing column length to 100 mm and particle size to 2.7 µm. Flow rates were recalculated using the USP equation F2=F1×(dc2²·dp1)/(dc1²·dp2). Two conditions were tested: 0.50 mL/min (injection 67 µL) and optimized 0.93 mL/min (67 µL).

Used Instrumentation

• Shimadzu LC-2030C Plus HPLC system (Prominence-i).
• Shim-pack VP-ODS column, 4.6×150 mm, 5 µm for original USP method.
• Shim-pack Velox C18 core-shell column, 4.6×100 mm, 2.7 µm for fast methods.
• UV detector set at 361 nm.
• Consumables: amber vials (1.5 mL), PTFE/silicone septa caps, 1 L solvent bottles with safety caps, PEEK fittings, 0.45 µm nylon syringe filters.

Main Results and Discussion

The original method yielded a retention time of 5.163 min. Using the Velox column at 0.50 mL/min reduced retention to 2.368 min but increased theoretical plates (N) by 81 % above the USP limit. Optimizing to 0.93 mL/min achieved a retention time of 1.297 min, with N within ±25 % of the original and resolution meeting system suitability (RSD <2.0 %, plate count and peak area stability confirmed over six injections). The fast method maintained UV sensitivity and reproducibility while cutting run time by ~75 % and solvent use proportionally.

Benefits and Practical Applications

• Significant reduction in analysis time increases sample throughput in QC laboratories.
• Lower mobile phase consumption reduces operational cost and environmental footprint.
• Maintained compliance with USP Chapter 621 ensures regulatory acceptance of modified methods.
• Core-shell technology can be adopted for other monograph analyses to enhance productivity.

Future Trends and Potential Applications

Emerging trends include wider adoption of UHPLC and core-shell columns for monograph transfers, integration of automated method scouting software, and implementation of greener solvents. The approach demonstrated here can be extended to other water-soluble vitamins and pharmaceutical compounds, driving further efficiency improvements in GMP laboratories.

Conclusion

The study successfully transferred the USP monograph method for cyanocobalamin to a high-speed protocol using Shim-pack Velox C18 and Shimadzu LC-2030C Plus. The optimized method delivered a four-fold decrease in run time, reduced solvent consumption, and met all system suitability criteria under USP Chapter 621, illustrating a robust strategy for method enhancement.

Reference

1. United States Pharmacopeia General Chapter 621, USP 40–NF 35, First Supplement.
2. United States Pharmacopeia Monograph, Cyanocobalamin, USP 40–NF 35, First Supplement.
3. Shimadzu Application News No. L464: Fast USP-Compliant HPLC Analysis, 2018.