High speed data acquisition and polarity switching MS/MS applied to water-soluble vitamin analysis using a novel multi-mode ODS separation

Significance of the Topic

Water-soluble vitamins are vital micronutrients required for numerous biochemical processes in humans. Accurate, high-throughput analysis of these polar compounds is critical for quality control in food and pharmaceutical industries. Conventional reversed-phase methods often fail to retain highly polar vitamins, leading to compromised resolution and sensitivity. The development of a rapid LC–MS/MS workflow addresses these limitations and aligns with regulatory requirements for dietary supplement and pharmaceutical testing.

Objectives and Study Overview

This work aimed to establish a simultaneous, sensitive, and fast LC–MS/MS method for nine water-soluble vitamins using a novel mixed-mode ODS column. By integrating high-speed polarity switching and rapid data acquisition, the study sought to eliminate the need for ion-pairing reagents while achieving robust quantitation within a single chromatographic run.

Methodology and Instrumentation

Chromatographic separation was performed on a UFLC system equipped with a Scherzo SM-C18 mixed-mode column (2.0 mm I.D. × 150 mm, 3 µm) containing ODS, anion, and cation exchange ligands, maintained at 40 °C. A binary gradient of 5 mmol/L ammonium formate with 0.1% formic acid (solvent A) and acetonitrile (solvent B) at 0.2 mL/min enabled elution within 10 minutes. Sample preparation involved simple dilution, brief heating, centrifugation, and filtration of tablet extracts.
The mass spectrometers employed were:

• Shimadzu LCMS-8030 triple quadrupole MS in MRM mode with 15 ms polarity switching, 15 000 u/s scan speed, and 1 ms pause time.
• Shimadzu LCMS-2020 single quadrupole MS in SIM mode.

Main Results and Discussion

The mixed-mode column exhibited tailored retention: anion exchange for nicotinic acid and ascorbic acid, weak cation exchange for nicotinamide, and hydrophobic interactions for riboflavin. All nine vitamins (thiamin, pyridoxine, nicotinic acid, nicotinamide, pantothenic acid, cyanocobalamin, riboflavin, biotin, folic acid) were baseline separated and detected in positive or negative ion modes as appropriate. Calibration curves demonstrated excellent linearity over three orders of magnitude (r2 > 0.99) with limits of quantitation around 0.5 pg/µL. Application to commercial supplement tablets yielded results consistent with label claims and regulatory standards.

Benefits and Practical Applications

This workflow offers:

• Simultaneous analysis of multiple polar vitamins without ion-pairing reagents.
• Fast run time (≤10 min) for high sample throughput.
• High sensitivity in the pg/µL range, suitable for regulatory compliance.

It is directly applicable to quality control laboratories in food, nutrition, and pharmaceutical sectors.

Future Trends and Opportunities

Future enhancements may include automated online sample preparation, extension to other polar nutrients and metabolites, and integration with ultrahigh-speed MS platforms. Advances in mixed-mode column chemistries and faster data acquisition will further increase throughput and analytical depth for complex polar analytes.

Conclusion

A high-speed LC–MS/MS method using a mixed-mode ODS column with fast polarity switching provides a robust, sensitive, and rapid solution for the simultaneous quantitation of nine water-soluble vitamins. This approach meets regulatory demands and improves laboratory efficiency without reliance on ion-pairing agents.

References

• Tanigawa T, Minohata T, Arakawa K, Watanabe J, Yazawa I, Herman A. High speed data acquisition and polarity switching MS/MS applied to water-soluble vitamin analysis using a novel multi-mode ODS separation. ASMS 2011 MP165.