Detection and Accurate Quantitation of 14 Water Soluble Vitamins and 14 Fat Soluble Vitamins in Supplements by LC-MS/MS Triple Quadrupole

Importance of the Topic

The accurate measurement of water-soluble B vitamins and fat-soluble vitamins in dietary supplements is critical for ensuring product quality, consumer safety, and compliance with regulatory standards.

Objectives and Study Overview

This study aimed to develop and validate two rapid, sensitive liquid chromatography–tandem mass spectrometry methods for the simultaneous quantitation of 14 water-soluble B vitamins and 14 fat-soluble vitamins in supplement matrices. The methods were demonstrated on multivitamin tablets to verify robustness and accuracy.

Methodology and Instrumentation

• Instrumentation:
  – Agilent 1290 Infinity II UHPLC system coupled to an Agilent 6470 triple‐quadrupole MS with AJS positive electrospray ionization.
• Chromatographic Conditions for Water-Soluble Vitamins:
  – Column: Poroshell 120 Phenyl-Hexyl, 3.0×100 mm, 2.7 µm at 30 °C.
  – Mobile phases: A: 5 mM ammonium formate/0.1% formic acid in water; B: 0.1% formic acid in methanol.
  – Flow: 0.50 mL/min; injection: 1 µL; gradient from 97% A/3% B to 97% B/3% A over 6.6 min.
• Chromatographic Conditions for Fat-Soluble Vitamins:
  – Column: Poroshell 120 SB-AQ, 2.1×150 mm, 2.7 µm at 45 °C.
  – Mobile phases: A: 0.1% formic acid in water; B: 0.1% formic acid in methanol.
  – Flow: 0.25 mL/min; injection: 1 µL; gradient from 20% A/80% B to 20% B/80% A over 12 min.
• MS Settings:
  – Dynamic MRM; stop times: 6.5 min (water-soluble) and 9.5 min (fat-soluble).
  – Drying gas: 270 °C/13 L/min; sheath gas: 375 °C/11 L/min; nebulizer: 40 psi; capillary: 2500 V; nozzle: 0 V for water-soluble.
  – Drying gas: 250 °C/9 L/min; sheath gas: 350 °C/12 L/min; capillary: 4000 V; nozzle: 1000 V for fat-soluble.
• Sample Preparation:
  – Water-soluble: acidified water/ACN with EDTA and vitamin C, heat at 90–95 °C for 25 min.
  – Fat-soluble: DMSO extraction at 55–65 °C, followed by ethanol addition and dilution.
  – Calibration: external standards, matrix-matched standards, and single-point standard addition.
• Acceptance Criteria:
  – Ion ratio within ±30% of standards; retention time matching native and labeled standards; calibration curve r² ≥ 0.99; post-spike or internal standard recoveries between 70% and 130%.

Main Results and Discussion

• All 28 target vitamins were separated and detected in under 10 minutes, with exemplary linearity (0.5–500 ng/mL) demonstrated for riboflavin and cholecalciferol.
• Multivitamin tablet analysis yielded corrected concentrations within 87%–115% of label claims, confirming method accuracy and precision.
• Post-matrix-spike recoveries validated effective compensation of matrix effects and reliable quantitation.

Benefits and Practical Applications

• Rapid, high-throughput workflows reduce assay time and operational costs.
• Simultaneous quantitation of a broad vitamin panel in a single run.
• Robust QC tool for supplement manufacturers and regulatory laboratories.

Future Trends and Applications

• Extension to additional micronutrients and potential degradation products.
• Integration with automated sample preparation and data-processing pipelines.
• Adoption of high-resolution MS for enhanced specificity and selectivity.
• Method transfer to routine QC settings and inter-laboratory standardization.

Conclusion

The validated LC-MS/MS methods provide accurate, sensitive, and efficient quantitation of 14 water-soluble B vitamins and 14 fat-soluble vitamins in supplement matrices, supporting quality assurance and regulatory compliance in the dietary supplement industry.

No formal references provided.