Bringing LC/MS to the Wellness Aisle: Quantitation of Water SolubleVitamins in Over-the-Counter Supplements

Significance of the topic

Dietary supplements containing water‑soluble vitamins are widely used for general health and wellness. Routine quality control of these products must address complex dosage forms (gummies, powders, drinks) and mixtures of analytes present at widely varying concentrations. Traditional assays (spectrophotometry, microbiological tests, LC‑UV) are often unsuitable for multi‑vitamin matrices because of limited specificity, sensitivity, throughput, or high variability. A robust, single‑run analytical workflow that combines specificity, sensitivity, and throughput is therefore valuable for manufacturers and QC laboratories.

Objectives and overview of the study

The study aimed to transfer a previously developed triple‑quadrupole LC/MS approach to a single‑quadrupole LC/MS platform to quantify multiple water‑soluble vitamins in over‑the‑counter supplements in one run. The target was to demonstrate adequate sensitivity, selectivity, linearity, and accuracy for routine QC of a representative commercial product (Emergen‑C Turmeric & Ginger gummies and fizzy drink mix), while simplifying workflow and lowering cost and instrument complexity.

Methodology

The sample set comprised an over‑the‑counter powdered fizzy drink mix and gummy formulation. Powdered samples were reconstituted to the manufacturer’s recommended dose volume and prepared as both filtered and unfiltered extracts to evaluate robustness. Key sample preparation steps were:

• Extraction with an acidic aqueous solvent containing 0.1% H3PO4, 5% acetonitrile, 0.5% EDTA and 0.5% ascorbic acid (ascorbic acid included to limit oxidation of labile vitamins).
• Initial agitation (20 minutes) and dilution to final volume.
• Heat treatment at 90–95 °C for 25 minutes to enhance release of riboflavin (vitamin B2).
• Centrifugation (13,000 rpm, 5 minutes) and additional dilution as required.
• Post‑spiking for recovery checks and injection in positive ionization mode.

Various dilution levels were prepared to cover the broad concentration range of vitamins present in the products and to challenge the method at LOD/LOQ levels.

Used instrumentation

The analytical system combined an Agilent Infinity III LC stack (Prime pump and multisampler) with a UV‑DAD detector and an Agilent Pro iQ single‑quadrupole mass spectrometer equipped with a MultiMode ion source (composite ESI+APCI). Selected ion monitoring (SIM) mode was used to maximize sensitivity for each target vitamin during a single chromatographic run.

Main results and discussion

The method simultaneously detected and quantified a panel of water‑soluble vitamins (including vitamin C, thiamine B1, riboflavin B2, niacin B3, pyridoxine B6, folate B9, cyanocobalamin B12 and pantothenic acid B5) in one analysis. Key findings included:

• Sensitivity: Limits of detection and quantitation were in the low parts‑per‑billion range for most analytes, enabling measurement well below typical label claims.
• Linearity: Calibration curves near LOD/LOQ exhibited excellent linearity (reported R2 ≈ 0.99), supporting reliable quantitation across a wide dynamic range. Example data demonstrated calibration for niacin from tens to hundreds of thousands of ppb.
• Accuracy and precision: Recoveries and measured amounts were consistent with label claims for the tested product. Replicate analyses of niacin (12 injections across filtered/unfiltered preparations) produced mean concentrations consistent with expected values and a relative standard deviation around 3.8%, indicating good repeatability.
• Robustness: Comparable results from filtered and unfiltered preparations showed the extraction and analysis procedure tolerates matrix variability typical of gummies and drink mixes.

The combination of a multimode source (ESI+APCI) and SIM acquisition on a single quadrupole allowed effective sensitivity and selectivity for a multi‑vitamin panel without resorting to tandem MS, demonstrating that single‑quadrupole instrumentation can meet QC requirements for many supplement analyses.

Benefits and practical applications

The presented workflow offers several advantages for routine QC of multivitamin supplements:

• Single‑run analysis of multiple water‑soluble vitamins reduces assay count, labor, and turnaround time compared with individual assays.
• Low‑ppb sensitivity and good selectivity in complex matrices improve accuracy of label‑claim verification.
• Use of a single‑quadrupole MS reduces instrument complexity and cost compared with triple‑quadrupole systems while retaining sufficient performance for many QC tasks.
• Compatibility with common dosage forms (gummies, powders, reconstitutable drinks) after a straightforward extraction and cleanup procedure.

Future trends and potential applications

Extensions and developments that would enhance applicability and robustness of the approach include:

• Formal method validation to regulatory guidance (accuracy, precision, matrix effects, stability, ruggedness) across multiple product types and brands.
• Incorporation of isotopically labeled internal standards for each analyte to further improve quantitative accuracy and correct for matrix suppression/enhancement.
• Automation of sample preparation workflows (liquid handling, heating/cooling steps) to increase throughput and reproducibility for high‑volume QC labs.
• Application to broader analyte panels (additional vitamins, metabolites, or contaminants) and to fat‑soluble vitamins with adapted extraction and chromatographic conditions.
• Interlaboratory studies and ring trials to demonstrate method transferability and support regulatory acceptance.

Conclusion

This study demonstrates that a single‑quadrupole LC/MS platform equipped with a multimode ESI+APCI source and operated in SIM mode can reliably identify and quantify a panel of water‑soluble vitamins in complex over‑the‑counter supplement matrices in a single run. The approach delivers low‑ppb sensitivity, strong linearity near LOD/LOQ, good repeatability, and agreement with label claims, offering an efficient, cost‑effective alternative for routine QC of multivitamin formulations.

References

1. Simultaneous Detection and Quantitation of 14 Water‑Soluble Vitamins in Supplement by LC/MS/MS Triple‑Quadrupole. Agilent application note (promotional material), Agilent Technologies, 2026. Document DE‑014962.