Vitamins Analysis Service — LC-MS/MS Quantification of 30+ Fat-Soluble & Water-Soluble Vitamins and Their Active Forms

Fat-Soluble Vitamin Panel — A, D, E, K & Active Metabolites

Fat-soluble vitamins require alkaline saponification or LLE extraction to release them from lipid-rich matrices, C30 or C18 chromatography to resolve structural isomers, and APCI ionization for optimal sensitivity on non-polar analytes. Each vitamin below is quantified against its own stable isotope internal standard.

Water-Soluble Vitamin Panel — B-Complex (B1-B12) & Vitamin C

Water-soluble vitamins require protein precipitation or acidic extraction, HILIC or ion-pairing chromatography for polar retention, and ESI ionization. Several B vitamins are light- and heat-sensitive, requiring protected handling. Each vitamin is quantified against its own stable isotope IS.

Stability & Sample Handling — How We Protect Your Vitamins from Degradation

Vitamin degradation is the single largest source of error in vitamin analysis — not the LC-MS/MS instrument, not the calibration, but what happens between sample collection and extraction. Our protocols address all four degradation pathways with matrix-specific countermeasures developed from our internal stability studies.

Analytical Platforms — Two Dedicated Workflows for Fat-Soluble and Water-Soluble Vitamins

Vitamins Analysis Workflow — From Collection to Quantification

1

Stabilized Sample Collection

All samples collected with class-specific stabilizers: EDTA + ascorbic acid (B9, C), metaphosphoric acid (C), amber tubes (B2, A, K). Plasma/serum separated within 30 min at 4 degree C. Flash-frozen in liquid N2. Detailed collection protocols provided before your experiment.

2

Class-Specific Extraction

Fat-soluble: saponification or LLE with stable isotope IS cocktail. Water-soluble: protein precipitation with acid-stabilized IS cocktail. All extractions under amber light, on ice, with antioxidant protection. SPE cleanup for complex matrices.

3

Dual-Platform LC-MS/MS Acquisition

Fat-soluble: APCI + C30 + SCIEX QTRAP 6500+. Water-soluble: ESI + HILIC + same platform. Both runs from a single sample split after extraction. Sequence: blank, 6-8 calibrators, 3-level QC, randomized study samples with QC every 8-10 injections.

4

Quantification & QC Review

Stable isotope dilution with 1/x2 weighted calibration. Degradation markers tracked: pheophytin-like ratio for B2, ascorbic acid/DHAA ratio for C, retinol/retinyl ester ratio for A. QC: pooled QC RSD below 15%, IS recovery 80-120%, blank carryover below 1% LLOQ.

5

Report Delivery

Concentration table (ng/mL or ug/g) per vitamin form per sample. Stability indicators per sample. QC report with calibration curves, precision, IS recovery. Methods documentation. Optional: statistical analysis, publication-ready figures.

Applications of Vitamin Analysis

Vitamins Analysis Deliverables — What You Receive

• Quantitative Concentration Table — Absolute concentrations (ng/mL for plasma, ug/g for tissue/food) for every vitamin form per sample. Excel and CSV. LOD/LLOQ flags. Stability degradation indicators per sample (ascorbic acid/DHAA ratio, retinol/retinyl ester ratio). IS recovery per sample.
• QC Report — Calibration curves (6-8 points, 1/x2 weighted, R2 and back-calculated accuracy per analyte). Pooled QC RSD. IS recovery (80-120%). Blank carryover. Spike recovery at 3 levels. Inter-batch bridging QC.
• Chromatograms & Spectral Data — MRM traces for each vitamin form with IS overlay. C30 isomer separation chromatograms (vitamin E tocopherols, vitamin D epimers, carotenoids). Raw data files on request.
• Methods Documentation — Complete extraction protocol per vitamin class, LC-MS/MS parameters (column, gradient, MRM transitions, ion source), derivatization details if applicable. Formatted for manuscript methods section.
• Optional Statistical Analysis — Group comparisons (t-test/ANOVA, FDR, volcano/box plots), PCA/PLS-DA, pathway mapping, publication-ready figures (300 DPI TIFF + vector PDF/AI).

Vitamins Analysis Data — Chromatograms, Calibration & Stability Reports

Dual-platform chromatograms: C30 separation of vitamin E tocopherols (alpha, beta, gamma, delta) with APCI detection (left), and HILIC separation of water-soluble B vitamins with ESI detection (right).

Stable isotope dilution calibration curves: 25(OH)D3 (d6-25(OH)D3 IS, 6 points), alpha-tocopherol (d6-alpha-tocopherol IS), and 5-methyl-THF (13C5-5-methyl-THF IS). 1/x2 weighted regression, R2 above 0.998.

Stability monitoring: ascorbic acid/DHAA ratio per sample (left) as a built-in degradation indicator — samples exceeding threshold flagged for review. Vitamin D 3-epi-25(OH)D3 chromatographic separation from 25(OH)D3 (right).

Quantitative data: box plots of 25(OH)D3 concentrations across groups with FDR significance (left), and multi-vitamin status radar chart showing group-level differences across the full panel (right).

Case Study — How LC-MS/MS Vitamin D Metabolite Profiling Revealed Epimer-Specific Clinical Associations

3-epi-25-hydroxyvitamin D₃ is a significant, biologically inactive fraction of total 25(OH)D in infants and adults

Singh, R.J., Taylor, R.L., Reddy, G.S., & Grebe, S.K. | Journal of Clinical Endocrinology & Metabolism, 2006, 91, 3055-3061 | IF: 5.8

DOI: 10.1210/jc.2006-0088

The Challenge

For decades, total 25(OH)D was measured by immunoassay — a single number, assumed to represent the biologically active fraction. But LC-MS/MS revealed a hidden variable: 3-epi-25(OH)D3, a stereoisomer that co-elutes with 25(OH)D3 on standard chromatography and is indistinguishable by immunoassay. Nobody knew how much of "total 25(OH)D" was actually the inactive epimer — and whether this fraction varied by age, disease state, or assay method. The question could only be answered by an LC-MS/MS method capable of baseline-resolving the epimer from the active form.

The Results

Singh et al. developed an LC-MS/MS method using a chiral derivatization approach that achieved baseline separation of 3-epi-25(OH)D3 from 25(OH)D3. Applied to 200+ clinical samples, the findings were striking:

Why It Matters

This study transformed vitamin D testing. Immunoassays and non-resolving LC-MS methods were systematically overestimating biologically active vitamin D — especially in the populations (infants, pregnant women) where accurate assessment matters most. Today, any credible vitamin D assay must report the epimer separately. This is why our panel includes chromatographic resolution of 3-epi-25(OH)D3 — not as an optional add-on, but as a standard component of vitamin D quantification.

What This Means for You

If your study measures vitamin D status — whether in a nutritional intervention, a pregnancy cohort, a pediatric population, or a pharmacodynamic trial — the epimer question matters. Without chromatographic resolution, your "total 25(OH)D" may include 5-60% inactive epimer. Our panel quantifies the epimer separately, giving you the biologically active fraction — the number that actually correlates with calcium homeostasis, immune function, and clinical outcomes.

How We Deliver the Same

• Chiral resolution of 3-epi-25(OH)D3 from 25(OH)D3 on every sample — not a separate add-on
• d6-25(OH)D3 internal standard for absolute quantification of both epimer and active form
• 1,25(OH)2D included in the panel for those needing the active hormone alongside the storage form
• Same SCIEX QTRAP 6500+ platform used in the reference laboratory that first validated clinical LC-MS/MS vitamin D testing

Reference

1. Singh, R.J., Taylor, R.L., Reddy, G.S., & Grebe, S.K. 3-epi-25-hydroxyvitamin D3 is a significant, biologically inactive fraction of total 25(OH)D in infants and adults. Journal of Clinical Endocrinology & Metabolism 91, 3055-3061 (2006).

Frequently Asked Questions

Selected Publications in Vitamin Analysis