Water-soluble vitamins, such as the B-complex group and vitamin C, are indispensable for sustaining metabolic functions, immune health, and neurological activity. Despite their physiological significance, accurate quantification of these vitamins remains analytically challenging due to their heterogeneous chemical profiles—marked by stark polarity variations and molecular instability—and the inherent complexity of biological matrices (e.g., dietary products, bodily fluids). Conventional analytical techniques frequently suffer from limited detection sensitivity and insufficient specificity, hindering precise measurement.
Liquid chromatography-mass spectrometry (LC-MS) has risen as the premier methodology for water-soluble vitamin analysis, combining chromatographic resolution with mass spectral precision to enable simultaneous multi-analyte detection. This review emphasizes the pivotal role of mass spectrometry within LC-MS systems, detailing technical refinements and strategic optimizations that enhance analytical performance. By addressing critical parameters such as ionization efficiency, fragmentation patterns, and matrix interference mitigation, this work underscores advancements that solidify LC-MS as the benchmark for reliable, high-throughput vitamin quantification in complex samples.
Chromatograms of the water-soluble vitamins generated using a standard solution under MS/MS analysis (Khaksari M et al., 2017).
Analytical Challenges and LC-MS Advantages in Water-Soluble Vitamin Profiling
1.1 Analytical Hurdles in Water-Soluble Vitamin Analysis
- Chemical Complexity: Water-soluble vitamins—including thiamine (B₁), riboflavin (B₂), pyridoxine (B₆), cobalamin (B₁₂), and ascorbic acid (C)—exhibit substantial structural heterogeneity. Their molecular weights span 176 Da (vitamin C) to 1355 Da (B₁₂), with logP values ranging from -2.5 (hydrophilic) to 2.5 (moderately lipophilic), complicating simultaneous analysis.
- Matrix Interference: Co-existing substances in biological and food samples, such as plasma proteins, dietary pigments, and carbohydrates, often obscure target analyte signals, compromising detection accuracy in conventional UV-based methods.
- Instability Constraints: Ascorbic acid is prone to oxidation, while thiamine degrades under light exposure, necessitating expedited processing under controlled light and temperature conditions during extraction.
1.2 LC-MS as a Paradigm-Shifting Solution
- Exceptional Sensitivity: LC-MS achieves detection thresholds at picomolar or lower concentrations, enabling precise quantification of water-soluble vitamins in biologically complex matrices such as blood and urine.
- Superior Chromatographic Resolution: The system effectively isolates target vitamers within intricate samples, minimizing matrix-derived interferences common in conventional assays and ensuring high-fidelity analytical outcomes.
- Multi-Analyte Quantification: Simultaneous detection of diverse vitamins—spanning varying polarities and molecular weights (e.g., B₁, B₂, B₆, B₁₂, and C)—is facilitated in a single analytical run.
- Structural Elucidation Capability: Beyond molecular weight determination, tandem mass spectrometry provides fragmentation patterns critical for identifying novel vitamin derivatives or metabolites.
- Expedited Workflow Efficiency: LC-MS significantly reduces analysis time compared to traditional methods, supporting high-throughput screening essential for clinical and nutritional research demands.
Chromatogram of water-soluble vitamins (Chunmei Geng et al., 2017)
Mass Spectrometric Detection Strategies for Water-Soluble Vitamin Analysis
2.1 Ionization Techniques and Source Optimization
Electrospray Ionization (ESI)
- Polarity Flexibility: ESI effectively accommodates polar analytes, enabling seamless switching between positive and negative ion modes.
- Positive Mode: Detects protonated ions (e.g., thiamine [B₁, m/z 265.1] and pyridoxine [B₆, m/z 170.1]).
- Negative Mode: Captures deprotonated ions (e.g., ascorbic acid [C, m/z 175.0] and folate [m/z 441.1]).
- Critical Parameters:
- Nebulizer gas pressure: 35 psi
- Desolvation temperature: 350°C
- Capillary voltage: ±3.5 kV
Atmospheric Pressure Chemical Ionization (APCI)
- Applications: Ideal for moderately hydrophobic species (e.g., vitamin B₃ precursors like nicotinic acid) but less efficient for highly polar vitamins.
2.2 Mass Analyzer Selection and Scanning Modes
| Analyzer | Scan Mode | Application | Example |
|---|---|---|---|
| Triple Quadrupole (QQQ) | MRM | High-sensitivity quantification | B₁₂: m/z 678.3 → 359.2 (CE 25 eV) |
| Time-of-Flight (TOF) | Full Scan | Untargeted profiling | Comprehensive vitamin/metabolite detection |
| Orbitrap | High-Resolution MS | Isomer differentiation | B₆ isomers (pyridoxal vs. pyridoxamine) |
2.3 Parameter Calibration for Enhanced Detection
Ion Transition Optimization
- Declustering Potential (DP): Adjust to maximize ion transmission (e.g., DP = 80 V for riboflavin [B₂]).
- Collision Energy (CE):
- Ascorbic acid: CE 12 eV (m/z 175 → 115)
- Thiamine: CE 18 eV (m/z 265 → 144)
Dynamic MRM (dMRM)
- Context-Specific CE Adjustment: Tailor collision energy to retention time (e.g., B₁₂ precursor at 10.5 min: CE 28 eV) to enhance signal-to-noise ratios.
LC-MS Analysis of Water-Soluble Vitamins: Principles and Optimization
3.1 Introduction to Analytical Challenges
Water-soluble vitamins (e.g., B-complex, C) present unique analytical hurdles due to their diverse polarity, matrix complexity (e.g., proteins, pigments), and instability. LC-MS addresses these challenges through integrated strategies spanning sample preparation, chromatographic separation, mass detection, and data processing.
3.2 Sample Pretreatment: Precision Extraction and Purification
Extraction Method Comparison
| Method | Applications | Advantages | Limitations |
|---|---|---|---|
| Liquid-Liquid Extraction (LLE) | Plasma, urine | Low cost, simplicity | Emulsification risks, variable recovery (e.g., ±15%) |
| Solid-Phase Extraction (SPE) | Dairy, complex foods | High selectivity (e.g., HLB columns for lipids) | Time-intensive elution optimization |
| QuEChERS | Fruits, vegetables | Rapid (<15 min), multi-analyte compatibility | Polar vitamin loss (e.g., ascorbic acid) |
| Ultrafiltration/Dialysis | Cell culture media | Retains small molecules | High equipment costs, low throughput |
Case Study: Extraction of Vitamin B3/B6 from Hair
- 500 mg of hair, purified and decontaminated by isopropanol, ground into powder by ball mill, vortex with 2 mL of methanol, and ultrasonic at 35 C.
- The supernatant was filtered through 0.45 μm PTFE membrane, dried by nitrogen blowing at 40°C, and redissolved in 50 μL methanol: water (50:50, v/v).
- Note: When the internal standard is added to the calibrator/quality control, the methanol extraction effect is better than that of the water/methanol mixture; The recovery rate and RSD need to be verified by experiments (Sallabi SM et al., 2021).
Stabilization Strategies
- Antioxidants: 0.1% EDTA + 1% metaphosphate for vitamin C.
- Light/Temperature Control: Dark, 4°C conditions for light-sensitive B₁.
- Rapid Processing: Limit degradation (e.g.,<2 hrs="" from="" sampling="" to="" loss="">20% after 4 hrs at RT).
3.3 Chromatographic Separation: Multi-Dimensional Resolution
Column Selection
| Column Type | Retention Mechanism | Applications | Parameters |
|---|---|---|---|
| HILIC | Hydrophilic interaction | Polar vitamins (C, B₂) | 2.1×150 mm, 1.7 μm, 40°C |
| C18 (Reversed-Phase) | Hydrophobic interaction | Moderately polar vitamins (B₁, B₆) | 2.1×100 mm, 1.8 μm, 35°C |
| Mixed-Mode | Hydrophobic/ionic exchange | Charged macromolecules (B₁₂) | 2.1×50 mm, 3 μm, pH 6.8 buffer |
Mobile Phase Optimization
- Additives: 0.1% formic acid (ESI+ enhancement); 5 mM ammonium acetate (ESI− stability).
- Gradient Program (HILIC, B vitamins):
| Time (min) | Acetonitrile (%) | Aqueous (%) |
|---|---|---|
| 0 | 95 | 5 |
| 5 | 80 | 20 |
| 10 | 60 | 40 |
| 15 | 95 | 5 |
- Outcome: Baseline separation of B₁ (8.2 min), B₂ (9.5 min), B₆ (10.8 min), B₁₂ (13.2 min); peak symmetry 0.95–1.05.
Chromatogram of B vitamins standards and internal standards at (500 μg/L) (Kahoun D et al., 2022).
3.4 Mass Spectrometric Detection: Precision and Sensitivity
Ionization Techniques
- Electrospray Ionization (ESI):
- Polarity switching: ESI+ for B₁ (m/z 265.1 [M+H]⁺), ESI− for C (m/z 175.0 [M−H]⁻).
- Source settings: Nebulizer gas 35 psi, dry gas 350°C, capillary ±3.5 kV.
- APCI: Limited utility for polar vitamins but effective for nonpolar derivatives (e.g., methyl nicotinate).
Analyzer Modes
| Analyzer | Scan Mode | Resolution | Application |
|---|---|---|---|
| Triple Quadrupole | MRM | Unit | High-sensitivity quantification (B₁₂: LOD 0.03 ng/mL) |
| Orbitrap | Full MS/dd-MS² | 70,000 | Isomer differentiation (B₆ forms) |
| TOF | All Ions MS/MS | 40,000 | Untargeted metabolomics |
Case study: Analysis of (B1, B2, B3, B5, B6, B7) by LC-MS/MS
- 50 μL sample (standard/quality control/whey)+10 μL internal standard, add 60 μL trichloroacetic acid (50 mg/mL) to precipitate protein, vortex for 30 seconds, and take the supernatant for injection.
- Chromatographic conditions: Zorbax C18 column (3.0×100 mm, 3.5 μm), mobile phase A(5 mM ammonium formate +0.1% formic acid water) and B(5 mM ammonium formate +0.1% formic acid methanol).
- Mass spectrometry conditions: positive ion ESI (450 C, 5500 V), MRM monitoring, curtain gas 20 psi, collision gas 40/60 psi.
- Verification parameters: the recovery rate is 80–120%, the precision RSD is less than 20% (isotope internal standard correction), and the detection limit/quantitative limit is shown in Supplementary Table S1 (calculated by Analyst 1.6.2) (Magan JB et al., 2020).
Dynamic Parameter Refinement
- Exclusion Lists: Suppress high-abundance matrix ions outside target retention windows.
- Segmented CE: Adjust collision energy by retention time (e.g., B₆: CE 18 eV at 8–10 min; CE 15 eV at 10–12 min).
3.5 Data Analysis and Validation
Quantitative Workflow
- Software: Skyline (targeted MRM), Compound Discoverer (untargeted profiling).
- Integration: ApexTrack algorithm with manual baseline correction for co-eluted/tailing peaks.
- Calibration: Isotope internal standards (e.g., B₁₂-d₄) or structural analogs (nicotinic acid).
Validation Metrics (Vitamin B₁ Example)
| Parameter | Requirement | Result |
|---|---|---|
| Linearity (R²) | ≥0.99 | 0.9995 |
| LOD (S/N ≥3) | – | 0.1 ng/mL |
| LOQ (S/N ≥10) | – | 0.5 ng/mL |
| Precision (RSD) | ≤15% | Intra-day: 3.2%; Inter-day: 6.8% |
| Recovery | 80–120% | 94.5% ± 4.1% |
3.6 Challenges and Innovations
Persistent Issues
- Matrix Effects: Phospholipids suppress B₂ signals (>30% ion inhibition).
- Dynamic Range: Vitamin C spans μM–mM concentrations in biological matrices.
Emerging Solutions
- Online SPE-LC-MS: Automated purification to minimize manual error.
- Derivatization: Dansyl chloride enhances B₆ sensitivity (10-fold improvement).
- AI-Driven Workflows: Machine learning predicts optimal LC-MS parameters.
- Portable Systems: Miniaturized MS for field-based vitamin monitoring.
LC-MS Method Development: Practical Approaches
4.1 Refinement of Chromatographic Parameters
Column Selection
- Reversed-Phase C18 Columns (e.g., 2.1 × 100 mm, 1.7 μm particle size): Balance resolution with analysis speed for broad-spectrum vitamin detection.
- Hydrophilic Interaction Liquid Chromatography (HILIC): Optimized for highly polar analytes (e.g., ascorbic acid) due to enhanced retention of water-soluble compounds.
Mobile Phase Gradient Optimization
- A tailored elution profile minimizes run time while resolving co-eluting peaks:
- Initial Phase (0 min): 95% aqueous (0.1% formic acid), 5% acetonitrile (0.1% formic acid).
- Linear Gradient (5 min): 80% aqueous, 20% acetonitrile.
- Final Gradient (10 min): 60% aqueous, 40% acetonitrile.
4.2 Advanced Sample Preparation Techniques
Biological Fluids (Plasma/Serum)
- Deproteinization: Precipitate proteins using acetonitrile-methanol (4:1 v/v), followed by centrifugation (15,000 ×g, 10 min).
- Solid-Phase Extraction (SPE): Oasis HLB cartridges efficiently remove phospholipids, achieving >90% recovery of hydrophilic vitamins (e.g., B-complex).
Food Matrices
- QuEChERS Workflow: Extract using 1% formic acid-acetonitrile, then purify with PSA (primary secondary amine) and C18 adsorbents to eliminate sugars, pigments, and lipids (ideal for B vitamins in produce).
- Real-Time Monitoring: Employ in vivo microdialysis probes for dynamic tracking of labile vitamins (e.g., ascorbic acid degradation kinetics).
Applications
- Reveal the storage conditions of water-soluble vitamins: LC-MS/MS analysis revealed that thiamine, pyridoxal, and ascorbic acid exhibited instability in serum under both ambient and refrigerated (2–8°C) conditions. In contrast, riboflavin and 5-methyltetrahydrofolate (5-MTHF) demonstrated limited stability at 2–8°C, with reliable detection windows of 48 and 72 hours, respectively. Cryogenic storage significantly enhanced preservation: all evaluated water-soluble vitamins remained stable at -20°C for 72 hours and at -80°C for up to 7 days. For whole blood samples (excluding nicotinamide), transient stability (2–4 hours) was achievable using ice bath preservation prior to centrifugation (Luo W et al., 2025).
- Biomarkers: Following chromatographic separation, analytes were ionized via electrospray ionization (ESI) and quantified using triple quadrupole mass spectrometry in multiple reaction monitoring (MRM) mode, targeting specific precursor-product ion transitions (e.g., m/z 123→80 for nicotinamide). The method exhibited sensitivity in the picogram-per-milligram range, demonstrated excellent linearity (R² >0.99), and achieved recovery rates exceeding 85%. Analysis of 40 human hair specimens revealed quantifiable nicotinamide levels ranging from 100 to 4,000 pg/mg, with concentration distributions categorized as low (30%), moderate (37.5%), and high (22.5%). These findings establish nicotinamide as a stable and reliably measurable B-vitamin biomarker in hair matrices, underscoring its utility for non-invasive nutritional assessments.
- Testing vitamin supplements: A liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based approach was employed for precise quantification of target analytes using multiple reaction monitoring (MRM). Organic solvent-based extraction (e.g., acetonitrile/methanol) facilitated sample preparation, while chromatographic separation was achieved using a reversed-phase C18 column with a gradient elution system (e.g., 0.1% formic acid in acetonitrile). Quantification relied on mass spectrometric detection of specific precursor-to-product ion transitions (e.g., m/z 401→383 for 25(OH)D3) under electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI) modes. Analytical results revealed nicotinamide (vitamin B3) concentrations in human hair ranging from 100 to 4,000 pg/mg, whereas vitamin B6 metabolites (e.g., pyridoxal phosphate, PLP) remained undetectable, likely due to low abundance or oxidative degradation, underscoring the method's selectivity for trace components (Chen P et al., 2007).
References
- Luo W, Wang D, Tang Y, Cheng Q, Ma X, Yu S, Qiu L. "Water-Soluble Vitamins Stability by Robust Liquid Chromatography-Mass Spectrometry." Ann Nutr Metab. 2025;81(1):32-40. doi: 10.1159/000541587
- Sallabi SM, Alhmoudi A, Alshekaili M, Shah I. "Determination of Vitamin B3 Vitamer (Nicotinamide) and Vitamin B6 Vitamers in Human Hair Using LC-MS/MS." Molecules. 2021 Jul 25;26(15):4487. doi: 10.3390/molecules26154487
- Magan JB, O'Callaghan TF, Zheng J, Zhang L, Mandal R, Hennessy D, Fenelon MA, Wishart DS, Kelly AL, McCarthy NA. "Effect of Diet on the Vitamin B Profile of Bovine Milk-Based Protein Ingredients." Foods. 2020 May 4;9(5):578. doi: 10.3390/foods9050578
- Kahoun D, Fojtíková P, Vácha F, Čížková M, Vodička R, Nováková E, Hypša V. "Development and validation of an LC-MS/MS method for determination of B vitamins and some its derivatives in whole blood." PLoS One. 2022 Jul 14;17(7):e0271444. doi: 10.1371/journal.pone.0271444
- Khaksari M, Mazzoleni LR, Ruan C, Kennedy RT, Minerick AR. "Data representing two separate LC-MS methods for detection and quantification of water-soluble and fat-soluble vitamins in tears and blood serum." Data Brief. 2017 Feb 16;11:316-330. doi: 10.1016/j.dib.2017.02.033
- Sallabi SM, Alhmoudi A, Alshekaili M, Shah I. "Determination of Vitamin B3 Vitamer (Nicotinamide) and Vitamin B6 Vitamers in Human Hair Using LC-MS/MS." Molecules. 2021 Jul 25;26(15):4487. doi: 10.3390/molecules26154487
- Chen P, Wolf WR. "LC/UV/MS-MRM for the simultaneous determination of water-soluble vitamins in multi-vitamin dietary supplements." Anal Bioanal Chem. 2007 Apr;387(7):2441-8. doi: 10.1007/s00216-006-0615-y
