Review on Analytical Methods of Water-Soluble Vitamins

Water-soluble vitamins, including B-complex groups (e.g., thiamine, riboflavin) and ascorbic acid (vitamin C), are indispensable for sustaining metabolic homeostasis and human health. Precise quantification of these micronutrients is critical for ensuring food safety, optimizing nutraceutical formulations, and advancing clinical diagnostics. Over decades, diverse methodologies have emerged to quantify these compounds, each presenting unique strengths and limitations.

This review systematically evaluates established and emerging analytical approaches, with a focus on the transformative impact of mass spectrometry (MS) as an indispensable analytical platform in modern vitamin analysis.

1.Spectrophotometric Analysis

1.1 Analytical Principle

Spectrophotometry quantifies compounds by measuring their absorption of light at specific wavelengths, leveraging unique molecular absorption spectra. Each substance exhibits characteristic wavelength-dependent absorption, enabling concentration determination via the Beer-Lambert Law: A=ε⋅c⋅l, where A is absorbance, ε is the molar absorptivity coefficient, c is concentration, and l is the path length. For instance, ascorbic acid (vitamin C) displays maximal absorption at 265 nm.

1.2 Detection Methodologies

Direct Analysis: Utilizes intrinsic absorption properties (e.g., riboflavin's fluorescence emission at 445 nm).
Derivatization: Enhances detectability via chemical reactions (e.g., thiamine quantification through ferricyanide-induced chromogenic complex formation).

1.3 Workflow

Sample Preparation: Extract vitamins using polar solvents (e.g., ethanol for vitamin C, aqueous buffers for B vitamins).
Chromogenic Reaction: Introduce reagents (e.g., 2,4-dinitrophenylhydrazine for vitamin C derivatization) to generate absorbance-specific products.
Absorbance Measurement: Analyze samples at predefined wavelengths using UV-Vis spectrophotometers.
Quantitative Calculation: Derive concentrations via calibration curves constructed from standard solutions.

1.4 Advantages

Cost-Effectiveness: Affordable instrumentation (e.g., basic models priced under ¥50,000).
Operational Simplicity: Minimal training required; protocols are straightforward and reproducible.
Broad Applicability: Effective for colorless analytes or chromophore-tagged substances across food, pharmaceutical, and clinical matrices.

1.5 Limitations

Sensitivity Constraints: Detection thresholds typically ≥1 μg/mL, limiting utility for trace-level analysis.
Spectral Interference: Co-absorbing compounds (e.g., plant pigments, polyphenols) may skew results, necessitating purification.
Matrix Effects: Competing reactions in complex samples (e.g., enzymatic degradation) can reduce recovery rates and precision.

2. HPLC

2.1 Analytical Principle

High-performance liquid chromatography (HPLC) separates sample components via differential interactions between a liquid mobile phase and a solid stationary phase. This method leverages distinct physicochemical properties (e.g., hydrophobicity, polarity) to achieve separation, followed by qualitative and quantitative detection. For vitamins, reversed-phase C18 columns are widely employed due to their efficacy in resolving hydrophobic compounds. Optimization of mobile phase composition (e.g., acetonitrile-phosphate buffer mixtures) and precise temperature control enhance resolution while minimizing degradation of labile vitamins like ascorbic acid (vitamin C). Detection modalities such as ultraviolet (UV) or fluorescence (FLD) ensure sensitivity and specificity.

2.2 Methodological Optimization

Mobile Phase Composition: A blend of acetonitrile and phosphate buffer (pH 2.8) enhances separation of hydrophilic vitamins (e.g., B₁, B₂). Acetonitrile facilitates rapid elution of hydrophobic analytes, while phosphate buffers stabilize pH for consistent retention times.
Column Temperature Control: Maintaining ~40°C minimizes oxidative degradation of thermolabile vitamins (e.g., vitamin C) and improves column efficiency (theoretical plates >10,000).
Detector Selection:
- UV Detection: Ideal for most vitamins, offering robust quantification based on UV absorption profiles.
- Fluorescence Detection (FLD): Provides superior sensitivity (detection limit ~0.1 μg/mL) for fluorophores like vitamin B₂.
Separation Mode:
- Reversed C18 column: suitable for the separation of polar vitamins (B₁, B₂, C), and 18% carbon loading can provide moderate retention (such as vitamin C retention factor k'=2.5).
- HILIC column: used for the analysis of strong polar vitamins (B₉, B₁₂), with acetonitrile-ammonium formate (pH 5.0) as the mobile phase, the retention time is shortened by 30%.

2.3 Sample Preparation Protocols

Solid-Phase Extraction (SPE)

Plasma Processing: Protein precipitation with acetonitrile (3:1 v/v), followed by centrifugation (10,000×g, 10 min).
SPE Workflow:
- Column activation: Methanol and water (3 mL each).
- Loading: Supernatant adjusted to pH 2.8.
- Elution: 80% methanol-water (recovery >90%).
- High-Throughput: 96-well SPE systems enable parallel processing of 48 samples in 30 minutes.
Online Purification: Dual-column systems (e.g., Agilent Zorbax SB-C18) with restricted surface packing reduce phospholipid interference, extending column lifespan by 50%.

Representative chromatogram of RP-HPLC/DAD analysis of water-soluble vitamin mixture at two wavelengths (Mateeva A et al., 2022).

2.4 Advantages

High Resolution: Theoretical plates >10,000 enable simultaneous quantification of multiple vitamins (e.g., B₁, B₂, B₆, C) in complex matrices.
Sensitivity: FLD achieves trace-level detection (e.g., 0.1 μg/mL for B₂).
Throughput: Rapid analysis with robust reproducibility across diverse samples (food, pharmaceuticals, biological fluids).

2.5 Limitations

Isomer Discrimination: Inability to differentiate structural isomers (e.g., vitamin B₁₂ forms) without complementary techniques (e.g., MS).
Matrix Complexity: Requires extensive pretreatment (e.g., SPE) for samples like plasma, increasing workflow duration (2–3 hours).
Operational Costs: High equipment expenses and need for skilled personnel to optimize parameters (mobile phase, temperature, flow rate).

3. Capillary Electrophoresis

Capillary electrophoresis (CE) has emerged as a pivotal technique for vitamin analysis, offering minimal sample requirements and superior resolving power. This review explores CE's operational principles, method advancements, and real-world applications, while addressing its challenges in complex matrix analyses.

3.1 Principles and Separation Modes

Fundamental Mechanism:
- Electrophoretic Mobility: Analyte migration velocity (v=μe×E) depends on charge-to-mass ratio (μe) and electric field strength (E).
- Electroosmotic Flow (EOF) Modulation: Achieved via capillary coatings (e.g., polyacrylamide) or buffer additives (e.g., cetyltrimethylammonium bromide, CTAB).
Innovative Separation Strategies:
- Micellar Electrokinetic Chromatography (MEKC):
- Micellar Phase: 1% sodium dodecyl sulfate (SDS) enhances resolution (e.g., pantothenic acid and biotin separation resolution: 0.8 → 1.5) (J. Chrom. A, 2022).
Nonaqueous CE (NACE):
- Solvent System: Acetonitrile-methanol (7:3) with 50 mM ammonium acetate optimizes lipid-soluble vitamin (A, D, E) separation.

3.2 Method Optimization and Performance

Buffer System Design

Vitamin Type	Buffer Composition	pH	Separation Voltage (kV)
Water-soluble B	20 mM borate + 50 mM SDS	9.3	25
Ascorbic acid (C)	30 mM phosphate + 10% methanol	2.5	20
Lipid-soluble A/D/E	50 mM CHES + 15 mM β-cyclodextrin	8.8	18
β-cyclodextrin (β-CD) enhances solubility of nonpolar vitamins.

Detection Innovations

Laser-Induced Fluorescence (LIF):
- Vitamin B₆ detection at 0.01 ng/mL (Ex/Em: 325/400 nm).
- Thiamine derivatization with naphthalene-2,3-dicarbaldehyde (NDA) enables fluorescence detection (Ex/Em: 420/520 nm).
CE-Mass Spectrometry (CE-MS):
- Sheath-liquid-assisted electrospray (3 μL/min) achieves 0.05 ng/mL sensitivity for vitamin B₁₂.

3.3 Advantages

Minimal Sample Consumption: Nanogram-scale requirements enable single-cell or rare-sample analysis.
High Resolution: Distinguishes structurally similar vitamins (e.g., B-complex isomers).
Versatile Detection: Compatible with UV, fluorescence, and MS detection for multi-modal analysis.
Microanalysis Proficiency: Detects trace analytes in pharmaceuticals, food, and environmental samples.

3.4 Limitations

Reproducibility Challenges: Relative standard deviation (RSD) >5% due to capillary surface variability, buffer composition, or temperature fluctuations.
Environmental Sensitivity: pH and temperature critically influence migration times, necessitating stringent control.
Throughput Constraints: Manual workflows and prolonged analysis times hinder large-scale adoption, despite advances in high-throughput CE (HTCE).
Complex Matrix Limitations: Requires coupling with techniques like liquid chromatography for highly heterogeneous samples.

4. Enzymatic Assays

Enzymatic assays are pivotal in biochemical and clinical diagnostics, leveraging enzyme-catalyzed reactions to quantify target analytes through measurable outputs such as optical shifts, luminescence, or electrochemical signals. These methods combine specificity, cost-efficiency, and operational simplicity, yet face constraints in sensitivity and applicability.

4.1 Fundamental Principles

Enzymatic techniques exploit the catalytic specificity of enzymes to induce detectable changes in substrates. Key mechanisms include:

Target-Specific Reactions

Vitamin B₁₂ (Cobalamin):
- Intrinsic Factor Binding: Competitive ELISA using intrinsic factor (IF) with high affinity (K_D = 10⁻¹⁰ M) detects free B₁₂ via alkaline phosphatase (ALP)-labeled IF, yielding a 0.1 ng/mL detection limit.
- Chromogenic Detection: ALP catalyzes 4-nitrophenyl phosphate (pNPP) hydrolysis, monitored at 405 nm.
Vitamin C (Ascorbic Acid):
- Oxidase-Based Sensing: Ascorbic acid oxidase converts vitamin C to dehydroascorbic acid, generating H₂O₂ detectable via amperometry (sensitivity: 0.5 μM).

Coenzyme-Dependent Systems

Vitamin B₆ (Pyridoxal Phosphate, PLP):
- Transaminase Coupling: Aspartate aminotransferase (AST) utilizes PLP to catalyze α-ketoglutarate conversion, quantified by NADH absorbance at 340 nm.
Vitamin B₂ (Riboflavin):
- Fluorometric Assay: Glutathione reductase (GR) reduces oxidized glutathione (GSSG) with FAD cofactor, emitting fluorescence (Ex/Em: 450/520 nm) proportional to B₂ concentration.

4.2 Advantages

Exceptional Selectivity: Enzymes like ascorbic acid oxidase minimize cross-reactivity, ensuring precise quantification even in complex matrices.
Operational Simplicity: Streamlined protocols require minimal instrumentation, ideal for point-of-care testing.
Cost-Effectiveness: Lower reagent and equipment costs compared to chromatographic or mass spectrometric methods.
Broad Utility: Applicable in food quality control, pharmaceutical testing, and environmental monitoring.

4.3 Limitations

Restricted Applicability: Limited to analytes with known enzymatic pathways (e.g., vitamins B₁₂, C), excluding broader metabolomic profiling.
Matrix Interference: Endogenous substances (e.g., drugs, metabolites) may inhibit enzymes, skewing results (e.g., false-positive signals in clinical samples).
Moderate Sensitivity: Detection thresholds (e.g., 0.5 μM for vitamin C) may necessitate supplementary techniques like LC-MS for trace analysis.

5. LC-MS/MS

LC-MS/MS excels in the precise quantification of water-soluble vitamins (e.g., B-complex and ascorbic acid) by integrating advanced chromatographic separation with high-sensitivity mass detection. This approach is indispensable for multi-analyte profiling in complex biological matrices.

5.1 Chromatographic System Design

Column Selection and Mobile Phase Optimization

Hydrophilic Interaction Liquid Chromatography (HILIC):
- Column: Waters XBridge BEH Amide (2.1 × 150 mm, 3.5 µm) for polar vitamins (B₉, C).
- Mobile Phase: Acetonitrile/10 mM ammonium formate (pH 3.5), gradient from 95% to 70% acetonitrile over 15 min.
Reversed-Phase Chromatography:
- Column: Agilent Zorbax Eclipse Plus C18 (2.1 × 100 mm, 1.8 µm) for hydrophobic analytes.
- Mobile Phase: 0.1% formic acid in water (A) and methanol (B), gradient from 5% to 95% B over 10 min.

Matrix Interference Mitigation

Online Purification: TurboFlow™ technology reduces phospholipid interference, lowering matrix effects from -50% to -5%.
Isotopic Internal Standards: Vitamin B₁₂-D₄ (m/z 682.3→151.1) enhances recovery rates (70–130% → 95–105%).

5.2 Mass Spectrometric Configuration

Ionization and Detection

Ion Sources:
- Electrospray Ionization (ESI): 3.5 kV voltage, 350°C nebulizer temperature for polar vitamins.
- Atmospheric Pressure Chemical Ionization (APCI): 4 µA discharge current for lipid-soluble vitamins.
Mass Analyzers:
- Triple Quadrupole (QqQ): Multiple reaction monitoring (MRM) mode with vitamin-specific transitions (e.g., B₁₂: m/z 678.3→147.1, CE 25 eV).
- High-Resolution MS (HRMS): Orbitrap (120,000 resolution) distinguishes isomers (e.g., vitamin K1/K2, Δm = 0.005 Da).

Select Service

Water Soluble Vitamins Analysis Service

Quantification of Fat-Soluble Vitamins

5.3 Method Validation and Analytical Performance

Sensitivity and Linearity

Vitamin	Quantitative Transition (m/z)	Linear Range (ng/mL)	LOD (ng/mL)	RSD (%)
B₁ (Thiamine)	265.1 → 122.1	0.1–50	0.03	2.1
C (Ascorbate)	175.0 → 115.0 (-ve)	1–200	0.5	3.5
25(OH)D₃	401.3 → 383.3	0.05–100	0.02	1.8

Chromatograms of nine water-soluble vitamins and their corresponding internal standards detected simultaneously using the HPLC-MS/MS method (Zhang M et al., 2024).

Precision and Accuracy

Intra-Day Precision: RSD < 3% for B₁, B₂, B₆ in serum.
Inter-Day Accuracy: >99% agreement with certified reference materials.

Throughput and Versatility

Multi-Analyte Detection: Simultaneous quantification of 12 vitamins within 20 minutes per run.
Clinical Utility: High-throughput screening for population studies, sports medicine, and deficiency diagnostics.

If you want to know more about the LC-MS method of water-soluble vitamins, please see "LC-MS for Water-Soluble Vitamin Analysis: A Focused Overview".

Experimental flowchart to investigate the stability of water-soluble vitamins (Luo W ET AL., 2025).

6. HRMS

6.1 Analytical Principle

High-resolution mass spectrometry (HRMS) enables precise determination of molecular and fragment ion masses (mass accuracy<5 ppm), coupled with resolving powers exceeding 100,000 to discriminate isotopic patterns and structural isomers. This capability facilitates robust qualitative and quantitative profiling of water-soluble vitamins in complex biological or food matrices.

6.2 Workflow Overview

Sample Preparation:
- Extraction: Proteins in plasma or food homogenates are precipitated using acetonitrile.
- Enrichment: Solid-phase extraction (SPE) with C18 columns selectively isolates target vitamins (e.g., B-complex vitamins).
Chromatographic Separation:
- Hydrophilic Interaction Chromatography (HILIC): Separates polar vitamins (e.g., folate [B₉], cobalamin [B₁₂]) using mobile phases like acetonitrile-ammonium formate buffers.
Ionization:
- Electrospray Ionization (ESI): Operated in positive/negative polarity modes (e.g., negative ion mode for ascorbic acid [m/z 175.0→115.0]).
Mass Analysis:
- Detection: High-resolution detectors (Orbitrap, time-of-flight [TOF]) acquire full-scan and data-dependent acquisition (DDA) spectra with sub-ppm mass accuracy (≤0.001 Da).
Data Interpretation:
- Spectral Matching: Fragment ions are cross-referenced against databases (e.g., mzCloud) to differentiate analogs (e.g., pyridoxine vs. pyridoxamine [B₆ variants]).

6.3 Advantages

Exceptional Sensitivity: Achieves detection limits as low as 0.001 ng/mL (e.g., serum vitamin B₁₂).
Superior Resolving Power: Distinguishes isotopic clusters (e.g., ¹³C in B₁₂) and structural isomers (e.g., ergocalciferol [D2] vs. cholecalciferol [D3]).
Untargeted Screening: Identifies novel derivatives (e.g., pyridoxal phosphate) in a single experimental run.

6.4 Limitations

Capital Intensity: Orbitrap systems require >¥3 million initial investment, with high maintenance costs.
Operational Complexity: Demands technical expertise for instrument operation and advanced software (e.g., Compound Discoverer) for data processing.
Matrix Challenges: Complex samples (e.g., whole blood) necessitate rigorous pretreatment to mitigate ion suppression (>30% signal loss).

Comparative Analysis of Vitamin Detection Methods

Parameter	Spectrophotometry	HPLC	Capillary Electrophoresis (CE)	LC-MS/MS	Enzymatic Assay	High-Resolution MS (HRMS)
Detection Limit	0.5–10 μg/mL	0.05–1 μg/mL	0.01–1 ng/mL	0.001–0.1 ng/mL	0.1–10 μM* (≈0.02–2 μg/mL)	0.001–0.01 ng/mL
Sample Volume	1–5 mL	10–100 μL	1–10 nL	10–50 μL	50–200 μL	10–50 μL
Analysis Time	5–30 min/sample	10–30 min/sample	5–15 min/sample	5–20 min/sample	10–30 min/sample	10–30 min/sample
Key Advantages	Low cost, simple operation	High sensitivity, multi-component detection	Ultra-trace analysis, high separation efficiency	Ultra-high sensitivity, multi-component analysis	High specificity, rapid screening	Ultra-high resolution, structural identification
Typical Applications	Rapid food screening (e.g., vitamin C in juices)	Pharmaceutical quality control (e.g., B-complex vitamins)	Single-cell/trace clinical detection (e.g., vitamin B₆ in cerebrospinal fluid)	Metabolomics research, clinical diagnostics (e.g., serum 25(OH)D₃)	Clinical POCT, food safety testing (e.g., vitamin B₁₂ in infant formula)	Novel metabolite discovery, precision medicine (e.g., vitamin K2 subtypes)

Some typical application scenarios

Spectrophotometry: Rapid screening of vitamin C/B₂ in fruit juice and dairy products.
HPLC: Quantification of B vitamins in multivitamin tablets.
CE: Trace detection of vitamin B₆ in cerebrospinal fluid (single cell sample).
LC-MS/MS: Clinical accurate diagnosis of serum 25(OH)D3.
Enzymatic method: instant detection of vitamin B₁₂ in infant formula milk powder.
HRMS: Study on Metabolic Pathway of Vitamin K₂ Subtype (MK-4/MK-7).

Want to know more about vitamin extraction methods, you can see "Comprehensive Evaluation of Vitamin Extraction Methodologies".