Riboflavin Analysis Service - Creative Proteomics

Q: What are the consequences of riboflavin deficiency in biological systems?

Riboflavin deficiency can lead to oral ulcers, angular stomatitis, skin lesions, eye congestion, and fatigue in humans. In livestock and poultry, it may cause growth retardation, poor feed conversion, dermatitis, and neurological issues.

Q: Why is it crucial to protect riboflavin samples from light during analysis?

Riboflavin is highly photosensitive, especially under ultraviolet light or alkaline conditions, where it can rapidly degrade into products like lumichrome and lumiflavin, leading to inaccurate quantification.

Q: What are the key advantages and limitations of Fluorescence Spectroscopy for riboflavin detection?

Fluorescence spectroscopy offers high sensitivity, with detection limits as low as 0.006μg, and is ideal for simple matrices like fortified foods. However, its specificity can be limited in complex samples due to potential interfering fluorescent compounds.

Q: How does the Glutathione Reductase Activity Coefficient (GRAC) relate to riboflavin status?

The GRAC assay measures the activity of the FAD-dependent enzyme glutathione reductase. An elevated activity coefficient indicates riboflavin deficiency, making it a functional and sensitive biomarker for assessing riboflavin nutritional status in biological systems.

Q: What are the primary considerations when choosing between HPLC and LC-MS/MS for riboflavin analysis?

The choice depends on required specificity, sensitivity, and sample complexity. LC-MS/MS provides superior selectivity for complex matrices (e.g., fermentation broths, multivitamin blends) and enables precise quantification of individual vitamins (riboflavin, FMN, FAD) using specific mass transitions. HPLC-FLD is a cost-effective and highly sensitive alternative for routine analysis of less complex samples.

Q: What are the stability concerns for riboflavin in pharmaceutical and food formulations?

Beyond light sensitivity, riboflavin's stability in formulations can be compromised by high temperatures, alkaline pH, and the presence of oxidizers. Its degradation leads to loss of potency, and in liquid formulations like milk, prolonged light exposure causes significant depletion.

Q: Can your service analyze riboflavin in animal feed and pet food matrices?

Yes, our methods are adaptable for analyzing riboflavin in animal feed, pet food, and related ingredients. We account for complex matrices and can help ensure nutritional claims meet required standards for livestock, poultry, and pets.

Q: What are the specifications for riboflacin reference standards used in your analyses?

We use high-purity reference standards, typically meeting specifications such as ≥99% purity, with controlled limits for parameters like specific rotation, related substances (e.g., light degradants), and residue on ignition, ensuring accurate and traceable quantification

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What Is Riboflavin Analysis?

Riboflavin, also known as vitamin B2, is an essential water-soluble vitamin that functions as a precursor to two key cofactors—flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). These cofactors participate in redox reactions that support energy metabolism, cellular respiration, and antioxidant defense. Because riboflavin is both fluorescent and photo-sensitive, it serves as an ideal marker for assessing nutrient stability in foods, biological samples, and fermentation products. Riboflavin analysis helps researchers quantify its concentration and evaluate conversion among its active forms, providing critical insights into nutritional quality and metabolic status.

Common Questions—and Why Testing Matters

How stable is riboflavin during processing? Light converts riboflavin to lumichrome and lumiflavin; monitoring both prevents under- or over-estimation.

Do I need FMN and FAD as well? Many applications track the full flavin pool; FMN/FAD often respond to processing and metabolism differently than free riboflavin.

Which method should I pick? HPLC with fluorescence is highly sensitive and cost-effective; LC-MS/MS adds selectivity for complex matrices and multi-vitamin panels.

Riboflavin Analysis Services: Targeted Testing for Riboflavin, FMN, and FAD

Service scope aligned to real use cases—food, dairy, supplements, bioprocess, and biological matrices.

Targeted LC–MS/MS quantification (riboflavin, FMN, FAD): Isotope-assisted calibration, matrix-matched curves, and MRM confirmation for precise, comparable results.

HPLC-FLD riboflavin testing: Fluorescence detection with analyte-specific wavelengths for routine assays in standard food and dairy matrices.

Photodegradation profiling (lumichrome, lumiflavin): Assess light sensitivity and packaging performance by tracking key riboflavin photoproducts.

B-vitamin multi-analyte panel (optional): Combine riboflavin with thiamine, niacin, pyridoxine, folate, pantothenate, and biotin for formulation and labeling needs.

Bioprocess monitoring for fermentation: Track riboflavin secretion or consumption and follow FMN/FAD dynamics to optimize media composition and harvest points.

Analyte Coverage for Riboflavin Analysis

Analyte	Category	Notes
Riboflavin (Vitamin B2)	Primary riboflavin vitamer	Fluorescent, photo-sensitive; baseline target in riboflavin panels.
Flavin Mononucleotide (Riboflavin-5'-phosphate, FMN)	Riboflavin coenzyme/vitamer	Phosphorylated flavin; benefits from biocompatible LC hardware.
Flavin Adenine Dinucleotide (FAD)	Riboflavin coenzyme/vitamer	Dimeric flavin cofactor; gentle extraction maintains integrity.
Lumichrome	Riboflavin photodegradation product	Marker of light-induced B2 loss.
Lumiflavin	Riboflavin photodegradation product	Secondary light-derived product; complements lumichrome tracking.
Thiamine (Vitamin B1)	Optional B-group vitamer	Common in multi-vitamin panels.
Thiamine Monophosphate (TMP)	Optional B1 phosphorylated vitamer	Included when B1 speciation is required.
Thiamine Pyrophosphate (TPP)	Optional B1 coenzyme form	Active coenzyme; optional in extended panels.
Niacin (Nicotinic Acid)	Optional B3 vitamer	Pair with nicotinamide for full B3 coverage.
Nicotinamide	Optional B3 vitamer	Amide form of B3; frequent in fortified products.
Pantothenic Acid (Vitamin B5)	Optional B-group vitamer	Often included for label verification.
Pyridoxine	Optional B6 vitamer	Select B6 forms per project scope.
Pyridoxal	Optional B6 vitamer	—
Pyridoxal-5'-phosphate (PLP)	Optional B6 coenzyme form	Add when coenzyme-level insight is needed.
Biotin (Vitamin B7)	Optional B-group vitamer	Low endogenous levels; MRM improves selectivity.
Folic Acid	Optional B9 vitamer (oxidized form)	Baseline B9 in fortified products.
5-Methyltetrahydrofolate (5-MTHF)	Optional B9 reduced folate	Include reduced folates only if required.
Tetrahydrofolate (THF)	Optional B9 reduced folate	Specialized requests for folate speciation.

Why Choose Our Riboflavin Analysis Service?

Trace-level detection.

LOD/LOQ in the pg/mL–ng/mL range (LC–MS/MS) and <10 ng/mL (HPLC-FLD), supporting low-abundance monitoring.

Matrix-adapted selectivity.

Analyte-specific MRM and fluorescence filters reduce signal suppression in complex samples.

Reliable quantification.

Internal standards and matrix-matched curves ensure 80–120% recovery with CVs typically ≤15% intra-batch.

Photo-degradation tracking.

Lumichrome/lumiflavin analysis reveals light-induced loss beyond total B2 values.

Vitamer-level resolution.

Separate values for riboflavin, FMN, and FAD support clearer formulation and process insights.

Instrumentation and Methods for Riboflavin Analysis

To support accurate vitamin B2 quantification and flavin profiling across diverse matrices, we apply two validated platforms based on sample complexity and reporting needs:

HPLC with Fluorescence Detection (HPLC-FLD)

Typical use case: Fortified foods, dairy, supplements, and clear liquid samples

Detection mode: Excitation at ~450 nm, emission at 520–530 nm (riboflavin-specific)

Column type: C18 reversed-phase

Benefits: High sensitivity, cost-effective, ideal for routine testing of riboflavin and FMN

Example platform: Agilent 1260 Infinity II Bio LC with FLD detector for biocompatible flow paths

Quality Controls Built-In

System suitability check before each run
Blank, spike recovery, and internal standard tracking

LC–MS/MS (MRM Mode)

Typical use case: Complex matrices (e.g., fermentation broth, multivitamin blends), coenzyme-level profiling

Ionization: Positive ESI (Electrospray Ionization)

Sample transitions (examples):

Riboflavin: m/z 377.1 → 243.1
FMN: m/z 457.1 → 243.1
FAD: m/z 786.2 → 440.1

Column type: UPLC C18, short gradients (3–10 min depending on panel)

Quantification strategy: Stable isotope–labeled internal standards + matrix-matched calibration

Example platform: Waters ACQUITY UPLC–Xevo TQ-S

Agilent 1260 Infinity II HPLC (Figure from Agilent)

Waters ACQUITY UPLC System (Figure from Waters)

Riboflavin Analysis Workflow | How We Quantify Vitamin B2 and Flavin Cofactors

Project Setup & Target Selection

Define required analytes—such as riboflavin, FMN, FAD—and select the appropriate detection panel based on your formulation or study goals.

Sample Intake & Light-Protected Storage

All samples are logged, stored under amber conditions, and reviewed for volume, matrix compatibility, and chain of custody.

Extraction & Preparation

Matrix-specific extraction methods (e.g., acid hydrolysis, protein removal, enzymatic treatment) are applied with minimal light exposure to preserve photolabile vitamers.

Instrumental Run (HPLC or LC–MS/MS)

Based on matrix complexity, we assign either fluorescence-based B-vitamin detection or MRM-based cofactor profiling. All systems are pre-validated with standard curves and QC runs.

Quantification & Quality Control

Internal standards and matrix-matched calibration enable robust vitamin B2 quantification. Each batch includes blanks, recovery samples, and system suitability tests.

Data Reporting & Interpretation

Deliverables include concentration tables, QC summary, method conditions, and analyte-specific flags—ready for direct use in product release or research evaluation.

How to Prepare and Submit Samples for Riboflavin Analysis

Sample Type	Minimum Amount	Container & Light Protection	Preparation Notes
Urine / Plasma / Serum (for B2 or FMN/FAD)	≥ 0.5 mL	Amber microvial on ice	Clearly label sample type and anticoagulant, if used.
Liquid Food / Beverage	≥ 50 mL	Amber glass or foil-wrapped	Record pH, preservatives, or clarifiers if added.
Milk / Formula / Dairy Products	≥ 100 g or mL	Opaque or amber container, chilled	Indicate fat content; homogenize before sending.
Dietary Supplement (Powder / Capsule)	≥ 20 g	Foil pouch or amber vial	Provide ingredient list or label claim if available.
Premix or Fortified Powder	≥ 20 g	Light-protected sealed bag	Note carrier composition and target vitamin concentration.
Fermentation Broth / Supernatant	≥ 50 mL	Amber bottle, chilled	Document medium composition and harvest timepoint.

Deliverables: What You Receive from Riboflavin Analysis

Concentration Report: Precise concentrations of riboflavin, FMN, and FAD in your samples.
Photodegradation Data: Light stability results including lumichrome and lumiflavin profiles.
Standard Curve: Calibration data with concentration vs. signal intensity and R² value.
Quality Control Summary: Recovery rates, internal standards, and data validation checks.
Methodology: Detailed analysis methods, calibration techniques, and detection limits.
Data Interpretation: Expert analysis of riboflavin levels and their relevance to your product.
Multi-Vitamin Panel Results: Optional B-vitamin concentrations (B1, B3, B6) in the same sample.
Process Optimization Recommendations: Tailored suggestions for improving stability or yield.
Raw Data Files: Access to chromatograms and mass spectra for full transparency.

LC-MS/MS MRM chromatogram showing ion intensity for Riboflavin, FMN, and FAD at specific m/z transitions.

LC-MS/MS MRM Data Plot

Standard curve for Vitamin B2 concentration vs signal intensity with a fitted line and R² value.

Standard Curve and Linearity Plot

Photodegradation plot showing Riboflavin, Lumichrome, and Lumiflavin concentration changes over time.

Photodegradation Process Plot

Stacked bar chart showing the concentration of Vitamins B1, B2, B3, B6, and B12 in various samples.

Multi-Dimensional B-Vitamin Analysis

Applications of Riboflavin Analysis

Nutritional Supplements

Quantifying riboflavin in dietary supplements to ensure compliance with regulatory standards and product specifications.

Food and Beverage Industry

Measuring riboflavin levels in fortified foods and beverages to ensure accurate labeling and nutritional content.

Pharmaceuticals

Monitoring riboflavin content and stability in vitamin and multivitamin formulations.

Bioprocessing and Fermentation

Tracking riboflavin production and consumption during fermentation to optimize media composition and yield.

Human Biological Samples

Analyzing riboflavin levels in plasma, urine, and serum to evaluate nutritional status and metabolic requirements.

Animal Nutrition

Determining riboflavin content in animal feed to ensure proper nutritional levels for livestock and pets.

What are the consequences of riboflavin deficiency in biological systems?

Riboflavin deficiency can lead to oral ulcers, angular stomatitis, skin lesions, eye congestion, and fatigue in humans. In livestock and poultry, it may cause growth retardation, poor feed conversion, dermatitis, and neurological issues.

Why is it crucial to protect riboflavin samples from light during analysis?

Riboflavin is highly photosensitive, especially under ultraviolet light or alkaline conditions, where it can rapidly degrade into products like lumichrome and lumiflavin, leading to inaccurate quantification.

What are the key advantages and limitations of Fluorescence Spectroscopy for riboflavin detection?

Fluorescence spectroscopy offers high sensitivity, with detection limits as low as 0.006μg, and is ideal for simple matrices like fortified foods. However, its specificity can be limited in complex samples due to potential interfering fluorescent compounds.

How does the Glutathione Reductase Activity Coefficient (GRAC) relate to riboflavin status?

The GRAC assay measures the activity of the FAD-dependent enzyme glutathione reductase. An elevated activity coefficient indicates riboflavin deficiency, making it a functional and sensitive biomarker for assessing riboflavin nutritional status in biological systems.

What are the primary considerations when choosing between HPLC and LC-MS/MS for riboflavin analysis?

The choice depends on required specificity, sensitivity, and sample complexity. LC-MS/MS provides superior selectivity for complex matrices (e.g., fermentation broths, multivitamin blends) and enables precise quantification of individual vitamins (riboflavin, FMN, FAD) using specific mass transitions. HPLC-FLD is a cost-effective and highly sensitive alternative for routine analysis of less complex samples.

What are the stability concerns for riboflavin in pharmaceutical and food formulations?

Beyond light sensitivity, riboflavin's stability in formulations can be compromised by high temperatures, alkaline pH, and the presence of oxidizers. Its degradation leads to loss of potency, and in liquid formulations like milk, prolonged light exposure causes significant depletion.

Can your service analyze riboflavin in animal feed and pet food matrices?

Yes, our methods are adaptable for analyzing riboflavin in animal feed, pet food, and related ingredients. We account for complex matrices and can help ensure nutritional claims meet required standards for livestock, poultry, and pets.

What are the specifications for riboflacin reference standards used in your analyses?

We use high-purity reference standards, typically meeting specifications such as ≥99% purity, with controlled limits for parameters like specific rotation, related substances (e.g., light degradants), and residue on ignition, ensuring accurate and traceable quantification

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Riboflavin Analysis Service for Vitamin B2, FMN, and FAD Quantification

What Is Riboflavin Analysis?

Common Questions—and Why Testing Matters

Riboflavin Analysis Services: Targeted Testing for Riboflavin, FMN, and FAD

Analyte Coverage for Riboflavin Analysis

Why Choose Our Riboflavin Analysis Service?

Instrumentation and Methods for Riboflavin Analysis

HPLC with Fluorescence Detection (HPLC-FLD)

Quality Controls Built-In

LC–MS/MS (MRM Mode)

Riboflavin Analysis Workflow | How We Quantify Vitamin B2 and Flavin Cofactors

How to Prepare and Submit Samples for Riboflavin Analysis

Deliverables: What You Receive from Riboflavin Analysis

Applications of Riboflavin Analysis