High-Precision Nicotinamide Adenine Dinucleotide (NAD) Analysis Service

Why NAD Analysis Matters for Your Research

Nicotinamide adenine dinucleotide (NAD) is a central coenzyme in cellular energy metabolism, redox reactions, and signaling pathways. Acting as a key electron carrier in glycolysis, the TCA cycle, and oxidative phosphorylation, NAD is essential for maintaining metabolic homeostasis. Beyond energy transfer, NAD serves as a substrate for sirtuins, PARPs, and CD38—enzymes critical for DNA repair, aging, and stress response.

Accurate quantification of NAD and its derivatives (NAD⁺, NADH, NADP⁺, NADPH) provides deep insights into cellular bioenergetics, oxidative stress, and disease-related metabolic reprogramming. Whether you are studying mitochondrial function, developing metabolic drugs, or exploring aging mechanisms, precise NAD profiling is indispensable.

Comprehensive NAD Analysis Services by Creative Proteomics

Our NAD analysis platform provides targeted and comprehensive metabolite quantification across multiple research applications:

• Absolute Quantification of NAD Species
  LC-MS/MS-based accurate measurement of NAD⁺, NADH, NADP⁺, and NADPH with isotope-labeled internal standards.
• Redox State Assessment
  Calculation of NAD⁺/NADH and NADP⁺/NADPH ratios to evaluate cellular oxidative stress and metabolic balance.
• Expanded Coenzyme Profiling
  Analysis of related nicotinamide metabolites and precursors (e.g., NMN, NR) for pathway interpretation.
• Integrated Metabolomics Options
  Combine NAD analysis with energy metabolism panels, TCA cycle intermediates, or redox-related pathways.
• Custom Experimental Design
  Flexible workflows tailored to your sample type, matrix complexity, and research objectives.

List of NAD-Related Metabolites Detected

Below is the comprehensive list of detectable compounds included in our NAD analysis service:

Detection can be extended to additional NAD salvage pathway metabolites upon request.

Why Choose Our NAD Analysis Service: Key Advantages

• Ultra-Sensitive Detection: LOD down to 10 fmol per injection, ideal for low-abundance samples.
• High Quantitative Accuracy: Calibration curve R² ≥ 0.995; CV < 10% for replicate assays.
• Wide Dynamic Range: Covers 5–6 orders of magnitude for diverse biological matrices.
• Isotope-Labeled Internal Standards: Guarantees absolute quantification and recovery correction.
• Broad Matrix Compatibility: Plasma, serum, tissue homogenates, cell lysates, fermentation broth.
• Integrated Data Analysis: Includes statistical evaluation, redox ratio visualization, and KEGG pathway mapping.

NAD Assay Technical Details & Coverage

We employ triple-quadrupole LC-MS/MS platforms—including SCIEX QTRAP® 6500+, Thermo TSQ Altis, and Waters Xevo TQ-S—combined with Multiple Reaction Monitoring (MRM) to achieve femtomole-level detection of NAD metabolites. Samples are separated on Vanquish UHPLC systems with reverse-phase C18 columns, providing <10-minute run times and high-resolution chromatographic performance.

Key features of our method:

• Instrumentation: Triple quadrupole MS + UHPLC
• Ionization: Positive ESI mode
• Quantification: Absolute, using isotope-labeled internal standards
• Typical Transitions: NAD⁺ (m/z 664 → 428), NADH (m/z 666 → 648)

SCIEX Triple Quad™ 6500+ (Figure from Sciex)

Waters Xevo TQ-s (Figure from Waters)

Vanquish UHPLC systems(Figure from Thermo)

Step-by-Step Workflow for NAD Analysis

1

Consultation & Experimental Design

We begin by understanding your research objectives and sample characteristics to design the most suitable analytical strategy.

• Platform Selection: Triple Quad or Orbitrap LC-MS/MS
• QC Planning: Calibration curves, isotope-labeled standards
• LOD/LOQ Definition: Based on biological range and project goals

2

Sample Preparation & Extraction

Extraction is performed using cold, acidified organic solvents to preserve NAD stability and prevent enzymatic degradation.

• Solvent: 80% methanol with 0.1% formic acid
• Temperature: Maintained at -20°C throughout
• Stabilization: Antioxidants and immediate quenching applied

3

LC-MS/MS Analysis

Targeted quantification is achieved through optimized chromatography and MRM for maximum specificity and sensitivity.

• Column: C18 RP (2.1 × 100 mm, 1.7 μm)
• Detection: Positive ESI, MRM transitions for NAD species
• Sensitivity: Down to 10 fmol per injection

4

Data Processing & Bioinformatics

Comprehensive analysis integrates raw data validation, normalization, and pathway interpretation.

• Software: Skyline for quantitative processing
• Normalization: Internal standard-based correction
• Advanced Insight: KEGG mapping, PCA, and statistical evaluation

5

Deliverables

We provide complete, publication-ready datasets with optional bioinformatics integration.

• Absolute Quantification: NAD⁺, NADH, NADP⁺, NADPH
• Redox Ratios: NAD⁺/NADH and NADP⁺/NADPH
• QC Report: Calibration data, chromatograms, precision metrics

How to Prepare Your Samples for NAD Quantification

Additional Guidelines

• Avoid preservatives and stabilizers that may interfere with LC-MS/MS analysis.
• Ship samples on dry ice; ensure leak-proof packaging.
• For custom matrices or limited samples, consult our technical team for tailored preparation instructions

Key Applications of NAD Analysis for Advanced Metabolism and Redox Research

Energy Metabolism and Mitochondrial Function

Monitor NAD/NADH ratios to evaluate oxidative phosphorylation, glycolysis, and TCA cycle activity. Essential for projects in metabolic disorders, exercise physiology, and bioenergetics.

Redox Biology and Oxidative Stress Studies

Quantify NADP⁺/NADPH to investigate antioxidant defense mechanisms and oxidative stress responses in cells, tissues, and disease models.

Aging and Longevity Research

Assess NAD decline and pathway changes in aging models, supporting interventions targeting sirtuins and PARP-mediated processes.

Drug Discovery and Therapeutic Development

Screen candidate molecules that influence NAD biosynthesis (e.g., NAMPT inhibitors) or salvage pathways. Applicable in metabolic disease and neurodegenerative disorder research.

Cancer Metabolism and Immunometabolism

Characterize altered NAD metabolism in tumors and immune cells to understand metabolic reprogramming and its impact on therapy resistance.

Systems Biology and Omics Integration

Combine NAD profiling with metabolomics, transcriptomics, and proteomics to construct comprehensive metabolic networks for pathway modeling.

Deliverables: What You Get from Our NAD Analysis Service

Our NAD analysis service provides comprehensive, publication-ready data packages to ensure your research outcomes are scientifically robust and ready for downstream interpretation.

Raw and Processed Data

• Original LC-MS/MS raw files (vendor format and mzML)
• Processed peak area tables with isotope-labeled internal standard correction

Quantitative Results

• Absolute concentrations of NAD⁺, NADH, NADP⁺, and NADPH in your samples
• Calculated redox ratios: NAD⁺/NADH and NADP⁺/NADPH
• Statistical outputs including mean, SD, and CV%

Quality Control Report

• Calibration curves with R² ≥ 0.995 for absolute quantification
• Validation of LOD/LOQ and recovery
• Technical reproducibility (CV ≤ 10%) and QC sample performance

Data Interpretation and Visualization

• Pathway mapping (KEGG-based) for NAD-related metabolism
• Optional PCA plots and heatmaps for group comparisons
• Custom statistical analysis upon request

All deliverables are provided in editable formats (Excel, PDF) and include both raw data and interpretative summaries.

Representative LC-MS/MS MRM chromatogram showing separation of NAD⁺, NADH, NADP⁺, and NADPH under optimized UHPLC conditions."

Calibration curve for NAD⁺ demonstrating excellent linearity (R² ≥ 0.995) across a wide concentration range.

Comparison of NAD⁺/NADH ratio between control and treated samples, reflecting changes in redox status.

Heatmap of NAD and related metabolites across different sample groups, highlighting metabolic differences.

Theacrine-Based Supplementation Improves Lipid Profiles and Maintains Cellular NAD+/NADH Status in Middle-Aged Adults

Background

Aging is strongly associated with dyslipidemia, reduced mitochondrial function, and declining nicotinamide adenine dinucleotide (NAD+) levels—key drivers of metabolic dysfunction and chronic inflammation. Researchers aimed to evaluate whether NAD3®, a theacrine-based supplement combining Wasabia japonica (isothiocyanates), theacrine, and cuprous niacin chelate, could improve serum lipid profiles and modulate NAD+ metabolism in peripheral blood mononuclear cells (PBMCs) over a 12-week intervention in middle-aged adults.

Challenge

The clinical team required:

• Accurate quantification of NAD+ metabolites in PBMCs to detect subtle cellular changes in redox balance.
• Reliable lipid panel analysis to measure total cholesterol, LDL, HDL, and triglycerides under fasting conditions.
• A robust method capable of detecting NAD+/NADH ratios with high sensitivity and reproducibility, despite limited PBMC sample yield.

Our Solution

Creative Proteomics provided targeted NAD+ metabolomics using LC-MS/MS (multiple reaction monitoring) with isotope-labeled internal standards for absolute quantification. Our approach included:

• Metabolite panel: NAD+, NADH, NAAD, and calculation of NAD+/NADH ratio.
• High-resolution chromatography: Waters Acquity UPLC with a reversed-phase C18 column.
• Precision mass spectrometry: Sciex QTRAP 6500 Plus, ensuring low detection limits (0.0025–40 nM range).
• Normalization using protein assays for PBMC lysates to enhance data comparability.
• Integration with serum lipid measurements for comprehensive interpretation.

Results & Findings

After 12 weeks:

• Serum Lipids:
• Total cholesterol ↓ 11.1%
• LDL cholesterol ↓ 15.2%
• LDL:HDL ratio ↓ 18.9% (All p < 0.05 vs. baseline)
• Cellular NAD+/NADH Ratio: Maintained in NAD3® group (+35%) versus decline in placebo group (−34%), p = 0.023 at Post.
• No significant changes in body weight, blood pressure, or clinical safety markers.

These data suggest NAD3® supplementation favorably impacts lipid metabolism and preserves cellular redox homeostasis, both critical hallmarks of healthy aging.

Targeted NAD+ metabolome data. Data are presented as means ± standard deviation values for 15 CTL and 13 NAD3 participants.

Reference

1. Roberts, Michael D., et al. "Enhance Trial: Effects of NAD3® on Hallmarks of Aging and Clinical Endpoints of Health in Middle Aged Adults: A Subset Analysis Focused on Blood Cell NAD+ Concentrations and Lipid Metabolism." Physiologia 2.1 (2022): 20–31.