Redox Cofactor Analysis Service

Redox Cofactors — What They Are and Why They Matter

Redox cofactors are the primary electron carriers that set the pace of metabolism and stress response: NAD⁺/NADH, NADP⁺/NADPH, GSH/GSSG, and FAD/FMN. Their absolute concentrations, ratios, and redox potentials (Eₕ) determine whether pathways run catabolic or anabolic, whether oxidative defenses hold, and whether engineered fluxes deliver the expected titer, rate, and yield.

Because these species are labile and matrix-sensitive, apparent shifts can be driven as much by sample handling as biology. Robust decisions therefore require true in-matrix preservation, selective chromatographic separation, and isotope-dilution quantification—not proxy readouts or single-enzyme kits. Measuring redox cofactors directly gives an early, quantitative window into pathway bottlenecks, antioxidant capacity, and process robustness across cultures, tissues, and complex broths.

What Problems We Solve for Our Clients

Understanding and maintaining redox balance is a central challenge in modern life sciences. Researchers working with microbial systems, mammalian cells, plants, or fermentation processes often encounter:

• Incomplete insight into metabolic efficiency — Even when cultures grow well, subtle imbalances in NAD⁺/NADH or NADP⁺/NADPH can limit flux through critical pathways.
• Uncertainty in oxidative stress evaluation — Conventional assays often fail to capture the real intracellular status of glutathione and other redox buffers.
• Variability between experiments or scales — Redox cofactors are highly labile; without strict stabilization and quantitative control, comparisons across batches or bioreactors can be unreliable.
• Limited reliability of indirect assays — Proxy measurements cannot substitute for direct quantification of cofactors at fmol–pmol sensitivity.
• Lack of defensible, publication-quality evidence — Many studies require robust, transparent data packages that document calibration, recovery, and QC acceptance rules.

By focusing on the direct, quantitative measurement of NAD(H), NADP(H), glutathione, flavins, CoA thioesters, and related metabolites, Creative Proteomics helps clients overcome these barriers and obtain data they can trust for research, process development, and comparative studies.

What Our Redox Cofactor Analysis Service Provides

Detected Redox Cofactors & Related Analytes (Configurable Panel)

Why Choose Our Redox Cofactor Analysis: Key Advantages

• Isotope-dilution accuracy: ¹³C/¹⁵N internal standards with matrix-matched calibration; weighted 1/x or 1/x² regression; documented r² ≥ 0.99.
• Actionable precision: Typical intra-batch RSD ≤ 10–15%; between-batch comparability monitored by pooled-QC trendlines and bracketed checks.
• Sensitivity aligned to biology: Indicative on-column LLOQ — NAD(P) couples: low-fmol; GSH/GSSG: low-pmol; FAD/FMN: sub- to low-pmol (matrix dependent).
• Low artifact risk: NEM-trapped thiols, oxygen/light minimization, pH-controlled extraction; carryover ≤ 20% of LLOQ or re-acquisition.
• Transparent QC logic: You receive transitions, retention windows, qualifier/quantifier ion ratios, recoveries, and acceptance criteria.

What Methods Are Used for Redox Cofactor Analysis

Waters ACQUITY UPLC System (Figure from Waters)

SCIEX Triple Quad™ 6500+ (Figure from Sciex)

Agilent 1260 Infinity II HPLC (Figure from Agilent)

Agilent 6495C Triple quadrupole (Figure from Agilent)

How Our Redox Cofactor Assay Works — Step-by-Step Process

1

Study design alignment – Define panel, matrices, QC accept/reject rules, and randomization plan.

2

Stabilized sampling – Provide submission kit, NEM-preloaded tubes for thiol workflows, and matrix-specific quench instructions.

3

Extraction & cleanup – Cold organic quench, pH-controlled buffers, light/oxygen minimization, optional SPE for complex matrices.

4

UHPLC–MS/MS acquisition – Scheduled MRM, internal standards, bracketed verification, pooled QC interleaving.

5

Processing & QC review – Calibration fit, precision, recovery, carryover checks; flagged items re-processed or re-acquired per rules.

6

Reporting & interpretation – Concentrations, ratios, Eₕ calculations (Nernst), effect sizes, and method/QC appendix.

Sample Requirements for Redox Cofactor Assay

Deliverables: What You Get from Our Redox Cofactor Analysis

• Quant results: Absolute concentrations for each target; ratios (NAD⁺/NADH, NADP⁺/NADPH, GSH/GSSG).
• Calculated metrics: Eₕ values for glutathione and pyridine nucleotides with documented assumptions (n, E°′, pH).
• QC dossier: Calibration curves and statistics (r², back-calculated bias), LLOQ/ULOQ, spike-recovery, precision, carryover, and pooled-QC trendlines.
• Data package: Raw vendor files, processed peak tables, transition lists, and a methods appendix (column, mobile phases, MRM transitions, source settings).
• Interpretation brief: Concise commentary on significant shifts, effect sizes, and study-design-aware caveats.
• Consultation: Optional review session to align findings with your next experimental step.

Calibration curve for NAD⁺ showing linear regression with r² ≥ 0.99, confirming accurate quantitation across the validated range.

Representative MRM chromatogram displaying separation of internal standard, NAD⁺, and NADH with clear peak resolution.

Representative MS/MS spectrum highlighting characteristic fragment ions for cofactor identification and confirmation.

Comparative bar plot illustrating control versus treatment group differences in redox ratio/Eₕ with error bars.