Glutathione Analysis Service - Creative Proteomics

Q: Can GSH/GSSG analysis be integrated into multi-omics studies?

Yes. The LC–MS/MS glutathione workflow can be combined with metabolomics, proteomics, or transcriptomics datasets to provide a more holistic view of redox biology and cellular stress responses.

Q: How does glutathione profiling support bioprocess or fermentation monitoring?

Measuring GSH/GSSG and related thiols provides early indicators of stress in microbial or cell-based production systems. Tracking redox shifts helps optimize culture conditions, maximize yield, and assess product stability.

Q: Can glutathione data be normalized to other biomarkers?

Absolutely. Researchers often integrate glutathione ratios with NADPH/NADP⁺ balance, ROS markers, or antioxidant enzyme activity to build a more robust redox profile.

Q: How do you distinguish biological variability from technical variability in glutathione measurements?

Biological replicates, isotope-labeled internal standards, and repeated QC checks are used to separate true biological differences from analytical noise, enabling more reliable biological interpretation.

Q: Is it possible to track glutathione dynamics over time?

Yes. Time-course studies can be performed to monitor dynamic changes in GSH/GSSG under stress, treatment, or process conditions, helping identify critical inflection points in redox balance.

Q: How can glutathione analysis contribute to food and nutrition research?

Accurate quantification of glutathione and related thiols in food and plant matrices helps evaluate antioxidant capacity, nutritional value, and shelf-life stability under different storage or processing conditions.

Q: Can glutathione analysis inform quality control in industrial applications?

Yes. Monitoring glutathione status in biopharmaceutical production or food processing pipelines provides a robust quality marker for ensuring batch-to-batch consistency and detecting oxidative degradation.

Q: How do results from GSH/GSSG analysis guide decision-making?

Reliable glutathione profiling helps researchers decide whether interventions reduce oxidative stress, whether bioprocesses are under control, or whether quality standards are being met—turning raw data into actionable insights.

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GSH/GSSG — What They Are and Why They Matter

Glutathione is the primary intracellular antioxidant, existing as reduced glutathione (GSH) and its oxidized form (GSSG). The GSH/GSSG ratio is a widely accepted indicator of oxidative stress and cellular redox balance. Accurate measurement of this ratio is essential in oxidative stress studies, metabolic research, process monitoring, and quality assessment across biological and industrial applications.

The challenge lies in GSH's instability and rapid oxidation during sample handling, which can produce misleading results if not properly stabilized. Without artifact control, researchers risk underestimating GSH, overestimating GSSG, and misinterpreting redox status.

By combining on-collection stabilization with isotope-dilution LC-MS/MS, Creative Proteomics ensures artifact-free, reproducible quantitation of GSH, GSSG, and their ratio. This enables scientists to obtain true redox profiles, make data-driven decisions, and maintain consistency across studies and sites.

What Problems We Solve for Our Clients

Unreliable Redox Measurements

Researchers often find their GSH results inconsistent because ex vivo oxidation during handling converts GSH to GSSG, leading to false redox ratios and misleading interpretations.

Matrix-Specific Interference

Plasma, tissues, microbial broths, and plant extracts each introduce different interferences and ion suppression that make standard methods inaccurate.

Data Inconsistency Across Studies

Without standardized stabilization and QC, results from different labs, batches, or time points often cannot be directly compared, slowing decision-making and reproducibility.

Sample Volume Limitations

Many projects are constrained by scarce or precious material (e.g., small tissue biopsies, limited plasma volume, rare cell populations). Standard assays may require more than what is available.

Opaque or Incomplete Reporting

Some services provide only concentration numbers without QC recoveries, calibration validation, or raw data, leaving researchers unsure whether results are trustworthy.

Detected Glutathiones & Related Analytes (Configurable Panel)

Analyte	Abbrev.	Measured Form	Representative MRM (Quant/Qual, m/z)	Typical LLOQ (nM)	Example Linear Range (nM)	Typical Precision (CV%)	Notes
Reduced Glutathione	GSH	Native	308 → 179 / 76	5–10	10–10,000	≤12	Rapid stabilization required to avoid ex vivo oxidation
Oxidized Glutathione	GSSG	Native	613 → 355 / 484	2–5	5–5,000	≤12	Used with GSH to compute ratio
GSH–NEM Adduct	GSH-NEM	Derivatized	433 → 304 / 84	1–5	5–10,000	≤10	Preferred under anti-artifact strategy
Total Glutathione	tGSH	Post-reduction	See GSH channel	5–10	10–10,000	≤12	Chemical reduction prior to measurement
γ-Glutamylcysteine	γ-Glu-Cys	Native	249 → 130 / 84	5–20	20–5,000	≤15	GSH biosynthetic precursor
Cysteine / Cystine	Cys/CySS	Native/Deriv.	122 → 76 / 59 (Cys); 241 → 152 (CySS)	5–20	20–5,000	≤15	Enables Cys/CySS ratio
NADPH / NADP⁺	NADPH/NADP⁺	Native	744 → 159 / 744 → 321 (ex.)	20–50	50–10,000	≤15	Optional cofactor readouts
Representative GSH Conjugates	GS-R	Native	Method-specific	10–50	50–10,000	≤15	Electrophile detox research (panel configurable)

Why Choose Our Glutathione Analysis Service: Key Advantages

Artifact Control: Immediate stabilization reduces ex vivo oxidation artifacts to <5%, ensuring that the reported GSH/GSSG ratio reflects the true biological state.
Quantitative Accuracy: Isotope-dilution LC-MS/MS achieves 85–115% recovery with relative error ≤15% across multiple matrices.
High Sensitivity: nM-level LLOQ allows reliable measurement from minimal input (50–100 µL plasma or 20–50 mg tissue).
Precision & Robustness: Intra-/inter-batch CV ≤15%, with dual ion-ratio and retention-time checks to prevent false positives.
Transparency: Full QC package including calibration plots, QC recoveries, blanks, and raw LC-MS/MS files guarantees traceability and reproducibility.
Scalable and Customizable: Custom panels and high-throughput options available for large-scale or multi-site studies.

Key Instrument Platforms for Accurate GSH/GSSG Quantification

Chromatography (UHPLC)

Column: C18 for native GSH/GSSG; HILIC for NEM-derivatized samples.
Column Dimensions: 2.1 × 50–100 mm, sub-2 µm for high resolution.
Mobile Phase: Water with formic acid or ammonium formate, paired with acetonitrile.
Injection Volume: 1–5 µL
Temperature: Column: 30–40 °C, Autosampler: 4–8 °C for stability.

Mass Spectrometry (Triple Quadrupole MS)

Ionization: ESI+ for maximum sensitivity.
Acquisition Mode: MRM (Multiple Reaction Monitoring) for high specificity and precision.
Resolution: Unit mass with dwell times tuned for optimal peak detection.
Desolvation Temperature: 350–450 °C for efficient ionization

Stabilization & Internal Standards

Stabilization: NEM alkylation with acidified quenching immediately after collection.
Internal Standards: ¹³C/¹⁵N-GSH and ¹³C/¹⁵N-GSSG for absolute quantification and matrix correction.

Calibration & Accuracy

Calibration: 6–8 point external calibration for accurate results (1/x weighting).
Identity Confirmation: Retention-time and ion-ratio checks ensure reliable quantitation.

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 GSH/GSSG Assay Works — Step-by-Step Process

Sample Collection & Stabilization

Sample collected in NEM-preloaded tubes or suitable containers. Immediate stabilization with acidified organic solvent.
Storage: Dry ice or ≤-70°C to prevent oxidation.

Sample Preparation

Thiol Alkylation using NEM to stabilize GSH.
Protein precipitation and centrifugation to remove proteins.
Filtration of supernatant through a 0.2 µm filter.

Chromatography (UHPLC)

Injection of the sample into UHPLC system.
Separation of GSH, GSSG, and other analytes using optimized mobile phases.

Mass Spectrometry (MS/MS)

Detection using MRM mode on Triple Quadrupole MS.
Quantification with stable-isotope internal standards.

Data Analysis & Reporting

Calibration Curve: 6–8 point external calibration for precise quantification.
Final report includes quantitative results, QC data, and raw data for transparency and reproducibility.

Sample Requirements & Handling Guidelines for Glutathione (GSH/GSSG) Analysis

Sample Type	Required Volume	Collection Method	Handling Instructions
Plasma/Serum	≥50–100 µL per analysis	Use K2-EDTA or heparin anticoagulant.	Stabilize immediately with NEM-preloaded tubes, store at ≤-70°C.
Whole Blood	≥100 µL per analysis	Collect in NEM-preloaded tubes or stabilize with NEM.	Store at ≤-70°C immediately after collection.
Tissue Samples	≥20–50 mg per analysis	Snap-freeze or use dry ice during transport.	Store at ≤-70°C prior to analysis.
Cell Pellets	≥0.5–2.0 × 10^6 cells per analysis	Pellet cells, quench with NEM stabilizer, and freeze.	Freeze at ≤-70°C immediately.
Microbial Cultures/Fermentation Broth	≥0.5–1.0 mL per analysis	Quench immediately with NEM and freeze.	Store at ≤-70°C.
Plant/Food Samples	≥50–100 mg per analysis	Homogenize and snap-freeze immediately or use dry ice.	Store at ≤-70°C prior to analysis.

Deliverables: What You Receive from Our Glutathione (GSH/GSSG) Analysis

Clear Quantitative Data: Reliable concentration values for GSH, GSSG, total glutathione (tGSH), and the GSH/GSSG ratio, presented in easy-to-read tables.
Extended Results (Optional): Additional metabolites such as γ-glutamylcysteine, cysteine/cystine, NADPH/NADP⁺, and selected glutathione conjugates if requested.
Quality-Checked Results: Every dataset includes QC validation, calibration curves, and reproducibility checks, so you can trust the numbers.
Raw and Processed Data: Access to both chromatograms & MS raw files plus processed datasets, ensuring transparency and enabling re-analysis.
Comprehensive Final Report: A client-ready report with results summary, QC notes, and key figures, designed for straightforward interpretation and easy integration into your research.

Box plot of reduced (GSH) and oxidized glutathione (GSSG) concentrations in different sample groups.

Quantitative comparison of GSH and GSSG levels across groups, showing distribution and variation.

Standard calibration curve of GSH with regression line and R² for LC–MS/MS quantification.

Representative 8-point calibration curve for GSH quantification by LC–MS/MS, demonstrating linearity.

UHPLC chromatogram with separated GSH and GSSG peaks for glutathione analysis.

UHPLC chromatogram showing clear separation of reduced glutathione (GSH) and oxidized glutathione (GSSG).

Tandem mass spectrometry spectra of GSH and GSSG showing major fragment ions.

MS/MS spectra of GSH (precursor m/z 308) and GSSG (precursor m/z 613) with annotated fragment ions.

Can GSH/GSSG analysis be integrated into multi-omics studies?

Yes. The LC–MS/MS glutathione workflow can be combined with metabolomics, proteomics, or transcriptomics datasets to provide a more holistic view of redox biology and cellular stress responses.

How does glutathione profiling support bioprocess or fermentation monitoring?

Measuring GSH/GSSG and related thiols provides early indicators of stress in microbial or cell-based production systems. Tracking redox shifts helps optimize culture conditions, maximize yield, and assess product stability.

Can glutathione data be normalized to other biomarkers?

Absolutely. Researchers often integrate glutathione ratios with NADPH/NADP⁺ balance, ROS markers, or antioxidant enzyme activity to build a more robust redox profile.

How do you distinguish biological variability from technical variability in glutathione measurements?

Biological replicates, isotope-labeled internal standards, and repeated QC checks are used to separate true biological differences from analytical noise, enabling more reliable biological interpretation.

Is it possible to track glutathione dynamics over time?

Yes. Time-course studies can be performed to monitor dynamic changes in GSH/GSSG under stress, treatment, or process conditions, helping identify critical inflection points in redox balance.

How can glutathione analysis contribute to food and nutrition research?

Accurate quantification of glutathione and related thiols in food and plant matrices helps evaluate antioxidant capacity, nutritional value, and shelf-life stability under different storage or processing conditions.

Can glutathione analysis inform quality control in industrial applications?

Yes. Monitoring glutathione status in biopharmaceutical production or food processing pipelines provides a robust quality marker for ensuring batch-to-batch consistency and detecting oxidative degradation.

How do results from GSH/GSSG analysis guide decision-making?

Reliable glutathione profiling helps researchers decide whether interventions reduce oxidative stress, whether bioprocesses are under control, or whether quality standards are being met—turning raw data into actionable insights.

High Levels of Oxidative Stress Early after HSCT Are Associated with Later Adverse Outcomes

Cook, E., Langenberg, L., Luebbering, N., Ibrahimova, A., Sabulski, A., Lake, K. E., … Davies, S. M.

Journal: Transplantation and Cellular Therapy

Year: 2024

Glycine supplementation can partially restore oxidative stress-associated glutathione deficiency in ageing cats

Ruparell, A., Alexander, J. E., Eyre, R., Carvell-Miller, L., Leung, Y. B., Evans, S. J., … Watson, P.

Journal: British Journal of Nutrition