Maleic Acid Analysis Service

Overview of Maleic Acid

Maleic acid (cis-butenedioic acid) is a dicarboxylic acid widely present in industrial, agricultural, and biological systems. As a critical intermediate in metabolic pathways, maleic acid serves functions in the tricarboxylic acid (TCA) cycle, nitrogen metabolism, and environmental biodegradation processes. In the industrial realm, it is utilized as a precursor in resins, coatings, and plasticizers, while in food safety and pharmaceutical development, trace detection of maleic acid is critical due to its potential toxicity when consumed in unregulated amounts.

Why Analyze Maleic Acid?

Accurate analysis of maleic acid is crucial across multiple sectors:

• Toxicology & Food Safety: Unintentional maleic acid adulteration in starch-based food products poses health risks. High-precision quantification ensures regulatory compliance.
• Metabolomics Research: Maleic acid is a diagnostic metabolite for mitochondrial dysfunction and can indicate disruptions in the TCA cycle or amino acid metabolism.
• Environmental Monitoring: Tracks pollutant biodegradation and assesses ecotoxicological effects.
• Industrial Quality Control: Verifies chemical integrity in product formulation, particularly in polymers and pharmaceuticals.

Maleic Acid Analysis Service Offered by Creative Proteomics

• Targeted Quantitative Maleic Acid Detection: Accurate quantification of maleic acid in biological fluids (serum, plasma, urine), tissues, food matrices, environmental samples, and industrial materials.Absolute concentration calculation using stable isotope-labeled internal standards. Ideal for pharmacokinetic, toxicology, and quality control studies.
• Maleic Acid Isomer Differentiation and Profiling: Differentiation and quantification of cis- (maleic acid) and trans- (fumaric acid) isomers. High-resolution chromatography combined with mass spectrometry ensures clear isomer separation and reliable detection.
• Related Organic Acids Profiling: Simultaneous detection of structurally related and pathway-associated organic acids, including succinic acid, malic acid, oxalic acid, and glutaric acid. Supports deeper investigation into TCA cycle activity, mitochondrial function, and metabolic disorders.
• Metabolic Pathway Mapping: Integration of maleic acid data into known metabolic networks (e.g., TCA cycle, glyoxylate and dicarboxylate metabolism). Enables pathway-level interpretation and biomarker discovery.
• Food and Feed Additive Screening: Sensitive detection of maleic acid as an unauthorized additive in food starches, gelatin-based snacks, or animal feeds.
• Environmental Monitoring and Degradation Analysis: Quantification of maleic acid in water, soil, and industrial waste samples. Useful in studies of environmental impact, pollutant degradation, and eco-toxicological risk assessment.

Detected Maleic Acid and Related Analytes

Advantages of Maleic Acid Assay

• Ultra-Low Detection Limits: LOD as low as 0.1 ng/mL, LOQ typically 0.5–1.0 ng/mL, enabling trace detection in complex matrices.
• High Precision & Reproducibility: Intra-assay CV < 5.0%, inter-assay CV < 8.0% ensures stable, repeatable results for large-scale studies.
• Broad Linear Dynamic Range: Quantifiable from 0.5 ng/mL to 10,000 ng/mL, suitable for low-dose exposure and high-concentration samples.
• Superior Isomer Separation: Full resolution (Rs > 1.8) of maleic and fumaric acid isomers using high-performance HPLC-MS/MS.
• Multi-Matrix Compatibility: Validated in 15+ matrices including plasma, urine, plant extracts, food starch, soil, and wastewater.
• Rich Analytical Outputs: Delivers chromatograms, peak tables, calibration curves, and optional bioinformatics (PCA, pathway mapping).

Workflow for Maleic Acid Analysis Service

Technology Platform for Maleic Acid Analysis Service

Agilent 6495C Triple Quadrupole LC-MS/MS: Offers ultra-high sensitivity and precision for low-abundance maleic acid detection in complex biological matrices.

Agilent 7890B-5977A GC-MS: Ideal for volatile and semi-volatile compounds; widely used in environmental, food, and industrial sample analysis.

Agilent 1260 Infinity II HPLC: Provides excellent chromatographic resolution for separating maleic acid from structural isomers like fumaric acid.

Agilent 6495C Triple Quadrupole (Figure from Agilent)

Agilent 7890B-5977A (Figure from Agilent)

Agilent 1260 Infinity II HPLC (Fig from Agilent)

Sample Requirements for Maleic Acid Analysis Service

FAQ of Maleic Acid Analysis Service

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Maleic Acid Analysis Service Case Study

Title: Determination of Organic Acids in Honey by Liquid Chromatography with Tandem Mass Spectrometry

Journal: Springer Science+Business Media, LLC, part of Springer Nature

Published: 2020

• Background
• Methods
• Results
• Conclusion
• Reference

Organic acids in honey, though comprising less than 0.5% of its constituents, significantly influence honey's color, flavor, pH, and antimicrobial and antioxidant properties. They also help identify the botanical origin of honey. The primary organic acid in honey is gluconic acid, produced mainly by bee glucose oxidase activity on nectar glucose and certain bacteria. Existing methods for quantifying organic acids, such as enzymatic assays, capillary zone electrophoresis, and chromatography, face challenges like long analysis times, low sensitivity, or complex sample preparation. Liquid chromatography–tandem mass spectrometry (LC-MS/MS) offers improved sensitivity and selectivity but had not previously been applied to honey organic acid analysis. This study aims to develop a rapid, sensitive LC-MS/MS method for determining organic acids in honey and to evaluate matrix effects, detection limits, and repeatability for key acids like gluconic, tartaric, malic, citric, and succinic acids.

This study employed a validated LC-MS/MS method for the quantification of five organic acids (gluconic, tartaric, malic, citric, and succinic acids) in honey. Analytical standards were prepared using ultrapure water, and honey samples from various countries were diluted appropriately. Gluconic acid samples underwent pH adjustment and hydrolysis prior to analysis. All samples were filtered through 0.45-μm membranes.

Separation was performed on an InertSustain AQ-C18 column using a gradient of formic acid (0.1%) and acetonitrile at 0.4 mL/min. Detection was conducted using electrospray ionization in negative mode on a TSQ Quantum MS system. Specific m/z transitions were monitored for each acid. Method validation included linearity, matrix effects, LOD/LOQ, and reproducibility using solvent and matrix-matched calibration curves, with results evaluated according to EU Commission Decision 2002/657/EC.

Table 1 Retention times and LC-MS/MS conditions for the target organic acids

Ion chromatograms of standard reagents (10 mg/kg) of the five target organic acids

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Creative Proteomics Services

Creative Proteomics can support similar research by offering:

• Targeted Metabolomics Analysis via LC-MS/MS for organic acids
• Custom Method Development & Validation (LOD, LOQ, linearity, matrix effects)
• Sample Preparation Optimization including hydrolysis and pH adjustment
• Complex Matrix Analysis (e.g., honey, food, biological fluids)
• Quantitative Analysis Using Standard Addition Method
• Multi-day Reproducibility Studies

These services are ideal for clients requiring precise quantification of trace-level metabolites in complex samples.

• Calibration curves for all target organic acids showed excellent linearity (R² = 0.985–0.999).
• Significant matrix effects were observed; thus, a standard addition method was used.
• LODs ranged from 0.005 to 0.70 mg/kg, demonstrating higher sensitivity (8–31×) than HPLC and enzymatic methods.
• Intra-day and inter-day RSDs (2.2–14.5%) met the acceptable limit (<15%), confirming method reliability.
• Among 25 honey samples, gluconic acid was the most abundant (2995.6 ± 1602.3 mg/kg; 64.6–99.8% of total acids), followed by malic and citric acids.
• Tartaric acid was least detected, present in only 5 samples.
• Significant correlations were found: citric-succinic acid (R = 0.91), malic-succinic (R = 0.79), and gluconic-citric (R = 0.83), suggesting biochemical linkage via bee enzyme activity and the TCA cycle.
• PCA explained 93.0% of variation; high organic acid levels co-occurred, and manuka honey showed distinct citric acid levels.

The LC-MS/MS method developed is sensitive, selective, and reproducible for organic acid quantification in honey. Gluconic acid dominated the profiles, and the observed acid correlations support their biosynthesis via bee enzymes and TCA cycle. This method holds potential for applications in food authenticity, metabolic studies, and contaminant evaluation.

Reference

1. Suto, Momoka, Hiroto Kawashima, and Yasunori Nakamura. "Determination of organic acids in honey by liquid chromatography with tandem mass spectrometry." Food Analytical Methods 13.12 (2020): 2249-2257. https://doi.org/10.1007/s12161-020-01845-w