Determination of Biogenic Amines in Fruit, Vegetables, and Chocolate Using Ion Chromatography with Suppressed Conductivity and Integrated Pulsed Amperometric Detections

Significance of the Topic

Biogenic amines are nitrogenous compounds that occur naturally in foods and play vital roles in human and animal physiology. However, elevated levels in food products can indicate spoilage and pose health risks such as hypotension, migraines, and allergic reactions. Accurate measurement of these amines in fruits, vegetables, and chocolate is essential for food safety, quality control, and regulatory compliance.

Objectives and Study Overview

This work extends a previously reported ion chromatography method to quantify ten underivatized biogenic amines in kiwi fruit, spinach, and various chocolate formulations. The aim was to evaluate an electrolytically generated methanesulfonic acid (MSA) gradient on a weak‐acid cation‐exchange column (IonPac CS18) with multiple detection modes—suppressed conductivity, integrated pulsed amperometric detection (IPAD), and UV—to achieve robust separation and sensitive quantitation without chemical derivatization.

Methodology and Instrumentation

Instrumentation comprised a Dionex ICS‐3000 system with dual pumps, EG Eluent Generator II for online MSA production, an IonPac CS18 analytical column (2×250 mm) with CG18 guard, a conductivity module with CSRS ULTRA II suppressor, an Au‐electrode IPAD cell, and a UV‐Vis detector at 276 nm. Eluent gradients ranged from 3 to 45 mM MSA at 0.30 mL/min and 40 °C. Standard solutions of ten amines were prepared in 3 mM MSA; stock concentrations were 1000 mg/L. Sample preparation involved acid extraction with 100 mM MSA or trichloroacetic acid, vortex homogenization, centrifugation, filtration, and dilution prior to injection.

Main Results and Discussion

Calibration curves (r2 >0.997) demonstrated linear ranges up to 20 mg/L and low µg/L detection limits. Suppressed conductivity provided the lowest LODs for charged amines, while IPAD enabled detection of neutral and aromatic amines; UV confirmed tyramine. In 70% cocoa chocolate, putrescine (6.9 mg/kg), histamine (3.3 mg/kg), serotonin (7.3 mg/kg), spermidine (9.8 mg/kg), and spermine (9.8 mg/kg) were quantified. Dark chocolate showed trace levels; milk chocolate was below detection. Spinach leaves contained up to 61 mg/kg cadaverine and 48.5 mg/kg spermidine; kiwi fruit yielded serotonin at 9.2 mg/kg and other amines at lower concentrations. Storage at 4 °C for 2–3 weeks led to significant degradation of histamine and spermidine in spinach and reduced putrescine in kiwi, highlighting amine instability under refrigeration.

Benefits and Practical Applications

The described method avoids time‐consuming derivatization, uses mild eluent conditions, and leverages online eluent generation for reproducibility. Multiple detection modes increase confidence in identification and quantification across diverse food matrices. This approach supports routine food safety testing, quality assurance, and shelf‐life studies in academic, industrial, and regulatory laboratories.

Future Trends and Potential Applications

Advances may include coupling with mass spectrometry for structural confirmation, miniaturized or portable IC systems for on‐site screening, automated sample preparation, and expanded application to fermented products and beverages. Emerging stationary phases and detector technologies will further lower detection limits and enhance throughput in food analysis.

Conclusion

An IonPac CS18 column with an electrolytically generated MSA gradient, combined with suppressed conductivity, IPAD, and UV detection, provides a robust, sensitive, and derivatization‐free method for comprehensive analysis of biogenic amines in fruits, vegetables, and chocolate. The protocol delivers reliable quantitation, facilitates shelf‐life monitoring, and can be integrated into routine food safety workflows.

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