Determination of Mono-, Di-, and Triphosphates and Citrate in Shrimp by Ion Chromatography

Importance of the Topic

Polyphosphates are routinely added to seafood to enhance water retention, texture, and appearance, but excessive or undeclared use may breach food safety regulations. Reliable quantification of mono-, di-, and triphosphates and citrate in shrimp is essential for quality control, regulatory compliance, and consumer safety.

Goals and Article Overview

This study develops and validates an ion chromatography (IC) method to measure orthophosphate, pyrophosphate, trimetaphosphate, triphosphate, and citrate in shrimp. The approach integrates reagent-free IC (RFIC) with an eluent generator and on-line sample clean-up to reduce manual preparation and improve reproducibility.

Methodology and Sample Preparation

Shrimp samples are homogenized, diluted with deionized water and 8 M KOH, sonicated, and filtered through a 0.45 µm syringe filter. On-line clean-up is performed using a Dionex InGuard HRP cartridge to trap hydrophobic matrix components before chromatographic separation. A gradient of potassium hydroxide eluent (30–50 mM) is generated in-line by the eluent generator, eliminating manual eluent preparation.

Used Instrumentation

• Thermo Scientific Dionex ICS-3000 or any RFIC system (DP dual pump, DC detector compartment, EG eluent generator, AS autosampler with cooling)
• Dionex InGuard HRP cartridge for on-line sample cleanup
• Dionex IonPac AS11 analytical column (4 × 250 mm) with AG11 guard (4 × 50 mm)
• Dionex IonPac UTAC-LP1 concentrator (4 × 35 mm) trap column
• Thermo Scientific Dionex EGC III KOH EluGen™ eluent generator with CR-ATC continuously regenerated trap column
• Thermo Scientific Dionex ASRS 300 suppressed conductivity detector (4 mm, external water mode, 130 mA)

Main Results and Discussion

The IC method achieves baseline separation of five analytes within 15 minutes. Calibration over 0.5–3.0 mg/L yielded correlation coefficients (r2) above 0.9989 for all species. Method precision on shrimp samples (n=5) showed RSD below 0.6%. Only orthophosphate was detected in an unspiked retail shrimp sample at 2.16 mg/L. Spike recovery studies (1 mg/L addition) demonstrated recoveries between 91.9% and 116% with RSD below 0.2%.

Long-term robustness was assessed by comparing retention times of the first and 300th injections with and without the InGuard HRP cartridge. Without the cartridge, retention times decreased significantly after 300 injections, indicating column fouling. In contrast, the on-line clean-up cartridge preserved column capacity and retention times, extending column lifetime.

Benefits and Practical Applications

• Automated on-line sample cleanup reduces manual handling and contamination risks.
• Reagent-free IC simplifies eluent management and improves run-to-run consistency.
• Fast analysis time (15 min) supports high sample throughput.
• High sensitivity and precision enable trace-level quantification for regulatory compliance in food quality laboratories.

Future Trends and Opportunities

Further developments may include expansion to larger polyphosphates, integration with mass spectrometric detection for structural elucidation, and on-line dilution or derivatization modules for complex matrices. Emerging column chemistries and suppressor technologies may further shorten analysis time and improve sensitivity.

Conclusion

The presented RFIC method with eluent generation and on-line InGuard HRP cleanup offers a robust, accurate, and efficient approach for quantifying small polyphosphates and citrate in shrimp. The method ensures regulatory compliance, high throughput, and extended column life.

References

1. Heitkemper, D. T.; Kaine, L. A.; Jackson, D. S.; Wolnik, K. A. Determination of Tripolyphosphate and Related Hydrolysis Products in Processed Shrimp. Proceedings of the 18th Annual Tropical and Subtropical Fisheries Technology Conference, 1993.
2. Thermo Scientific Application Note 71. Determination of Polyphosphates Using Ion Chromatography with Suppressed Conductivity Detection. LPN 034629-03, 2002.
3. Thermo Scientific Application Update 172. Determination of Polyphosphates Using Ion Chromatography. LPN 2496, 2010.
4. Dafflon, O.; Scheurer, L.; Gobet, H.; Bosset, J. O. Polyphosphate Determination in Seafood and Processed Cheese Using High-Performance Anion-Exchange Chromatography after Phosphatase Inhibition. Mitt. Lebensm. Hyg. 94, 127–135 (2003).
5. Kaufmann, A.; Maden, K.; Leisser, W.; Matera, M.; Gude, T. Analysis of Polyphosphates in Fish and Shrimp Tissues by Two Different Ion Chromatography Methods: Implications on False-Negative and -Positive Findings. Food Addit. Contam. 22, 1073–1082 (2005).