Speciation of zinc in microliter volumes of plant sap by capillary HPLC-ICP-MS

Significance of the Topic

Zinc is an indispensable micronutrient in plants, acting as a structural and functional cofactor in over 300 enzymes and regulating key processes such as gene expression, protein stabilization, photosynthesis and CO₂ fixation. Both zinc deficiency and excess result in impaired growth, chlorosis, necrosis and reduced crop yields, posing significant challenges for food security. Detailed speciation of zinc in plant fluids can illuminate nutrient transport mechanisms, improve agronomic practices and enhance our understanding of plant physiology.

Objectives and Overview of the Study

This study aimed to develop and apply a capillary HPLC–ICP-MS method with online preconcentration to separate, detect and identify low-molecular-weight zinc complexes in microliter volumes of pea (Pisum sativum) post-phloem sap. By combining sensitive elemental detection with high-resolution ESI-MS/MS, the work sought to overcome challenges related to the low abundance and instability of metal complexes in biological matrices.

Methodology and Instrumentation

• Sample Collection and Preparation: Liquid endosperm was extracted from developing pea pods via a glass capillary and peristaltic pump, immediately frozen in liquid nitrogen, stored at –20 °C, thawed and diluted 1:2 with acetonitrile, then centrifuged to obtain a clear supernatant.
• Chromatographic Separation: An Agilent 1100 capillary HPLC with a 100 µL loading loop and a SeQuant ZIC-HILIC guard column performed online preconcentration. After a wash step (90% acetonitrile/10 mM ammonium formate, pH 5.5), analytes were back-flushed onto a ZIC-HILIC capillary column (150 mm × 0.3 mm, 3.5 µm) using a gradient from 90% to 35% acetonitrile at 4 µL/min.
• ICP-MS Detection: An Agilent 7700x equipped with the capillary LC interface (G3680A) and ORS3 collision cell in helium mode monitored ⁶⁴Zn and ⁶⁶Zn isotopes (dwell time 60 ms). The system achieved a 75 ng/L detection limit for ⁶⁴Zn (~6 fmol Zn–nicotianamine) and 70–80% column recovery.
• ESI-MS/MS Identification: An LTQ Orbitrap Velos operated in positive mode (3.0 kV, vaporizer 120 °C, capillary 280 °C, resolution 100 000 at m/z 400) provided accurate mass and isotopic pattern confirmation of zinc complexes.

Key Results and Discussion

Two distinct zinc species were resolved and identified in pea post-phloem sap: zinc–nicotianamine (m/z 366.06389) and zinc-bis-histidine (m/z 373.05957). Retention times in capillary ZIC-HILIC ICP-MS matched the extracted ion chromatograms in ESI-MS, confirming coelution and identity. Mass accuracy deviations were within ±0.5 ppm. The online preconcentration achieved sharp peaks and high sensitivity despite sample volumes below 100 µL.

Benefits and Practical Applications

• The capillary HPLC–ICP-MS approach enables trace-level speciation in limited-volume plant fluids without extensive offline concentration steps.
• Helps characterize dynamic metal ligand pools in vivo, supporting research in plant nutrition, environmental monitoring and biofortification strategies.

Future Trends and Potential Applications

Advances in microflow chromatography and high-resolution mass spectrometry will further enhance sensitivity and speciation capabilities in plant and environmental matrices. Integration with imaging and isotopic labeling could map zinc distribution at the cellular level. The methodology can be extended to other essential or toxic metals, enabling comprehensive metallomic profiling under diverse growth and stress conditions.

Conclusion

This work demonstrates a robust capillary HPLC–ICP-MS method with online preconcentration and ESI-MS/MS identification for zinc speciation in microliter volumes of plant sap. Two key zinc complexes, nicotianamine and bis-histidine, were confidently identified, showcasing the approach’s utility for studying metal biochemistry in scarce biological samples.

Reference

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