Elsevier

Journal of Chromatography A

Volume 1428, 8 January 2016, Pages 346-351
Journal of Chromatography A

Direct electrokinetic injection of inorganic cations from whole fruits and vegetables for capillary electrophoresis analysis

https://doi.org/10.1016/j.chroma.2015.08.012Get rights and content

Highlights

  • A direct injection method was developed to introduce sample in the capillary electrokinetically.

  • A high viscosity polymer was added to BGE to obtain precise and repeatable injections.

  • Quantitative analysis of inorganic cations in fruits and vegetables was achieved successfully.

  • The results obtained by CE were in close agreement (r2 = 0.98) with the traditional ICP-MS analysis.

  • The developed method was used to determine inorganic cations across different varieties of apples.

Abstract

A novel approach for the direct injection from plant tissues without any sample pre-treatment has been developed by simply placing a small piece of the tissue into a capillary electrophoresis vial followed by application of a voltage for electrokinetic injection. Separations of sodium, potassium, calcium and magnesium were achieved using a BGE comprising 10 mM imidazole and 2.5 mM 18-crown-6-ether at pH 4.5. The addition of 2% (m/v) hydroxypropylmethyl cellulose to the separation buffer allowed for precise and accurate electrokinetic injection of ions from the plant material by halting the movement of tissue fluid into the capillary. This method provides both qualitative and quantitative data of inorganic cations, with quantitation in zucchini, mushroom and apple samples in agreement with Sector Field Inductively Coupled Plasma Mass Spectrometric analysis (r2 = 0.98, n = 9). This method provides a new way for rapid, quantitative analysis by eliminating sample preparation procedures, and has great potential for a range of applications in plant science and food chemistry.

Introduction

Minerals and vitamins are essential nutrients required for a healthy functioning body [1], [2] with vegetables and fruits being an important source [3]. The relative abundance of minerals in different foods vary significantly [4] and is of interest to a health conscious public [5], [6]. Furthermore, seasonal variations [7], [8] and growing [9] and storage conditions can impact the nutrient levels and hence quality of a food [10]. Therefore, efficient methods for the analysis of minerals in food are necessary.

Determination of inorganic mineral cations, such as Ca2+, Na+, Mg2+ and K+ in fruits and vegetables is typically achieved by atomic spectroscopic methods [7] including inductively coupled plasma-mass spectrometry (ICP-MS) [11], but alternatives such as ion chromatography [12] and capillary electrophoresis (CE) [13] have also been reported. In all cases sample preparation is required and typically involves drying and pulverizing the sample followed by acid digestion and dilution [2], [3], [4], [14], [15]. Fukushi et al. reported an electrophoresis method for free calcium in vegetable that was slightly simpler, but still required boiling pulverized vegetable for 15–20 min, cooling, filtering and making to volume prior to analysis [16]. Sample preparation is not only time consuming and labor intensive but also provides opportunity for sample contamination and analyte loss. A simpler method for direct analysis of plant tissue is highly desirable.

Methods for direct analysis of tissues of biological or clinical interest have emerged over the last decade [17], [18], [19], [20]. For example, the direct determination of drugs in tissue samples have been achieved using mass spectrometry (MS) in combination with matrix-assisted laser desorption/ionization (MALDI)-MS [14]. MS methods are typically limited to providing qualitative information of the analytes. Quantitative information in direct analysis of a bulk sample was obtained by MS in combination with internal extractive electrospray ionization. The capillary tip was placed inside the sample and a solvent was introduced into the sample matrix to extract the analytes at high voltage (±4.5 kV) for direct injection into the MS [20]. The signal intensities were highly dependent on the position of the ESI capillary in the sample with slight changes in capillary position resulting in differences in the injected sample volume, compromising repeatability. The approach also required samples to be precisely cut to ensure a uniform size and shape to achieve reproducible results, which combined with the solvent required for the extraction of analytes from the sample matrix, complicates the method.

Analytical separation techniques offer the possibility of separating target analytes based on their physicochemical properties, avoiding the reliance on the resolving power of the mass spectrometer. CE is known for its ability to perform rapid separations with very small sample volumes, and there are two reports in which analytes have been directly injected from tissue samples. For example, Oguri et al. reported the direct sampling from rat's brain using CE in combination with laser induced fluorescence (LIF) for the analysis of taurine [21]. Electrokinetic injection was performed by piercing a rat's brain with the capillary and allowed for the determination of both intra- and extra-cellular taurine, an advantage in comparison with microdialysis only extra-cellular taurine can be sampled. However, this approach only provided qualitative information, as it was not possible to control the amount of sample injected. Also, sampling was achieved only from the surface to minimize the accidental release of taurine from damaged tissues. The use of a tapered capillary was suggested as a way to minimize damage and for sampling deep inside the brain. This approach was subsequently employed by Wang et al. who etched the capillary to a sharp point using HF and used this to detect the anticancer drug doxorubicin in human liver tissue [22]. For direct sampling from thin slices of liver tissues, a negative pressure of −7.6 kPa for 2s was applied. However, etching is a hazardous process and the resulting fragile capillary is likely to break when directly sampling from more solid samples such as many plant tissues. This method also required tissues to be cut into very thin slices (5 μm) to prevent large injections, thus making it technically demanding and unsuitable for analysis of intact plant tissues. In addition to this, electrokinetic injection of intracellular content of single cells using CE in combination with laser induced fluorescence (LIF) has also been demonstrated [23], [24] illustrating the potential of CE to provide information on biological systems.

In this paper, our aim was to develop a simple and robust method for the direct injection of ions from plant tissue, improving analytical simplicity by eliminating the requirement for sample treatment and hence reducing the risk of contamination. Direct analysis only requiring tissue to be cut and placed in a CE vial, when implemented in a more portable platform and extended to other analytes, may form the basis for rapid on-site analysis of food products to inform agricultural production and nutrition as well as food safety.

Section snippets

Chemicals

Imidazole, 18-crown-6 ether, sodium chloride, potassium chloride, hydoxypropyl methyl cellulose (HPMC) (viscosity 3500–5600 cP, 2% in H2O, 20 °C), sodium hydroxide, acetic acid and nitric acid were all purchased from Sigma Aldrich (Sydney, Australia). Calcium chloride dihydrate was from Univar (New South Wales, Australia). Magnesium chloride hexahydrate was from BDH Laboratory Supplies (Poole, England).

Instrumentation

A Hewlett Packard 3D CE (Waldbron, Germany) instrument equipped with a diode array UV

Results and discussion

Sample preparation is often a complex, time consuming, labor intensive and hence expensive step which can be avoided in CE by injecting directly from samples, provided this is practically feasible and can be done in a controlled manner. To evaluate the feasibility of directly injecting from fruit and vegetables for CE analysis, a piece of zucchini was cut into a 5 mm3 piece, placed directly in a 1.5 mL CE vial and positioned in the instrument (Fig. 1). Electrokinetic injection was performed by

Conclusion

A novel, fast and inexpensive method for the determination of cations from the direct injection of fruits and vegetables into a capillary electrophoresis system is demonstrated. The approach has broad applicability to a range of fruits and vegetables, and comparison of the concentration of three cations (K+, Ca2+, Mg2+) in three different matrices (apple, mushroom, zucchini) with quantitative data found to correlate well with ICP-MS. Differences between sample matrix mean that a matched

Acknowledgements

M.C.B would like to acknowledge the ARC for funding through a QEII Fellowship (DP0984745) and Future Fellowship (FT130100101). U.K would like to acknowledge Edith Cowan University for the mobility grant and providing the opportunity to travel to Tasmania and conduct research at the University of Tasmania. Access to ICP-MS instrumentation was supported through ARC LIEF LE0989539.

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