Utilizando electroforesis capilar para cuantificar ácidos orgánicos a partir de Tejidos Vegetales: Un caso de prueba del examen<em> Coffea arabica</em> Semillas
Summary
Este artículo presenta un método para la detección y cuantificación de ácidos orgánicos a partir de material vegetal utilizando electroforesis capilar zonal libre. Un ejemplo de la aplicación potencial de este método, la determinación de los efectos de una fermentación secundaria en los niveles de ácido orgánicos en semillas de café, se proporciona.
Abstract
Los ácidos carboxílicos son los ácidos orgánicos que contienen uno o más terminales carboxilo (COOH) grupos funcionales. ácidos carboxílicos de cadena corta (SCCAs; ácidos carboxílicos que contienen de tres a seis átomos de carbono), tales como malato y citrato, son fundamentales para el buen funcionamiento de muchos sistemas biológicos, donde funcionan en la respiración celular y pueden servir como indicadores de la salud de las células. En los alimentos, el contenido de ácido orgánico puede tener un impacto significativo en el sabor, con un aumento de los niveles de SCCA que resulta en un sabor "ácido" o amargo. Debido a esto, los métodos para el análisis rápido de los niveles de ácido orgánicos son de particular interés para las industrias de alimentos y bebidas. Desafortunadamente, sin embargo, la mayoría de los métodos utilizados para la cuantificación SCCA dependen de protocolos consumen mucho tiempo que requieren la derivatización de las muestras con reactivos peligrosos, seguido de cromatografía costoso y / o espectrometría de masas. Este método detalla un método alternativo para la detección y cuantificación de orgAnic ácidos a partir de material vegetal y las muestras de alimentos utilizando electroforesis capilar de zona libre (CZE), a veces denominado simplemente electroforesis capilar (CE). CZE proporciona un método rentable para medir SCCAs con un bajo límite de detección (0,005 mg / ml). En este artículo se detalla la extracción y cuantificación de SCCAs a partir de muestras de plantas. Mientras que el método proporcionado se centra en la medición de SCCAs de granos de café, el método proporcionado se puede aplicar a múltiples materiales de alimentos de origen vegetal.
Introduction
Carboxylic acids are organic compounds containing one or more terminal carboxyl functional groups, each attached to an R-group containing one or more carbons (R-C[O]OH). Short chain, low molecular weight carboxylic acids (short chain carboxylic acids, SCCAs) containing between one and six carbons, are essential components of cellular respiration, and function in several biochemical pathways necessary for cell growth and development. SCCAs play critical roles in cellular metabolism1, cell signaling2, and organismal responses to the environment (such as antibiosis3). Because of this, SCCAs can serve as useful indicators of disruptions to cellular metabolism, plant stress responses4,5, and fruit quality6,7. To date, SCCAs have been quantified primarily through chromatographic techniques such as high performance liquid chromatography (HPLC) or gas chromatography-mass spectroscopy (GC-MS). While these methods, are capable of achieving very low limits of detection, they can be expensive, require the derivatization of target SCCAs using caustic and/or toxic reagents, and include lengthy separation runs on the GC or HPLC. Because of this, interest in the use of free zonal capillary electrophoresis (CZE), which does not require sample derivatization, to quantify organic acids has steadily increased8.
Free zonal capillary electrophoresis (CZE) is a chromatographic separation methodology that, due to its high number of theoretical plates, speed, and relative ease-of-use, is increasingly replacing both GC-MS and high-pressure liquid chromatography as an analytical method for the quantification (particularly for quality control purposes) of anions, cations, amino acids, carbohydrates, and short chain carboxylic acids (SCCAs)8,9,10. CZE-based separation of small molecules, including SCCAs, is based two primary principles: the electrophoretic movement of charged ions in an electrical field established across the buffer filling the capillary; and the electro-osmotic movement of the entire buffer system from one end of the capillary to the other, generally towards the negative electrode. In this system, small molecules move towards the negative electrode at varying speeds, with the speed of each molecule determined by the ratio of the net charge of the molecule to the molecular mass. As the movement of each individual molecule in this system is dependent on the charge state of the molecule and the overall rate of electro-osmotic flow (which is itself based on the ion content of the buffer used to fill the capillary), the buffer pH and ionic composition heavily impact the degree to which molecules can be efficiently separated using CZE. Because of this, SCCAs, with their relatively high charge-to-mass ratios, are ideal targets for CZE-based separation. Metabolites separated using CZE can be detected using a variety of methods, including UV absorbance, spectral absorbance (which is generally performed using a photo-diode array [PDA]), and/or mass spectroscopy (CE-MS or CE-MS/MS)8. The diversity of separation and detection methods provided by CZE makes it an extremely flexible and adaptable technique. Because of this, CZE has been increasingly applied as a standard method of analysis in the areas of food safety and quality11,12, pharmaceutical research13, and environmental monitoring13,14.
Capillary electrophoresis has been used to detect and quantify short chain carboxylic acids for nearly two decades13. The resolving power (particularly for small, charged molecules), short run time, and low per sample cost of CZE analyses make CZE an ideal technique for the separation and quantification of SCCAs13. This method presents a protocol to utilize CZE to measure the concentration of organic acids from plant tissues. Example data was generated through the successful implementation of this protocol to measure the change in organic acid levels in coffee seeds following a secondary fermentation treatment. The protocol details the critical steps and common errors of CZE-based separation of SCCAs, and discusses the means by which this protocol can be successfully applied to quantify SCCAs in additional plant tissues.
Protocol
Representative Results
Discussion
Al igual que con cualquier técnica analítica, hay varios factores críticos que pueden afectar significativamente la calidad y la fiabilidad de los datos generados. En primer lugar, es importante procesar las muestras de manera eficiente, con un mínimo de ciclos de congelación / descongelación. Congelación y descongelación repetida pueden comprometer la composición química de la muestra antes de su procesamiento o análisis. En segundo lugar, es fundamental para aplicar las medidas de este protocolo para todas …
Disclosures
The authors have nothing to disclose.
Acknowledgements
The authors would like to acknowledge the financial support of this project by The J.M. Smucker company.
Materials
| Ceramic Moarter and Pestle | Coorstek | 60310 | |
| Beckman Coulter P/ACE MDQ CE system | Beckman Coulter | Various | |
| Glass sample vials | Fisher Inc. | 033917D | |
| 1.5 ml microcentrifuge tubes | Fisher Inc. | 02-681-5 | |
| LC/MS grade water | Fisher Inc. | W6-1 | Milli-Q water (18.2 MΩ.cm) is also acceptable |
| 15 ml glass tube/ Teflon lined cap | Fisher Inc. | 14-93331A | |
| Parafilm M | Fisher Inc. | 13-374-12 | |
| CElixirOA detection Kit pH 5.4 | MicroSolv | 06100-5.4 | |
| BD Safety-Lok syringes | Fisher Inc. | 14-829-32 | |
| 17 mm Target Syringe filter, PTFE | Fisher Inc. | 3377154 | |
| 32 Karat, V. 8.0 control software | Beckman Coulter | 285512 | |
| capillary electrophoresis (CE) sample vials | Beckman Coulter | 144980 | |
| caps for CE vials | Beckman Coulter | 144648 | |
| Liquid Nitrogen | N/A | N/A | Liquid nitrogen is available from most facilities services |
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