Determination of Phytohormones in Plant Extracts Using In-matrix Ethyl Chloroformate Derivatization and DLLME–GC–MS - - Chromatography Online
Determination of Phytohormones in Plant Extracts Using In-matrix Ethyl Chloroformate Derivatization and DLLME–GC–MS


LCGC Europe
Volume 26, Issue 6, pp. 310-325

A fast and simple sample preparation method using in-matrix derivatization and dispersive liquid–liquid microextraction (DLLME) for the simultaneous determination of 11 phytohormones in plants by gas chromatography–mass spectrometry (GC–MS) was developed. In this derivatization–extraction procedure, phytohormones in aqueous samples were derivatized with ethyl chloroformate (ECF) and extracted by DLLME simultaneously using ethanol–pyridine (4:1, v:v), both as derivatization catalyst and DLLME dispersant. This proposed rapid and convenient method was also successfully applied for analysing the phytohormones in rice seed callus and cucumber fuits, indicating other wide applications in other plant tissues.



The first phytohormone, auxin, was discovered in 1926, and since then an increasing number of naturally-occurring or synthetic molecules with plant-growth regulation activities have been reported, including abscisic acid (ABA); salicylic acid (SA); gibberellic acid (GA).phenylacetic acid (PAA); 1-naphthylacetic acid (NAA); and 2,4-dichlorophenoxyacetic acid (2,4-D). Numerous aspects of physiological processes in plants — such as seed germination, shoot elongation, and organogenesis — are manipulated delicately by corresponding phytohormones (1,2). Some phytohormones are also involved in the adaptive behaviour of plants in response to environmental and biological stresses (3–6). Many studies have shown evidence that synergistic, as well as antagonistic, actions occur between different phytohormones in plants (7–9). In addition, signalling crosstalks between several phytohormones in regulating plant development are reported instead of their individual effect (10,11). It is therefore necessary to develop reliable methods for the simultaneous monitoring of different phytohormones during physiological processes (12,13).

Many analytical procedures have been developed to determine the importance of phytohormones in plants simultaneously (12–15), however, it is still an analytical challenge. This is because there are low concentrations of phytohormones in plants and the sample matrix is complex. Most analytical methods to determine phytohormones rely heavily on high performance liquid chromatography (HPLC) (14,15); gas chromatography (GC) (16,17); and capillary electrophoresis (CE) (18) for separation. HPLC with tandem mass spectrometry (HPLC–MS–MS) is suitable for phytohormone analysis (14, 19), but requires expensive equipment and is generally expensive. Gas chromatography coupled with mass spectrometry (GC–MS) is preferred because of its cost-effectiveness and improved separation, however, it always requires a derivatization step to improve the volatility and sensitivity of some phytohormones (17, 20).

Despite great advances in instrumentation, most analytical instruments cannot handle sample matrices directly. Sample preparation steps are commonly introduced to transfer analytes into a form that is pre-purified, concentrated, and compatible. Solid-phase extraction (SPE), combining integrated purification and concentration, is most commonly used as the sample pretreatment technique for phytohormones determination (16,19).

However, SPE is laborious, time-consuming, and requires a larger volume of sample (scarce in most plant physiological research projects) because of its low enrichment factor. These problems can be resolved using solid-phase microextraction (SPME) (15) and liquid-phase microextraction (LPME) (13). However, the high cost of SPME fibre and the operational difficulties of LPME mean that they are not widely adopted by other researchers. In addition, SPME and LPME require special conditions and long extraction times for equilibrium — during which the degradation of several labile phytohormones may potentially occur (15).

In recent years, a rapid and simple method termed dispersive liquid–liquid microextraction (DLLME) has been developed by Assadi and co-workers (21). DLLME has now been introduced in the extraction of polybrominated diphenyl ethers (PBDEs), organophosphorus pesticides (OPPs), and other organic pollutants from aqueous samples. It has a high extraction efficiency, as well as being convenient and inexpensive (22,23). With several years of development, in situ derivatization combined with DLLME has also been applied to GC (GC–MS) analysis of polar compounds such as fatty acids, chlorophenols, and anilines (24–26). Compared with post-derivatization that requires special conditions that will introduce extra steps in the sample preparation procedure, DLLME with in situ derivatization is both cost-effective and convenient. In addition, in situ derivatization can reduce the hydrophilicity of polar analytes and thus can enhance extraction efficiency.

Among the derivatizing reagents studied, alkyl chloroformate (ACF) was superior for derivatization of amines, fatty acids, phenoic acids, and amino acids in bio-fluid matrix while leaving sugars and other related compounds unaffected (27). ACF also exhibited superiority when used as an in situ derivatization reagent for DLLME. The organic catalyst (namely alcohol, pyridine, acetonitrile, and other water-miscible solvents) can spontaneously act as dispersant. Simultaneous derivatization and DLLME using ethyl chloroformate (ECF) as the derivatizing agent were first reported by Pusvaskiene for the analysis of fatty acids (24). In a previous study, simultaneous derivatization and DLLME using methyl chloroformate (MCF) as the derivatization reagent was successfully applied for the GC–MS analysis of alkylphenols (APs) in river water samples (28). These two successful applications implied that ACF in combination with DLLME could provide an efficient method for derivatization and extraction of phytohormones that contained carboxylic and hydroxyl groups from plant tissue extracts for GC–MS analysis.

In this study, an in-matrix ECF derivatization and DLLME for the determination of 11 phytohormones in plant tissue extracts is reported for the first time. This study emphasizes the use of an organic catalyst [in this instance ethanol and pyridine at the ratio of 4:1 (v:v)] as the dispersant. Some key parameters — including the amount of catalyst–dispersant, ECF and extraction solvent, pH, and ionic strength — that might affect both derivatization and DLLME were thoroughly investigated and optimized. The established method was validated for an analysis of cucumber fruit extract and was applied to the monitoring of phytohormones in rice seed callus.


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