Fruity phytohormones: Labelled for HPLC with fluorescence detection
- Published: Jan 6, 2015
- Author: Steve Down
- Channels: HPLC
Plants produce a wide range of hormones that are important for their development and stress responses but they are often supplemented by agricultural phytohormones that are applied by growers to speed up the growth process, aid plant propagation from cuttings, or provide specific protection against particular pests.
However, their propensity to leak into soil and water courses, as well as loiter in the harvested fruits, has driven the regulatory authorities to set maximum residue limits for them. The reasons for this cautionary approach are clear: many phytohormones can have strong effects on animals and humans, such as impaired reproduction and development, carcinogenicity and neurotoxicity.
A range of methods have been developed by different research groups for the analysis of multiple phytohormones, many of them based on GC, LC or CE coupled to ultraviolet of fluorescence detectors or mass spectrometry. The diverse chemical structures can cause difficulties but LC/MS has proven successful for the analysis of trace amounts of phytohormones. Nevertheless, its widespread adoption has been restricted by the need for expensive instrumentation and isotopic internal standards.
An alternative method has been proposed by Chinese scientists who stayed with HPLC but turned to fluorescence detection. Their procedure is suitable for phytohormones containing a carboxylic acid group, of which there are many, and they illustrated it with a group of eight common hormones that are found in fruits.
The new method was described in Journal of Separation Science by Guoliang Li and colleagues from Qufu Normal University, Guangdong Ocean University, the Northwest Institute of Plateau Biology, and Shan Dong Career Development College, Jining. It involves a derivatisation step using a (benzoacridinone)ethyl p-toluenesulphonate reagent that reacts with the target phytohormones to give fluorescent ester derivatives.
The eight phytohormones selected for analysis were jasmonic acid, 12-oxophytodienoic acid, indole-3-acetic acid, 3-indolybutyric acid, 3-indolepropionic acid, gibberellin A3, 1-naphthylacetic acid and 2-naphthylacetic acid. None of them are naturally fluorescent, hence the need for derivatisation. Four HPLC columns were compared for separation of the derivatives, the best being a commercial C18 column with a gradient of acetonitrile in water as the mobile phase. All eight hormones were well separated within 16 minutes and detected at 405 nm following excitation at 250 nm. The detection limits were 0.19-0.44 ng/mL and the other analytical data such as linearity, accuracy and repeatability were all acceptable.
Variable levels in fruits
In order to look for phytohormones in several fruits, the researchers developed an extraction protocol based on ultrasound-assisted dispersive liquid-liquid microextraction after which the hormones were derivatised for analysis.
Their concentrations in nectarines, lychees, cherries, apples and grapes varied enormously. For instance, gibberellin A3 ranged from 1.34 ng/g in cherries to 21.71 ng/g in lychees whereas the amounts of 3-indolepropionic acid were more similar at 0.65-1.23 ng/g. Four of the hormones, indole-3-acetic acid, 3-indolybutyric acid, 1-naphthylacetic acid and 2-naphthylacetic acid, were absent from all fruits.
The new method is better than two other published HPLC-fluorescence methods in that it is faster, with a total analysis time of 50 versus 80 minutes, and has lower detection limits. It is far faster than two other published procedures, based on HPLC with electrochemical detection and LC/MS/MS, which take several hours, while still beating them on sensitivity.
Journal of Separation Science 2015 (Article in Press): "Determination of multiple phytohormones in fruits by high-performance liquid chromatography with fluorescence detection using dispersive liquid–liquid microextraction followed by precolumn fluorescent labelling"
Article by Steve Down
The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.
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