Accepted Manuscript Title: Simultaneous determination of plant hormones in peach based on dispersive liquid-liquid microextraction coupled with liquid chromatography- ion trap mass spectrometry Author: Qiaomei Lu Wenmin Zhang Jia Gao Minghua Lu Lan Zhang Jianrong Li PII: DOI: Reference:
S1570-0232(15)00217-2 http://dx.doi.org/doi:10.1016/j.jchromb.2015.04.014 CHROMB 19405
To appear in:
Journal of Chromatography B
Received date: Revised date: Accepted date:
31-7-2014 2-4-2015 9-4-2015
Please cite this article as: Q. Lu, W. Zhang, J. Gao, M. Lu, L. Zhang, J. Li, Simultaneous determination of plant hormones in peach based on dispersive liquidliquid microextraction coupled with liquid chromatography- ion trap mass spectrometry, Journal of Chromatography B (2015), http://dx.doi.org/10.1016/j.jchromb.2015.04.014 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Simultaneous determination of plant hormones in peach based on dispersive liquid-liquid microextraction coupled with liquid
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chromatography- ion trap mass spectrometry
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Qiaomei Lu a, b, Wenmin Zhang b, Jia Gao b, Minghua Lu a, b , Lan Zhang a, b*, Jianrong Li c, d* Analytical and Testing Center, Fuzhou University, Fuzhou, Fujian, 350002, China
b
Ministry of Education Key Laboratory of Analysis and Detection for Food Safety, College of
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a
c
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Chemistry, Fuzhou University, Fuzhou, 350002, Fujian, China
Food Safety Key laboratory of Zhejiang Province, College of Food Science and Biotechnology,
d
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Zhejiang Gongshang University, Hangzhou, Zhejiang, 310035, China
Food Safety Key laboratory of Liaoning Province, Bohai University, Jinzhou, Liaoning, 121013,
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China
Corresponding author: Lan Zhang, Jianrong Li Postal address: College of Chemistry, Fuzhou University, Fuzhou 350002, Fujian, China Fax: 86-591-87893207 Tel: 86-591-87800172 E-mail: [email protected] (Lan Zhang); [email protected] (Jianrong Li)
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ABSTRACT Fruit development is influenced greatly by endogenous hormones including salicylic acid (SA) and abscisic acid (ABA). Mass spectrometry with high sensitivity has become a
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routine technology to analyze hormones. However, pretreatment of plant samples remains a
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difficult problem. Thus, dispersive liquid-liquid microextraction (DLLME) was used to
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concentrate trace plant hormones before liquid chromatography-ion trap mass spectrometry (LC-ITMS) analysis. Standard curves were linear within the ranges of 0.5-50, 0.2-20 ng/mL
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for SA and ABA, respectively. The correlation coefficients were greater than 0.9995 with recoveries above 87.5%. The limits of detection were 0.2 ng/mL for SA and 0.1 ng/mL for
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ABA in spiked water solution, respectively (injection 20 μL). The successful analysis of SA and ABA in fruit samples indicated our DLLME-LC-ITMS approach was efficient, allowing
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reliable quantification of both two compounds from very small amounts of plant material. Moreover, this research revealed the relationship between SA and ABA content and
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development of peach fruit at different growth stages.
Keywords: Dispersive liquid-liquid microextraction; High performance liquid chromatography; Mass spectrometry; Salicylic acid; Abscisic acid; Plant hormone
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1. Introduction Fruit development and maturation is a normal physiological phenomenon in plants which is affected by external environmental conditions (including light, moisture, and
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temperature) as well as internal factors (such as plant hormones). Plant hormones are a
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group of trace endogenous signal molecules [1, 2]. Abscisic acid (ABA) is known to trigger
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various biological functions involving the acceleration of abscission, induction of dormancy, inhibition of seed germination and increasing of stress resistance, etc [3]. Salicylic acid (SA),
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which was confirmed as a new plant hormone in 1992, is also an endogenous signal substance. Many physiological processes such as seed development, fruits ripening,
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horticultural products preservation and environmental stress responses are stimulated and controlled by SA [4].
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The role of SA or ABA in influencing fruit development is well documented [5-11]. Endogenous SA content increased sharply at the early stage of fruit development in Ya-li
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pear [5], apple [6] and banana [7], followed by marked augmentation of fruit size and weight. Additionally, the contents of ABA changed continuously in Roxburgh rose [8] and Nai [9] at the different developing stages of above seeds and fruits. When the amount of ABA reached its peak, serious physiological fruit dropping began in young loquat [10] and sweet cherry fruit [11]. Since endogenous SA and ABA are involved in fruit development, exogenous application is employed to increase fruit yield and quality in agriculture. There is little research on the relationship between content change of endogenous plant hormones and peach development. A recent study reported on content change of ABA in peach fruit development [12]. Nevertheless, research on simultaneous analysis of SA and ABA is
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lacking. Under physiological conditions, SA and ABA, like other plant hormones, are present at very low concentrations against a background of abundant primary and secondary
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metabolites. Therefore, analytical approaches to detect SA and ABA must be extremely
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selective and sensitive. Compared with conventional methods involving high performance
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liquid chromatography, gas chromatography, and gas chromatography-mass spectrometry, liquid chromatograph-mass spectrometry (LC-MS) has become increasingly popular for
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plant hormones detection [13-16], especially liquid chromatography/electrospray ionization tandem mass spectrometry such as ion trap MS (LC/ESI-ITMS). This technique combines
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the efficient separation capability of LC with the great power of structural characterization of MS. Furthermore, the distinct MS/MS function possessed in IT can isolate the desired
specificity.
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signal from possible interfering peaks and improve both analytical sensitivity and
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Sample preparation is required before LC-MS analysis, especially for the
determination of ultra-trace plant hormones in a complex plant matrix. An ideal cleanup technique will isolate target compounds from the matrix, reduce or even eliminate the background interferences coexisting in the sample and concentrate trace analytes to facilitate their quantitative analysis. Therefore, sample preparation can be the bottleneck in modern analyses. In recent years, a variety of new microextration techniques have gradually replaced the traditional ones (i.e., liquid-phase and solid-phase extraction). For instance, plant hormones analysis using solid-phase microextraction (SPME) has been reported [17, 18]. Hollow fiber-based liquid-phase extraction (HF-LPME) has been used to
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measure several plant hormones in coconut [19]. Dispersive liquid-liquid microextraction (DLLME) has been proposed by Assadi and his coworkers in 2006 [20]. In contrast with SPME or HF-LPME, DLLME requires no special interface when coupled with HPLC, and
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its simple operation makes it a popular technique. Moreover, this method has been reported
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to extract hormones from plant material in previous research with satisfactory results [21,
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22].
The aims of the present work were: (1) To adopt DLLME for extraction and enrichment
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of SA and ABA, then to simplified plant hormone pretreatment and enlarge application range of DLLME. (2) To reveal the content changes of SA and ABA in development of
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peach fruit. We aim to understand better the crosstalk of these two hormones in fruit
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2. Materials and methods
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physiological processes.
2.1. Reagents and chemicals
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ABA standard (purity>98%) was purchased from J&K Chemical (Shanghai, China).
SA standard (purity>99.5%) was obtained from Fuchen Chemical Reagents (Tianjin, China). The above standards were dissolved individually in acetonitrile (ACN) at a stock concentration of 2.0 mg/mL and stored at 4 ℃. Working standard solutions of lower
concentrations were prepared by diluting stock solutions with ACN prior to use. HPLC-grade ACN and methanol were obtained from Merck (Darmstadt, Germany). Other reagents used were of analytical reagent grade (Shanghai Chemical Reagents Corp., Shanghai, China). Distilled water was sourced from a Milli-Q SP Reagent water system (Millipore, Bedford, USA). All the solvents were passed through a 0.45 μm cellulose filter
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(Xinya Purification Apparatus Factory, Shanghai, China) before use. Peach samples (Prunus persica cv. NinhBinh) were kindly provided by Sciences Research Institute of Pomology of Fujian Academy of Agricultural (Fujian, China). During
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the fruit growth period, samples were picked at regular time for hormone determination,
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trees and stored quickly at -80 ℃ for following experiments.
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and each sample was tested in triplicate. And the fruits were obtained from the same five
2.2. LC/ESI-ITMS analysis
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Plant hormones analyses were performed with a LC/ESI-ITMS system containing an 1100 Series LC (Agilent, USA) consisting of an autosampler, a quaternary pump and a
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degasser. Separation of SA and ABA was performed on an Eclipse XDB-C18 reversed-phase column (5 μm, 3.0 × 250 mm, Agilent) with a flow rate of 0.6 mL/min and column
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temperature of 25 ℃. Binary solvent system consisting of ACN/water (40: 60, v/v, %) was adopted for an isocratic chromatographic separation. The injection volume was 20 μL for
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each analysis. An Esquire 3000 Ion Trap MSn (Bruker Daltonik GmbH, Germany) was equipped with an ESI source and operated in the negative ion mode. The ESI conditions were as follows: capillary voltage 3.5 kV; end plate offset voltage –500 V; capillary exit voltage 100 V; nebulizer pressure 40 psi; drying gas flow 8 L/min and temperature 350 ℃.
Nitrogen was used as nebulizer and drying gas. The ITMS was operated in full scan and multiple reaction monitoring (MRM) modes, scanning at 50–280 m/z range. ChemStation software was used for instrument control, data acquisition and data processing. 2.3. Procedure for sample preparation Fresh peaches were divided into two parts of pulp and core immediately after collection,
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and the former was stored at -80 ℃.Before analysis, frozen pulp material was pulverized with liquid nitrogen using a pestle and mortar. 250 mg fine powdered sample was weighed accurately and transferred to a 7 mL-microcentrifuge tube. An aliquot of 1.5 mL of
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methanol-water-acetic acid extraction solution (80:19:1, v/v/v, %, stored at 4 ℃ prior to use)
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containing 1 mmol/L 3, 5-di-tert-butyl-4-hydroxytoluol as an antioxidant was added.
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Subsequently, the extracting mixture was kept at 4 ℃ overnight. Then the mixture was centrifuged at 8000 rpm for 5 min at 4 ℃ and the supernatant was transferred to a 4 mL vial.
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The residue was re-extracted once with 0.5 mL of above extractant for 1 h. Both supernatants were combined together and the methanol was removed under a stream of N2 at
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ambient temperature. Finally, the solution was diluted and adjusted to 5.0 mL with acidized
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2.4. DLLME enrichment
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water (pH 3.0 with 0.1 mol/L HCl) for DLLME.
The above 5.0 mL water solution (pH 3.0 adjusted with 0.1 mol/L HCl) was placed in a
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conical test tube, spiking with 0.1 μg/mL analytes of interest. The extraction solvent (30 μL
CHCl3) and the disperser solvent (800 μL tetrahydrofuran, THF) were mixed and added to
the sample solution. Very quickly, a cloudy solution consisting of many dispersed fine droplets of CHCl3 was observed. After the mixture was shaken gently for 0.1 min and centrifuged at 4500 rpm for 3.0 min, the sedimented phase (about 25 μL) in the bottom of the conical tube was withdrawn with a 50 μL LC syringe (Shanghai Gaoge Industrial and Trading Co. Ltd., China) and then placed into a vial insert fitted with polymeric feet (Agilent, USA). The CHCl3 phase containing target plant hormones were immediately subjected to LC-MS analysis.
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3. Results and discussion 3.1. Optimization of chromatographic conditions As is known, HPLC separation is affected greatly by a number of variables including
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the mobile phase, proportion of organic solvent, pump flow rate, etc., therefore, preliminary
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studies were carried out to obtain optimum chromatographic conditions with 0.1 µg/mL
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standard mixture. Based on our experiments, ACN and H2O were selected as the mobile phase due to the well-shaped peaks and better chromatographic behavior. After examining
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three kinds of chromatographic columns (2.1×150 mm, 3.0×250 mm, 4.6×150 mm, C18, particle size 5 µm), we found that SA and ABA were separated well using the 3.0×250 mm
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chromatographic column. Since ACN percentage, column flow rate and column temperature were supposed to have joint influence on the separation resolution (Rs) and retention time
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(tR), an orthogonal experiment was designed to optimize the parameters (Table 1). The minimal Rs (Rs min) and the maximal tR (tR max) were final assessment indexes. From the
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results of orthogonal experiments, 25 ℃ column temperature, 0.6 mL/min flow rate, and
40% acetonitrile in mobile phase were chosen in consideration of short tR, good Rs and
satisfactory peak shape.
3.2. ESI-IT-MS/MS detection
Tandem mass spectrometry (IT-MS/MS) was used as the detector after HPLC separation.
IT-MS/MS can provide information of precursor ion, product ion as well as fragmentation pathway, so it is a powerful quantitative technique. Additionally, SA and ABA studied in our work exist in plant matrix at small concentrations, I think it very necessary to adopt IT-MS/MS for fast qualitative and quantitative analysis of target compounds.
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Due to the presence of a carboxyl group in both SA and ABA, very sensitive MS measurements could be obtained using ESI (-) mode. Ions used to confirm SA were m/z 137 [M-H]-, and m/z 93 [M-H-COO]-. The precursor ion for ABA was m/z 263 [M-H]-. The
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MS/MS spectrum of ABA showed fragments at m/z 219 and 153, corresponding to the
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successive losses of CO2 (remaining m/z 219) and C5H6 (remaining m/z 153), respectively
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(Fig. 1). Namely, m/z transitions 137>93 for SA, and 263>153, 219 for ABA were monitored. Based on the LC retention time and the precursor ion-to-product ion transitions
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of the authentic standards, these two hormones in real sample were also identified. 3.3. Optimization of DLLME process
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Generally, parameters influencing the extraction efficiency of DLLME, including extraction solvent, disperser solvent, sample pH, salinity, and extraction time were tested in
plot the curves.
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sequence. All experiments were performed in triplicate and means of results were used to
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3.3.1 Selection of extraction solvent and disperser solvent The extraction solvent in DLLME should have special requirements, such as low
solubility in water, high affinity to analytes, high density and good chromatographic behavior [23]. With respect to these criteria, many kinds of water immiscible organic solvents, like chlorobenzene (C6H5Cl, 1.10 g/mL), chloroform (CHCl3, 1.48 g/mL),
dichloromethane (CH2Cl2, 1.33 g/mL) and tetrachloroethylene (C2Cl4, 1.63 g/mL) were tested, respectively. Enrichment factors for the two analytes exhibited different tendencies, and C2Cl4 provided low extraction efficiency (Fig. 2A). The extraction effect of SA was investigated emphatically, and the performance with CHCl3 was obviously better than that
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with C6H5Cl. Thus, CHCl3 was chosen as the most suitable extraction solvent for both analytes in one analysis, with other conditions being 50 µL extraction solvent, 700 μL THF as the disperser solvent (see below), extraction time 0.1 min, centrifugation at 4500 rpm for
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3.0 min.
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In principle, the disperser solvent should be miscible with both the extraction solvent
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and the aqueous sample. The emulsifying effectiveness of various solvents such as ACN, methanol, acetone and ethanol in cloudy system were studied. As shown in Fig. 2B, the
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emulsification of ACN and methanol was not satisfactory with low extraction efficiency. In contrast, a fine emulsion was formed in the presence of THF or acetone. For instance, fixing
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CHCl3 as extraction solvent, the extraction efficiency of SA was 1.2 times higher using acetone as disperser solvent than that using THF. However, the enrichment effect of ABA
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was 1.3 times higher with THF than that with acetone. Finally, THF was selected to disperse sample solution for the following experiments due to its less toxicity and better extraction
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performance.
3.3.2. Optimization of sedimented organic phase volume In DLLME, the volume of sedimented organic phase directly affects concentration of the
analytes and thus the extraction efficiency. We found that the volume of sedimented phase was influenced by the extraction solvent, disperser solvent and NaCl amount. Extraction efficiency enhances firstly as an increase volume of extraction solvent. When extraction solvent is excessive, extraction efficiency decreases due to dilution effect. Adequate amount of disperser solvent can disperse extraction solvent completely for extraction; however, large consumption of disperser solvent will decrease extraction performance because of analytes
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solubility in water solution. Generally, as NaCl amount increases, the solubility of analytes in sample solution decreases, leading to an increase in extraction efficiency. Therefore, an orthogonal experiment design was conducted to optimize these factors. 40-70 µL CHCl3 and
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500-800 µL THF were optimized in Table 2. The ionic strength of the solution was modified
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by adding NaCl ranging from 0 to 4.5%. Results showed that signal response of SA and
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ABA reached maximum with 30 µL CHCl3, 800 µL THF and no NaCl. 3.3.3. Selection of pH and extraction time
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Development of a DLLME method requires optimum extraction conditions including pH value and extraction time to be established. For this purpose, pH values ranging from 2.0-5.0
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were investigated. ABA with a pKa of 6.8 is considered to be in non-ionic state at pH 3.0, which was beneficial for extraction. SA with a pKa of 3.1 is expected to be approximately
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50% ionized at pH 3.0. According to our experimental results, peak areas for both SA and ABA reached highest values when the sample solution was controlled at pH 3.0 (data not
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shown). Extraction times (0.1, 3, 10, 30 and 60 min) were then evaluated. There was no obvious influence of extraction time on peak areas, indicating that extraction in DLLME was very fast. Therefore, 0.1 min was used as extraction time in the subsequent experiments. 3.4 Evaluation of method performance 3.4.1. Working curve in spiked water For the purpose of quantitative analysis, the characteristic MRM ions used for quantitation were listed in Table 3. Calibration curves based on the external standard method were plotted from a series of standard mixtures of different concentrations in spiked water. As can be seen from Table 3, all curves exhibited good linearity, with correlation
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coefficients (R) >0.9995. To make the results more convincing, the goodness of fit for regression line was used for linearity evaluation. F-test and t-test were applied respectively (α=0.05). Data shown that nice linearity was obtained with F-statistic >Fα and t-statistic >tα/2
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(Table 4).
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Based on a signal-to-noise ratio (S/N) of 3, the limits of detection (LODs) were 0.2
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ng/mL for SA and 0.1 ng/mL for ABA in spiked water solution, respectively (20 μL injection). The low LODs were not only attributed to the high sensitivity and selectivity of
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ITMS, but also to the good enrichment effect of DLLME approach. Such high sensitivity was enough to detect SA and ABA in plant samples effectively. In addition, both the
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linearity and LODs we obtained were superior to those reported previously [22] (linearity: 0.2-100 μg/mL; SA LOD: 0.5 μg/mL; ABA LOD: 0.2 μg/mL). The contrast is clear evidence
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of the utility of DLLME-LC-ITMS for this application. 3.4.2. Method repeatability
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To verify the precision of the proposed method, repeatability experiments were
conducted. The relative standard deviations (RSDs) for intra-day and inter-day precision
were evaluated (n=5). When 0.01 μg/mL standard solutions were extracted under optimal
conditions, RSDs of the intra-day peak area precision were less than 4.50%, while inter-day RSDs were no more than 7.03%. The satisfactory precision suggested that this DLLME method possessed high repeatability. In short, the main advantages of our DLLME method are high speed, satisfactory analyte enrichment, good repeatability and organic solvent usage at the μL level.
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3.5. Analysis of SA and ABA in peach fruit Our method was used to measure SA and ABA in peach. Fruit development was divided into the first fast growth period (stages Ⅰ-Ⅲ, picking fruits every two weeks), the slow
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growth period (namely, stone hard period, stages Ⅳ-Ⅵ, picking fruits every two weeks),
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and the second fast growth period (stages Ⅶ-Ⅺ, picking fruits every week). The fruit
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developmental periods and growth features were summarized in Table 5. Take the first fast growth period as an example: it lasted 35 days after flowering, with an obvious increase in
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fruit volume and size. Additionally, there are three physiological fruit-dropping stages in peach development. Fresh peach pulp was selected as real sample analysis as described in
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Section 2.3. Frozen pulp material was grinded and 250 mg powdered sample was used, extracted with aqueous methanol, and then enriched by our DLLME procedure.
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3.5.1. Dynamic change of SA in peach
The amount of SA was comparatively higher at stages Ⅰand Ⅱ of the first fast growth
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period than those at stages Ⅲ-Ⅸ (Fig. 3A). That is, SA content reached 20.2 ng/g (stage Ⅰ,
peak 1) during early fruit development. The concentration of SA decreased as the fruit developed. During the second fast growth period, SA increased rapidly at mid-ripe (stage Ⅹ, 31.0 ng/g, peak 2) and full ripe (stage Ⅺ) periods. The content change of SA and peak
of fruit growth appeared concurrently, indicating there was some relationship between SA and fruit development. In agreement with other research [5, 6], our work clearly demonstrated that SA was involved in fruit growth regulation. Experiments also revealed that SA amounts in pre-harvest fruit drop were higher than those of contemporaneous normal ones. For example, at stage Ⅳ, SA in dropped peach reached 26.2 ng/g (data not
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shown). 3.5.2. Dynamic change of ABA in peach The content changes of ABA at different developmental stages of peach were determined
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by our DLLME-HPLC/ESI-ITMS assay. We observed two small peaks and one larger spike
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in ABA content during the whole growth duration of peach (Fig. 3B). Namely, ABA amount
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peaked at stage Ⅱ (peak 1) then decreased, followed by obvious augmentation of peach fruit size and weight. Similar trend was appeared at stage Ⅶ, with the peak value of 19.2 ng/g
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(peak 2). The remarkable third peak at stage Ⅹ (144.7 ng/g) occurred during the mid-ripe period. Afterwards, full ripening (i.e. stage Ⅺ) was initiated and presumably promoted.
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Therefore, the accumulation of endogenous ABA was considered to be an important signal to accelerate fruit ripening. Stages Ⅱ, Ⅳ and Ⅶ corresponded to the physiological
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fruit-dropping periods, which suggested the increase of ABA would lead to fruit drop. The above assumptions were consistent with those reported in Roxburgh rose [8], and loquat
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[10].
3.5.3. Recovery experiment
To examine the reliability of the proposed assay, peach pulp at stage Ⅲ of first fast
growth period was selected for a recovery experiment. After initial sample preparation and extraction, the dilution solution was used as the sample matrix with little signal response of SA and ABA. Known amounts of analytes (0.8, 5.0, 10.0 ng/mL) were added to matrix prior to DLLME extraction and total SA and ABA were quantified by LC-MS/MS. For each addition level, three replicate tests were carried out, and the results were listed in Table 6. Recoveries were in the range of 87.5-119.0%, with RSDs less than 12.6%, suggesting that
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this method was acceptable to be a routine analytical protocol for plant hormones. 4. Conclusion Both SA and ABA were indentified by ITMS and MRM technique. The MS/MS had the
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advantages of high sensitivity and powerful qualitative ability, which had increasing become
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a versatile tool to analyze trace plant hormones. The present work offered a new purification
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protocol to extract SA and ABA from plant tissues. The DLLME method was faster and simpler, reliable with a low amount of plant material, and did not require much organic
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solvents. Moreover, the enrichment effect of this technology afforded low level LODs of SA and ABA, making it possible to analyze these two hormones from plant samples with
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convenience and sensitivity.
Our methodology enabled analyzing SA and ABA contents in peach fruit. Furthermore,
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physiological phenomena such as increased volume and weight of peach and fruit drop were related to the endogenous contents of the above two hormones. Our results suggested that
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SA and ABA play important roles in fruit growth and development, which will provide a better understanding of multiple plant hormone crosstalk and interplay in physiological processes.
Acknowledgment
The authors are grateful to the National Nature Sciences Foundation of China (21275029), the National Basic Research Program of China (No.2010CB732403), National Key Technologies R&D Program of China during the 12th Five-Year Plan Period (2012BAD29B06), the Natural Science Foundation of Fujian Province (2010J05021), the
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Program for Changjiang Scholars and Innovative Research Team in University (No.
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58 (2010) 2763-2770.
[22] G. Vishal, K. Manoj, B. Harshad, R C.R.K., S. Abhiram, J. Bhavanath, Simultaneous determination of different endogenetic plant growth regulators in common green seaweeds using dispersive liquid-liquid microextraction method, Plant Physiol. Biochem. 49 (2011) 1259-1263.
[23] X. H. Zang, Q. H. Wu, M. Y. Zhang, G. H. Xi, Z. Wang, Developments of dispersive liquid-liquid microextraction technique, Chin. J. Anal. Chem. 37 (2009) 161-168.
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Figure legends
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Fig. 1 MS/MS spectra of SA and ABA.
an
263 > 219, 153) and concentration was 0.1 μg/mL.
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The CID voltages were 0.7 V (SA, transition 137 > 93) and 0.6 V (ABA, transition
Fig. 2 Selection of extraction solvent (A) and disperser solvent (B).
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Sample concentration was 0.1 μg/mL. Other conditions in Fig. 2A were 50 µL extraction solvent, 700 μL THF, extraction time 0.1 min, centrifugation at 4500 rpm
te
d
for 3.0 min. Other conditions in Fig. 2B were 50 µL CHCl3, 700 μL disperser solvent,
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extraction time 0.1 min, centrifugation at 4500 rpm for 3.0 min.
Fig. 3 SA (A) and ABA (B) contents at different development stages of peach. Values are means of three replicate samples.
19
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Table 1. Optimization of HPLC conditions column temperature, ℃
flow rate,
ACN proportion,
mL min-1
%
Test indexes Rs min
35
0.7
35
5.62
2
30
0.6
35
5.46
3
25
0.5
35
5.69
4
30
0.7
40
5
25
0.6
40
6
35
0.5
40
7
25
0.7
45
8
35
0.6
9
30
0.5
2.70 3.15 3.86
cr
1
tR max
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Exp#
2.38
4.61
2.88
4.93
3.30
4.25
2.15
45
4.35
2.50
45
4.52
3.01
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d
M
an
us
1.40
20
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Table 2. Optimization of DLLME sedimented phase volume Exp#
CHCl3 volume, μL
THF volume, μL
NaCl amount, %
Signal response SA
ABA
30
500
4.5
255408
71838
2
30
600
3.0
243417
69749
3
30
700
1.5
270940
71268
4
30
800
0
5
40
500
3.0
6
40
600
7
40
700
8
40
800
9
50
500
10
50
11
50
12
50
13
60
14
60
15 16
cr
ip t
1
122800
213844
36727
4.5
213217
76644
0
194497
88379
1.5
134030
68929
1.5
69198
34169
600
0
115029
51529
700
4.5
53004
34633
3.0
62477
49145
500
0
46027
48051
600
1.5
46700
68929
60
700
3.0
37094
40155
60
800
4.5
28066
41262
d
M
an
us
377522
Ac ce p
te
800
21
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Table 3. Analytical performance data for two analytes in spiked water
SA
Linear range
(m/z)
(ng/mL)
93.0 219.0, 153.0
Calibration curve
R
LOD (ng/mL)
0.5-50
Y = 656275x+982.2
0.9995
0.2
0.2-20
Y = 4734251x+571.3
0.9999
0.1
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d
M
an
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cr
ABA
Quantitation
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Analyte
22
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Table 4. The goodness of fit for regression lines of SA and ABA (α=0.05) yi
ŷi
1
0.0005
917
1310.37
2
0.0008
1391
1507.25
3
0.001
2034
1638.51
4
0.002
2781
5
0.008
6268
6
0.01
7475
7
0.02
13567
8
0.05
33999
SSE
900846.71
SSR
SST
864438498.00
r
0.9995
F-statistic
5751.51
t-statistic
75.84
No.
xi
1
ABA
2294.78
cr
6232.43 7544.98
us
14107.73 33795.98
863537651.29
an M
n
8
F0.05
7.71
t0.025
2.7765
yi
ŷi
0.0002
1558
1518.14
0.0005
2908
2938.42
Ac ce p
2
ip t
xi
d
SA
No.
te
Analyte
3
0.0008
4389
4358.69
4
0.001
4999
5305.54
5
0.002
10015
10039.79
6
0.008
39457
38445.30
7
0.01
47249
47913.80
8
0.02
95201
95256.31
SSE
1566573.75
SSR
7713453780.25
SST
7715020354.00
r
0.9999
n
8
F-statistic
29542.64
F0.05
7.71
t-statistic
171.88
t0.025
2.7765
23
Page 23 of 29
Table 5. Peach fruit growth period and some data of growth rates in diameter Growth period
Days of growth lasted
the first fast growth period
stages Ⅰ- Ⅲ
28
the slow growth period (stone hard period)
stages Ⅳ- Ⅵ
28
the second fast growth period
stages Ⅶ- Ⅺ
period
flowering
Fruit (cm) Transverse
The first
diameter
cr
Days after
35
Vertical
Kernel (cm)
Transverse
us
Growth
ip t
Developmental period
Vertical
diameter
diameter
diameter
1.60±0.17
0.46±0.09
0.76±0.11
Stage Ⅰ
7
0.95±0.15
period
Stage Ⅱ
21
1.64±.027
2.02±0.32
0.61±0.13
0.94±0.15
Stage Ⅲ
35
2.02±0.31
2.71±0.39
0.91±0.18
1.21±0.17
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d
M
an
fast growth
25
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Table 6. Recoveries of SA and ABA in spiked samples (n = 3)
(ng/mL)
(ng/mL)
（%）
0.8
0.70±0.05
87.5±6.2
5.0
4.56±0.11
91.2±2.2
10.0
9.79±0.40
97.9±4.0
0.8
0.95±0.01
119.0±1.2
5.0
4.53±0.08
90.6±1.6
10.0
10.89±1.26
SA
108.9±12.6
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te
d
M
an
ABA
Recovery
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Found
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Added
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Compound
26
Page 25 of 29
Highlights specifications: (1) DLLME assay was adopted to concentrate salicylic acid and abscisic acid.
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(3) Small amounts of real material were required for analysis.
ip t
(2) This DLLME-LC-ITMS approach was sensitive with LODs at ng/mL level.
Ac ce p
te
d
M
an
us
(4) Content changes of salicylic acid and abscisic acid in peach were revealed.
27
Page 26 of 29
Ac
ce
pt
ed
M
an
us
cr
i
Figure1
Page 27 of 29
Ac
ce
pt
ed
M
an
us
cr
i
Figure2
Page 28 of 29
Ac
ce
pt
ed
M
an
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cr
i
Figure 3
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S1570-0232(15)00217-2 http://dx.doi.org/doi:10.1016/j.jchromb.2015.04.014 CHROMB 19405
To appear in:
Journal of Chromatography B
Received date: Revised date: Accepted date:
31-7-2014 2-4-2015 9-4-2015
Please cite this article as: Q. Lu, W. Zhang, J. Gao, M. Lu, L. Zhang, J. Li, Simultaneous determination of plant hormones in peach based on dispersive liquidliquid microextraction coupled with liquid chromatography- ion trap mass spectrometry, Journal of Chromatography B (2015), http://dx.doi.org/10.1016/j.jchromb.2015.04.014 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Simultaneous determination of plant hormones in peach based on dispersive liquid-liquid microextraction coupled with liquid
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chromatography- ion trap mass spectrometry
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Qiaomei Lu a, b, Wenmin Zhang b, Jia Gao b, Minghua Lu a, b , Lan Zhang a, b*, Jianrong Li c, d* Analytical and Testing Center, Fuzhou University, Fuzhou, Fujian, 350002, China
b
Ministry of Education Key Laboratory of Analysis and Detection for Food Safety, College of
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a
c
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Chemistry, Fuzhou University, Fuzhou, 350002, Fujian, China
Food Safety Key laboratory of Zhejiang Province, College of Food Science and Biotechnology,
d
M
Zhejiang Gongshang University, Hangzhou, Zhejiang, 310035, China
Food Safety Key laboratory of Liaoning Province, Bohai University, Jinzhou, Liaoning, 121013,
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d
China
Corresponding author: Lan Zhang, Jianrong Li Postal address: College of Chemistry, Fuzhou University, Fuzhou 350002, Fujian, China Fax: 86-591-87893207 Tel: 86-591-87800172 E-mail: [email protected] (Lan Zhang); [email protected] (Jianrong Li)
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ABSTRACT Fruit development is influenced greatly by endogenous hormones including salicylic acid (SA) and abscisic acid (ABA). Mass spectrometry with high sensitivity has become a
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routine technology to analyze hormones. However, pretreatment of plant samples remains a
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difficult problem. Thus, dispersive liquid-liquid microextraction (DLLME) was used to
us
concentrate trace plant hormones before liquid chromatography-ion trap mass spectrometry (LC-ITMS) analysis. Standard curves were linear within the ranges of 0.5-50, 0.2-20 ng/mL
an
for SA and ABA, respectively. The correlation coefficients were greater than 0.9995 with recoveries above 87.5%. The limits of detection were 0.2 ng/mL for SA and 0.1 ng/mL for
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ABA in spiked water solution, respectively (injection 20 μL). The successful analysis of SA and ABA in fruit samples indicated our DLLME-LC-ITMS approach was efficient, allowing
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d
reliable quantification of both two compounds from very small amounts of plant material. Moreover, this research revealed the relationship between SA and ABA content and
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development of peach fruit at different growth stages.
Keywords: Dispersive liquid-liquid microextraction; High performance liquid chromatography; Mass spectrometry; Salicylic acid; Abscisic acid; Plant hormone
2
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1. Introduction Fruit development and maturation is a normal physiological phenomenon in plants which is affected by external environmental conditions (including light, moisture, and
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temperature) as well as internal factors (such as plant hormones). Plant hormones are a
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group of trace endogenous signal molecules [1, 2]. Abscisic acid (ABA) is known to trigger
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various biological functions involving the acceleration of abscission, induction of dormancy, inhibition of seed germination and increasing of stress resistance, etc [3]. Salicylic acid (SA),
an
which was confirmed as a new plant hormone in 1992, is also an endogenous signal substance. Many physiological processes such as seed development, fruits ripening,
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horticultural products preservation and environmental stress responses are stimulated and controlled by SA [4].
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The role of SA or ABA in influencing fruit development is well documented [5-11]. Endogenous SA content increased sharply at the early stage of fruit development in Ya-li
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pear [5], apple [6] and banana [7], followed by marked augmentation of fruit size and weight. Additionally, the contents of ABA changed continuously in Roxburgh rose [8] and Nai [9] at the different developing stages of above seeds and fruits. When the amount of ABA reached its peak, serious physiological fruit dropping began in young loquat [10] and sweet cherry fruit [11]. Since endogenous SA and ABA are involved in fruit development, exogenous application is employed to increase fruit yield and quality in agriculture. There is little research on the relationship between content change of endogenous plant hormones and peach development. A recent study reported on content change of ABA in peach fruit development [12]. Nevertheless, research on simultaneous analysis of SA and ABA is
3
Page 3 of 29
lacking. Under physiological conditions, SA and ABA, like other plant hormones, are present at very low concentrations against a background of abundant primary and secondary
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metabolites. Therefore, analytical approaches to detect SA and ABA must be extremely
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selective and sensitive. Compared with conventional methods involving high performance
us
liquid chromatography, gas chromatography, and gas chromatography-mass spectrometry, liquid chromatograph-mass spectrometry (LC-MS) has become increasingly popular for
an
plant hormones detection [13-16], especially liquid chromatography/electrospray ionization tandem mass spectrometry such as ion trap MS (LC/ESI-ITMS). This technique combines
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the efficient separation capability of LC with the great power of structural characterization of MS. Furthermore, the distinct MS/MS function possessed in IT can isolate the desired
specificity.
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d
signal from possible interfering peaks and improve both analytical sensitivity and
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Sample preparation is required before LC-MS analysis, especially for the
determination of ultra-trace plant hormones in a complex plant matrix. An ideal cleanup technique will isolate target compounds from the matrix, reduce or even eliminate the background interferences coexisting in the sample and concentrate trace analytes to facilitate their quantitative analysis. Therefore, sample preparation can be the bottleneck in modern analyses. In recent years, a variety of new microextration techniques have gradually replaced the traditional ones (i.e., liquid-phase and solid-phase extraction). For instance, plant hormones analysis using solid-phase microextraction (SPME) has been reported [17, 18]. Hollow fiber-based liquid-phase extraction (HF-LPME) has been used to
4
Page 4 of 29
measure several plant hormones in coconut [19]. Dispersive liquid-liquid microextraction (DLLME) has been proposed by Assadi and his coworkers in 2006 [20]. In contrast with SPME or HF-LPME, DLLME requires no special interface when coupled with HPLC, and
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its simple operation makes it a popular technique. Moreover, this method has been reported
cr
to extract hormones from plant material in previous research with satisfactory results [21,
us
22].
The aims of the present work were: (1) To adopt DLLME for extraction and enrichment
an
of SA and ABA, then to simplified plant hormone pretreatment and enlarge application range of DLLME. (2) To reveal the content changes of SA and ABA in development of
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peach fruit. We aim to understand better the crosstalk of these two hormones in fruit
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2. Materials and methods
d
physiological processes.
2.1. Reagents and chemicals
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ABA standard (purity>98%) was purchased from J&K Chemical (Shanghai, China).
SA standard (purity>99.5%) was obtained from Fuchen Chemical Reagents (Tianjin, China). The above standards were dissolved individually in acetonitrile (ACN) at a stock concentration of 2.0 mg/mL and stored at 4 ℃. Working standard solutions of lower
concentrations were prepared by diluting stock solutions with ACN prior to use. HPLC-grade ACN and methanol were obtained from Merck (Darmstadt, Germany). Other reagents used were of analytical reagent grade (Shanghai Chemical Reagents Corp., Shanghai, China). Distilled water was sourced from a Milli-Q SP Reagent water system (Millipore, Bedford, USA). All the solvents were passed through a 0.45 μm cellulose filter
5
Page 5 of 29
(Xinya Purification Apparatus Factory, Shanghai, China) before use. Peach samples (Prunus persica cv. NinhBinh) were kindly provided by Sciences Research Institute of Pomology of Fujian Academy of Agricultural (Fujian, China). During
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the fruit growth period, samples were picked at regular time for hormone determination,
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trees and stored quickly at -80 ℃ for following experiments.
cr
and each sample was tested in triplicate. And the fruits were obtained from the same five
2.2. LC/ESI-ITMS analysis
an
Plant hormones analyses were performed with a LC/ESI-ITMS system containing an 1100 Series LC (Agilent, USA) consisting of an autosampler, a quaternary pump and a
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degasser. Separation of SA and ABA was performed on an Eclipse XDB-C18 reversed-phase column (5 μm, 3.0 × 250 mm, Agilent) with a flow rate of 0.6 mL/min and column
te
d
temperature of 25 ℃. Binary solvent system consisting of ACN/water (40: 60, v/v, %) was adopted for an isocratic chromatographic separation. The injection volume was 20 μL for
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each analysis. An Esquire 3000 Ion Trap MSn (Bruker Daltonik GmbH, Germany) was equipped with an ESI source and operated in the negative ion mode. The ESI conditions were as follows: capillary voltage 3.5 kV; end plate offset voltage –500 V; capillary exit voltage 100 V; nebulizer pressure 40 psi; drying gas flow 8 L/min and temperature 350 ℃.
Nitrogen was used as nebulizer and drying gas. The ITMS was operated in full scan and multiple reaction monitoring (MRM) modes, scanning at 50–280 m/z range. ChemStation software was used for instrument control, data acquisition and data processing. 2.3. Procedure for sample preparation Fresh peaches were divided into two parts of pulp and core immediately after collection,
6
Page 6 of 29
and the former was stored at -80 ℃.Before analysis, frozen pulp material was pulverized with liquid nitrogen using a pestle and mortar. 250 mg fine powdered sample was weighed accurately and transferred to a 7 mL-microcentrifuge tube. An aliquot of 1.5 mL of
ip t
methanol-water-acetic acid extraction solution (80:19:1, v/v/v, %, stored at 4 ℃ prior to use)
cr
containing 1 mmol/L 3, 5-di-tert-butyl-4-hydroxytoluol as an antioxidant was added.
us
Subsequently, the extracting mixture was kept at 4 ℃ overnight. Then the mixture was centrifuged at 8000 rpm for 5 min at 4 ℃ and the supernatant was transferred to a 4 mL vial.
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The residue was re-extracted once with 0.5 mL of above extractant for 1 h. Both supernatants were combined together and the methanol was removed under a stream of N2 at
M
ambient temperature. Finally, the solution was diluted and adjusted to 5.0 mL with acidized
te
2.4. DLLME enrichment
d
water (pH 3.0 with 0.1 mol/L HCl) for DLLME.
The above 5.0 mL water solution (pH 3.0 adjusted with 0.1 mol/L HCl) was placed in a
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conical test tube, spiking with 0.1 μg/mL analytes of interest. The extraction solvent (30 μL
CHCl3) and the disperser solvent (800 μL tetrahydrofuran, THF) were mixed and added to
the sample solution. Very quickly, a cloudy solution consisting of many dispersed fine droplets of CHCl3 was observed. After the mixture was shaken gently for 0.1 min and centrifuged at 4500 rpm for 3.0 min, the sedimented phase (about 25 μL) in the bottom of the conical tube was withdrawn with a 50 μL LC syringe (Shanghai Gaoge Industrial and Trading Co. Ltd., China) and then placed into a vial insert fitted with polymeric feet (Agilent, USA). The CHCl3 phase containing target plant hormones were immediately subjected to LC-MS analysis.
7
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3. Results and discussion 3.1. Optimization of chromatographic conditions As is known, HPLC separation is affected greatly by a number of variables including
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the mobile phase, proportion of organic solvent, pump flow rate, etc., therefore, preliminary
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studies were carried out to obtain optimum chromatographic conditions with 0.1 µg/mL
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standard mixture. Based on our experiments, ACN and H2O were selected as the mobile phase due to the well-shaped peaks and better chromatographic behavior. After examining
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three kinds of chromatographic columns (2.1×150 mm, 3.0×250 mm, 4.6×150 mm, C18, particle size 5 µm), we found that SA and ABA were separated well using the 3.0×250 mm
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chromatographic column. Since ACN percentage, column flow rate and column temperature were supposed to have joint influence on the separation resolution (Rs) and retention time
te
d
(tR), an orthogonal experiment was designed to optimize the parameters (Table 1). The minimal Rs (Rs min) and the maximal tR (tR max) were final assessment indexes. From the
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results of orthogonal experiments, 25 ℃ column temperature, 0.6 mL/min flow rate, and
40% acetonitrile in mobile phase were chosen in consideration of short tR, good Rs and
satisfactory peak shape.
3.2. ESI-IT-MS/MS detection
Tandem mass spectrometry (IT-MS/MS) was used as the detector after HPLC separation.
IT-MS/MS can provide information of precursor ion, product ion as well as fragmentation pathway, so it is a powerful quantitative technique. Additionally, SA and ABA studied in our work exist in plant matrix at small concentrations, I think it very necessary to adopt IT-MS/MS for fast qualitative and quantitative analysis of target compounds.
8
Page 8 of 29
Due to the presence of a carboxyl group in both SA and ABA, very sensitive MS measurements could be obtained using ESI (-) mode. Ions used to confirm SA were m/z 137 [M-H]-, and m/z 93 [M-H-COO]-. The precursor ion for ABA was m/z 263 [M-H]-. The
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MS/MS spectrum of ABA showed fragments at m/z 219 and 153, corresponding to the
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successive losses of CO2 (remaining m/z 219) and C5H6 (remaining m/z 153), respectively
us
(Fig. 1). Namely, m/z transitions 137>93 for SA, and 263>153, 219 for ABA were monitored. Based on the LC retention time and the precursor ion-to-product ion transitions
an
of the authentic standards, these two hormones in real sample were also identified. 3.3. Optimization of DLLME process
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Generally, parameters influencing the extraction efficiency of DLLME, including extraction solvent, disperser solvent, sample pH, salinity, and extraction time were tested in
plot the curves.
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sequence. All experiments were performed in triplicate and means of results were used to
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3.3.1 Selection of extraction solvent and disperser solvent The extraction solvent in DLLME should have special requirements, such as low
solubility in water, high affinity to analytes, high density and good chromatographic behavior [23]. With respect to these criteria, many kinds of water immiscible organic solvents, like chlorobenzene (C6H5Cl, 1.10 g/mL), chloroform (CHCl3, 1.48 g/mL),
dichloromethane (CH2Cl2, 1.33 g/mL) and tetrachloroethylene (C2Cl4, 1.63 g/mL) were tested, respectively. Enrichment factors for the two analytes exhibited different tendencies, and C2Cl4 provided low extraction efficiency (Fig. 2A). The extraction effect of SA was investigated emphatically, and the performance with CHCl3 was obviously better than that
9
Page 9 of 29
with C6H5Cl. Thus, CHCl3 was chosen as the most suitable extraction solvent for both analytes in one analysis, with other conditions being 50 µL extraction solvent, 700 μL THF as the disperser solvent (see below), extraction time 0.1 min, centrifugation at 4500 rpm for
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3.0 min.
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In principle, the disperser solvent should be miscible with both the extraction solvent
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and the aqueous sample. The emulsifying effectiveness of various solvents such as ACN, methanol, acetone and ethanol in cloudy system were studied. As shown in Fig. 2B, the
an
emulsification of ACN and methanol was not satisfactory with low extraction efficiency. In contrast, a fine emulsion was formed in the presence of THF or acetone. For instance, fixing
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CHCl3 as extraction solvent, the extraction efficiency of SA was 1.2 times higher using acetone as disperser solvent than that using THF. However, the enrichment effect of ABA
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was 1.3 times higher with THF than that with acetone. Finally, THF was selected to disperse sample solution for the following experiments due to its less toxicity and better extraction
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performance.
3.3.2. Optimization of sedimented organic phase volume In DLLME, the volume of sedimented organic phase directly affects concentration of the
analytes and thus the extraction efficiency. We found that the volume of sedimented phase was influenced by the extraction solvent, disperser solvent and NaCl amount. Extraction efficiency enhances firstly as an increase volume of extraction solvent. When extraction solvent is excessive, extraction efficiency decreases due to dilution effect. Adequate amount of disperser solvent can disperse extraction solvent completely for extraction; however, large consumption of disperser solvent will decrease extraction performance because of analytes
10
Page 10 of 29
solubility in water solution. Generally, as NaCl amount increases, the solubility of analytes in sample solution decreases, leading to an increase in extraction efficiency. Therefore, an orthogonal experiment design was conducted to optimize these factors. 40-70 µL CHCl3 and
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500-800 µL THF were optimized in Table 2. The ionic strength of the solution was modified
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by adding NaCl ranging from 0 to 4.5%. Results showed that signal response of SA and
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ABA reached maximum with 30 µL CHCl3, 800 µL THF and no NaCl. 3.3.3. Selection of pH and extraction time
an
Development of a DLLME method requires optimum extraction conditions including pH value and extraction time to be established. For this purpose, pH values ranging from 2.0-5.0
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were investigated. ABA with a pKa of 6.8 is considered to be in non-ionic state at pH 3.0, which was beneficial for extraction. SA with a pKa of 3.1 is expected to be approximately
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d
50% ionized at pH 3.0. According to our experimental results, peak areas for both SA and ABA reached highest values when the sample solution was controlled at pH 3.0 (data not
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shown). Extraction times (0.1, 3, 10, 30 and 60 min) were then evaluated. There was no obvious influence of extraction time on peak areas, indicating that extraction in DLLME was very fast. Therefore, 0.1 min was used as extraction time in the subsequent experiments. 3.4 Evaluation of method performance 3.4.1. Working curve in spiked water For the purpose of quantitative analysis, the characteristic MRM ions used for quantitation were listed in Table 3. Calibration curves based on the external standard method were plotted from a series of standard mixtures of different concentrations in spiked water. As can be seen from Table 3, all curves exhibited good linearity, with correlation
11
Page 11 of 29
coefficients (R) >0.9995. To make the results more convincing, the goodness of fit for regression line was used for linearity evaluation. F-test and t-test were applied respectively (α=0.05). Data shown that nice linearity was obtained with F-statistic >Fα and t-statistic >tα/2
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(Table 4).
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Based on a signal-to-noise ratio (S/N) of 3, the limits of detection (LODs) were 0.2
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ng/mL for SA and 0.1 ng/mL for ABA in spiked water solution, respectively (20 μL injection). The low LODs were not only attributed to the high sensitivity and selectivity of
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ITMS, but also to the good enrichment effect of DLLME approach. Such high sensitivity was enough to detect SA and ABA in plant samples effectively. In addition, both the
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linearity and LODs we obtained were superior to those reported previously [22] (linearity: 0.2-100 μg/mL; SA LOD: 0.5 μg/mL; ABA LOD: 0.2 μg/mL). The contrast is clear evidence
te
d
of the utility of DLLME-LC-ITMS for this application. 3.4.2. Method repeatability
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To verify the precision of the proposed method, repeatability experiments were
conducted. The relative standard deviations (RSDs) for intra-day and inter-day precision
were evaluated (n=5). When 0.01 μg/mL standard solutions were extracted under optimal
conditions, RSDs of the intra-day peak area precision were less than 4.50%, while inter-day RSDs were no more than 7.03%. The satisfactory precision suggested that this DLLME method possessed high repeatability. In short, the main advantages of our DLLME method are high speed, satisfactory analyte enrichment, good repeatability and organic solvent usage at the μL level.
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3.5. Analysis of SA and ABA in peach fruit Our method was used to measure SA and ABA in peach. Fruit development was divided into the first fast growth period (stages Ⅰ-Ⅲ, picking fruits every two weeks), the slow
ip t
growth period (namely, stone hard period, stages Ⅳ-Ⅵ, picking fruits every two weeks),
cr
and the second fast growth period (stages Ⅶ-Ⅺ, picking fruits every week). The fruit
us
developmental periods and growth features were summarized in Table 5. Take the first fast growth period as an example: it lasted 35 days after flowering, with an obvious increase in
an
fruit volume and size. Additionally, there are three physiological fruit-dropping stages in peach development. Fresh peach pulp was selected as real sample analysis as described in
M
Section 2.3. Frozen pulp material was grinded and 250 mg powdered sample was used, extracted with aqueous methanol, and then enriched by our DLLME procedure.
te
d
3.5.1. Dynamic change of SA in peach
The amount of SA was comparatively higher at stages Ⅰand Ⅱ of the first fast growth
Ac ce p
period than those at stages Ⅲ-Ⅸ (Fig. 3A). That is, SA content reached 20.2 ng/g (stage Ⅰ,
peak 1) during early fruit development. The concentration of SA decreased as the fruit developed. During the second fast growth period, SA increased rapidly at mid-ripe (stage Ⅹ, 31.0 ng/g, peak 2) and full ripe (stage Ⅺ) periods. The content change of SA and peak
of fruit growth appeared concurrently, indicating there was some relationship between SA and fruit development. In agreement with other research [5, 6], our work clearly demonstrated that SA was involved in fruit growth regulation. Experiments also revealed that SA amounts in pre-harvest fruit drop were higher than those of contemporaneous normal ones. For example, at stage Ⅳ, SA in dropped peach reached 26.2 ng/g (data not
13
Page 13 of 29
shown). 3.5.2. Dynamic change of ABA in peach The content changes of ABA at different developmental stages of peach were determined
ip t
by our DLLME-HPLC/ESI-ITMS assay. We observed two small peaks and one larger spike
cr
in ABA content during the whole growth duration of peach (Fig. 3B). Namely, ABA amount
us
peaked at stage Ⅱ (peak 1) then decreased, followed by obvious augmentation of peach fruit size and weight. Similar trend was appeared at stage Ⅶ, with the peak value of 19.2 ng/g
an
(peak 2). The remarkable third peak at stage Ⅹ (144.7 ng/g) occurred during the mid-ripe period. Afterwards, full ripening (i.e. stage Ⅺ) was initiated and presumably promoted.
M
Therefore, the accumulation of endogenous ABA was considered to be an important signal to accelerate fruit ripening. Stages Ⅱ, Ⅳ and Ⅶ corresponded to the physiological
te
d
fruit-dropping periods, which suggested the increase of ABA would lead to fruit drop. The above assumptions were consistent with those reported in Roxburgh rose [8], and loquat
Ac ce p
[10].
3.5.3. Recovery experiment
To examine the reliability of the proposed assay, peach pulp at stage Ⅲ of first fast
growth period was selected for a recovery experiment. After initial sample preparation and extraction, the dilution solution was used as the sample matrix with little signal response of SA and ABA. Known amounts of analytes (0.8, 5.0, 10.0 ng/mL) were added to matrix prior to DLLME extraction and total SA and ABA were quantified by LC-MS/MS. For each addition level, three replicate tests were carried out, and the results were listed in Table 6. Recoveries were in the range of 87.5-119.0%, with RSDs less than 12.6%, suggesting that
14
Page 14 of 29
this method was acceptable to be a routine analytical protocol for plant hormones. 4. Conclusion Both SA and ABA were indentified by ITMS and MRM technique. The MS/MS had the
ip t
advantages of high sensitivity and powerful qualitative ability, which had increasing become
cr
a versatile tool to analyze trace plant hormones. The present work offered a new purification
us
protocol to extract SA and ABA from plant tissues. The DLLME method was faster and simpler, reliable with a low amount of plant material, and did not require much organic
an
solvents. Moreover, the enrichment effect of this technology afforded low level LODs of SA and ABA, making it possible to analyze these two hormones from plant samples with
M
convenience and sensitivity.
Our methodology enabled analyzing SA and ABA contents in peach fruit. Furthermore,
te
d
physiological phenomena such as increased volume and weight of peach and fruit drop were related to the endogenous contents of the above two hormones. Our results suggested that
Ac ce p
SA and ABA play important roles in fruit growth and development, which will provide a better understanding of multiple plant hormone crosstalk and interplay in physiological processes.
Acknowledgment
The authors are grateful to the National Nature Sciences Foundation of China (21275029), the National Basic Research Program of China (No.2010CB732403), National Key Technologies R&D Program of China during the 12th Five-Year Plan Period (2012BAD29B06), the Natural Science Foundation of Fujian Province (2010J05021), the
15
Page 15 of 29
Program for Changjiang Scholars and Innovative Research Team in University (No.
References
us
[1] H. S. Li, Modern plant physiology, Beijing, 2nd ed., 2006.
cr
ip t
IRT1116), and Agilent Technologies.
[2] P. McSteen, Y. Zhao, Plant hormones and signaling: common themes and new
an
developments, Dev. Cell 14 (2008) 467-473.
Opin. Plant Biol. 6 (2003) 470-479.
M
[3] A. Himmelbach, Y. Yang, E. Grill, Relay and control of abscisic acid signaling, Curr.
[4] J. P. Métraux, Recent breakthroughs in the study of salicylic acid biosynthesis, Trends in
te
d
Plant Science 7 (2002) 332-334.
[5] Z. X. Tian, Y. X. Zhang, Y. J. Yu, J. F. Xu, Effects of salicylic acid on PG, PME and
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respiration in Ya-li pears, J. Fruit Sci. 19 (2002) 381-384. [6] Z. X. Tian, Y. X. Zhang, Studies on the effect of salicylic acid on ripening of “Starkrimson” apple fruits, Acta Horticulture Sinica 28 (2001) 557-559. [7] M. K. Srivastava, U. N. Dwivedi, Delayed ripening of banana fruit by salicylic acid, Plant Sci. 158 (2000) 87-96.
[8] W. G. Fan, H. M. An, G. Q. Liu, S. T. He, J. P. Liu, Changes of endogenous hormones contents in fruit, seeds and their effects on the fruit development of Rosa roxburghii, Sci. Agric. Sinica 37 (2004) 728-733. [9] Z. X. Guo, D. M. Pan, L. X. Lv, Studies on the mechanism of fruit growth in Nai
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(Prunus salicina Lindl var.), Chin. Agric. Sci. Bull. 22 (2006) 330-334. [10] J. X. Cai, G. Q. Liu, W. Chen, Changes in endogenous polyamine and hormone contents during different fruit developing stages in loquat, Chin. Agric. Sci. Bull. 23 (2007)
ip t
317-322.
cr
[11] B. H. Liu, Y. M. Jiang, F. T. Peng, J. Sui, F. X. Zhao, H. Y. Wang, The dynamic
us
changes of endogenous hormones in sweet cherry (Prunus avium L.) pulp, Acta Horticulture Sinaca 34 (2007) 1535-1538.
an
[12] Y. Q. Cao, P. Leng, Z. G. Yan, J. Ren, Role of abscisic acid in fruit ripening of peach, Acta Horticulture Sinaca 36 (2009) 1037-1042.
M
[13] S. Croubels, A. Maes, K. Baert, P. D. Backer, Quantitative determination of salicylic acid and metabolites in animal tissues by liquid chromatography-tandem mass spectrometry,
te
d
Anal. Chim. Acta 529 (2005) 179-187.
[14] Q. M. Lu, L. Zhang, T. W. Chen, M. H. Lu, G. N. Chen, Content change of endogenous
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plant hormones under salt stress by liquid chromatography-tandem mass spectrometry, Sci. in China Series B: Chemistry 39 (2009) 785-792. [15] Y. Izumi, A. Okazawa, T. Bamba, A. Kobayashi, E. Fukusaki. Development of a method for comprehensive and quantitative analysis of plant hormones by highly sensitive nanoflow liquid chromatography-electrospray ionization-ion trap mass spectrometry, Anal. Chim. Acta 648 (2009) 215-225. [16] X. Q. Pan, X. M. Wang, Profiling of plant hormones by mass spectrometry, J. Chromatogr. B 877 (2009) 2806-2813. [17] H. T. Liu, Y. F. Li, T. G. Luan, C. Y. Lan, W. S. Shu, Simultaneous determination of
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phytohormones in plant extracts using SPME and HPLC, Chromatographia 66 (2007) 515-520. [18] C. Zadra, A. Borgogni, C. Marucchini, Quantification of jasmonic acid by SPME in
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tomato plants stressed by ozone, J. Agric. Food Chem. 54 (2006) 9317-9321.
cr
[19] Y. L. Wu, B. Hu, Simultaneous determination of several phytohormones in natural
us
coconut juice by hollow fiber-based liquid-liquid-liquid microextraction-high performance liquid chromatography, J. Chromatogr. A 1216 (2009) 7657-7663.
an
[20] M. Rezaee, Y. Assadi, M. R. M. Hosseini, E. Aghaee, F. Ahmadi, S. Berijani,
J. Chromatogr. A 1116 (2006) 1-9.
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Determination of organic compounds in water using dispersive liquid-liquid microextraction,
[21] Q. M. Lu, L. H. Chen, M. H. Lu, G. N. Chen, L. Zhang, Extraction and analysis of plants
using
dispersive
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in
liquid-Liquid
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followed
by
te
auxins
high-performance liquid chromatography with fluorescence detection, J. Agric. Food Chem.
Ac ce p
58 (2010) 2763-2770.
[22] G. Vishal, K. Manoj, B. Harshad, R C.R.K., S. Abhiram, J. Bhavanath, Simultaneous determination of different endogenetic plant growth regulators in common green seaweeds using dispersive liquid-liquid microextraction method, Plant Physiol. Biochem. 49 (2011) 1259-1263.
[23] X. H. Zang, Q. H. Wu, M. Y. Zhang, G. H. Xi, Z. Wang, Developments of dispersive liquid-liquid microextraction technique, Chin. J. Anal. Chem. 37 (2009) 161-168.
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Page 18 of 29
ip t
Figure legends
cr
Fig. 1 MS/MS spectra of SA and ABA.
an
263 > 219, 153) and concentration was 0.1 μg/mL.
us
The CID voltages were 0.7 V (SA, transition 137 > 93) and 0.6 V (ABA, transition
Fig. 2 Selection of extraction solvent (A) and disperser solvent (B).
M
Sample concentration was 0.1 μg/mL. Other conditions in Fig. 2A were 50 µL extraction solvent, 700 μL THF, extraction time 0.1 min, centrifugation at 4500 rpm
te
d
for 3.0 min. Other conditions in Fig. 2B were 50 µL CHCl3, 700 μL disperser solvent,
Ac ce p
extraction time 0.1 min, centrifugation at 4500 rpm for 3.0 min.
Fig. 3 SA (A) and ABA (B) contents at different development stages of peach. Values are means of three replicate samples.
19
Page 19 of 29
Table 1. Optimization of HPLC conditions column temperature, ℃
flow rate,
ACN proportion,
mL min-1
%
Test indexes Rs min
35
0.7
35
5.62
2
30
0.6
35
5.46
3
25
0.5
35
5.69
4
30
0.7
40
5
25
0.6
40
6
35
0.5
40
7
25
0.7
45
8
35
0.6
9
30
0.5
2.70 3.15 3.86
cr
1
tR max
ip t
Exp#
2.38
4.61
2.88
4.93
3.30
4.25
2.15
45
4.35
2.50
45
4.52
3.01
Ac ce p
te
d
M
an
us
1.40
20
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Table 2. Optimization of DLLME sedimented phase volume Exp#
CHCl3 volume, μL
THF volume, μL
NaCl amount, %
Signal response SA
ABA
30
500
4.5
255408
71838
2
30
600
3.0
243417
69749
3
30
700
1.5
270940
71268
4
30
800
0
5
40
500
3.0
6
40
600
7
40
700
8
40
800
9
50
500
10
50
11
50
12
50
13
60
14
60
15 16
cr
ip t
1
122800
213844
36727
4.5
213217
76644
0
194497
88379
1.5
134030
68929
1.5
69198
34169
600
0
115029
51529
700
4.5
53004
34633
3.0
62477
49145
500
0
46027
48051
600
1.5
46700
68929
60
700
3.0
37094
40155
60
800
4.5
28066
41262
d
M
an
us
377522
Ac ce p
te
800
21
Page 21 of 29
Table 3. Analytical performance data for two analytes in spiked water
SA
Linear range
(m/z)
(ng/mL)
93.0 219.0, 153.0
Calibration curve
R
LOD (ng/mL)
0.5-50
Y = 656275x+982.2
0.9995
0.2
0.2-20
Y = 4734251x+571.3
0.9999
0.1
Ac ce p
te
d
M
an
us
cr
ABA
Quantitation
ip t
Analyte
22
Page 22 of 29
Table 4. The goodness of fit for regression lines of SA and ABA (α=0.05) yi
ŷi
1
0.0005
917
1310.37
2
0.0008
1391
1507.25
3
0.001
2034
1638.51
4
0.002
2781
5
0.008
6268
6
0.01
7475
7
0.02
13567
8
0.05
33999
SSE
900846.71
SSR
SST
864438498.00
r
0.9995
F-statistic
5751.51
t-statistic
75.84
No.
xi
1
ABA
2294.78
cr
6232.43 7544.98
us
14107.73 33795.98
863537651.29
an M
n
8
F0.05
7.71
t0.025
2.7765
yi
ŷi
0.0002
1558
1518.14
0.0005
2908
2938.42
Ac ce p
2
ip t
xi
d
SA
No.
te
Analyte
3
0.0008
4389
4358.69
4
0.001
4999
5305.54
5
0.002
10015
10039.79
6
0.008
39457
38445.30
7
0.01
47249
47913.80
8
0.02
95201
95256.31
SSE
1566573.75
SSR
7713453780.25
SST
7715020354.00
r
0.9999
n
8
F-statistic
29542.64
F0.05
7.71
t-statistic
171.88
t0.025
2.7765
23
Page 23 of 29
Table 5. Peach fruit growth period and some data of growth rates in diameter Growth period
Days of growth lasted
the first fast growth period
stages Ⅰ- Ⅲ
28
the slow growth period (stone hard period)
stages Ⅳ- Ⅵ
28
the second fast growth period
stages Ⅶ- Ⅺ
period
flowering
Fruit (cm) Transverse
The first
diameter
cr
Days after
35
Vertical
Kernel (cm)
Transverse
us
Growth
ip t
Developmental period
Vertical
diameter
diameter
diameter
1.60±0.17
0.46±0.09
0.76±0.11
Stage Ⅰ
7
0.95±0.15
period
Stage Ⅱ
21
1.64±.027
2.02±0.32
0.61±0.13
0.94±0.15
Stage Ⅲ
35
2.02±0.31
2.71±0.39
0.91±0.18
1.21±0.17
Ac ce p
te
d
M
an
fast growth
25
Page 24 of 29
Table 6. Recoveries of SA and ABA in spiked samples (n = 3)
(ng/mL)
(ng/mL)
（%）
0.8
0.70±0.05
87.5±6.2
5.0
4.56±0.11
91.2±2.2
10.0
9.79±0.40
97.9±4.0
0.8
0.95±0.01
119.0±1.2
5.0
4.53±0.08
90.6±1.6
10.0
10.89±1.26
SA
108.9±12.6
Ac ce p
te
d
M
an
ABA
Recovery
ip t
Found
cr
Added
us
Compound
26
Page 25 of 29
Highlights specifications: (1) DLLME assay was adopted to concentrate salicylic acid and abscisic acid.
cr
(3) Small amounts of real material were required for analysis.
ip t
(2) This DLLME-LC-ITMS approach was sensitive with LODs at ng/mL level.
Ac ce p
te
d
M
an
us
(4) Content changes of salicylic acid and abscisic acid in peach were revealed.
27
Page 26 of 29
Ac
ce
pt
ed
M
an
us
cr
i
Figure1
Page 27 of 29
Ac
ce
pt
ed
M
an
us
cr
i
Figure2
Page 28 of 29
Ac
ce
pt
ed
M
an
us
cr
i
Figure 3
Page 29 of 29
