A selective pretreatment method for determination of endogenous active brassinosteroids in plant tissues: double layered solid phase extraction combined with boronate affinity polymer monolith microextraction
© Ding et al.; licensee BioMed Central Ltd. 2013
Received: 20 February 2013
Accepted: 10 April 2013
Published: 18 April 2013
Brassinosteriods (BRs), a group of important phytohormones, have various effects on plant growth and development. However, their physiological functions in plants have not been fully understood to date. Endogenous BRs in plant tissue are extremely low and the elucidation of BRs functions relies on sensitive detection method. Reported methods for the determination of BRs required large amount of plant tissue, tedious pretreatment process, and were lack of selectivity. Therefore, development of a simple and selective method for the sensitive quantification of BRs is highly needed.
We established a pretreatment method of BRs in plant tissues by employing double layered solid phase extraction (DL/SPE) combined with boronate affinity polymer monolith microextraction (BA/PMME). After the initial depigmentation with DL/SPE cartridge, BA/PMME was employed to selectively extract BRs from sample matrix. Uniquely, most sample matrix was successfully removed by BA monolith purification. Using this method, BRs was determined by liquid chromatography-mass spectrometry (LC-MS). Endogenous active BRs could be detected in only 1 g fresh weigh (FW) leaves or 0.5 g FW flower tissues.
A DL/SPE-BA/PMME pretreatment method for the determination of endogenous brassinosteroids in plant tissues was developed and validated. The proposed method was sensitive and selective. Besides, it may be further developed for the determination of other BRs including their precursors and conjugates.
Brassinosterods (BRs), confirmed as the sixth plant hormone, are a group of naturally occurring polyhydroxy steroids. Since the first BR was discovered in 1970, around 60 natural occurrence of BRs have been reported with wide occurrence in the plant kingdom. BRs have multiple functions on various physiological and metabolic processes and normally occur in extremely low concentration. Many biologists have been dedicated to the researches on signal transduction, biosynthesis, degradation and metabolic pathway of BRs[3–5]. The studies of BRs functions rely on the availability of selective and sensitive method for the quantification of endogenous BRs in plant tissues.
As for phytohormone analysis, liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) is a dominant analytical technique due to their high selectivity and sensitivity[6–8]. However, the concentrations of endogenous BRs in plant tissue are extremely low, and matrix of plant extracts are complicated. Therefore, direct analysis of BRs in plant matrix by LC-ESI-MS/MS is unpractical. To circumvent this problem, it is necessary to develop an effective pretreatment method to eliminate the interference of the sample matrix. Up to date, liquid-liquid extraction (LLE), solid phase extraction (SPE), magnetic solid phase extraction (MSPE), solid phase microextraction (SPME), HPLC purification or their combinations were extensively employed in BR pretreatment[9–13]. However, most of the previously reported methods were tedious, solvent-consuming, and required large amount of plant materials (more than 5 g fresh weight (FW)). Besides, BRs were normally isolated from plant extract based on hydrophobic or hydrophilic interaction; thus it was inevitable that some other similar components would be co-extracted with BRs, which burdens the following liquid chromatographic separation and suppresses the response signal of target analytes during mass spectrometry analysis. Therefore, it’s essential to develop a selective and sensitive sample pretreatment method.
Results and discussion
Five active forms of BRs (BL, CS, 28-norBL, 28-homoBL, 28-norCS) were chosen as targets to evaluate the performance of our analytical method. Among them, BL, CS and 28-norCS represented the most important BRs because of their wide distribution as well as their potential biological activity.
Optimization of PMME conditions
BA/PMME is the core step in the whole sample preparation procedure. In order to obtain high extraction efficiency and good purity of BRs in plant matrix, conditions of PMME process were optimized, including pH of sampling solution, acetonitrile content in sampling solution, sampling rate, type of desorption solvent, acetonitrile content in desorption solvent and desorption rate.
BRs are hydrophobic molecules with polar groups; thus acetonitrile content in sampling solution would affect the solubility of BRs in aqueous buffer, while high content of acetonitrile might affect the reaction efficiency of BRs with boronate affinity monolith. Therefore, acetonitrile content in sampling buffer was investigated in the range of 0-80% (v/v). As shown in Figure 4B, the extraction efficiencies increased from 0 to 20%, and then dropped with the increase of acetonitrile content from 20% to 80%, which suggested that with 20% acetonitrile content in the sampling solution, BRs were endowed with fair solubility and the boronic acid-BR reaction efficiencies were acceptable. So 20% acetonitrile was adopted in the following experiments.
Since the boronate affinity extraction was accomplished through covalent bond, the reaction efficiency might be related to the sampling flow rate. The flow rate was investigated in the range of 50–150 μL/min. The result indicated that the sampling flow rate has minor influence on the extraction efficiencies (Figure 4C), and higher flow rate may cause the damage of the column due to the high pressure. Therefore, the flow rate of 100 μL/min was selected.
The effect of flow rate on desorption efficiency was also investigated in the range of 50–150 μL/min (Figure 5D). No remarkable effect of the flow rate was observed onto the desorption efficiencies. And the desorption rate of 100 μL/min was selected.
Taken together, sample was dissolved in acetonitrile/buffer (1/4, v/v, pH 9.0, 1 mL), and loaded onto the boronate affinity monolith at the flow rate of 100 μL/min. After washing with the washing solution of acetonitrile/buffer (1/4, v/v, pH 9.0, 0.5 mL), the extracted BRs were desorbed with 3% H2O2 in acetonitrile/water (9/1, v/v, 0.6 mL) at the flow rate of 100 μL/min.
Comparison of two pretreatment methods
Linearity, LODs and LOQs of BRs
Linear range ng/mL (ng)
LOD ng/mL (pg)
LOQ ng/mL (pg)
Precisions (intra- and inter-day) and recoveries of BRs in O. sativa seedlings (1 g FW)
Intraday precision (RSD, %, n = 4)
Interday precision (RSD, %, n = 3)
Recovery (%, n = 4)
Low (1 ng/g)
Medium (5 ng/g)
High (25 ng/g)
Low (1 ng/g)
Medium (5 ng/g)
High (25 ng/g)
Low (1 ng/g)
Medium (5 ng/g)
High (25 ng/g)
Determination of BRs in real samples
Amount of endogenous BRs in different plant tissues
O. sativa YTA shoot
O. sativa YTB shoot
B. napus L. shoot
B. napus L. flower bud
B. napus L. flower
15.84 ± 0.78
89.80 ± 7.41
1.86 ± 0.11
1.33 ± 0.22
0.30 ± 0.05
13.54 ± 0.20
11.42 ± 1.54
6.14 ± 0.18
5.13 ± 0.15
12.36 ± 0.56
In this study, a DL/SPE-BA/PMME-HPLC-ESI-MS/MS method for the determination of endogenous BRs in plant tissues was developed. Coupled with the previous established DL/SPE, DL/SPE-BA/PMME exhibited good purification efficiency towards BR. Compared with previously reported methods, the method proposed in this study is highly selective and solvent-saving. In addition, the endogenous BRs can be detected in 1 g (FW) leaves or 0.5 g (FW) flower tissues.
It is worth noting that the method developed here is not limited to the quantification of the above mentioned five BRs. Theoretically, all the BRs, along with their precursors and conjugates, could be analyzed using the same method because all these cis-diol-containing targets possess the similar specific affinity towards BA monoliths.
Reagents and materials
BR standards:28-norbrassinolide (28-norBL), 28-norcastasterone (28-norCS), brassinolide (BL), castasterone (CS), 28-homobrassinolide (28-homoBL), and stable isotope-labeled standards, [2H3]brassinolide and [2H3]castasterone, were the current commercially available BR standards, and were all purchased from Olchemim Ltd. (Olomouc, Czech Republic).
Acetonitrile and methanol with HPLC grade were purchased from Tedia Co. (Fairfield, OH, USA) and Merck (Darmstadt, Germany), respectively. Formic acid (FA, AR), NaCl, anhydrous MgSO4 (AR), Disodium hydrogen orthophosphate (AR), Azobisisobutyronitrile (AIBN), poly(ethylene glycol) (PEG) with molecular weights of 20,000 were bought from Sinopharm Chemical Reagent (Shanghai, China). 3-acrylamidophenylboronic acid (AAPBA) was purchased from J&K Scientific Ltd. (Beijing, China). Deionized water was obtained from a Millipore Milli-Q water purification system (Milford, MA, USA).
HiCapt GCB/PSA double layered SPE cartridge (100 mg GCB/500 mg PSA, 6 mL) was obtained from Weltech Co. (Wuhan, China). Poly(AAPBA-co-EDMA) boronate affinity monolith was prepared according to the previous work. Briefly, 30 mg functional monomer AAPBA, 70 mg cross-linker EDMA, 265 mg methanol, 35 mg PEG 20000 and 1 wt% AIBN was homogeneously mixed in a centrifugetube and degassed by ultra-sonication. Then the prepolymerization solution was filled into fused-silica capillaries (530 mm ID) which were activated and modified with 3-(triethoxysilyl)propylmethacrylate. After sealing at both ends with silica rubber, the capillaries were polymerization at 60°C for 16 h. At last, the prepared monolith was washed with methanol with a pump.
Rice (O. sativa, O. sativ a YTA and O. sativa YTB) shoots were harvested upon 4 months growing in the field. Three-month-old rape (B. napus) leaves and flowers were also harvested from the field. All the plant materials were weighted, immediately frozen in liquid nitrogen, and then stored at −80°C till analysis.
DL/SPE-BA/PMME process for plant samples
Sample pretreatment process was shown in Figure 3. Plant tissue (1 g FW leaves, or 0.5 g FW flower tissue) was frozen in liquid nitrogen and grounded into fine powder with a mortar and pestle, and then transferred into a 10-mL centrifuge tube. Stable isotope labeled BRs [2H3]BL (2 ng) and [2H3]CS (2 ng) were added into the mixture followed by extraction with acetonitrile (5 mL/g) overnight at −20°C. The extraction, dehydration and DL/SPE were performed according to previous reported method. Briefly, the acetonitrile exacted sample was centrifuged at 10,000 rpm under 4°C for 10 min. Then the supernatant was collected and the rest plant residue was re-extracted with 1 mL acetonitrile. After combining the two parts of solution, NaCl (250 mg/g FW) was added and vortexed for several minutes to induce phase separation. Anhydrous MgSO4 (500 mg/g FW) was added into the upper layered acetonitrile to remove residual water. After centrifugation at 10,000 rpm under 4°C for 10 min, the supernatant was collected and passed through a GCB/PSA double layered SPE cartridge (100 mg/ 500 mg, GCB/PSA) which was pre-conditioned with acetonitrile (6 mL), and the eluate was collected. The residues of BRs on the SPE cartridge were desorbed with methanol:acetonitrile (1:1, v/v, 2 mL). Then the two parts of the eluates were combined and evaporated under mild nitrogen stream followed by reconstituting in acetonitrile/buffer (1/4, v/v, pH 9.0, 1 mL). The reconstituted solution was loaded onto the previously activated boronate affinity monolith at the flow rate of 100 μL/min. After washing with acetonitrile/buffer (1/4, v/v, pH 9.0, 0.5 mL), the extracted BRs were desorbed with 3% H2O2 in acetonitrile/water (9/1, v/v, 0.6 mL) at the flow rate of 100 μL/min. The desorption solution was collected and evaporated under mild nitrogen stream followed by re-dissolving in 100 μL methanol/H2O ( 65/35, v/v), and 80 μL of the solution was used for the quantification of BRs by HPLC-ESI-MS/MS.
Instrument and analytical conditions
The instrument and analytical conditions were identical to the previous work. Analysis of BRs were performed on a HPLC-ESI-MS/MS system consisting of a AB SCIEX 3200 QTRAP MS/MS (Applied Biosystems, Foster City, CA, USA) with an ESI source (Turbo Ionspray), and a Shimadzu LC-20 AD HPLC system (Tokyo, Japan), which was equipped with two LC-20 AD pumps, a SIL-20A auto sampler, a CTO-20 AC column thermostat, and a DGU-20A3 degasser. The separation was achieved on a shim-pack ODS column (15 cm × 2.0 mm id, 5 μm, Shimadzu, Tokyo, Japan). The column oven temperature was set at 40°C. Binary mobile phase was used. Solvent A is 0.1% formic acid in water (v/v) and solvent B is acetonitrile. A gradient of 50 min 30–65% B, 10 min 100% B, 10 min 30% B at a flow rate of 0.2 mL/min was used. Data acquisition and processing were achieved with AB SCIEX Analyst 1.5 software.
All BRs were quantified by multiple reaction monitoring (MRM) mode in positive mode. The optimal ESI source conditions were as follows: turbo heater temperature (TEM) 400°C, ion spray voltage 5500 V, curtain gas 40 psi, nebulizing gas (gas 1) 50 psi and heated gas (gas 2) 80 psi. The collision energy (CE) and entrance potential (EP) was separately set at 20 V and 10 V. The mass transitions of BRs, optimal declustering potential (DP) and collision cell exit potential (CXP) were the same as our previously study (see supporting information).
The authors thank the financial support from the National Natural Science Foundation of China (91017013, 91217309, 31070327, 21205091, 21228501), the Fundamental Research Funds for the Central Universities.
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