Dispersive Matrix Solid-Phase Extraction Method Coupled with High

Article Cite This: J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Dispersive Matrix Solid-Phase Extraction Method Coupled with High Performance Liquid Chromatography-Tandem Mass Spectrometry for Ultrasensitive Quantification of Endogenous Brassinosteroids in Minute Plants and Its Application for Geographical Distribution Study Yuxuan Li,†,‡,1 Ting Deng,†,‡,1 Chunfeng Duan,*,† Lanxiu Ni,†,‡ Nan Wang,†,‡ and Yafeng Guan*,† J. Agric. Food Chem. Downloaded from pubs.acs.org by WASHINGTON UNIV on 03/05/19. For personal use only.

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CAS Key Laboratory of Separation Sciences for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhongshan Road, Dalian 116023, P.R. China ‡ University of Chinese Academy of Sciences, Beijing 100039, P.R. China S Supporting Information *

ABSTRACT: An ultrasensitive analysis method for quantification of endogenous brassinosteroids in fresh minute plants was developed based on dispersive matrix solid-phase extraction coupled with high performance liquid chromatography-tandem mass spectrometry. During the dispersive matrix solid-phase extraction, plant samples were first ground with solid sorbent (dispersant) in one microcentrifuge tube and then centrifuged after adding extraction solvent and cleanup materials (another type of sorbent). Three protocols based on dispersive matrix solid-phase extraction were compared and discussed for plant samples with different matrix complexity. The choice of any protocol was a compromise of increasing purification efficiency and decreasing sample loss. Under optimized conditions, the limits of detection were 1.38−6.75 pg mL−1 for five brassinosteroids in the oilseed rape samples. The intraday and interday precisions were in the range of 0.8%−9.8% and 4.6%−17.3%, respectively. The proposed method was successfully applied to detection of endogenous brassinosteroids in milligram oilseed rape (2.0 mg) and submilligram Arabidopsis thaliana seedlings (0.5 mg). Finally, the geographical distribution of five endogenous brassinosteroids of Brassica napus L. oilseed rape in different provinces of origin in the Yangtze River basin was described. KEYWORDS: dispersive matrix solid-phase extraction, endogenous brassinosteroids, oilseed rape, Arabidopsis thaliana seedlings, geographical distribution

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INTRODUCTION Brassinosteroids (BRs) have been considered as the sixth class of plant hormones since first found by Mitchell et al. in 1970.1 They have extraordinary biological effects on plant growth and development, such as cell division and differentiation,2,3 root growth, development and symbiosis,4,5 cold resistance,6 and stress responses.7 To understand the physiological mechanism of BRs in plants, it is important to determine the content and distribution of BRs in different plant tissues. Thus, it is necessary to develop sensitive and reliable methods for quantification of endogenous BRs in minute plant samples. Because of the extremely low concentration of endogenous BRs in plants (only 1−100 ng g−1 fresh weight (FW) in pollen and immature seeds and even lower in the mature stems and leaves8) and the lack of identifiable functional groups for detection, quantification of BRs in plants is a great challenge. Nowadays the widely used analytical methods for BRs include gas chromatography−mass spectrometry (GC-MS),9 liquid chromatography coupled with tandem mass spectrometry (LCMS/MS),10−13 and high or ultraperformance liquid chromatography (HPLC/UPLC) coupled with ultraviolet (UV) or fluorescence detectors.14,15 Among them, liquid chromatography-tandem mass spectrometry (LC-MS/MS) in multiple reaction monitoring (MRM) mode is one of the most suitable and predominant analytical methods for phytohormones © XXXX American Chemical Society

analysis, due to the efficient separation performance of LC and high selectivity and sensitivity of MS/MS.10,13 Before the instrumental analysis, sample preparation is essential to extract and enrich BRs from the plants and remove impurities, which has important influence on the overall performance of the analytical method because of the complex matrix of plant samples. With wide application of solid phase extraction (SPE) for sample preparation of target molecules and ions,16−24 it has been used for plant sample preparation,25 as well as its variations including solid-phase microextraction (SPME),26 immunoaffinity extraction,27 magnetic solid-phase extraction28 and so on. For the analysis of BRs, two or more extraction processes are usually acquired to remove the complicated matrix. Izumi et al.29 compared the method using both Oasis HLB and Oasis MCX column with that using only Oasis HLB column. Much better results were obtained when using two SPE columns for purification and enrichment. Ding et al.30 successfully developed a pretreatment method based on the combination of double layered solid phase extraction and boronate affinity polymer monolith microReceived: December 26, 2018 Revised: February 13, 2019 Accepted: February 22, 2019

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DOI: 10.1021/acs.jafc.8b07224 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

Figure 1. Schematic illustration of the DMSPE-HPLC-MS/MS method (A) for oilseed rape; (B) for Arabidopsis thaliana seedlings.

extraction. Schäfer et al.31 used multistep solid phase extraction combined with UPLC-ESI-MS/MS for the analysis of more than 100 plant hormones (including their primary and secondary metabolites) in different plant organs. Our previous work reported a pipet-tip SPE (PT-SPE) method to quantify BRs in minute plant samples without further SPE step.13 However, these methods usually acquired long-time even overnight solvent extraction before solid phase-based extraction. Since first reported in 1989 by Barker,32 matrix solid-phase dispersion (MSPD) has been widely used for pretreatment of viscous, semisolid, and solid samples, such as food, drug, and plant tissues.33,34 The samples were ruptured completely due to the shear force generated by mechanical grinding, which promoted the dissolution and extraction of the targets.35 Compared with SPE and SPME, MSPD procedure provides simultaneous disruption and homogenization of samples, together with extraction and purification in only a single step.36 Since solid sample was directly mixed with dispersant and ground together in MSPD process, the longtime solvent extraction could be avoided.37,38 In our present work, MSPD was successfully used for extraction of gibberellins (GAs) in Arabidopsis thaliana samples.39 In another work, an in-line MSPD-tandem SPE method was developed and coupled with HPLC-MS/MS for quantification of six endogenous BRs in 50 mg FW of rice samples.10 An issue of MSPD is the inevitable sample loss in the transfer process of the ground semidry mixture from mortar to the empty SPE cartridge, which decreases the sensitivity of measurement, especially for minute samples of several or sub mg. In our latest work,12 a microscale MSPD method was proposed, in which the dispersant and the plants were ground together in one microcentrifuge tube, followed by solvent elution in the same tube without transfer of the sample. This method was successfully applied to analysis of GAs in Arabidopsis thaliana samples. However, for other low-content hormones such as BRs, further cleanup is still demanded because of the limited selectivity of MSPD. On the other hand, dispersive solid-phase extraction (dSPE) has been used as a fast, reliable pretreatment method mainly for the pesticide residue.40 The adsorbent is directly dispersed in the extractant of sample for cleanup, followed by a simple centrifugation to separate the two phases. From this

aspect, d-SPE is a good substitute for the elution step of MSPD that needs packing the ground mixture of dispersant and samples into the cartridge with solvent elution. Therefore, a new extraction method, named dispersive matrix solid-phase extraction (DMSPE) is proposed based on the combination of MSPD and d-SPE. During the DMSPE, solid samples are first ground with solid sorbent (dispersant) in one microcentrifuge tube and then centrifuged after adding extraction solvent and cleanup materials, i.e., another type of sorbent. The DMSPE combines the mechanical grinding of sample and sorbent (the front portion of MSPD) with the fast cleanup by centrifugation (the back portion of d-SPE). This combination avoids the sample transfer and packing step in MSPD and the longtime solvent extraction in d-SPE, which will minimize the sample loss and simplify the sample preparation process. In this work, a sample preparation method based on DMSPE was developed for BRs detection in milligram or submilligram FW plant samples. The plant segments and dispersant were ground together, followed by extraction and cleanup under centrifugation condition. Different protocols of DMSPE for different plant matrix were discussed in detail. After DMSPE, 4-borono-N,N,N-trimethylbenzenaminium iodide (BTBA) was used for derivatization of BRs, which showed high MS response in our previous work.13 The proposed DMSPE method was coupled with HPLC-MS/MS for quantification of five endogenous BRs in milligram (FW) oilseed rape and submilligram (FW) Arabidopsis thaliana seedlings. Finally, the analytical method was applied to geographical profile of endogenous BRs of oilseed rape in different provinces in the Yangtze River basin.

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MATERIALS AND METHODS

Chemicals and Reagents. 24-epibrassinolide (24-epiBL), 24epicastasterone (24-epiCS), 6-deoxo-24-epicastasterone (d-epiCS), teasterone (TE) and typhasterol (TY) of purity >98% were purchased from Olchemim Ltd. (Olomouc, Czech Republic). Stable isotope internal standards [2H3] 24-epibrassinolide (d3-24-epiBL) and [2H3] 24-epicastasterone (d3-24-epiCS) of purity >95% were acquired from Olchemim Ltd. (Olomouc, Czech Republic). Stock solutions of all of the brassinosteroids and d3-brassinosteroids were prepared at 100 μg mL−1 in acetonitrile (ACN) and then stored at −20 °C. 4-BoronoN,N,N-trimethylbenzenaminium iodide (BTBA) was the product synthesized by Wylton Chem Co. (Guiyang, China). C18 silica sorbent (50 μm, Dikma, U.S.A.) and C8 silica sorbent (50 μm, Daiso, B


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Figure 2. Three pretreatment protocols of DMSPE method. Conditions: mass ratio, plant sample:C8:MAX:MCX was 1:4:1:1; extraction solution, 200 μL of 80% (v/v) ACN; vibration time, 5 min; centrifugation time, 10 min. Japan) were used for this work. Mixed mode anion exchange absorbent (MAX) and mixed mode cation exchange absorbent (MCX) were bought from CNW (Duesseldorf, Germany). ACN of HPLC grade from Tedia Inc. (Fairfield, OH, U.S.A.) and analyticalgrade pyridine from Kermel Chemical Reagent Co. (Tianjin, China) were used. Formic acid (chromatographic grade) was taken from Sigma (St. Louis, U.S.A.). Wahaha purified water (Hangzhou, China) was used throughout the experiments in this work. Sample Collection. Oilseed rape was supported by Shengpu and Suhu Seed Industry Co. Ltd. (Jiangsu, China). The seed samples were stored at room temperature with dry and airtight conditions. Seeds of Arabidopsis thaliana ecotype Columbia-0 (Col-0) were supplied from Beijing Institute of Botany, Chinese Academy of Sciences. The seeds were pretreated and sown according to the previous report.40 The 12day-old seedlings were collected for this work. Sample Preparation Process. The sample preparation of oilseed rape by DMSPE was employed as described in Figure 1A. Briefly, each sample was premixed and homogenized using a tissue homogenizer (Jintan District Xicheng Xinrui Instrument Factory, Jiangsu, China) before DMSPE. The homogeneous oilseed rape samples (2.0 mg FW) were mixed with 8 mg of C8 silica sorbent prewashed with methanol (10 mL g−1) in a prechilled microcentrifuge tube (2 mL, Eppendorf AG, Hamburg, Germany). The mixture was ground with a homemade cylindrical quartz pestle (80 mm × 3 mm i.d.) at once under liquid nitrogen condition. For accurate quantification of BRs, d3-24-epiBL (5 pg) and d3-24-epiCS (5 pg) were added as internal standards (IS) to the tube containing the frozen plant material. Then 200 μL of cold ACN (80% v/v, water) was added, and the samples were centrifuged at 10 000 rpm for 10 min at 4 °C. A total of 2 mg of MAX and 2 mg of MCX were added to the collected supernatant. Then the samples were vibrated at 1500 rpm for 5 min and centrifuged at 10 000 rpm for 10 min at 4 °C. The supernatant was collected and then evaporated for dryness using a mild nitrogen stream, and then reconstituted for the subsequent derivatization. With minor modification of the preparation process of oilseed rape above, the Arabidopsis thaliana seedlings (0.5 mg FW) were first mixed in a prechilled microcentrifuge tube with 2 mg of C8 silica sorbent that was prewashed with methanol (10 mL g−1). Immediately, the mixture was ground using a homemade cylindrical quartz pestle in liquid nitrogen. Isotope internal standards were also used. Then 200 μL of cold ACN (80% v/v, water), 0.5 mg of MAX, and 0.5 mg of MCX were added. The mixture was vibrated at 1500 rpm for 5 min and centrifuged at 10 000 rpm for 10 min at 4 °C. The supernatant was collected and then evaporated for dryness using a mild nitrogen

stream and then reconstituted for the subsequent derivatization (Figure 1B). A total of 50 μL of 1.0 mg mL−1 BTBA derivatization solution that contained 2% (v/v) pyridine in acetonitrile was used for the reconstitution in the end of DMSPE. The derivatization was carried out at 80 °C for 30 min. The derivatives were filtered through a syringe microfilter (Nylon, 0.2 μm, Thermo Fisher Scientific, San Jose, U.S.A.), and 5 μL of the sample volume was injected for HPLCMS/MS analysis. Instruments and Analytical Conditions. HPLC-MS/MS analysis was performed on an Agilent 1200 HPLC system coupled with an Agilent 6460 triple quadrupole mass spectrometer (Agilent Technologies, U.S.A.). A C18 analytical column (Phenomenex Kinetex C18, 100 mm × 2.1 mm i.d, 2.6 μm) was used for the separation of BRs derivatives. The mobile phase for the separation was a binary mixture consisting 0.1% formic acid in water as eluent A and 0.1% formic acid in ACN as eluent B. The gradient elution program was used and set as follows: 0−0.01 min, 45% B; 0.01−0.02, 45%−80% B; 0.02−4 min, 80%−85% B; 4−6 min, 85%−100% B; 6−8 min, 100% B; 8−8.10 min, 100%−45% B; 8.10−18 min, 45% B. 0.4 mL min−1 of the flow rate and 35 °C of the column oven temperature were adopted. The tandem MS analyses were carried out with an electrospray ionization (ESI) interface in the positive ion mode. Quantification and confirmation of BRs derivatives were performed by MRM mode. The ESI source conditions after optimization were set as follows: capillary voltage 3.5 kV, nebulizer 35 psi, nozzle voltage 500 V, sheath gas temperature 250 °C, sheath gas flow 11 L min−1, drying gas flow 12 L min−1, drying gas temperature 300 °C.

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RESULTS AND DISCUSSION Principle of DMSPE Sample Preparation. The DMSPE method proposed here combined MSPD (grinding of sample and dispersant) with d-SPE (centrifugation of dispersive sorbents in extraction solvent). The mixture of plant sample and dispersant was ground in one microcentrifuge tube, in which solvent was added for extraction/elution under centrifugation. Obviously, the manual sample transfer step from mortar to the empty SPE cartridge was avoided, leading to a decrease in sample loss. For further purification, another kind of sorbent could be added in the same tube before centrifugation (Protocol A) or added in the supernatant obtained after centrifugation (Protocol B). When two kinds of C


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Figure 3. Effects of three pretreatment based on DMSPE method on the recoveries of BRs (n = 3) in Arabidopsis thaliana seedling samples (A) and the rape seed samples (B). Conditions: BRs spiked at 0.2 ng mL−1; the recovery was calculated by (A − A0)/A1 × 100%, where A was the peak area of BRs spiked plant sample, A0 was the peak area of plant sample, and A1 was the peak area of standard BR solution without sample preparation. Other conditions were the same as Figure 2.

Figure 4. Optimization of DMSPE (n = 3). (A) The ratio of samples and MAX/MCX sorbent; (B) volume of extraction solvent; (C) vibration time; (D) centrifugation time. Conditions: oilseed rape samples spiked 0.2 ng mL−1 BRs; the recovery was calculated by the formula as described in Figure 3.

sorbents used for cleanup, they could be added simultaneously (Protocol A or B) or successively with a centrifugation after each adding (Protocol C). In this work, nonpolar dispersant was used for the extraction by grinding with plant samples, while ion-exchange sorbent was adopted for further cleanup. The nonpolar dispersant such as C8 or C18 could eliminate lipids, pigments and other nonpolar substances, while the ionexchange sorbent such as MAX and MCX could remove polar and ionizable impurities that had adverse effect on the ionization of BRs for MS detection.10

To investigate the extraction and purification efficiency of the three protocols of DMSPE, two kinds of plants were tested, i.e., Arabidopsis thaliana seedling and oilseed rape (that was more complex with high content of grease). The DMSPE procedures of these protocols were listed in Figure 2. All protocols adopted C8 dispersant for grinding with plant samples. In Protocol A (Figure 2A), MAX and MCX sorbents (mass ratio of 1:1) were added together directly after adding extraction solvent without any sample transfer. Then the mixture was centrifuged for extraction and purification. In Protocol B (Figure 2B), the supernatant obtained after D


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Journal of Agricultural and Food Chemistry Table 1. Linearity, LODs, LOQs of BRs and Matrix Effect of 5 BRs (n = 3) analytes

IS

linear range (ng mL−1)

R2

LOD (pg mL−1)a

LOQ (pg mL−1)b

ME (%)

24-epiBL 24-epiCS d-epiCS TE TY

d3-24-epiBL d3-24-epiCS d3-24-epiCS d3-24-epiCS d3-24-epiCS

0.005∼1 0.005∼5 0.005∼5 0.005∼5 0.01∼5

0.9918 0.9942 0.9953 0.9912 0.9923

2.76 2.58 1.38 6.75 5.36

9.21 8.59 4.60 22.50 17.86

114.1 88.6 83.6 93.8 82.4

a LODs were calculated as S/N = 3 on the basis of the analytical signals at the 0.005 ng mL−1 level for 24-epiBL, 24-epiCS, d-epiCS, and TE, and 0.01 ng mL−1 level for TY. bLOQs were calculated as S/N = 10 on the basis of the analytical signals at the 0.005 ng mL−1 level for 24-epiBL, 24epiCS, d-epiCS, and TE, and 0.01 ng mL−1 level for TY.

In DMSPE, the extraction solvent added to the mixture of plant sample and C8 dispersant was used to extract the BRs from the mixture to the organic solvent. Different volumes of 80% (v/v) ACN were used for comparison. As shown in Figure 4B, the optimized recoveries of all the five targeted BRs were obtained by using 200 μL of extraction solvent. Vibration and centrifugation were important steps in DMSPE. The times for vibration and centrifugation may affect the efficiency of extraction and purification. Distinct vibration time of 3, 5, 10, 15, 20 min and centrifugation time of 1, 3, 5, 10 min were investigated, respectively. It can be seen that 5 min of vibration and 10 min of centrifugation were enough to obtain high extraction and purification efficiency (Figure 4C and D). Since plant hormones are generally temperaturesensitive, the vibration and centrifugation were carried out at 4 °C (Figure S2). Evaluation of Matrix Effect. For complex plant samples, matrix interferences may affect ionization efficiency of the targets in the ESI process. Matrix effect (ME) is a general factor to evaluate the effects of such residual matrix interferences after sample pretreatment. It was defined that ME was the ratio of the peak area of BRs spiked (10 pg) into the oilseed rape segments (2.0 mg FW) after sample preparation to that of BRs in standard solution. The formula for ME value was described as follows: [(a − b)/c × 100%]. In the formula, a stands for the peak area of BRs in the pretreated plant sample after spiking, b stands for the peak area of BRs in the blank plant sample, and c is the peak area of standard BR solution. Before the separation by LC, derivatization was conducted for all samples. As shown in Table 1, ME values were in the range of 82.4% and 114.1%, indicating an effective removal of interferences by the DMSPE method. Method Validation. The proposed analytical method for BRs was validated including limits of detection (LODs), limits of quantification (LOQs), and dynamic linear range. The results were summarized in Table 1. To assess the linearity of the method, oilseed rape samples with BRs spiked at 9 concentrations (0, 0.005, 0.01, 0.02, 0.05, 0.1, 0.5, 1 and 5 ng mL−1 of five BRs with 0.1 ng mL−1 stable isotope internal standards) were provided in triplicate, labeled by BTBA and then analyzed by DMSPE-HPLC-MS/MS method. Using internal standard method, the calibration curves were set up by plotting the peak area ratio (analyte/IS) as Y axis and the BRs concentrations (0.005−5 ng mL−1) as X axis. Under the optimized conditions, good linearities were obtained with correlation coefficients (R2) ranging from 0.9912 to 0.9953. The LODs and LOQs for the 5 BRs were in the range of 1.38−6.75 pg mL−1 and 4.60−22.50 pg mL−1, respectively. It facilitated analysis of endogenous BRs in several or sub milligram plant samples (FW).

centrifugation was transferred to a new tube, in which MAX and MCX sorbents (mass ratio of 1:1) were added together. Different from Protocol B, in Protocol C (Figure 2C), after adding of MAX to the supernatant, a centrifugation was carried out and the resulted supernatant was transferred to another tube, in which MCX sorbent was added for further purification. The results obtained were showed in Figure 3. For Arabidopsis thaliana seedling (Figure 3A), the highest recoveries of five BRs were obtained by Protocol A, while for oilseed rape (Figure 3B), Protocol B showed the best results. In Protocol A, the extraction, elution and purification were performed in the same tube, which provided minimal samples loss. However, the purification effect was fair because that the cleanup material (MAX and MCX) was mixed with extraction sorbents (C8) in the d-SPE process. In Protocol B, the purification effect was better than Protocol A since the dispersive cleanup material was added to the supernatant (plant extractant) in a new tube without extraction sorbents. Therefore, it can be explained that for relatively simple plant matrix such as Arabidopsis thaliana seedling, the protocol with lowest sample loss and acceptable purification effect was preferred (Protocol A). While for the complex matrix such as oilseed rape, the protocol with high purification efficiency was required, such as Protocol B and C. Because that sample transfer happened more in Protocol C, Protocol B showed better results for oilseed rape since its lower sample loss. These results suggested that the proposed DMSPE method is suitable for different plant matrix, and the choice of any protocol of DMSPE is based on the compromise of increasing purification efficiency and decreasing sample loss. Optimization of DMSPE Conditions. The parameters of DMSPE that affected the extraction efficiency of all targeted BRs were assessed, including the type of dispersant, the ratio of the samples to MAX or MCX absorbent, the volume of elution solvent, the time and temperature of vibration and centrifugation. C8 and C18 sorbent were the most widely used reverse dispersant to eliminate the nonpolar matrix interference. Figure S1 shows the effect of C8 and C18 sorbent on the recovery of BRs by DMSPE. It can be seen that better recoveries of BRs were obtained for C8 than C18 dispersant. This is probably resulted from the stronger hydrophobic effect of C18 sorbent that made the targeted BRs harder to be eluted, especially for d-epiCS that has the strongest hydrophobicity in all five BRs. A mass ratio of 1:4 for samples to C8 dispersant was adopted as referred by MSPD methods.10,13 In this work, equal mass of MAX and MCX sorbents were used for cleanup, and different ratios (2:1, 1:1, 2:3, and 1:2) of sample to MAX sorbent were evaluated (Figure 4A). The highest recoveries of BRs were achieved when the ratio of 1:1 was used; therefore, the ratio of 1:1 was used in the following experiments. E


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Table 2. Precisions (Intra- and Inter-Day) and Recoveries of BRs in 2.0 mg of FW of Oilseed Rape Segments (n = 3) intraday precision

interday precision

recovery

(CV, %, n = 3)

(CV, %, n = 3)

(%, n = 3)

analytes

0.5 ng/g

2 ng/g

10 ng/g

0.5 ng/g

2 ng/g

10 ng/g

0.5 ng/g

2 ng/g

10 ng/g

24-epiBL 24-epiCS d-epiCS TE TY

2.8 7.8 5.4 5.2 9.8

4.8 0.8 5.4 2.3 2.5

1.8 2.1 3.2 5.7 5.2

17.3 14.9 12.1 13.0 16.9

6.0 8.7 9.0 4.6 12.6

7.1 5.6 7.1 8.9 8.5

117.4 85.8 84.9 72.0 92.3

115.7 81.1 91.8 75.1 83.0

112.4 79.4 97.3 81.9 91.2

Table 3. Endogenous BRs Detected in Plant Samples (Mean ± SD, ng g−1 FW) sample

type

1 2 3 4 5 6 7 8 9 10 11 12 13

Brassica napus L. oilseed rapea

province of origin Anhui

Shaanxi Jiangsu Hubei Sichuan

Arabidopsis thaliana seedlingsb

24-epiBL 0.26 ± 0.41 ± 0.25 ± 0.36 ± 0.24 ± 0.30 ± 0.27 ± 0.19 ± 0.12 ± 0.35 ± 0.57 ± 0.45 ± n.d.c

24-epiCS

0.05 0.04 0.09 0.02 0.04 0.06 0.08 0.01 0.07 0.11 0.15 0.06

0.07 ± 0.06 ± 0.10 ± n.d.c 0.07 ± 0.07 ± n.d.c 0.10 ± 0.09 ± n.d.c n.d.c 0.06 ± 0.21 ±

0.02 0.01 0.03 0.01 0.03 0.01 0.03

0.02 0.03

d-epiCS 5.17 ± 0.66 4.35 ± 0.53 6.48 ± 0.58 4.16 ± 0.33 6.12 ± 0.58 4.05 ± 0.19 4.52 ± 0.22 17.31 ± 1.12 6.89 ± 0.69 2.54 ± 0.22 4.09 ± 0.38 6.10 ± 0.65 10.57 ± 1.87

TE 1.18 ± 0.78 ± 0.99 ± 1.10 ± 0.20 ± 0.46 ± 2.41 ± 0.29 ± 0.50 ± 0.52 ± 1.25 ± 0.48 ± n.d.c

TY 0.19 0.07 0.16 0.13 0.02 0.04 0.32 0.03 0.09 0.06 0.07 0.08

0.83 ± 0.47 ± 0.50 ± 0.75 ± 0.60 ± 0.42 ± 0.76 ± 4.00 ± 0.82 ± n.d. 0.74 ± 0.49 ± n.d.c

0.44 0.12 0.13 0.08 0.16 0.14 0.17 0.15 0.29 0.16 0.18

a

2.0 mg FW of sample used. b0.5 mg FW of sample used. cn.d., not detected.

Figure 5. MRM chromatograms of unspiked and spiked Arabidopsis thaliana seedlings samples by DMSPE-HPLC-MS/MS analysis.

neglectable sample loss, which promoted the ability of accurate quantification of BRs in tiny and complex plant samples. A comparison of methods between the present and other previously published articles was shown in Table S2. Analysis of Endogenous BRs in Submilligram Arabidopsis thaliana Samples. Generally, subgram or several milligrams of plant samples were required for the determination of endogenous BRs.10,41 While in this work, submilligram of Arabidopsis thaliana seedlings (0.5 mg) was tested, and two BRs (24-epiCS and d-epiCS) were successfully detected as shown in Table 3. The MRM chromatograms of

In the matrix of 2.0 mg of FW of oilseed rape segments, the absolute recoveries were gained by the formula [(d − b)/c × 100%], where d is for the peak area of BRs in the spiked plant samples after pretreatment, b and c are the same as mentioned above. Under three different spiking levels (0.5, 2, and 10 ng g−1), the absolute recoveries were in the range of 72.0% to 117.4%, and the coefficient of variances (CVs) were below 17.3% (Table 2). For the two isotope-labeled internal standards, their absolute recoveries in the present method were 115.3% and 98.2%, respectively (Table S1). These results suggested that the sample utilization was highly efficient with F


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Figure 6. Geographical distribution of five endogenous BRs of Brassica napus L. oilseed rape originated from different provinces in Yangtze River basin.

five endogenous BRs in spiked and unspiked Arabidopsis thaliana samples were described in Figure 5. These results confirmed that the DMSPE with minimized sample loss and acceptable purification efficiency (Protocol A) was benefit to increasing sensitivity for tiny samples, showing good potential of application for spatiotemporal distribution study of BRs in other tiny plant organs and tissues. Geographical Distribution of Endogenous BRs of Brassica napus L. Oilseed Rape. The proposed method was employed for the study of the geographical distribution of endogenous BRs of Brassica napus L. oilseed rape, which is mainly used for extracting edible rapeseed oil. The samples in different provinces of origin in the Yangtze River basin including Sichuan, Shaanxi, Hubei, Anhui and Jiangsu Province were adopted and compared in detail. For oilseed rape, 2.0 mg FW of each sample was used, which was similar to the mass of one single seed of oilseed rape (about 2−4 mg). In order to obtain the typical data of BRs contents in different places of origin, each sample was premixed and homogenized before sample preparation. The results were summarized in Table 3, and the geographical distribution of five BRs content is described in Figure 6. It can be seen that the concentration of d-epiCS was higher than that of other four BRs in all Brassica napus L. oilseed rape samples. Moreover, the d-epiCS content of Brassia napus L. oilseed rape showed obvious distinction in different places of origin. The highest concentration of d-epiCS appeared in Hubei Province. This is probably resulted from the climate difference (i.e., temperature, humidity, illumination) that affected the synthesis of BRs in the plants. According to the current planting situation of Brassia napus L. in China,42 the highest output of Brassica napus L. is located in Hubei Province, which is in accordance with the geographical distribution of d-epiCS of Brassia napus L. oilseed rape in this work. This suggests that the content of d-epiCS in oilseed rape may be related to the yield of Brassia napus L. In conclusion, a simple and efficient sample preparation based on DMSPE was established for quantification of endogenous BRs in minute plant samples. Three protocols based on DMSPE were compared for plant samples with different matrix complexity. The choice of any protocol was a

compromise of increasing purification efficiency and decreasing sample loss. The DMSPE coupled with HPLC-MS/MS method showed high sensitivity, high selectivity, nice reproducibility and low sample consumption (submilligrams Arabidopsis thaliana seedlings, or one single seed of oilseed rape). Based on the proposed method, a geographical distribution of the content of BRs in different provinces in Yangtze River basin was obtained, which showed obvious distinction of the d-epiCS contents in Brassia napus L. oilseed rape from different origin. The study herein provides an important inference about the d-epiCS contents for further research on the cultivating new varieties and increasing yield of Brassia napus L.. It is foreseeable that the proposed method can be applicable to quantify BRs in other micro-organs or tissues with complex matrices. It will probably facilitate the researches in the fields of physiological pathway and dynamics transfer processes of BRs with spatiotemporal specificity in an individual plant.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.8b07224. Effect of solid dispersant of DMSPE on the recoveries of BRs (Figure S1), effect of the temperature of vibration and centrifugation on the recoveries of BRs (Figure S2), absolute recoveries of d3-24-epiBL and d3-24-epiCS in the 2.0 mg FW of oilseed rape samples (Table S1), and method comparison of the present with others (Table S2) (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Phone/Fax: +86-411-8437 9590. ORCID

Yafeng Guan: 0000-0002-5424-927X Author Contributions 1

G

Y.L. and T.D. contributed equally to this work. DOI: 10.1021/acs.jafc.8b07224 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry Funding

chromatography tandem mass spectrometry. Journal of Chromatography A 2016, 1456, 105−112. (14) Gamoh, K.; Omote, K.; Okamoto, N.; Takatsuto, S. Highperformance Liquid-chromatography of Brassinosteroids in Plants with Derivatization Using 9-Phenanthreneboronic Acid. J. Chromatogr. 1989, 469, 424−428. (15) Lv, T.; Zhao, X.-E.; Zhu, S.; Ji, Z.; Chen, G.; Sun, Z.; Song, C.; You, J.; Suo, Y. Development of an Efficient HPLC Fluorescence Detection Method for Brassinolide by Ultrasonic-Assisted Dispersive Liquid-Liquid Microextraction Coupled with Derivatization. Chromatographia 2014, 77 (23−24), 1653−1660. (16) Behbahani, M.; Esrafili, A.; Bagheri, S.; Radfar, S.; Bojdi, M. K.; Bagheri, A. Modified nanoporous carbon as a novel sorbent before solvent-based de-emulsification dispersive liquid-liquid microextraction for ultra-trace detection of cadmium by flame atomic absorption spectrophotometry. Measurement 2014, 51, 174−181. (17) Behbahani, M.; Bide, Y.; Bagheri, S.; Salarian, M.; Omidi, F.; Nabid, M. R. A pH responsive nanogel composed of magnetite, silica and poly(4-vinylpyridine) for extraction of Cd(II), Cu(II), Ni(II) and Pb(II). Microchim. Acta 2016, 183 (1), 111−121. (18) Behbahani, M.; Tapeh, N. A. G.; Mahyari, M.; Pourali, A. R.; Amin, B. G.; Shaabani, A. Monitoring of trace amounts of heavy metals in different food and water samples by flame atomic absorption spectrophotometer after preconcentration by amine-functionalized graphene nanosheet. Environ. Monit. Assess. 2014, 186 (11), 7245− 7257. (19) Omidi, F.; Behbahani, M.; Abandansari, H. S.; Sedighi, A.; Shahtaheri, S. J. Application of molecular imprinted polymer nanoparticles as a selective solid phase extraction for preconcentration and trace determination of 2,4-dichlorophenoxyacetic acid in the human urine and different water samples. Journal of Environmental Health Science and Engineering 2014, 12, 137. (20) Behbahani, M.; Najafi, M.; Amini, M. M.; Sadeghi, O.; Bagheri, A.; Salarian, M. Dithizone-modified nanoporous fructose as a novel sorbent for solid-phase extraction of ultra-trace levels of heavy metals. Microchim. Acta 2013, 180 (9−10), 911−920. (21) Ghambarian, M.; Behbahani, M.; Esrafili, A.; Sobhi, H. R. Application of a dispersive solid-phase extraction method using an amino-based silica-coated nanomagnetic sorbent for the trace quantification of chlorophenoxyacetic acids in water samples. J. Sep. Sci. 2017, 40 (17), 3479−3486. (22) Omidi, F.; Behbahani, M.; Samadi, S.; Sedighi, A.; Shahtaheri, S. J. Coupling of Molecular Imprinted Polymer Nanoparticles by High Performance Liquid Chromatography as an Efficient Technique for Sensitive and Selective Trace Determination of 4-Chloro-2-Methylphenoxy Acetic Acid in Complex Matrices. Iranian Journal of Public Health 2014, 43 (5), 645−657. (23) Behbahani, M.; Abolhasani, J.; Amini, M. M.; Sadeghi, O.; Omidi, F.; Bagheri, A.; Salarian, M. Application of mercapto ordered carbohydrate-derived porous carbons for trace detection of cadmium and copper ions in agricultural products. Food Chem. 2015, 173, 1207−1212. (24) Behbahani, M.; Veisi, A.; Omidi, F.; Badi, M. Y.; Noghrehabadi, A.; Esrafili, A.; Sobhi, H. R. The conjunction of a new ultrasonicassisted dispersive solid-phase extraction method with HPLC-DAD for the trace determination of diazinon in biological and water media. New J. Chem. 2018, 42 (6), 4289−4296. (25) Tarkowska, D.; Novak, O.; Oklestkova, J.; Strnad, M. The determination of 22 natural brassinosteroids in a minute sample of plant tissue by UHPLC-ESI-MS/MS. Anal. Bioanal. Chem. 2016, 408 (24), 6799−6812. (26) Pan, J.; Hu, Y.; Liang, T.; Li, G. Preparation of solid-phase microextraction fibers by in-mold coating strategy for derivatization analysis of 24-epibrassinolide in pollen samples. Journal of Chromatography A 2012, 1262, 49−55. (27) Oklestkova, J.; Tarkowska, D.; Eyer, L.; Elbert, T.; Marek, A.; Smrzova, Z.; Novak, O.; Franek, M.; Zhabinskii, V. N.; Strnad, M. Immunoaffinity chromatography combined with tandem mass

The financial support by the National Natural Science Foundation of China (Grant Nos. 21505135) is gratefully acknowledged. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We also thank Chenguang Bao from National Marine Environmental Monitoring Center for the help of map drawing.

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DEDICATION Dedicated to the 70th anniversary of Dalian Institute of Chemical Physics, CAS. REFERENCES

(1) Mitchell, J. W.; Mandava, N.; Worley, J. F.; Plimmer, J. R.; Smith, M. V. Brassinsa New Family of Plant Hormones from Rape Pollen. Nature 1970, 225, 1065. (2) Kang, Y. H.; Breda, A.; Hardtke, C. S. Brassinosteroid signaling directs formative cell divisions and protophloem differentiation in Arabidopsis root meristems. Development 2017, 144 (2), 272−280. (3) Du, J.; Yin, H.; Zhang, S.; Wei, Z.; Zhao, B.; Zhang, J.; Gou, X.; Lin, H.; Li, J. Somatic Embryogenesis Receptor Kinases Control Root Development Mainly via Brassinosteroid-Independent Actions in Arabidopsis thaliana. J. Integr. Plant Biol. 2012, 54 (6), 388−399. (4) Wei, Z.; Li, J. Brassinosteroids Regulate Root Growth, Development, and Symbiosis. Mol. Plant 2016, 9 (1), 86−100. (5) Lv, B.; Tian, H.; Zhang, F.; Liu, J.; Lu, S.; Bai, M.; Li, C.; Ding, Z. Brassinosteroids regulate root growth by controlling reactive oxygen species homeostasis and dual effect on ethylene synthesis in Arabidopsis. PLoS Genet. 2018, 14 (1), No. e1007144. (6) Ereminaa, M.; U, S. J.; Rathnayakea, A. I.; Castellanosb, M.; Khana, M.; Kuglerc, K. G.; Mayb, S. T.; Mayerc, K. F. X.; Rozhona, W.; Poppenbergera, B. Brassinosteroids participate in the control of basal and acquired freezing tolerance of plants. Proc. Natl. Acad. Sci. U. S. A. 2017, 114 (6), E1038−E1039. (7) Divi, U. K.; Rahman, T.; Krishna, P. Gene expression and functional analyses in brassinosteroid-mediated stress tolerance. Plant Biotechnology Journal 2016, 14 (1), 419−432. (8) Bajguz, A.; Tretyn, A. The chemical characteristic and distribution of brassinosteroids in plants. Phytochemistry 2003, 62 (7), 1027−1046. (9) Joo, S.-H.; Jang, M.-S.; Kim, M. K.; Lee, J.-E.; Kim, S.-K. Biosynthetic relationship between C-28-brassinosteroids and C-29brassinosteroids in rice (Oryza sativa) seedlings. Phytochemistry 2015, 111, 84−90. (10) Wang, L.; Duan, C.; Wu, D.; Guan, Y. Quantification of endogenous brassinosteroids in sub-gram plant tissues by in-line matrix solid-phase dispersion-tandem solid phase extraction coupled with high performance liquid chromatography-tandem mass spectrometry. Journal of Chromatography A 2014, 1359, 44−51. (11) Wu, Q.; Wu, D.; Shen, Z.; Duan, C.; Guan, Y. Quantification of endogenous brassinosteroids in plant by on-line two-dimensional microscale solid phase extraction-on column derivatization coupled with high performance liquid chromatography-tandem mass spectrometry. Journal of Chromatography A 2013, 1297, 56−63. (12) Deng, T.; Wu, D.; Duan, C.; Yan, X.; Du, Y.; Zou, J.; Guan, Y. Spatial Profiling of Gibberellins in a Single Leaf Based on Microscale Matrix Solid-Phase Dispersion and Precolumn Derivatization Coupled with Ultraperformance Liquid Chromatography-Tandem Mass Spectrometry. Anal. Chem. 2017, 89 (17), 9537−9543. (13) Deng, T.; Wu, D.; Duan, C.; Guan, Y. Ultrasensitive quantification of endogenous brassinosteroids in milligram fresh plant with a quaternary ammonium derivatization reagent by pipettetip solid-phase extraction coupled with ultra-high-performance liquid H


Article

Journal of Agricultural and Food Chemistry spectrometry: A new tool for the selective capture and analysis of brassinosteroid plant hormones. Talanta 2017, 170, 432−440. (28) Luo, X. T.; Cai, B. D.; Chen, X.; Feng, Y. Q. Improved methodology for analysis of multiple phytohormones using sequential magnetic solid-phase extraction coupled with liquid chromatographytandem mass spectrometry. Anal. Chim. Acta 2017, 983, 112−120. (29) Izumi, Y.; Okazawa, A.; Bamba, T.; Kobayashi, A.; Fukusaki, E. 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 2009, 648 (2), 215−225. (30) Ding, J.; Mao, L. J.; Yuan, B. F.; Feng, Y. Q. 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. Plant Methods 2013, 9, 13. (31) Schafer, M.; Brutting, C.; Baldwin, I. T.; Kallenbach, M. Highthroughput quantification of more than 100 primary- and secondarymetabolites, and phytohormones by a single solid-phase extraction based sample preparation with analysis by UHPLC-HESI-MS/MS. Plant Methods 2016, 12, 30. (32) Barker, S. A.; Long, A. R.; Short, C. R. Isolation of Drug Residues from Tissues by Solid-Phase Dispersion. J. Chromatogr. 1989, 475, 353−361. (33) Barker, S. A. Matrix solid-phase dispersion. Journal of Chromatography A 2000, 885 (1−2), 115−127. (34) Kristenson, E. M.; Ramos, L.; Brinkman, U. A. T. Recent advances in matrix solid-phase dispersion. TrAC, Trends Anal. Chem. 2006, 25 (2), 96−111. (35) Wu, Q.; Wang, L.; Wu, D.; Duan, C.; Guan, Y. Recent advances in sample preparation methods of plant hormones. Sepu 2014, 32 (4), 319−329. (36) Barker, S. A. Applications of matrix solid-phase dispersion in food analysis. Journal of Chromatography A 2000, 880 (1−2), 63−68. (37) Zhang, L. Y.; Wang, C. Y.; Li, Z. T.; Zhao, C. J.; Zhang, H. Q.; Zhang, D. J. Extraction of acetanilides in rice using ionic liquid-based matrix solid phase dispersion-solvent flotation. Food Chem. 2018, 245, 1190−1195. (38) Garcia-Rodriguez, D.; Cela-Torrijos, R.; Lorenzo-Ferreira, R. A.; Carro-Diaz, A. M. Analysis of pesticide residues in seaweeds using matrix solid-phase dispersion and gas chromatography-mass spectrometry detection. Food Chem. 2012, 135 (1), 259−267. (39) Wang, L.; Wu, Q.; Duan, C.; Wu, D.; Guan, Y. Determination of gibberellins in Arabidopsis thaliana by matrix solid-phase dispersion extraction and high performance liquid chromatography-tandem mass spectrometry. Chinese Journal of Chromatography 2011, 29 (9), 923− 6. (40) Shi, J.; Li, J.; Wang, Y.; Fan, J. Analysis of 112 pesticide residues in vegetables using dispersive-solid phase extraction and gas chromatographytriple quadrupole mass spectrometry. Sepu 2012, 30 (6), 602−612. (41) Luo, X. T.; Cai, B. D.; Yu, L.; Ding, J.; Feng, Y. Q. Sensitive determination of brassinosteroids by solid phase boronate affinity labeling coupled with liquid chromatography-tandem mass spectrometry. Journal of chromatography A 2018, 1546, 10−17. (42) Li, N.; Yang, T. Development status and strategies of rapeseed industry in China. Science and Technology of Cereals, Oils and Foods 2009, 17 (2), 34−36.

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Dispersive Matrix Solid-Phase Extraction Method Coupled with High

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