- Email: [email protected]
Improved methodology for analysis of multiple phytohormones using sequential magnetic solid-phase extraction coupled with liquid chromatography-tandem mass spectrometry
Accepted Manuscript Improved methodology for analysis of multiple phytohormones using sequential magnetic solid-phase extraction coupled with liquid chromatography-tandem mass spectrometry Xiao-Tong Luo, Bao-Dong Cai, Xi Chen, Yu-Qi Feng PII:
S0003-2670(17)30723-7
DOI:
10.1016/j.aca.2017.06.019
Reference:
ACA 235265
To appear in:
Analytica Chimica Acta
Received Date: 22 February 2017 Revised Date:
3 June 2017
Accepted Date: 11 June 2017
Please cite this article as: X.-T. Luo, B.-D. Cai, X. Chen, Y.-Q. Feng, Improved methodology for analysis of multiple phytohormones using sequential magnetic solid-phase extraction coupled with liquid chromatography-tandem mass spectrometry, Analytica Chimica Acta (2017), doi: 10.1016/ j.aca.2017.06.019. 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.
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Improved methodology for analysis of multiple phytohormones using
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sequential magnetic solid-phase extraction coupled with
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liquid chromatography-tandem mass spectrometry
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Xiao-Tong Luo a ※, Bao-Dong Cai a ※, Xi Chen b, Yu-Qi Feng a *
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a
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Education), Department of Chemistry, Wuhan University, Wuhan 430072, China
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b
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Wuhan Institute of Biotechnology, Wuhan 430075, China
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Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of
These authors contributed equally to this work.
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*Corresponding author. Tel: +86-27-68755595; fax: +86-27-68755595; Email:
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[email protected]
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Abstract Phytohormones are special small molecules that play important role in plant
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growth and development at trace levels. Quantification of multiple phytohormones
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will be great helpful for researches about cross-talks that plant hormones regulate the
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responses of plants against both biotic and abiotic stresses by means of synergistic or
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antagonistic interactions. In the current study, we developed a method for profiling of
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phytohormones in one small sample, including indole-3-acetic acid, abscisic acid,
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jasmonic acid, gibberellins, cytokinins and brassinosteroids. These phytohormones
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mentioned above were firstly purified and separated by sequential magnetic
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solid-phase extraction (MSPE) and then analyzed by ultra-high performance liquid
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chromatography-electrospray tandem mass spectrometry (UHPLC-MS/MS). Under
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the optimized extraction conditions, good linearity was obtained with correlation
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coefficients (r) ranging from 0.9961 to 0.9998. The limits of detection (LODs, S/N =
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3) were ranged from 0.45 to 126.1 pg mL-1. The recoveries were between 85.0% and
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116.2%. The relative standard deviations (RSDs) were ranged from 2.7% to 16.1%.
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With the proposed strategy, 23 phytohormones could be purified and analyzed from a
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single plant extract. Finally, 16 phytohormones could be detected in 100 mg (fresh
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weight) flower of Brassica napus L, including IAA, ABA, JA, 4 GAs, 3 BRs and 6
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CKs with the concentration ranged from 0.09 to 305.23 ng g-1.
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Key words: Phytohormones; Magnetic solid-phase extraction; Mass spectrometry
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1. Introduction Phytohormones are of vital importance at trace amount level for the growth and
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development of plants and protective response against stress. [1, 2]. They are grouped
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into several classes, including abscisic acid (ABA), auxins, gibberellins (GAs),
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cytokinins (CKs), ethylene (ET), brassinosteroids (BRs), jasmonates and salicylates
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according to their structures and physiological functions [3, 4]. Although each class of
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phytohormones has been identified for their specific biological functions, they usually
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collectively regulate the plant responses against biotic and abiotic stress by means of
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synergistic effect or antagonistic effect [5-7]. It is referred to as cross-talk of
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phytohormones which is universally present in plant physiological activities [5].
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Because the cross-talks are complex and refined, the molecular mechanisms of them
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remain elusive. For better understanding the cross-talk mechanisms of phytohormones,
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simultaneous determination of multi-class phytohormones is needed.
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The challenges to simultaneous determination of multi-class phytohormones are
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originated not only from their low abundance in plants (pmol per gram fresh weight,
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pmol g−1 FW) and the complex matrix of plants, but also from the difficulties of
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simultaneous extraction and purification of multi-class phytohormones due to their
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diverse chemical properties. Although a variety of analytical methods have been
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developed for single-class phytohormones by GC-MS or LC-MS [8-16], few methods
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were reported for the simultaneous determination of multi-class phytohormones
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[17-19].
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Up to now, many sample preparation methods have been developed for
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enrichment and purification of multiple phytohormones, such as immune affinity
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extraction (SPE) [19, 25-27], polymer monolith microextraction (PMME) [28],
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magnetic solid-phase extraction (MSPE) [29] and so on. Thanks to these sample
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preparation methods, Kojima et al. developed a multi-step strategy for determination
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of 43 phytohormones in rice including auxins, ABA, gibberellins and CKs [17]. Liu et
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al. described a method for simultaneous analysis of CKs and acidic phytohormones
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(auxins, ABA and GAs), in which a binary SPE was employed for purification and
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enrichment of phytohormones [19]. CKs and acidic phytohormones were eluted from
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different SPE cartridges respectively. The two fractions of elution were combined for
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UHPLC-MS/MS analysis. More recently, a TiO2-based MSPE was developed for
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profiling of multiple phytohormones, including indole-3-acetic acid (IAA), ABA, JA,
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GAs and CKs through hydrophilic interaction and coordination [18]. All target
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analytes mentioned above were assigned to acidic phytohormones (IAA, ABA, JA and
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GAs) and CKs. Nevertheless, none of the methods could be used for simultaneous
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analysis of acidic phytohormones, CKs and BRs.
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Recently, Tong et.al revealed a previously unknown mechanism underlying BR
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and GA crosstalk depending on tissues and hormone levels, which greatly advanced
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our understanding of hormone actions in crop plants, appearing much different from
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that in Arabidopsis thaliana [6]. It is indispensable to get more information of
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phytohormones from the same sample to illustrate the complex relationship between
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phytohormones. However, it is still a challenge for simultaneous analysis of multiple
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class phytohormones in one small sample, including acidic phytohormones, CKs and
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BRs. Also, it is a bottleneck that simultaneous purification of multiple phytohormones
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with different structures and chemical properties from one crude plant extract. In the current study, we developed a method for simultaneous analysis of
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multiple phytohormones including acidic phytohormones (IAA, ABA, JA, GAs), BRs
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and CKs by sequential MSPE coupled with UHPLC-MS/MS. The classification, name
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and structures of all the targets were shown in Table S1. In our proposed strategy, the
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crude plant extract was firstly processed by a TiO2-based MSPE [18]. Subsequently,
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the second MSPE was employed for further purification of BRs while the separation
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of BRs from acidic hormones and CK could be realized. Finally, the developed
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method was successfully applied to detect multiple phytohomones in flower of
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Brassica napus L .
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2. Experimental
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2.1. Chemicals and reagents
Phytohormone standards: indole-3-acetic acid (IAA), abscisic acid (ABA),
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jasmonic acid (JA), gibberellic acids (GA1, GA3, GA4, GA7, GA12, GA24, GA53)，
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28-norbrassinolide (28-norBL), 28-norcastasterone (28-norCS), 28-homobrassinolde
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(28-homoBL), brassinolide (BL), castasterone (CS); N6-isopentenyladenine (iP),
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N6-isopentenyladenine riboside (iPR), N6-isopentenyladenine 9-glucoside (iP9G),
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trans-zeatin (tZ), trans-zeatin-riboside (tZR), trans-zeatin 9-glucoside (tZ9G),
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dihydrozeatin (DHZ), dihydrozeatin riboside (DHZR) and stable isotope-labeled
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standards: [2H2]IAA, [2H6]ABA, [2H2]GA1, [2H2]GA3, [2H2]GA4, [2H2]GA12,
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[2H2]GA24, [2H2]GA53, [2H3]BL, [2H3]CS, [2H6]iP, [2H6]iPR, [2H6]iP9G, [2H5]tZ,
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[2H5]tZR, [2H5]tZ9G, [2H3]DHZ, [2H3]DHZR were all purchased from Olchemim Ltd.
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(Olomouc, Czech Republic). Ferric chloride (FeCl3·6H2O), sodium acetate (NaAc), ethylene glycol (EG),
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1,2-ethylenediamine (ETH), ethanol (EtOH), tetraethyl orthosilicate (TEOS),
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ammonia hydrate (NH3·H2O, 25%, aqueous solution), ammonium hexafluorotitanate
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((NH4)2TiF6),
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2,2-azobis(2-methyl-propionitrile) (AIBN) were all purchased from Sinopharm
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Chemical Reagent (Shanghai, China). 3-(Methacryloxy)propyl trimethoxysilane
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(MPS) was bought from Wuhan University Silicone New Material (Wuhan, China),
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while ethylene glycol dimethacrylate (EGDMA) was bought from Sigma–Aldrich (St.
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Louis, MO, USA). 4-(N,N-dimethyamino) phenylboronic acid (DMAPBA) was
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purchased from J&K Scientific Ltd (Beijing, China). AIBN was recrystallized from
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ethanol, and other reagents were of analytical grade and used directly without further
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purification. Acetonitrile (ACN, HPLC grade) was obtained from Tedia Co. (Fairfield,
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OH, USA). Ultra-pure water used throughout the study was purified by Milli-Q
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system (Milford, MA, USA).
pyridine,
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2.2.
Preparation
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Fe3O4@SiO2@Poly(DMAPBA-co-EGDMA)
polymer
particles
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MPS-modified Fe3O4 particles were prepared according to our previous method
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[29]. Subsequently, Fe3O4@SiO2@Poly(DMAPBA-co-EGDMA) particles were
ACCEPTED MANUSCRIPT synthesized by the distillation–precipitation polymerization method proposed by Yang
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et al [30]. Briefly, DMAPBA (0.25 g) was dissolved in 200 mL ACN/pyridine (50/1,
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v/v) in a 500-mL two-necked round bottom flask equipped with a distillation
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apparatus and a stirring device. Then, MPS-modified Fe3O4@SiO2 (0.50 g), EGDMA
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(2.25 g) and AIBN (0.02 g) were successively added. The mixture was heated to
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boiling within 30 min and kept boiling until approximately half of the acetonitrile was
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distilled out (occurring within 2 h). As soon as the mixture cooled down to room
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temperature, Fe3O4@SiO2@Poly(DMAPBA-co-EGDMA) particles were separated by
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the aid of a magnet, and washed several times with water and methanol. Finally, the
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resultant magnetic polymers were dried in a vacuum at 60oC.
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2.3. Plant materials
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Three-month-old Brassica napus L. flowers were provided by Dr Jia Liu from
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Oil Crops Research Institute, Chinese Academy of Agricultural Sciences (Wuhan,
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China). The plant materials were harvested, weighted, immediately frozen in liquid
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nitrogen, and stored at -80oC.
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2.4. Sample pretreatment
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The general process of sample pretreatment was shown in Fig. 1. Plant sample
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tissues (100 mg FW) were frozen in liquid nitrogen, ground into powder and then
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transferred into a 1.5 ml centrifuge tube. [2H2]IAA (1.0 ng), [2H6]ABA (1.0 ng), stable
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isotope labeled GAs (0.4 ng), [2H3]BL (1.0 ng), [2H3]CS (1.0 ng) and stable isotope
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labeled CKs (0.1 ng) were added to the samples, serving as internal standards for the
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quantification. Next, ACN (0.5 mL) was added into the mixture. After extraction at
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below 0oC for 20 min. Subsequently, sequential MSPE was carried out. Firstly,
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TiO2-based MSPE was employed for enrichment of all target analytes according to
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our previous work with minor modification [18]. Briefly, the supernatant was added
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into a 2-mL vial containing Fe3O4@TiO2 (50 mg) magnetic nanoparticles pretreated
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with ACN (1.0 mL). Before the supernatant was discarded with the help of magnetic
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field, the mixture was vortexed vigorously for 1 min. Afterwards, the magnetic
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sorbent, which adsorbed analytes, was washed by ACN (1.0 mL) twice. Then, 1 mL
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20% ACN (v/v) aqueous solution containing 1% NH3·H2O (v/v) was added as elution
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solvent, in which 20 mg Fe3O4@SiO2@Poly(DMAPBA-co-EGDMA) dispersed.
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After vigorously vortexed for 5 min, acidic phytohormones, CKs and BRs were all
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eluted. Meanwhile, BRs, which contain cis-hydroxyl group, were re-enriched by
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Fe3O4@SiO2@Poly(DMAPBA-co-EGDMA) through boronate affinity interaction.
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With the aid of a magnet, acidic phytohormones and CKs in elution were collected
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and evaporated to dryness under a mild nitrogen gas flow at 35°C. Finally, the sample
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was re-dissolved in 50 µL 30% MeOH, and 10 µL was injected for the quantification
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of acidic phytohormones and CKs by UHPLC-ESI-MS/MS. The BR adsorbed
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magnetic nanoparticles were washed orderly by 1 mL 50% ACN and 1 mL ACN.
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After vortexed for 5 min, BRs were desorbed with 3% H2O2 in 90% ACN aqueous
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(v/v, 1 mL). The desorption solution was collected and evaporated under mild
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nitrogen stream. Finally, the sample was re-dissolving in 50 µL 30% MeOH and 10
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µL of the solution was injected for the quantification of BRs by UHPLC-ESI-MS/MS.
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2.5. Instruments and analytical conditions Analysis of phytohormones was performed on a UHPLC-ESI-MS/MS system
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consisting of a AB SCIEX 4500 triple quadrupole mass spectrometer (Foster City, CA,
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USA) with an electrospray ionization source (Turbo Ionspray), a Shimadzu LC-30AD
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HPLC system (Kyoto, Japan) with two 30AD pumps, a SIL-30AC auto sampler, a
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CTO-30A thermostat column compartment, and a DGU-20A5R degasser. Data
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acquisition and processing were performed using AB SCIEX Analyst 1.6 software
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(Foster City, CA, USA).
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The UHPLC separation was performed at 40oC on a Shim-pack XR-ODS Ш
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column (75 mm × 2.0 mm i.d., 1.6 µm) purchased from Shimadzu (Kyoto, Japan). A
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27-min gradient of 0.05% FA in H2O (A) and 100% ACN (B) was employed for the
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separation of acidic phytohormones and CKs with a flow rate of 0.4 mL min-1. A
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linear gradient with the following proportions (v/v) of solvent B was applied: 0-2 min
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at 5%, 2-12 min from 5% to 25%, 12-20 min from 25% to 80%, 20-22 min at 80%,
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22-24 min from 80%-5%, followed by 3 min of re-equilibration at 5%. For separating
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BRs, 14-min gradient of 0.05% FA in H2O (A) and 100% ACN (B) was employed
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with a flow rate of 0.4 mL min-1. A linear gradient with the following proportions (v/v)
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of solvent B was applied: 0-6 min from 30% to 65%, 6-7 min from 65% to 85%, 7-10
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min at 85%, 10-11 min from 85%-30%, followed by 3 min of re-equilibration at 30%.
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Multiple reaction monitoring (MRM) and the appropriate product ions were
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chosen to quantify phytohormones. The optimized conditions for selective MRM
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experiments of acidic phytohormones, CKs and BRs were listed in Table S2. And
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other optimized conditions of MRM experiments were as follows: curtain gas, 40 psi;
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ion spray voltage, 5500 V; turbo heater temperature (TEM), 450oC; nebulizing gas
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(Gas 1), 55 psi; heated gas (Gas 2), 45 psi.
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3. Results and discussion
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3.1. Evaluation of Fe3O4@SiO2@Poly(DMAPBA-co-EGDMA)
Boronate affinity chromatography is a powerful tool for selective separation and
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enrichment of cis-diol containing compounds. In this study, adenosine (A) and
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deoxyadenosine (dA) were selected as model compounds to investigate the selectivity
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of
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Fe3O4@SiO2@Poly(DMAPBA-co-EGDMA), cis-diol adenosine was hardly detected
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in residual solution, while deoxyadenosine was detected with the similar signal
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strength from standard solution (Fig. S1). It meant that adenosine could be extracted
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by the prepared Fe3O4@SiO2@Poly(DMAPBA-co-EGDMA) while deoxyadenosine
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could not. And the average efficiency of adenosine elution from the beads (n = 3) was
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102.5% (RSD = 7.4%). This result demonstrated high specificity of the prepared
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magnetic nanoparticles towards cis-diol-containing molecules, further indicating that
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the
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successfully.
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After
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nanoparticles
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Next, the extraction capacity of the boronate affinity magnetic nanoparticles,
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which was 849.3 µg g-1, was evaluated by using adenosine as model compound. The
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reproducibility of preparation was also investigated by estimating the extraction
ACCEPTED MANUSCRIPT capacity of three batches of the magnetic polymer adsorbents. As the results shown in
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Table 1, the relative standard deviation (RSD) of the extraction capacity was only
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6.6%, suggesting good reproducibility of the preparation method.
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3.2 Enrichment of BRs with boronate affinity MSPE
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As BRs and CK nucleosides (iPR, tZR, DHZR) all contain cis-diol, they could
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all be extracted by the prepared boronate affinity magnetic adsorbents. However, it
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was better that CK nucleosides could not be extracted in further purification of BRs.
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Because the H2O2 contained in BRs desorption solution could result in CK
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degradation. To ensure BRs be extracted through adsorbents while CK nucleosides
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not, we investigated if pH of sampling solution could influence extraction efficiency
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of BRs and CK nucleosides first. BRs were extracted in 50% ACN (v/v) with pH from
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3.0 to 10.0, while CK nucleosides were extracted in 10% ACN (v/v) with pH from 3.0
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to 10.0. After extracted, the magnetic adsorbents were washed with sampling solution.
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Then, BRs were eluted with 90% ACN (v/v) containing 3% H2O2 (v/v) while CK
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nucleosides were eluted with 10% ACN (v/v) containing 1% FA (v/v). Finally, the
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eluted analytes were analyzed. As shown in Fig. 2A and Fig. 2B, the recoveries of
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both CK nucleosides and BRs were low when pH under 8. While pH up to 9 and 10,
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the recoveries were increased sharply. It indicated that both CK nucleosides and BRs
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were inclined to be extracted by Fe3O4@SiO2@Poly(DMAPBA-co-EGDMA) when
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the sampling solution was alkaline. Meanwhile, it demonstrated that it was hard to
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separate CK nucleosides and BRs relying on pH in sampling solution.
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Then, ACN content in sampling solution was also investigated in the range of 0
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dramatically as ACN content increased from 0 to 20% and CK nucleosides were
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hardly extracted when ACN content was over 20%. However, the recoveries of BRs
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increased with the increase of ACN content in the range of 5% to 20% and decreased
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with the increase of ACN content in the range of 20% to 80% (Fig. 2D).that the
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existence of ACN in sampling solution was negative for boronate affinity reaction,
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therefore resulted in reduced recoveries of CKs with increasing content of ACN. As
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BRs are hydrophobic compounds, they have better solubility with 20% or more ACN
242
content in sampling solution. So, it resulted in increasing recoveries. However, if
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ACN content further increased, a decreasing recovery could be observed. Furthermore,
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the increasing recoveries of BRs in the range of 80% to 90% may be contributed to
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hydrophilic interaction. Therefore, we assumed that BRs could be selectively
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extracted with 20% ACN while CK nucleosides could not.
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Inspired by the experiment above, we concluded that CK nucleosides and BRs
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could be separated in alkaline aqueous solution with 20% ACN (v/v). For easy
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operation, the solution which contained 20% ACN and 1% NH3·H2O (v/v) was
250
employed. However, the pH of chosen solution was approximately 11，which was not
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investigated above. Hence, the extract efficiency of BRs and CKs was detected. First,
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BRs and CKs (CK free bases, nucleosides and glucosides) were dissolved in sampling
253
solution, in which 20% ACN and 1% NH3·H2O (v/v) contained. Then, it was
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extracted by the prepared boronate affinity magnetic nanoparticles. After extraction,
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the residual solution was collected and the magnetic nanoparticles were washed by
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ACCEPTED MANUSCRIPT sampling solution. BRs were eluted by 90% ACN (v/v) together with 3% H2O2 (v/v)
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from the magnetic nanoparticles. As shown in Fig. 3A, most of CKs were detected in
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the residual solution while BRs could not (data not shown). Meanwhile, the recoveries
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of BRs were detected more than 90% as shown in Fig. 3B. These results suggested
260
that, with the proposed solution containing 20% ACN and 1% NH3·H2O (v/v), only
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BRs could be extracted by the prepared boronate affinity magnetic nanoparticles.
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3.3. Comparison of two pretreatment methods
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Recently, we developed a TiO2-based MSPE method for simultaneous analysis of
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acidic phytohormones (carboxyl compounds) and CKs [18]. The method was simple,
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rapid and effective. The TiO2 coated magnetic nanoparticles could adsorb acidic
266
phytohormones, CKs and BRs through hydrophilic interaction and coordination [14,
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18]. However, BRs could not be detected in plant sample (spiked with BR standards)
268
through the proposed one-step sample preparation method because of the matrix
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interference. Now the newly developed sequential MSPE method could solve this
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problem. To compare the two pretreatment methods, the aspects of recoveries,
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selectivity and desorption efficiencies were investigated.
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First, the recoveries of BRs using TiO2-based MSPE and the sequential MSPE
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were investigated in spiked sample matrices. As shown in Fig. 4, the recoveries of
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BRs using TiO2-based MSPE were between 55% and 69%, which was similar to the
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recoveries of the sequential MSPE. This further suggested that the BRs eluted from
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TiO2 magnetic nanoparticles were mostly extracted by boronate affinity adsorbent.
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The result was consistent with that in standard solution (Fig. 3B), indicating that
ACCEPTED MANUSCRIPT 278
sample matrix had little influence on BRs enrichment using the prepared adsorbents. To improve the selectivity, the boronate affinity MSPE was employed for the
280
further purification of BRs after TiO2-based MSPE in the current study. The
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performance of these two methods (TiO2-based MSPE and sequential MSPE) was
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compared. Fig. 5A represented TIC chromatogram of BR standards (4 ng for each
283
standards) dissolved in ACN treated with sequential MSPE, plant sample (spiked with
284
4 ng for each BR standards) treated with TiO2-based MSPE and plant sample (spiked
285
with 4 ng for each BR standards) treated with sequential MSPE. Fig. 5B represented
286
the chromatogram of extracted m/z from scan mode of BL. Based on the TIC (Fig. 5A)
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and extracted chromatograms (Fig. 5B and Fig. S2), the purification performance of
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sequential MSPE method was much better than that of TiO2-based MSPE. And it was
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similar to the BR standards without plant matrix, suggesting high selectivity and
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powerful ability of removing matrix of our newly developed method.
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During boronate affinity MSPE procedure, 20% ACN containing 1% NH3·H2O
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(v/v) was employed as sampling solution which could also be used as desorption
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solution for TiO2-based MSPE. Therefore, the desorption efficiencies using 20% ACN
294
and 20% ACN containing 1% NH3·H2O as desorption solution during TiO2-based
295
MSPE were compared. Acidic phytohormones and CKs spiked in matrix was
296
extracted by Fe3O4@TiO2. 20% ACN and 20% ACN containing 1% NH3·H2O were
297
employed as desorption solution. The results showed that using 20% ACN containing
298
1% NH3·H2O as desorption solution ensured high recoveries for dual carboxyl
299
compounds (GA12, GA24, GA53); for other phytohormones, the recoveries were
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ACCEPTED MANUSCRIPT similar (Fig. 6). In addition, it could simplify sample pretreatment by dispersing
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Fe3O4@SiO2@Poly(DMAPBA-co-EGDMA) in 20% ACN (v/v) containing 1%
302
NH3·H2O. Thus, the desorption of all phytohotmones from Fe3O4@TiO2 as well as
303
selective enrichment of BRs by boronate affinity MSPE could be simultaneously
304
completed.
305
3.4. Method validation
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The linearity of the developed method was evaluated by preparing 11-point
307
calibration curves in 30% MeOH aqueous solution for the different phytohormones in
308
triplicate. The calibration curves were constructed by plotting the peak area ratio
309
(analytes/I.S.) versus the phytohormones concentrations. As listed in Table 2, good
310
linearity was obtained with correlation coefficients (r) ranging from 0.9961 to 0.9998.
311
The limits of detection (LODs) in standard solution and matrix were calculated as the
312
signal to noise ratios being 3:1. The results showed that LODs in standard solution
313
and matrix ranged from 0.45 to 126.1 pg mL-1 and 1.2 to 196.1 pg mL-1 respectively,
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indicating that it was quite sensitive for profiling of endogenous phytohormones in
315
plant samples.
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In addition, the effect of plant matrix on the quantification of endogenous
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phytohormones was evaluated by spiking phytohormone standards into plant sample.
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The results showed that the spiked phytohormones could be successfully determined
319
with 85.0%–116.2% recoveries and the relative standard deviations (RSDs) were
320
2.7%–16.1% (Table 3).
321
Finally, the developed sequential MSPE method was used for analysis of
ACCEPTED MANUSCRIPT multiple phytohormones in flower of Brassica napus L. As listed in Table 4,
323
phytohormones analysis could be completed with 100 mg (FW) flower of Brassica
324
napus L and 16 phytohormoes were successfully detected, including 6 CKs, IAA,
325
ABA, JA and 4 GAs and 3 BRs. The MRM chromatograms of 16 detected
326
endogenous phytohormones were shown in Fig. 7. According to previous reports, the
327
contents of CKs were ranged from 0.03-15.40 ng g-1 FW and the contents of BRs were
328
ranged from 5.54-89.80 ng g-1 FW in Brassica napus flowers [13, 31-33]. And in this
329
work, as shown in Table 4, the contents of CKs were ranged from 0.09-5.38 ng g-1 FW
330
and the contents of BRs were ranged from 3.25-10.43 ng g-1 FW. The phytohormones
331
contents detected by this method were in the range of the contents detected before,
332
which validated the method further.
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4. Conclusion
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In this study, magnetic poly (4-(N,N-dimethyamino) phenylboronic acid ̶ co ̶
335
ethylene glycol dimethacrylate) particles were prepared by distillation precipitation
336
polymerization and was employed for class-selective enrichment of phytohormones
337
coupled with TiO2-based MSPE. The sequential MSPE was more selective for BRs
338
than TiO2-based MSPE. By combination with UHPLC-MS/MS, a rapid and selective
339
analytical method for the analysis of phytohormones, including acidic phytohormones
340
(IAA, ABA, JA, GAs), BRs and CKs was established. Finally, 16 phytohormones
341
could be detected in flower of Brassica napus L .
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Acknowledgements This work was supported by the National Natural Science Foundation of China
346
(91217309, 31670373), and the Natural Science Foundation of Hubei Province, China
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(2014CFA002), the Fundamental Research Funds for the Central Universities.
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References
350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391
[1] J. Fu, X. Sun, J. Wang, J. Chu, C. Yan, Progress in quantitative analysis of plant hormones, Chin. Sci. Bull., 56 (2011) 355-366. [2] L. Van Meulebroek, J.V. Bussche, K. Steppe, L. Vanhaecke, Ultra-high performance liquid chromatography coupled to high resolution Orbitrap mass spectrometry for metabolomic profiling of the endogenous phytohormonal status of the tomato plant, J Chromatogr A, 1260 (2012) 67-80.
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[3] P. Davies, The Plant Hormones: Their Nature, Occurrence, and Functions, in: P. Davies (Ed.) Plant Hormones, Springer Netherlands1995, pp. 1-12.
[4] M. Umehara, A. Hanada, S. Yoshida, K. Akiyama, T. Arite, N. Takeda-Kamiya, H. Magome, Y. Kamiya, K. Shirasu, K. Yoneyama, J. Kyozuka, S. Yamaguchi, Inhibition of shoot branching by new terpenoid plant hormones, Nature, 455 (2008) 195-200.
[5] E.A. Schmelz, J. Engelberth, H.T. Alborn, P. O'Donnell, M. Sammons, H. Toshima, J.H. Tumlinson,
SC
Simultaneous analysis of phytohormones, phytotoxins, and volatile organic compounds in plants, Proceedings of the National Academy of Sciences, 100 (2003) 10552-10557.
[6] H. Tong, Y. Xiao, D. Liu, S. Gao, L. Liu, Y. Yin, Y. Jin, Q. Qian, C. Chu, Brassinosteroid regulates cell
M AN U
elongation by modulating gibberellin metabolism in rice, The Plant cell, 26 (2014) 4376-4393. [7] S. Gao, J. Fang, F. Xu, W. Wang, X. Sun, J. Chu, B. Cai, Y. Feng, C. Chu, CYTOKININ OXIDASE/DEHYDROGENASE4 Integrates Cytokinin and Auxin Signaling to Control Rice Crown Root Formation, Plant physiology, 165 (2014) 1035-1046.
[8] M. Manzi, A. Gomez-Cadenas, V. Arbona, Rapid and reproducible determination of active gibberellins in citrus tissues by UPLC/ESI-MS/MS, Plant Physiol Biochem, 94 (2015) 1-9. [9] Y.-H. Hao, Z. Zhang, L. Wang, C. Liu, A.-W. Lei, B.-F. Yuan, Y.-Q. Feng, Stable isotope labeling assisted
TE D
liquid chromatography–electrospray tandem mass spectrometry for quantitative analysis of endogenous gibberellins, Talanta, 144 (2015) 341-348. [10] Y.Q. Huang, J.Q. Liu, H. Gong, J. Yang, Y. Li, Y.Q. Feng, Use of isotope mass probes for metabolic analysis of the jasmonate biosynthetic pathway, Analyst, 136 (2011) 1515-1522. [11] T. Henriksen, R.K. Juhler, B. Svensmark, N.B. Cech, The relative influences of acidity and polarity
EP
on responsiveness of small organic molecules to analysis with negative ion electrospray ionization mass spectrometry (ESI-MS), J Am Soc Mass Spectrom, 16 (2005) 446-455. [12] C. Birkemeyer, A. Kolasa, J. Kopka, Comprehensive chemical derivatization for gas
AC C
chromatography-mass spectrometry-based multi-targeted profiling of the major phytohormones, J Chromatogr A, 993 (2003) 89-102. [13] B.-D. Cai, J.-X. Zhu, Z.-G. Shi, B.-F. Yuan, Y.-Q. Feng, A simple sample preparation approach based on hydrophilic solid-phase extraction coupled with liquid chromatography–tandem mass spectrometry for determination of endogenous cytokinins, Journal Of Chromatography B, 942-943 (2013) 31-36. [14] J. Ding, J.-H. Wu, J.-F. Liu, B.-F. Yuan, Y.-Q. Feng, Improved methodology for assaying brassinosteroids in plant tissues using magnetic hydrophilic material for both extraction and derivatization, Plant Methods, 10 (2014) 39. [15] P. Xin, J. Yan, J. Fan, J. Chu, C. Yan, A dual role of boronate affinity in high-sensitivity detection of vicinal diol brassinosteroids from sub-gram plant tissues via UPLC-MS/MS, The Analyst, 138 (2013) 1342-1345. [16] D. Li, Z. Guo, Y. Chen, Direct Derivatization and Quantitation of Ultra-trace Gibberellins in Sub-milligram Fresh Plant Organs, Molecular Plant, 9
175-177.
ACCEPTED MANUSCRIPT [17] M. Kojima, T. Kamada-Nobusada, H. Komatsu, K. Takei, T. Kuroha, M. Mizutani, M. Ashikari, M. Ueguchi-Tanaka, M. Matsuoka, K. Suzuki, H. Sakakibara, Highly sensitive and high-throughput analysis of plant hormones using MS-probe modification and liquid chromatography-tandem mass spectrometry: an application for hormone profiling in Oryza sativa, Plant Cell Physiol, 50 (2009) 1201-1214. [18] B.D. Cai, J. Yin, Y.H. Hao, Y.N. Li, B.F. Yuan, Y.Q. Feng, Profiling of phytohormones in rice under elevated cadmium concentration levels by magnetic solid-phase extraction coupled with liquid
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chromatography tandem mass spectrometry, Journal of chromatography. A, 1406 (2015) 78-86.
[19] S. Liu, W. Chen, L. Qu, Y. Gai, X. Jiang, Simultaneous determination of 24 or more acidic and alkaline phytohormones in femtomole quantities of plant tissues by high-performance liquid chromatography–electrospray ionization–ion trap mass spectrometry, Anal Bioanal Chem, 405 (2013) 1257-1266.
[20] E. Hauserova, J. Swaczynova, K. Dolezal, R. Lenobel, I. Popa, M. Hajduch, D. Vydra, K. Fuksova, M.
SC
Strnad, Batch immunoextraction method for efficient purification of aromatic cytokinins, J Chromatogr A, 1100 (2005) 116-125.
[21] P. Ulvskov, J. Marcussen, P. Seiden, C. Olsen, Immunoaffinity purification using monoclonal Alaska peas, Planta, 188 (1992) 182-189.
M AN U
antibodies for the isolation of indole auxins from elongation zones of epicotyls of red-light-grown [22] Y. Liang, X. Zhu, M. Zhao, H. Liu, Sensitive quantification of isoprenoid cytokinins in plants by selective immunoaffinity purification and high performance liquid chromatography–quadrupole-time of flight mass spectrometry, Methods, 56 (2012) 174-179.
[23] X.Q. Pan, R. Welti, X.M. Wang, Quantitative analysis of major plant hormones in crude plant extracts by high-performance liquid chromatography-mass spectrometry, Nat Protoc, 5 (2010)
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986-992.
[24] M.-L. Chen, X.-M. Fu, J.-Q. Liu, T.-T. Ye, S.-Y. Hou, Y.-Q. Huang, B.-F. Yuan, Y. Wu, Y.-Q. Feng, Highly sensitive and quantitative profiling of acidic phytohormones using derivatization approach coupled with nano-LC–ESI-Q-TOF-MS analysis, J Chromatogr B, 905 (2012) 67-74. [25] G.U. Balcke, V. Handrick, N. Bergau, M. Fichtner, A. Henning, H. Stellmach, A. Tissier, B. Hause, A.
EP
Frolov, An UPLC-MS/MS method for highly sensitive high-throughput analysis of phytohormones in plant tissues, Plant Methods, 8 (2012) 47. [26] K. Cui, Y. Lin, X. Zhou, S. Li, H. Liu, F. Zeng, F. Zhu, G. Ouyang, Z. Zeng, Comparison of sample pretreatment methods for the determination of multiple phytohormones in plant samples by liquid
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392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435
chromatography–electrospray ionization-tandem mass spectrometry, Microchem J, 121 (2015) 25-31. [27] Z. Ma, L. Ge, A.S. Lee, J.W. Yong, S.N. Tan, E.S. Ong, Simultaneous analysis of different classes of phytohormones in coconut (Cocos nucifera L.) water using high-performance liquid chromatography and liquid chromatography-tandem mass spectrometry after solid-phase extraction, Anal Chim Acta, 610 (2008) 274-281.
[28] Z. Liu, F. Wei, Y.-Q. Feng, Determination of cytokinins in plant samples by polymer monolith microextraction coupled with hydrophilic interaction chromatography-tandem mass spectrometry, Analytical Methods, 2 (2010) 1676-1685. [29] Z. Liu, B.D. Cai, Y.Q. Feng, Rapid determination of endogenous cytokinins in plant samples by combination of magnetic solid phase extraction with hydrophilic interaction chromatography-tandem mass spectrometry, J. Chromatogr. B, 891 (2012) 27-35. [30] G. Liu, H. Wang, X. Yang, Synthesis of pH-sensitive hollow polymer microspheres with movable
ACCEPTED MANUSCRIPT magnetic core, Polymer, 50 (2009) 2578-2586. [31] J. Ding, L.-J. Mao, S.-T. Wang, B.-F. Yuan, Y.-Q. Feng, Determination of Endogenous Brassinosteroids in Plant Tissues Using Solid-phase Extraction with Double Layered Cartridge Followed by High-performance Liquid Chromatography-Tandem Mass Spectrometry, Phytochemical Analysis, 24 (2013) 386-394. [32] J. Ding, L.-J. Mao, B.-F. Yuan, Y.-Q. Feng, 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, 9 (2013).
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[33] J. Ding, L.-J. Mao, N. Guo, L. Yu, Y.-Q. Feng, Determination of endogenous brassinosteroids using sequential magnetic solid phase extraction followed by in situ derivatization/desorption method coupled with liquid chromatography–tandem mass spectrometry, Journal of Chromatography A, 1446 (2016) 103-113.
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ACCEPTED MANUSCRIPT Figure captions
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Fig. 1. Scheme of the sequential magnetic solid-phase extraction.
451
Fig. 2. Effect of pH in sampling solution on CK nucleosides (A) and BRs (B)
452
extraction efficiencies; effect of ACN content in sampling solution on CK nucleosides
453
(C) and BRs (D). BRs (4 ng) and CK nucleosides (iPR, tZR, DHZR, 0.2 ng for each
454
compound) were dissolved in sampling solution (1 mL) for investigation. Internal
455
standards (I.S.) were added into the enriched samples just before UHPLC–MS/MS
456
analysis.
457
Fig. 3. Recoveries of BRs and CKs in the entire sample preparation procedure. BRs (4
458
ng for each compound) and CKs (0.2 ng for each compound) were dissolved in ACN
459
(1 mL) for investigation. Internal standards (I.S.) were added into the enriched
460
samples just before UHPLC–MS/MS analysis.
461
Fig. 4. Comparison of recoveries of BRs between TiO2-based MSPE and the proposed
462
sequential MPSE. BRs (4 ng for each compound) were dissolved in ACN (1 mL) for
463
investigation. Internal standards (I.S.) were added into the enriched samples just
464
before UHPLC–MS/MS analysis.
465
Fig. 5. Comparison of purification performance of BRs between TiO2-based MSPE
466
and the proposed sequential MSPE through TIC chromatogram (A) and extracted m/z
467
chromatogram from scan mode of BL (B).
468
Fig. 6. Comparison of desorption efficiencies of acidic phytohormones and CKs by
469
using 20% ACN and 20% ACN containing 1% NH3·H2O (v/v) as desorption solvent.
470
IAA, ABA, JA (1 ng for each compound), GAs (0.4 ng for each compound) and CKs
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472
standards (I.S.) were added into the enriched samples just before UHPLC–MS/MS
473
analysis.
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Fig. 7. MRM chromatograms of endogenous phytohormones detected in 100 mg
475
flower of Brassica napus L.
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Table
1.
The
extraction
capacity
of
three
batches
of
489
Fe3O4@SiO2@Poly(DMAPBA-co-EGDMA) adsorbents (20 mg magnetic material
490
and 5 µg adenosine were spiked).
Ш
RSD (%)
849.3
745.5
814.5
6.6
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II
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492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521
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Extraction capacity (µg g-1)
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Retention Time (min)
Linear dynamic range (ng mL-1)
Slope
Intercept
iP iPR
5.90 9.49
0.01 – 30 0.01 – 30
0.2199 0.3089
0.0012 0.0015
iP9G
7.15
0.01 – 30
0.7626
0.0045
tZ
1.53
0.01 – 30
0.4452
tZR
5.38
0.01 – 30
tZ9G
2.64
DHZ
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Table 2. Retention time, linearity and LODs of target phytohormones.
r value
LODs in standard solution (pg mL-1)
0.9989 0.9979
2.34 0.98
0.9966
0.88
0.0017
0.9992
1.96
0.2468
0.0003
0.9996
1.22
7.9 1.2
0.01 – 30
2.5654
0.0030
0.9983
0.45
22.1
1.60
0.01 – 30
0.2553
0.0017
0.9987
2.48
2.6
DHZR
5.40
0.01 – 30
0.3630
0.0011
0.9961
2.36
3.8
IAA
10.06
0.10 – 100
0.0343
0.0094
0.9972
27.0
97.4
ABA
12.81
0.20 – 100
0.0504
0.0021
0.9987
59.6
45.6
JA
14.25
0.05 – 100
0.0490
0.0457
0.9998
23.6
16.4
GA1
9.10
0.05 – 30
0.1809
0.0068
0.9975
21.0
55.6
GA3
8.92
0.05 – 30
0.5223
0.0203
0.9969
6.3
57.7
GA4
15.48
0.05 – 30
0.1307
0.0083
0.9994
15.4
135.9
GA7
15.36
0.05 – 30
1.4796
0.0462
0.9988
12.5
9.5
GA12
17.50
0.05 – 30
0.2095
0.0035
0.9991
6.1
128.9
GA24
15.80
0.05 – 30
0.2375
0.0324
0.9971
1.9
52.2
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Regression line
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LODs in matrix* (pg g-1 FW) 3.1 3.0 5.8
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0.05 – 30
0.2030
0.0015
0.9975
10.6
89.6
28-norCS
4.16
0.5 – 100
0.3102
-0.0220
0.9990
119.0
152.9
28-norBL
3.63
0.5 – 100
0.0659
0.0093
0.9987
81.1
124.0
CS
4.91
0.5 – 100
0.0737
0.0108
0.9995
126.1
167.1
BL
4.31
0.5 – 100
0.0669
-0.0078
0.9991
83.3
112.9
0.2253
-0.0054
114.5
196.1
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0.9985
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Detected (ng)
Recovery (%)
RSD (%)
iP
0.1
0.11 ± 0.004
109.2
3.6
iPR
0.1
0.11 ± 0.003
114.6
2.7
iP9G
0.1
0.11 ± 0.006
108.3
5.7
tZ
0.1
0.11 ± 0.006
109.0
5.5
tZR
0.1
0.10 ± 0.006
104.5
5.8
tZ9G
0.1
0.11 ± 0.009
107.6
8.3
DHZ
0.1
0.11 ± 0.006
109.7
5.1
DHZR
0.1
0.11 ± 0.007
113.3
5.9
IAA
1.0
1.00 ± 0.16
99.8
15.9
ABA
1.0
1.06 ± 0.17
105.9
15.8
JA
1.0
1.13 ± 0.15
113.4
13.0
GA1
0.4
0.37 ± 0.06
91.6
16.1
GA3
0.4
0.36 ± 0.04
89.7
11.3
GA4
0.4
0.40 ± 0.04
100.6
8.8
GA7
0.4
0.41 ± 0.03
102.7
7.6
GA12
0.4
0.34 ± 0.01
85.0
4.1
GA24
0.4
0.41 ± 0.05
103.0
11.1
GA53
0.4
0.46 ± 0.03
116.2
6.9
28-norCS
1.0
1.11 ± 0.15
111.3
15.3
1.0
1.07 ± 0.09
107.5
9.1
1.0
0.95 ± 0.16
94.8
15.8
28-norBL
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Analytes
Spiked phytohormone standards (ng)
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Table 3. Recoveries of phytohormones spiked to sample matrices (n=3).
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525 526 527
BL
1.0
1.06 ± 0.12
106.4
12.1
28-homoBL
1.0
0.98 ± 0.16
98.2
16.0
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Table 4. Contents of endogenous phytohormones detected in 100 mg flower of Brassica napus L. Analyte
iP
iPR
tZ
tZR
DHZ
DHZR
IAA
ABA
0.09 ± 0.01
1.16 ± 0.06
0.20 ± 0.03
5.38 ± 0.69
0.14 ± 0.02
0.17 ± 0.02
38.18 ± 5.09
53.83 ± 1.27
JA
GA4
GA12
GA24
GA53
28-norCS
CS
BL
305.23 ± 7.94
9.09 ± 0.70
2.83 ± 0.10
14.35 ± 0.97
0.35 ± 0.04
3.39 ± 0.48
3.25 ± 0.19
10.43 ± 0.79
Analyte Content -1
531 532 533 534 535 536 537
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Fig. 1.
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Fig. 2.
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546 547
549 550 551
Fig. 3
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Fig. 4.
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559 560 561
Fig. 5.
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Fig. 6
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Fig. 7
ACCEPTED MANUSCRIPT A simple and efficient method was developed for simultaneous analysis of multiple phytohormones including IAA, ABA, JA, GAs, BRs and CKs. IAA, ABA, JA, GAs, BRs and CKs were purified by sequential magnetic
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solid-phase extraction. 16 endogenous phytohormones could be detected in 100 mg (fresh weight) flower
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Supporting Information
2
for
Improved methodology for analysis of multiple
4
phytohormones using sequential magnetic solid-phase
5
extraction coupled with
6
liquid chromatography-tandem mass spectrometry
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3
7
Xiao-Tong Luo a ※, Bao-Dong Cai a ※, Xi Chen b, Yu-Qi Feng a *
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a
11
Education), Department of Chemistry, Wuhan University, Wuhan 430072, China
12
b
13
※These
14
*Corresponding author. Tel: +86-27-68755595; fax: +86-27-68755595; Email:
15
[email protected]
17 18 19 20 21 22
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Wuhan Institute of Biotechnology, Wuhan 430075, China
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authors contributed equally to this work.
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Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of
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Fig. S1. Selectively enrichment of adenosine by prepared boronate affinity magnetic
25
adsorbent.
28
29
30
31
32
33
34
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Fig. S2. Comparison of purification performance of BRs between TiO2-based MSPE
37
and the proposed sequential MSPE through extracted m/z chromatogram from scan
38
mode of CS (A), 28-norBL (B), 28-norCS (C) and 28-homoBL (D).
41 42 43 44 45 46 47 48 49 50
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ACCEPTED MANUSCRIPT Table S1. The classification, name and structures of target phytohormones.
IAA
indole-3-acetic acid (IAA)
ABA
abscisic acid (ABA)
JA
jasmonic acid (JA)
Structure
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GA4
GA7
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GA24
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GA12
castasterone (CS)
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brassinolide (BL)
28-norbrassinolide (28-norBL)
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28-norcastasterone (28-norCS)
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28-homobrassinolde (28-homoBL)
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isopentenyladenine (iP)
isopentenyladenine riboside (iPR)
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CK
isopentenyladenine 9-glucoside (iP9G)
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trans-zeatin-riboside (tZR)
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trans-zeatin (tZ)
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trans-zeatin 9-glucoside (tZ9G)
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dihydrozeatin (DHZ)
dihydrozeatin riboside (DHZR)
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ACCEPTED MANUSCRIPT Table S2. Optimized parameters of multiple reaction monitoring (MRM) mode for analysis of 23 phytohormones.
Scan mode
Precursor ion
Product ion
DP
CE
(V)
(V)
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Compounds
+
204.1
118.9/136.0
iPR
+
336.1
136.0/204.1
iP9G
+
366.1
136.0/204.1
tZ
+
220.1
118.9/136.0
tZR
+
352.2
136.0/220.0
85/85
45/27
tZ9G
+
382.1
136.0/220.0
105/105
49/30
DHZ
+
DHZR
+
IAA
+
ABA
-
JA
-
45/23
90/90
44/26
80/80
45/29
80/80
45/25
M AN U 222.1
118.9/136.0
95/95
50/29
354.1
136.0/222.0
80/80
50/29
176.1
102.9/130.1
60/60
58/25
263.0
202.7/218.6
-80/-80
-23/-19
209.0
41.0/58.8
-65/-65
-25/-19
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GA1
90/90
SC
iP
-
347.1
228.6/272.7
-104/-104
-43/-34
-
345.0
142.6/238.6
-75/-75
-44/-22
-
331.1
212.6/256.7
-83/-83
-42/-32
-
329.0
210.6/222.7
-75/-75
-35/-25
-
331.1
286.7/312.9
-66/-66
-37/-37
-
345.1
256.7/300.8
-50/-50
-38/-31
GA53
-
347.1
302.9/328.9
-47/-47
-38/-38
28-norCS
+
451.3
415.2/433.2
104/104
25/16
28-norBL
+
467.3
413.3/431.3
108/108
21/17.5
CS
+
465.4
429.3/447.3
106/106
25/16
BL
+
481.3
315.4/319.1
115/115
23/23
28-homoBL
+
495.3
441.2/459.2
120/120
18/18
GA3
GA7 GA12
AC C
GA24
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GA4
The product ions in bold were used for quantification.
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N6-isopentenyladenine (iP), N6-isopentenyladenine riboside (iPR), N6-isopentenyladenine 9-glucoside (iP9G), trans-zeatin (tZ), trans-zeatin-riboside (tZR), trans-zeatin 9-glucoside (tZ9G), dihydrozeatin (DHZ), dihydrozeatin riboside (DHZR), indole-3-acetic acid (IAA), abscisic acid (ABA), jasmonic acid (JA), Gibberellins (GA1, GA3, GA4, GA7, GA12, GA24, GA53), 28-norbrassinolide (28-norBL), 28-norcastasterone (28-norCS), castasterone (CS), brassinolide (BL), 28-homobrassinolde (28-homoBL).
S0003-2670(17)30723-7
DOI:
10.1016/j.aca.2017.06.019
Reference:
ACA 235265
To appear in:
Analytica Chimica Acta
Received Date: 22 February 2017 Revised Date:
3 June 2017
Accepted Date: 11 June 2017
Please cite this article as: X.-T. Luo, B.-D. Cai, X. Chen, Y.-Q. Feng, Improved methodology for analysis of multiple phytohormones using sequential magnetic solid-phase extraction coupled with liquid chromatography-tandem mass spectrometry, Analytica Chimica Acta (2017), doi: 10.1016/ j.aca.2017.06.019. 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.
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ACCEPTED MANUSCRIPT
Improved methodology for analysis of multiple phytohormones using
2
sequential magnetic solid-phase extraction coupled with
3
liquid chromatography-tandem mass spectrometry
4
Xiao-Tong Luo a ※, Bao-Dong Cai a ※, Xi Chen b, Yu-Qi Feng a *
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a
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Education), Department of Chemistry, Wuhan University, Wuhan 430072, China
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b
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Wuhan Institute of Biotechnology, Wuhan 430075, China
※
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Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of
These authors contributed equally to this work.
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*Corresponding author. Tel: +86-27-68755595; fax: +86-27-68755595; Email:
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[email protected]
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Abstract Phytohormones are special small molecules that play important role in plant
16
growth and development at trace levels. Quantification of multiple phytohormones
17
will be great helpful for researches about cross-talks that plant hormones regulate the
18
responses of plants against both biotic and abiotic stresses by means of synergistic or
19
antagonistic interactions. In the current study, we developed a method for profiling of
20
phytohormones in one small sample, including indole-3-acetic acid, abscisic acid,
21
jasmonic acid, gibberellins, cytokinins and brassinosteroids. These phytohormones
22
mentioned above were firstly purified and separated by sequential magnetic
23
solid-phase extraction (MSPE) and then analyzed by ultra-high performance liquid
24
chromatography-electrospray tandem mass spectrometry (UHPLC-MS/MS). Under
25
the optimized extraction conditions, good linearity was obtained with correlation
26
coefficients (r) ranging from 0.9961 to 0.9998. The limits of detection (LODs, S/N =
27
3) were ranged from 0.45 to 126.1 pg mL-1. The recoveries were between 85.0% and
28
116.2%. The relative standard deviations (RSDs) were ranged from 2.7% to 16.1%.
29
With the proposed strategy, 23 phytohormones could be purified and analyzed from a
30
single plant extract. Finally, 16 phytohormones could be detected in 100 mg (fresh
31
weight) flower of Brassica napus L, including IAA, ABA, JA, 4 GAs, 3 BRs and 6
32
CKs with the concentration ranged from 0.09 to 305.23 ng g-1.
33
Key words: Phytohormones; Magnetic solid-phase extraction; Mass spectrometry
34
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1. Introduction Phytohormones are of vital importance at trace amount level for the growth and
37
development of plants and protective response against stress. [1, 2]. They are grouped
38
into several classes, including abscisic acid (ABA), auxins, gibberellins (GAs),
39
cytokinins (CKs), ethylene (ET), brassinosteroids (BRs), jasmonates and salicylates
40
according to their structures and physiological functions [3, 4]. Although each class of
41
phytohormones has been identified for their specific biological functions, they usually
42
collectively regulate the plant responses against biotic and abiotic stress by means of
43
synergistic effect or antagonistic effect [5-7]. It is referred to as cross-talk of
44
phytohormones which is universally present in plant physiological activities [5].
45
Because the cross-talks are complex and refined, the molecular mechanisms of them
46
remain elusive. For better understanding the cross-talk mechanisms of phytohormones,
47
simultaneous determination of multi-class phytohormones is needed.
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The challenges to simultaneous determination of multi-class phytohormones are
49
originated not only from their low abundance in plants (pmol per gram fresh weight,
50
pmol g−1 FW) and the complex matrix of plants, but also from the difficulties of
51
simultaneous extraction and purification of multi-class phytohormones due to their
52
diverse chemical properties. Although a variety of analytical methods have been
53
developed for single-class phytohormones by GC-MS or LC-MS [8-16], few methods
54
were reported for the simultaneous determination of multi-class phytohormones
55
[17-19].
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Up to now, many sample preparation methods have been developed for
57
enrichment and purification of multiple phytohormones, such as immune affinity
ACCEPTED MANUSCRIPT purification (IAP) [20-22], liquid-liquid extraction (LLE) [23, 24], solid-phase
59
extraction (SPE) [19, 25-27], polymer monolith microextraction (PMME) [28],
60
magnetic solid-phase extraction (MSPE) [29] and so on. Thanks to these sample
61
preparation methods, Kojima et al. developed a multi-step strategy for determination
62
of 43 phytohormones in rice including auxins, ABA, gibberellins and CKs [17]. Liu et
63
al. described a method for simultaneous analysis of CKs and acidic phytohormones
64
(auxins, ABA and GAs), in which a binary SPE was employed for purification and
65
enrichment of phytohormones [19]. CKs and acidic phytohormones were eluted from
66
different SPE cartridges respectively. The two fractions of elution were combined for
67
UHPLC-MS/MS analysis. More recently, a TiO2-based MSPE was developed for
68
profiling of multiple phytohormones, including indole-3-acetic acid (IAA), ABA, JA,
69
GAs and CKs through hydrophilic interaction and coordination [18]. All target
70
analytes mentioned above were assigned to acidic phytohormones (IAA, ABA, JA and
71
GAs) and CKs. Nevertheless, none of the methods could be used for simultaneous
72
analysis of acidic phytohormones, CKs and BRs.
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Recently, Tong et.al revealed a previously unknown mechanism underlying BR
74
and GA crosstalk depending on tissues and hormone levels, which greatly advanced
75
our understanding of hormone actions in crop plants, appearing much different from
76
that in Arabidopsis thaliana [6]. It is indispensable to get more information of
77
phytohormones from the same sample to illustrate the complex relationship between
78
phytohormones. However, it is still a challenge for simultaneous analysis of multiple
79
class phytohormones in one small sample, including acidic phytohormones, CKs and
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BRs. Also, it is a bottleneck that simultaneous purification of multiple phytohormones
81
with different structures and chemical properties from one crude plant extract. In the current study, we developed a method for simultaneous analysis of
83
multiple phytohormones including acidic phytohormones (IAA, ABA, JA, GAs), BRs
84
and CKs by sequential MSPE coupled with UHPLC-MS/MS. The classification, name
85
and structures of all the targets were shown in Table S1. In our proposed strategy, the
86
crude plant extract was firstly processed by a TiO2-based MSPE [18]. Subsequently,
87
the second MSPE was employed for further purification of BRs while the separation
88
of BRs from acidic hormones and CK could be realized. Finally, the developed
89
method was successfully applied to detect multiple phytohomones in flower of
90
Brassica napus L .
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2. Experimental
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2.1. Chemicals and reagents
Phytohormone standards: indole-3-acetic acid (IAA), abscisic acid (ABA),
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jasmonic acid (JA), gibberellic acids (GA1, GA3, GA4, GA7, GA12, GA24, GA53)，
96
28-norbrassinolide (28-norBL), 28-norcastasterone (28-norCS), 28-homobrassinolde
97
(28-homoBL), brassinolide (BL), castasterone (CS); N6-isopentenyladenine (iP),
98
N6-isopentenyladenine riboside (iPR), N6-isopentenyladenine 9-glucoside (iP9G),
99
trans-zeatin (tZ), trans-zeatin-riboside (tZR), trans-zeatin 9-glucoside (tZ9G),
100
dihydrozeatin (DHZ), dihydrozeatin riboside (DHZR) and stable isotope-labeled
101
standards: [2H2]IAA, [2H6]ABA, [2H2]GA1, [2H2]GA3, [2H2]GA4, [2H2]GA12,
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[2H2]GA24, [2H2]GA53, [2H3]BL, [2H3]CS, [2H6]iP, [2H6]iPR, [2H6]iP9G, [2H5]tZ,
103
[2H5]tZR, [2H5]tZ9G, [2H3]DHZ, [2H3]DHZR were all purchased from Olchemim Ltd.
104
(Olomouc, Czech Republic). Ferric chloride (FeCl3·6H2O), sodium acetate (NaAc), ethylene glycol (EG),
106
1,2-ethylenediamine (ETH), ethanol (EtOH), tetraethyl orthosilicate (TEOS),
107
ammonia hydrate (NH3·H2O, 25%, aqueous solution), ammonium hexafluorotitanate
108
((NH4)2TiF6),
109
2,2-azobis(2-methyl-propionitrile) (AIBN) were all purchased from Sinopharm
110
Chemical Reagent (Shanghai, China). 3-(Methacryloxy)propyl trimethoxysilane
111
(MPS) was bought from Wuhan University Silicone New Material (Wuhan, China),
112
while ethylene glycol dimethacrylate (EGDMA) was bought from Sigma–Aldrich (St.
113
Louis, MO, USA). 4-(N,N-dimethyamino) phenylboronic acid (DMAPBA) was
114
purchased from J&K Scientific Ltd (Beijing, China). AIBN was recrystallized from
115
ethanol, and other reagents were of analytical grade and used directly without further
116
purification. Acetonitrile (ACN, HPLC grade) was obtained from Tedia Co. (Fairfield,
117
OH, USA). Ultra-pure water used throughout the study was purified by Milli-Q
118
system (Milford, MA, USA).
pyridine,
formic
acid
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(H3BO3),
(FA,
88%),
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boric
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2.2.
Preparation
of
magnetic
121
Fe3O4@SiO2@Poly(DMAPBA-co-EGDMA)
polymer
particles
122
MPS-modified Fe3O4 particles were prepared according to our previous method
123
[29]. Subsequently, Fe3O4@SiO2@Poly(DMAPBA-co-EGDMA) particles were
ACCEPTED MANUSCRIPT synthesized by the distillation–precipitation polymerization method proposed by Yang
125
et al [30]. Briefly, DMAPBA (0.25 g) was dissolved in 200 mL ACN/pyridine (50/1,
126
v/v) in a 500-mL two-necked round bottom flask equipped with a distillation
127
apparatus and a stirring device. Then, MPS-modified Fe3O4@SiO2 (0.50 g), EGDMA
128
(2.25 g) and AIBN (0.02 g) were successively added. The mixture was heated to
129
boiling within 30 min and kept boiling until approximately half of the acetonitrile was
130
distilled out (occurring within 2 h). As soon as the mixture cooled down to room
131
temperature, Fe3O4@SiO2@Poly(DMAPBA-co-EGDMA) particles were separated by
132
the aid of a magnet, and washed several times with water and methanol. Finally, the
133
resultant magnetic polymers were dried in a vacuum at 60oC.
134
2.3. Plant materials
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Three-month-old Brassica napus L. flowers were provided by Dr Jia Liu from
136
Oil Crops Research Institute, Chinese Academy of Agricultural Sciences (Wuhan,
137
China). The plant materials were harvested, weighted, immediately frozen in liquid
138
nitrogen, and stored at -80oC.
139
2.4. Sample pretreatment
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The general process of sample pretreatment was shown in Fig. 1. Plant sample
141
tissues (100 mg FW) were frozen in liquid nitrogen, ground into powder and then
142
transferred into a 1.5 ml centrifuge tube. [2H2]IAA (1.0 ng), [2H6]ABA (1.0 ng), stable
143
isotope labeled GAs (0.4 ng), [2H3]BL (1.0 ng), [2H3]CS (1.0 ng) and stable isotope
144
labeled CKs (0.1 ng) were added to the samples, serving as internal standards for the
145
quantification. Next, ACN (0.5 mL) was added into the mixture. After extraction at
ACCEPTED MANUSCRIPT -20oC all night long, the supernatant was collected after centrifugation at 10,000 × g
147
below 0oC for 20 min. Subsequently, sequential MSPE was carried out. Firstly,
148
TiO2-based MSPE was employed for enrichment of all target analytes according to
149
our previous work with minor modification [18]. Briefly, the supernatant was added
150
into a 2-mL vial containing Fe3O4@TiO2 (50 mg) magnetic nanoparticles pretreated
151
with ACN (1.0 mL). Before the supernatant was discarded with the help of magnetic
152
field, the mixture was vortexed vigorously for 1 min. Afterwards, the magnetic
153
sorbent, which adsorbed analytes, was washed by ACN (1.0 mL) twice. Then, 1 mL
154
20% ACN (v/v) aqueous solution containing 1% NH3·H2O (v/v) was added as elution
155
solvent, in which 20 mg Fe3O4@SiO2@Poly(DMAPBA-co-EGDMA) dispersed.
156
After vigorously vortexed for 5 min, acidic phytohormones, CKs and BRs were all
157
eluted. Meanwhile, BRs, which contain cis-hydroxyl group, were re-enriched by
158
Fe3O4@SiO2@Poly(DMAPBA-co-EGDMA) through boronate affinity interaction.
159
With the aid of a magnet, acidic phytohormones and CKs in elution were collected
160
and evaporated to dryness under a mild nitrogen gas flow at 35°C. Finally, the sample
161
was re-dissolved in 50 µL 30% MeOH, and 10 µL was injected for the quantification
162
of acidic phytohormones and CKs by UHPLC-ESI-MS/MS. The BR adsorbed
163
magnetic nanoparticles were washed orderly by 1 mL 50% ACN and 1 mL ACN.
164
After vortexed for 5 min, BRs were desorbed with 3% H2O2 in 90% ACN aqueous
165
(v/v, 1 mL). The desorption solution was collected and evaporated under mild
166
nitrogen stream. Finally, the sample was re-dissolving in 50 µL 30% MeOH and 10
167
µL of the solution was injected for the quantification of BRs by UHPLC-ESI-MS/MS.
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ACCEPTED MANUSCRIPT 168
2.5. Instruments and analytical conditions Analysis of phytohormones was performed on a UHPLC-ESI-MS/MS system
170
consisting of a AB SCIEX 4500 triple quadrupole mass spectrometer (Foster City, CA,
171
USA) with an electrospray ionization source (Turbo Ionspray), a Shimadzu LC-30AD
172
HPLC system (Kyoto, Japan) with two 30AD pumps, a SIL-30AC auto sampler, a
173
CTO-30A thermostat column compartment, and a DGU-20A5R degasser. Data
174
acquisition and processing were performed using AB SCIEX Analyst 1.6 software
175
(Foster City, CA, USA).
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The UHPLC separation was performed at 40oC on a Shim-pack XR-ODS Ш
177
column (75 mm × 2.0 mm i.d., 1.6 µm) purchased from Shimadzu (Kyoto, Japan). A
178
27-min gradient of 0.05% FA in H2O (A) and 100% ACN (B) was employed for the
179
separation of acidic phytohormones and CKs with a flow rate of 0.4 mL min-1. A
180
linear gradient with the following proportions (v/v) of solvent B was applied: 0-2 min
181
at 5%, 2-12 min from 5% to 25%, 12-20 min from 25% to 80%, 20-22 min at 80%,
182
22-24 min from 80%-5%, followed by 3 min of re-equilibration at 5%. For separating
183
BRs, 14-min gradient of 0.05% FA in H2O (A) and 100% ACN (B) was employed
184
with a flow rate of 0.4 mL min-1. A linear gradient with the following proportions (v/v)
185
of solvent B was applied: 0-6 min from 30% to 65%, 6-7 min from 65% to 85%, 7-10
186
min at 85%, 10-11 min from 85%-30%, followed by 3 min of re-equilibration at 30%.
187
Multiple reaction monitoring (MRM) and the appropriate product ions were
188
chosen to quantify phytohormones. The optimized conditions for selective MRM
189
experiments of acidic phytohormones, CKs and BRs were listed in Table S2. And
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other optimized conditions of MRM experiments were as follows: curtain gas, 40 psi;
191
ion spray voltage, 5500 V; turbo heater temperature (TEM), 450oC; nebulizing gas
192
(Gas 1), 55 psi; heated gas (Gas 2), 45 psi.
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3. Results and discussion
195
3.1. Evaluation of Fe3O4@SiO2@Poly(DMAPBA-co-EGDMA)
Boronate affinity chromatography is a powerful tool for selective separation and
197
enrichment of cis-diol containing compounds. In this study, adenosine (A) and
198
deoxyadenosine (dA) were selected as model compounds to investigate the selectivity
199
of
200
Fe3O4@SiO2@Poly(DMAPBA-co-EGDMA), cis-diol adenosine was hardly detected
201
in residual solution, while deoxyadenosine was detected with the similar signal
202
strength from standard solution (Fig. S1). It meant that adenosine could be extracted
203
by the prepared Fe3O4@SiO2@Poly(DMAPBA-co-EGDMA) while deoxyadenosine
204
could not. And the average efficiency of adenosine elution from the beads (n = 3) was
205
102.5% (RSD = 7.4%). This result demonstrated high specificity of the prepared
206
magnetic nanoparticles towards cis-diol-containing molecules, further indicating that
207
the
208
successfully.
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After
extracted
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Fe3O4@SiO2@Poly(DMAPBA-co-EGDMA)
nanoparticles
were
prepared
209
Next, the extraction capacity of the boronate affinity magnetic nanoparticles,
210
which was 849.3 µg g-1, was evaluated by using adenosine as model compound. The
211
reproducibility of preparation was also investigated by estimating the extraction
ACCEPTED MANUSCRIPT capacity of three batches of the magnetic polymer adsorbents. As the results shown in
213
Table 1, the relative standard deviation (RSD) of the extraction capacity was only
214
6.6%, suggesting good reproducibility of the preparation method.
215
3.2 Enrichment of BRs with boronate affinity MSPE
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As BRs and CK nucleosides (iPR, tZR, DHZR) all contain cis-diol, they could
217
all be extracted by the prepared boronate affinity magnetic adsorbents. However, it
218
was better that CK nucleosides could not be extracted in further purification of BRs.
219
Because the H2O2 contained in BRs desorption solution could result in CK
220
degradation. To ensure BRs be extracted through adsorbents while CK nucleosides
221
not, we investigated if pH of sampling solution could influence extraction efficiency
222
of BRs and CK nucleosides first. BRs were extracted in 50% ACN (v/v) with pH from
223
3.0 to 10.0, while CK nucleosides were extracted in 10% ACN (v/v) with pH from 3.0
224
to 10.0. After extracted, the magnetic adsorbents were washed with sampling solution.
225
Then, BRs were eluted with 90% ACN (v/v) containing 3% H2O2 (v/v) while CK
226
nucleosides were eluted with 10% ACN (v/v) containing 1% FA (v/v). Finally, the
227
eluted analytes were analyzed. As shown in Fig. 2A and Fig. 2B, the recoveries of
228
both CK nucleosides and BRs were low when pH under 8. While pH up to 9 and 10,
229
the recoveries were increased sharply. It indicated that both CK nucleosides and BRs
230
were inclined to be extracted by Fe3O4@SiO2@Poly(DMAPBA-co-EGDMA) when
231
the sampling solution was alkaline. Meanwhile, it demonstrated that it was hard to
232
separate CK nucleosides and BRs relying on pH in sampling solution.
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Then, ACN content in sampling solution was also investigated in the range of 0
ACCEPTED MANUSCRIPT to 90% (v/v). As shown in Fig. 2C, recoveries of CK nucleosides decreased
235
dramatically as ACN content increased from 0 to 20% and CK nucleosides were
236
hardly extracted when ACN content was over 20%. However, the recoveries of BRs
237
increased with the increase of ACN content in the range of 5% to 20% and decreased
238
with the increase of ACN content in the range of 20% to 80% (Fig. 2D).that the
239
existence of ACN in sampling solution was negative for boronate affinity reaction,
240
therefore resulted in reduced recoveries of CKs with increasing content of ACN. As
241
BRs are hydrophobic compounds, they have better solubility with 20% or more ACN
242
content in sampling solution. So, it resulted in increasing recoveries. However, if
243
ACN content further increased, a decreasing recovery could be observed. Furthermore,
244
the increasing recoveries of BRs in the range of 80% to 90% may be contributed to
245
hydrophilic interaction. Therefore, we assumed that BRs could be selectively
246
extracted with 20% ACN while CK nucleosides could not.
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Inspired by the experiment above, we concluded that CK nucleosides and BRs
248
could be separated in alkaline aqueous solution with 20% ACN (v/v). For easy
249
operation, the solution which contained 20% ACN and 1% NH3·H2O (v/v) was
250
employed. However, the pH of chosen solution was approximately 11，which was not
251
investigated above. Hence, the extract efficiency of BRs and CKs was detected. First,
252
BRs and CKs (CK free bases, nucleosides and glucosides) were dissolved in sampling
253
solution, in which 20% ACN and 1% NH3·H2O (v/v) contained. Then, it was
254
extracted by the prepared boronate affinity magnetic nanoparticles. After extraction,
255
the residual solution was collected and the magnetic nanoparticles were washed by
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ACCEPTED MANUSCRIPT sampling solution. BRs were eluted by 90% ACN (v/v) together with 3% H2O2 (v/v)
257
from the magnetic nanoparticles. As shown in Fig. 3A, most of CKs were detected in
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the residual solution while BRs could not (data not shown). Meanwhile, the recoveries
259
of BRs were detected more than 90% as shown in Fig. 3B. These results suggested
260
that, with the proposed solution containing 20% ACN and 1% NH3·H2O (v/v), only
261
BRs could be extracted by the prepared boronate affinity magnetic nanoparticles.
262
3.3. Comparison of two pretreatment methods
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Recently, we developed a TiO2-based MSPE method for simultaneous analysis of
264
acidic phytohormones (carboxyl compounds) and CKs [18]. The method was simple,
265
rapid and effective. The TiO2 coated magnetic nanoparticles could adsorb acidic
266
phytohormones, CKs and BRs through hydrophilic interaction and coordination [14,
267
18]. However, BRs could not be detected in plant sample (spiked with BR standards)
268
through the proposed one-step sample preparation method because of the matrix
269
interference. Now the newly developed sequential MSPE method could solve this
270
problem. To compare the two pretreatment methods, the aspects of recoveries,
271
selectivity and desorption efficiencies were investigated.
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First, the recoveries of BRs using TiO2-based MSPE and the sequential MSPE
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were investigated in spiked sample matrices. As shown in Fig. 4, the recoveries of
274
BRs using TiO2-based MSPE were between 55% and 69%, which was similar to the
275
recoveries of the sequential MSPE. This further suggested that the BRs eluted from
276
TiO2 magnetic nanoparticles were mostly extracted by boronate affinity adsorbent.
277
The result was consistent with that in standard solution (Fig. 3B), indicating that
ACCEPTED MANUSCRIPT 278
sample matrix had little influence on BRs enrichment using the prepared adsorbents. To improve the selectivity, the boronate affinity MSPE was employed for the
280
further purification of BRs after TiO2-based MSPE in the current study. The
281
performance of these two methods (TiO2-based MSPE and sequential MSPE) was
282
compared. Fig. 5A represented TIC chromatogram of BR standards (4 ng for each
283
standards) dissolved in ACN treated with sequential MSPE, plant sample (spiked with
284
4 ng for each BR standards) treated with TiO2-based MSPE and plant sample (spiked
285
with 4 ng for each BR standards) treated with sequential MSPE. Fig. 5B represented
286
the chromatogram of extracted m/z from scan mode of BL. Based on the TIC (Fig. 5A)
287
and extracted chromatograms (Fig. 5B and Fig. S2), the purification performance of
288
sequential MSPE method was much better than that of TiO2-based MSPE. And it was
289
similar to the BR standards without plant matrix, suggesting high selectivity and
290
powerful ability of removing matrix of our newly developed method.
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During boronate affinity MSPE procedure, 20% ACN containing 1% NH3·H2O
292
(v/v) was employed as sampling solution which could also be used as desorption
293
solution for TiO2-based MSPE. Therefore, the desorption efficiencies using 20% ACN
294
and 20% ACN containing 1% NH3·H2O as desorption solution during TiO2-based
295
MSPE were compared. Acidic phytohormones and CKs spiked in matrix was
296
extracted by Fe3O4@TiO2. 20% ACN and 20% ACN containing 1% NH3·H2O were
297
employed as desorption solution. The results showed that using 20% ACN containing
298
1% NH3·H2O as desorption solution ensured high recoveries for dual carboxyl
299
compounds (GA12, GA24, GA53); for other phytohormones, the recoveries were
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301
Fe3O4@SiO2@Poly(DMAPBA-co-EGDMA) in 20% ACN (v/v) containing 1%
302
NH3·H2O. Thus, the desorption of all phytohotmones from Fe3O4@TiO2 as well as
303
selective enrichment of BRs by boronate affinity MSPE could be simultaneously
304
completed.
305
3.4. Method validation
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The linearity of the developed method was evaluated by preparing 11-point
307
calibration curves in 30% MeOH aqueous solution for the different phytohormones in
308
triplicate. The calibration curves were constructed by plotting the peak area ratio
309
(analytes/I.S.) versus the phytohormones concentrations. As listed in Table 2, good
310
linearity was obtained with correlation coefficients (r) ranging from 0.9961 to 0.9998.
311
The limits of detection (LODs) in standard solution and matrix were calculated as the
312
signal to noise ratios being 3:1. The results showed that LODs in standard solution
313
and matrix ranged from 0.45 to 126.1 pg mL-1 and 1.2 to 196.1 pg mL-1 respectively,
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indicating that it was quite sensitive for profiling of endogenous phytohormones in
315
plant samples.
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In addition, the effect of plant matrix on the quantification of endogenous
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phytohormones was evaluated by spiking phytohormone standards into plant sample.
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The results showed that the spiked phytohormones could be successfully determined
319
with 85.0%–116.2% recoveries and the relative standard deviations (RSDs) were
320
2.7%–16.1% (Table 3).
321
Finally, the developed sequential MSPE method was used for analysis of
ACCEPTED MANUSCRIPT multiple phytohormones in flower of Brassica napus L. As listed in Table 4,
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phytohormones analysis could be completed with 100 mg (FW) flower of Brassica
324
napus L and 16 phytohormoes were successfully detected, including 6 CKs, IAA,
325
ABA, JA and 4 GAs and 3 BRs. The MRM chromatograms of 16 detected
326
endogenous phytohormones were shown in Fig. 7. According to previous reports, the
327
contents of CKs were ranged from 0.03-15.40 ng g-1 FW and the contents of BRs were
328
ranged from 5.54-89.80 ng g-1 FW in Brassica napus flowers [13, 31-33]. And in this
329
work, as shown in Table 4, the contents of CKs were ranged from 0.09-5.38 ng g-1 FW
330
and the contents of BRs were ranged from 3.25-10.43 ng g-1 FW. The phytohormones
331
contents detected by this method were in the range of the contents detected before,
332
which validated the method further.
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4. Conclusion
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In this study, magnetic poly (4-(N,N-dimethyamino) phenylboronic acid ̶ co ̶
335
ethylene glycol dimethacrylate) particles were prepared by distillation precipitation
336
polymerization and was employed for class-selective enrichment of phytohormones
337
coupled with TiO2-based MSPE. The sequential MSPE was more selective for BRs
338
than TiO2-based MSPE. By combination with UHPLC-MS/MS, a rapid and selective
339
analytical method for the analysis of phytohormones, including acidic phytohormones
340
(IAA, ABA, JA, GAs), BRs and CKs was established. Finally, 16 phytohormones
341
could be detected in flower of Brassica napus L .
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Acknowledgements This work was supported by the National Natural Science Foundation of China
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(91217309, 31670373), and the Natural Science Foundation of Hubei Province, China
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(2014CFA002), the Fundamental Research Funds for the Central Universities.
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References
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[1] J. Fu, X. Sun, J. Wang, J. Chu, C. Yan, Progress in quantitative analysis of plant hormones, Chin. Sci. Bull., 56 (2011) 355-366. [2] L. Van Meulebroek, J.V. Bussche, K. Steppe, L. Vanhaecke, Ultra-high performance liquid chromatography coupled to high resolution Orbitrap mass spectrometry for metabolomic profiling of the endogenous phytohormonal status of the tomato plant, J Chromatogr A, 1260 (2012) 67-80.
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[3] P. Davies, The Plant Hormones: Their Nature, Occurrence, and Functions, in: P. Davies (Ed.) Plant Hormones, Springer Netherlands1995, pp. 1-12.
[4] M. Umehara, A. Hanada, S. Yoshida, K. Akiyama, T. Arite, N. Takeda-Kamiya, H. Magome, Y. Kamiya, K. Shirasu, K. Yoneyama, J. Kyozuka, S. Yamaguchi, Inhibition of shoot branching by new terpenoid plant hormones, Nature, 455 (2008) 195-200.
[5] E.A. Schmelz, J. Engelberth, H.T. Alborn, P. O'Donnell, M. Sammons, H. Toshima, J.H. Tumlinson,
SC
Simultaneous analysis of phytohormones, phytotoxins, and volatile organic compounds in plants, Proceedings of the National Academy of Sciences, 100 (2003) 10552-10557.
[6] H. Tong, Y. Xiao, D. Liu, S. Gao, L. Liu, Y. Yin, Y. Jin, Q. Qian, C. Chu, Brassinosteroid regulates cell
M AN U
elongation by modulating gibberellin metabolism in rice, The Plant cell, 26 (2014) 4376-4393. [7] S. Gao, J. Fang, F. Xu, W. Wang, X. Sun, J. Chu, B. Cai, Y. Feng, C. Chu, CYTOKININ OXIDASE/DEHYDROGENASE4 Integrates Cytokinin and Auxin Signaling to Control Rice Crown Root Formation, Plant physiology, 165 (2014) 1035-1046.
[8] M. Manzi, A. Gomez-Cadenas, V. Arbona, Rapid and reproducible determination of active gibberellins in citrus tissues by UPLC/ESI-MS/MS, Plant Physiol Biochem, 94 (2015) 1-9. [9] Y.-H. Hao, Z. Zhang, L. Wang, C. Liu, A.-W. Lei, B.-F. Yuan, Y.-Q. Feng, Stable isotope labeling assisted
TE D
liquid chromatography–electrospray tandem mass spectrometry for quantitative analysis of endogenous gibberellins, Talanta, 144 (2015) 341-348. [10] Y.Q. Huang, J.Q. Liu, H. Gong, J. Yang, Y. Li, Y.Q. Feng, Use of isotope mass probes for metabolic analysis of the jasmonate biosynthetic pathway, Analyst, 136 (2011) 1515-1522. [11] T. Henriksen, R.K. Juhler, B. Svensmark, N.B. Cech, The relative influences of acidity and polarity
EP
on responsiveness of small organic molecules to analysis with negative ion electrospray ionization mass spectrometry (ESI-MS), J Am Soc Mass Spectrom, 16 (2005) 446-455. [12] C. Birkemeyer, A. Kolasa, J. Kopka, Comprehensive chemical derivatization for gas
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chromatography-mass spectrometry-based multi-targeted profiling of the major phytohormones, J Chromatogr A, 993 (2003) 89-102. [13] B.-D. Cai, J.-X. Zhu, Z.-G. Shi, B.-F. Yuan, Y.-Q. Feng, A simple sample preparation approach based on hydrophilic solid-phase extraction coupled with liquid chromatography–tandem mass spectrometry for determination of endogenous cytokinins, Journal Of Chromatography B, 942-943 (2013) 31-36. [14] J. Ding, J.-H. Wu, J.-F. Liu, B.-F. Yuan, Y.-Q. Feng, Improved methodology for assaying brassinosteroids in plant tissues using magnetic hydrophilic material for both extraction and derivatization, Plant Methods, 10 (2014) 39. [15] P. Xin, J. Yan, J. Fan, J. Chu, C. Yan, A dual role of boronate affinity in high-sensitivity detection of vicinal diol brassinosteroids from sub-gram plant tissues via UPLC-MS/MS, The Analyst, 138 (2013) 1342-1345. [16] D. Li, Z. Guo, Y. Chen, Direct Derivatization and Quantitation of Ultra-trace Gibberellins in Sub-milligram Fresh Plant Organs, Molecular Plant, 9
175-177.
ACCEPTED MANUSCRIPT [17] M. Kojima, T. Kamada-Nobusada, H. Komatsu, K. Takei, T. Kuroha, M. Mizutani, M. Ashikari, M. Ueguchi-Tanaka, M. Matsuoka, K. Suzuki, H. Sakakibara, Highly sensitive and high-throughput analysis of plant hormones using MS-probe modification and liquid chromatography-tandem mass spectrometry: an application for hormone profiling in Oryza sativa, Plant Cell Physiol, 50 (2009) 1201-1214. [18] B.D. Cai, J. Yin, Y.H. Hao, Y.N. Li, B.F. Yuan, Y.Q. Feng, Profiling of phytohormones in rice under elevated cadmium concentration levels by magnetic solid-phase extraction coupled with liquid
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chromatography tandem mass spectrometry, Journal of chromatography. A, 1406 (2015) 78-86.
[19] S. Liu, W. Chen, L. Qu, Y. Gai, X. Jiang, Simultaneous determination of 24 or more acidic and alkaline phytohormones in femtomole quantities of plant tissues by high-performance liquid chromatography–electrospray ionization–ion trap mass spectrometry, Anal Bioanal Chem, 405 (2013) 1257-1266.
[20] E. Hauserova, J. Swaczynova, K. Dolezal, R. Lenobel, I. Popa, M. Hajduch, D. Vydra, K. Fuksova, M.
SC
Strnad, Batch immunoextraction method for efficient purification of aromatic cytokinins, J Chromatogr A, 1100 (2005) 116-125.
[21] P. Ulvskov, J. Marcussen, P. Seiden, C. Olsen, Immunoaffinity purification using monoclonal Alaska peas, Planta, 188 (1992) 182-189.
M AN U
antibodies for the isolation of indole auxins from elongation zones of epicotyls of red-light-grown [22] Y. Liang, X. Zhu, M. Zhao, H. Liu, Sensitive quantification of isoprenoid cytokinins in plants by selective immunoaffinity purification and high performance liquid chromatography–quadrupole-time of flight mass spectrometry, Methods, 56 (2012) 174-179.
[23] X.Q. Pan, R. Welti, X.M. Wang, Quantitative analysis of major plant hormones in crude plant extracts by high-performance liquid chromatography-mass spectrometry, Nat Protoc, 5 (2010)
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986-992.
[24] M.-L. Chen, X.-M. Fu, J.-Q. Liu, T.-T. Ye, S.-Y. Hou, Y.-Q. Huang, B.-F. Yuan, Y. Wu, Y.-Q. Feng, Highly sensitive and quantitative profiling of acidic phytohormones using derivatization approach coupled with nano-LC–ESI-Q-TOF-MS analysis, J Chromatogr B, 905 (2012) 67-74. [25] G.U. Balcke, V. Handrick, N. Bergau, M. Fichtner, A. Henning, H. Stellmach, A. Tissier, B. Hause, A.
EP
Frolov, An UPLC-MS/MS method for highly sensitive high-throughput analysis of phytohormones in plant tissues, Plant Methods, 8 (2012) 47. [26] K. Cui, Y. Lin, X. Zhou, S. Li, H. Liu, F. Zeng, F. Zhu, G. Ouyang, Z. Zeng, Comparison of sample pretreatment methods for the determination of multiple phytohormones in plant samples by liquid
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392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435
chromatography–electrospray ionization-tandem mass spectrometry, Microchem J, 121 (2015) 25-31. [27] Z. Ma, L. Ge, A.S. Lee, J.W. Yong, S.N. Tan, E.S. Ong, Simultaneous analysis of different classes of phytohormones in coconut (Cocos nucifera L.) water using high-performance liquid chromatography and liquid chromatography-tandem mass spectrometry after solid-phase extraction, Anal Chim Acta, 610 (2008) 274-281.
[28] Z. Liu, F. Wei, Y.-Q. Feng, Determination of cytokinins in plant samples by polymer monolith microextraction coupled with hydrophilic interaction chromatography-tandem mass spectrometry, Analytical Methods, 2 (2010) 1676-1685. [29] Z. Liu, B.D. Cai, Y.Q. Feng, Rapid determination of endogenous cytokinins in plant samples by combination of magnetic solid phase extraction with hydrophilic interaction chromatography-tandem mass spectrometry, J. Chromatogr. B, 891 (2012) 27-35. [30] G. Liu, H. Wang, X. Yang, Synthesis of pH-sensitive hollow polymer microspheres with movable
ACCEPTED MANUSCRIPT magnetic core, Polymer, 50 (2009) 2578-2586. [31] J. Ding, L.-J. Mao, S.-T. Wang, B.-F. Yuan, Y.-Q. Feng, Determination of Endogenous Brassinosteroids in Plant Tissues Using Solid-phase Extraction with Double Layered Cartridge Followed by High-performance Liquid Chromatography-Tandem Mass Spectrometry, Phytochemical Analysis, 24 (2013) 386-394. [32] J. Ding, L.-J. Mao, B.-F. Yuan, Y.-Q. Feng, 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, 9 (2013).
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[33] J. Ding, L.-J. Mao, N. Guo, L. Yu, Y.-Q. Feng, Determination of endogenous brassinosteroids using sequential magnetic solid phase extraction followed by in situ derivatization/desorption method coupled with liquid chromatography–tandem mass spectrometry, Journal of Chromatography A, 1446 (2016) 103-113.
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ACCEPTED MANUSCRIPT Figure captions
450
Fig. 1. Scheme of the sequential magnetic solid-phase extraction.
451
Fig. 2. Effect of pH in sampling solution on CK nucleosides (A) and BRs (B)
452
extraction efficiencies; effect of ACN content in sampling solution on CK nucleosides
453
(C) and BRs (D). BRs (4 ng) and CK nucleosides (iPR, tZR, DHZR, 0.2 ng for each
454
compound) were dissolved in sampling solution (1 mL) for investigation. Internal
455
standards (I.S.) were added into the enriched samples just before UHPLC–MS/MS
456
analysis.
457
Fig. 3. Recoveries of BRs and CKs in the entire sample preparation procedure. BRs (4
458
ng for each compound) and CKs (0.2 ng for each compound) were dissolved in ACN
459
(1 mL) for investigation. Internal standards (I.S.) were added into the enriched
460
samples just before UHPLC–MS/MS analysis.
461
Fig. 4. Comparison of recoveries of BRs between TiO2-based MSPE and the proposed
462
sequential MPSE. BRs (4 ng for each compound) were dissolved in ACN (1 mL) for
463
investigation. Internal standards (I.S.) were added into the enriched samples just
464
before UHPLC–MS/MS analysis.
465
Fig. 5. Comparison of purification performance of BRs between TiO2-based MSPE
466
and the proposed sequential MSPE through TIC chromatogram (A) and extracted m/z
467
chromatogram from scan mode of BL (B).
468
Fig. 6. Comparison of desorption efficiencies of acidic phytohormones and CKs by
469
using 20% ACN and 20% ACN containing 1% NH3·H2O (v/v) as desorption solvent.
470
IAA, ABA, JA (1 ng for each compound), GAs (0.4 ng for each compound) and CKs
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472
standards (I.S.) were added into the enriched samples just before UHPLC–MS/MS
473
analysis.
474
Fig. 7. MRM chromatograms of endogenous phytohormones detected in 100 mg
475
flower of Brassica napus L.
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485 486 487
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Table
1.
The
extraction
capacity
of
three
batches
of
489
Fe3O4@SiO2@Poly(DMAPBA-co-EGDMA) adsorbents (20 mg magnetic material
490
and 5 µg adenosine were spiked).
Ш
RSD (%)
849.3
745.5
814.5
6.6
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II
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492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521
І
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Extraction capacity (µg g-1)
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Retention Time (min)
Linear dynamic range (ng mL-1)
Slope
Intercept
iP iPR
5.90 9.49
0.01 – 30 0.01 – 30
0.2199 0.3089
0.0012 0.0015
iP9G
7.15
0.01 – 30
0.7626
0.0045
tZ
1.53
0.01 – 30
0.4452
tZR
5.38
0.01 – 30
tZ9G
2.64
DHZ
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Table 2. Retention time, linearity and LODs of target phytohormones.
r value
LODs in standard solution (pg mL-1)
0.9989 0.9979
2.34 0.98
0.9966
0.88
0.0017
0.9992
1.96
0.2468
0.0003
0.9996
1.22
7.9 1.2
0.01 – 30
2.5654
0.0030
0.9983
0.45
22.1
1.60
0.01 – 30
0.2553
0.0017
0.9987
2.48
2.6
DHZR
5.40
0.01 – 30
0.3630
0.0011
0.9961
2.36
3.8
IAA
10.06
0.10 – 100
0.0343
0.0094
0.9972
27.0
97.4
ABA
12.81
0.20 – 100
0.0504
0.0021
0.9987
59.6
45.6
JA
14.25
0.05 – 100
0.0490
0.0457
0.9998
23.6
16.4
GA1
9.10
0.05 – 30
0.1809
0.0068
0.9975
21.0
55.6
GA3
8.92
0.05 – 30
0.5223
0.0203
0.9969
6.3
57.7
GA4
15.48
0.05 – 30
0.1307
0.0083
0.9994
15.4
135.9
GA7
15.36
0.05 – 30
1.4796
0.0462
0.9988
12.5
9.5
GA12
17.50
0.05 – 30
0.2095
0.0035
0.9991
6.1
128.9
GA24
15.80
0.05 – 30
0.2375
0.0324
0.9971
1.9
52.2
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LODs in matrix* (pg g-1 FW) 3.1 3.0 5.8
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0.05 – 30
0.2030
0.0015
0.9975
10.6
89.6
28-norCS
4.16
0.5 – 100
0.3102
-0.0220
0.9990
119.0
152.9
28-norBL
3.63
0.5 – 100
0.0659
0.0093
0.9987
81.1
124.0
CS
4.91
0.5 – 100
0.0737
0.0108
0.9995
126.1
167.1
BL
4.31
0.5 – 100
0.0669
-0.0078
0.9991
83.3
112.9
0.2253
-0.0054
114.5
196.1
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28-homoBL 5.06 0.5 – 100 * The extract of flower of Brassica napus L served as matrix.
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GA53
0.9985
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Detected (ng)
Recovery (%)
RSD (%)
iP
0.1
0.11 ± 0.004
109.2
3.6
iPR
0.1
0.11 ± 0.003
114.6
2.7
iP9G
0.1
0.11 ± 0.006
108.3
5.7
tZ
0.1
0.11 ± 0.006
109.0
5.5
tZR
0.1
0.10 ± 0.006
104.5
5.8
tZ9G
0.1
0.11 ± 0.009
107.6
8.3
DHZ
0.1
0.11 ± 0.006
109.7
5.1
DHZR
0.1
0.11 ± 0.007
113.3
5.9
IAA
1.0
1.00 ± 0.16
99.8
15.9
ABA
1.0
1.06 ± 0.17
105.9
15.8
JA
1.0
1.13 ± 0.15
113.4
13.0
GA1
0.4
0.37 ± 0.06
91.6
16.1
GA3
0.4
0.36 ± 0.04
89.7
11.3
GA4
0.4
0.40 ± 0.04
100.6
8.8
GA7
0.4
0.41 ± 0.03
102.7
7.6
GA12
0.4
0.34 ± 0.01
85.0
4.1
GA24
0.4
0.41 ± 0.05
103.0
11.1
GA53
0.4
0.46 ± 0.03
116.2
6.9
28-norCS
1.0
1.11 ± 0.15
111.3
15.3
1.0
1.07 ± 0.09
107.5
9.1
1.0
0.95 ± 0.16
94.8
15.8
28-norBL
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Analytes
Spiked phytohormone standards (ng)
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Table 3. Recoveries of phytohormones spiked to sample matrices (n=3).
EP
525 526 527
BL
1.0
1.06 ± 0.12
106.4
12.1
28-homoBL
1.0
0.98 ± 0.16
98.2
16.0
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Table 4. Contents of endogenous phytohormones detected in 100 mg flower of Brassica napus L. Analyte
iP
iPR
tZ
tZR
DHZ
DHZR
IAA
ABA
0.09 ± 0.01
1.16 ± 0.06
0.20 ± 0.03
5.38 ± 0.69
0.14 ± 0.02
0.17 ± 0.02
38.18 ± 5.09
53.83 ± 1.27
JA
GA4
GA12
GA24
GA53
28-norCS
CS
BL
305.23 ± 7.94
9.09 ± 0.70
2.83 ± 0.10
14.35 ± 0.97
0.35 ± 0.04
3.39 ± 0.48
3.25 ± 0.19
10.43 ± 0.79
Analyte Content -1
531 532 533 534 535 536 537
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530
Values are means ± SD (n=3);
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Fig. 1.
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Fig. 2.
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542
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546 547
549 550 551
Fig. 3
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548
552 553 554 555
Fig. 4.
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556 557
559 560 561
Fig. 5.
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558
562 563 564
Fig. 6
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565 566
Fig. 7
ACCEPTED MANUSCRIPT A simple and efficient method was developed for simultaneous analysis of multiple phytohormones including IAA, ABA, JA, GAs, BRs and CKs. IAA, ABA, JA, GAs, BRs and CKs were purified by sequential magnetic
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solid-phase extraction. 16 endogenous phytohormones could be detected in 100 mg (fresh weight) flower
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of Brassica napus L.
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Supporting Information
2
for
Improved methodology for analysis of multiple
4
phytohormones using sequential magnetic solid-phase
5
extraction coupled with
6
liquid chromatography-tandem mass spectrometry
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3
7
Xiao-Tong Luo a ※, Bao-Dong Cai a ※, Xi Chen b, Yu-Qi Feng a *
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a
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Education), Department of Chemistry, Wuhan University, Wuhan 430072, China
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b
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※These
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*Corresponding author. Tel: +86-27-68755595; fax: +86-27-68755595; Email:
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[email protected]
17 18 19 20 21 22
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Wuhan Institute of Biotechnology, Wuhan 430075, China
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authors contributed equally to this work.
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Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of
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23
Fig. S1. Selectively enrichment of adenosine by prepared boronate affinity magnetic
25
adsorbent.
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29
30
31
32
33
34
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27
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Fig. S2. Comparison of purification performance of BRs between TiO2-based MSPE
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and the proposed sequential MSPE through extracted m/z chromatogram from scan
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mode of CS (A), 28-norBL (B), 28-norCS (C) and 28-homoBL (D).
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ACCEPTED MANUSCRIPT Table S1. The classification, name and structures of target phytohormones.
IAA
indole-3-acetic acid (IAA)
ABA
abscisic acid (ABA)
JA
jasmonic acid (JA)
Structure
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Compound & abbreviation
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Classification
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GA1
GA
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GA3
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GA4
GA7
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GA53
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GA24
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GA12
castasterone (CS)
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BR
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brassinolide (BL)
28-norbrassinolide (28-norBL)
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28-norcastasterone (28-norCS)
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28-homobrassinolde (28-homoBL)
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isopentenyladenine (iP)
isopentenyladenine riboside (iPR)
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CK
isopentenyladenine 9-glucoside (iP9G)
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trans-zeatin-riboside (tZR)
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trans-zeatin (tZ)
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trans-zeatin 9-glucoside (tZ9G)
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dihydrozeatin (DHZ)
dihydrozeatin riboside (DHZR)
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ACCEPTED MANUSCRIPT Table S2. Optimized parameters of multiple reaction monitoring (MRM) mode for analysis of 23 phytohormones.
Scan mode
Precursor ion
Product ion
DP
CE
(V)
(V)
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Compounds
+
204.1
118.9/136.0
iPR
+
336.1
136.0/204.1
iP9G
+
366.1
136.0/204.1
tZ
+
220.1
118.9/136.0
tZR
+
352.2
136.0/220.0
85/85
45/27
tZ9G
+
382.1
136.0/220.0
105/105
49/30
DHZ
+
DHZR
+
IAA
+
ABA
-
JA
-
45/23
90/90
44/26
80/80
45/29
80/80
45/25
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118.9/136.0
95/95
50/29
354.1
136.0/222.0
80/80
50/29
176.1
102.9/130.1
60/60
58/25
263.0
202.7/218.6
-80/-80
-23/-19
209.0
41.0/58.8
-65/-65
-25/-19
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GA1
90/90
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iP
-
347.1
228.6/272.7
-104/-104
-43/-34
-
345.0
142.6/238.6
-75/-75
-44/-22
-
331.1
212.6/256.7
-83/-83
-42/-32
-
329.0
210.6/222.7
-75/-75
-35/-25
-
331.1
286.7/312.9
-66/-66
-37/-37
-
345.1
256.7/300.8
-50/-50
-38/-31
GA53
-
347.1
302.9/328.9
-47/-47
-38/-38
28-norCS
+
451.3
415.2/433.2
104/104
25/16
28-norBL
+
467.3
413.3/431.3
108/108
21/17.5
CS
+
465.4
429.3/447.3
106/106
25/16
BL
+
481.3
315.4/319.1
115/115
23/23
28-homoBL
+
495.3
441.2/459.2
120/120
18/18
GA3
GA7 GA12
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GA24
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GA4
The product ions in bold were used for quantification.
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N6-isopentenyladenine (iP), N6-isopentenyladenine riboside (iPR), N6-isopentenyladenine 9-glucoside (iP9G), trans-zeatin (tZ), trans-zeatin-riboside (tZR), trans-zeatin 9-glucoside (tZ9G), dihydrozeatin (DHZ), dihydrozeatin riboside (DHZR), indole-3-acetic acid (IAA), abscisic acid (ABA), jasmonic acid (JA), Gibberellins (GA1, GA3, GA4, GA7, GA12, GA24, GA53), 28-norbrassinolide (28-norBL), 28-norcastasterone (28-norCS), castasterone (CS), brassinolide (BL), 28-homobrassinolde (28-homoBL).