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Sequential solvent induced phase transition extraction for profiling of endogenous phytohormones in plants by liquid chromatography-mass spectrometry
Journal of Chromatography B, 1004 (2015) 23–29
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Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Sequential solvent induced phase transition extraction for profiling of endogenous phytohormones in plants by liquid chromatography-mass spectrometry Bao-Dong Cai, Er-Cui Ye, Bi-Feng Yuan, Yu-Qi Feng ∗ Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), Department of Chemistry, Wuhan University, Wuhan 430072, China
a r t i c l e
i n f o
Article history: Received 5 May 2015 Received in revised form 10 September 2015 Accepted 20 September 2015 Available online 28 September 2015 Keywords: Phytohormones Sample pretreatment SIPTE UPLC–MS/MS
a b s t r a c t In the current study, a novel method for high-throughput and sensitive determination of 12 phytohormones in plants was developed by using sequential solvent induced phase transition extraction (SIPTE) coupled with ultra–performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS). In sequential SIPTE, 0.1% formic acid (v/v) and 50 mM NaHCO3 aqueous solution were used for enrichment and purification of alkaline and acidic phytohormones from the acetonitrile extract of plant tissues in sequence, in which hydrophobic solvent (toluene) was added to the acetonitrile aqueous mixture for driving the phase separation. Under optimized sequential SIPTE conditions, the phytohormones in acetonitrile extract of plant tissues could be effectively enriched and purified, which was in favor of the following UPLC–MS/MS analysis with less matrix effect. The phytohormones could be detected using the developed sequential SIPTE–UPLC–MS/MS method with the limits of the detection (LODs) ranging from 0.56 to 438.60 pg mL−1 and linear range over 2 orders of magnitude with correlation coefficients (r) > 0.9970. The relative recoveries of the detected phytohormones were in the range of 85.1–114.6%. Finally, the proposed method was applied to simultaneous determination of endogenous phytohormones in different tissues of model plants (Oryza sativa and Arabidopsis thaliana) with small amount of sample size (5 mg, fresh weight). The proposed method may be suitable for studying the distribution of phytohormones in model plants. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Phytohormones are a collection of small molecules which play vital roles in the regulation of plant growth, development and responses to biotic and abiotic stress [1,2]. They are generally grouped into several major classes including auxin, cytokinins (CKs), abscisic acid (ABA), jasmonates (JA), salicylic acid (SA), gibberellins (GAs), brassinasteroids (BRs) and ethylene (ET) according to their structure and physiological function. Although each class of phytohormones has characteristic biological effects, multiple phytohormones regulate physiological activities by synergistic or antagonistic actions referred to cross-talk [3,4]. To better understand molecular mechanisms and interactions of phytohormones, basic information about the concentration, diversification, spacial and temporal distribution of phytohormones at the organ
∗ Corresponding author. Fax: +86 27 68755595. E-mail address: [email protected] (Y.-Q. Feng). http://dx.doi.org/10.1016/j.jchromb.2015.09.031 1570-0232/© 2015 Elsevier B.V. All rights reserved.
, cellular and sub-cellular level is essential. Therefore, the simultaneous determination of multiple phytohormones would be greatly helpful for the progress. Many methods for determination of phytohormones have been developed and are summarized in several reviews [5–7]. Bioassay and immunoassay are the traditional method, but cross-reaction often results in reduced accuracy [8]. Gas chromatography–mass spectrometry (GC–MS) is endowed with high separation resolution capability and sensitivity, which plays a vital role in identification and quantification of phytohormones in early time [9–11]. On the other hand, it requires a chemical derivatization procedure before analysis and degradation of the thermally labile compounds may occur because of high temperature. In the past decade, liquid chromatography–tandem mass spectrometry (LC–MS/MS) is widely used for determination of multiple phytohormones with multiple reaction monitoring (MRM) mode because of its sensitivity and selectivity [12–16]. Typically, naturally occurring phytohormones are present at trace amounts in plants [7]. For routine analysis, organic solvent
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Fig. 1. The structures of 12 phytohormones.
is usually employed for extracting phytohormones from plant tissues [5]. However, the crude plant extract commonly contains large amounts of impurities such as lipids and hydrophobic matrix substances, which heavily affect the accurate quantification [17]. Therefore, sample preparation is essential before LC–MS analysis. Progresses in sample preparation for analysis of phytohormones have been made, including polymer monolith microextraction (PMME), solid-phase extraction (SPE), magnetic solid-phase extraction (MSPE), liquid–liquid extraction (LLE) [18–23]. However, most of them focused on the purification and enrichment of a single class, or a few classes of phytohormones (e.g. carboxylated phytohormones) [22,24,25]. Simultaneous enrichment of multiple phytohormones is still a challenging issue due to their chemical diversity. In this respect, multiple SPE was widely employed for selective purification and enrichment of acidic and alkaline phytohormones. Acidic and alkaline phytohormones were often isolated into different fractions and then were detected respectively [15,26,27] or together [28]. However, these multiple SPE based methods were tedious and time-consuming. Recently, Meulebroek et al. developed a generic extraction protocol and an UPLC–Orbitrap-MS based method for analysis of eight phytohormones, in which crude plant extract was just passed through a 30 kDa Amicon® Ultra centrifugal filter unit prior to LC–MS analysis [29]. Pan et al. used dichloromethane to extract and purify seven major classes phytohormones from plant extract before LC–MS analysis [5,19]. Although these proposed protocols are simple and have been applied for phytohormones analysis in plant, their efficiency of enrichment and purification should be further improved. Phase transition extraction is a separation technique derived from the fact that hydrophilic and water-miscible acetonitrile could be separated from water under induced conditions such as saltingout [30,31] and cooling at subzero temperatures [31,32]. It makes acetonitrile based LLE possible. More recently, Liu et al. proposed an improved phase transition extraction approach, hydrophobic solvent induced phase transition extraction (SIPTE), for extracting drugs from plasma, in which hydrophilic acetonitrile was used as extractant with high partition coefficient for analytes, while hydrophobic solvent such as chloroform could drive phase transition of acetonitrile aqueous mixture [33]. The proposed SIPTE exhibited high recovery rate, high pre-concentration ratio and less matrix effect. In the current work, we developed a sequential SIPTE for rapid and sensitive analysis of multiple phytohormones by
Table 1 Linearity, LODs and LOQs of phytohormones. Analytes
iP iPR iP9G tZ tZR tZ9G DHZ DHZR IAA ABA JA SA
Linear dynamic range
Regression line
(ng mL−1 )
Slope
Intercept
0.01–10 0.01–10 0.01–10 0.01–10 0.01–10 0.01–10 0.01–10 0.01–10 0.3–10 0.1–10 0.1–10 1.0–100
2.8066 3.8011 9.0771 4.7316 2.7343 29.3317 2.4921 4.3856 0.3512 0.7438 1.0856 0.0637
0.0020 −0.0075 −0.0185 −0.0014 −0.0043 −0.0715 −0.0121 −0.0108 0.0172 0.0249 0.0087 0.1097
LODs
LOQs
r Value
(pg mL−1 )
(pg mL−1 )
0.9993 0.9992 0.9987 0.9992 0.9970 0.9977 0.9990 0.9988 0.9990 0.9990 0.9971 0.9985
1.84 1.15 1.04 1.99 0.85 0.56 1.65 0.63 113.64 42.02 18.36 438.60
6.13 3.85 3.47 6.62 2.84 1.88 5.49 2.11 378.79 140.06 61.20 1461.99
Fig. 2. The effect of the added amount of toluene on recoveries of CKs. CK standards were spiked at 0.4 ng mL−1 . Internal standards (I.S.) were added into the enriched samples just before UPLC–MS/MS analysis.
UPLC–MS/MS. In our proposed sequential SIPTE, aqueous solutions at different pH were used for selective enrichment of alkaline CKs and acidic phythormones (Fig. 1), respectively, from plant
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acetonitrile extract. The phase separation of acetonitrile aqueous solution occurred with the assistance of toluene. Subsequently, the selectively enriched phytohormones were detected with UPLC–MS/MS in a single run. Using our proposed protocol, the quantification of endogenous phytohormones in different plant tissues (5 mg, fresh weight) was achieved.
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2.2. Plant materials Oryza sativa was grown in green house at 30 ◦ C under 16 h light/8 h dark photoperiods for two weeks. Arabidopsis thaliana was kindly provided by Wuhan Botanical Garden, Chinese Academy of Sciences (Wuhan, China). All the plant materials were harvested, weighed, immediately frozen in liquid nitrogen, and stored at −80 ◦ C.
2. Experimental 2.3. Sample pretreatment 2.1. Chemicals and reagents Phytohormone standards: indole-3-acetic acid (IAA), abscisic acid (ABA), jasmonic acid (JA), salicylic acid (SA), N6 isopentenyladenine (iP), 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), and stable isotope-labeled standards: [2 H2 ]IAA, [2 H6 ]ABA, [2 H4 ]SA, [2 H6 ]iP, [2 H6 ]iPR, [2 H6 ]iP9G, [2 H5 ]tZ, [2 H5 ]tZR, [2 H5 ]tZ9G, [2 H3 ]DHZ, [2 H3 ]DHZR were purchased from Olchemim (Olomouc, Czech Republic). Formic acid (FA, 88%), sodium bicarbonate, toluene, chloroform, butyl acetate, ethyl acetate, n-octyl alcohol, dichloromethane, n-hexane and cyclohexane were all purchased from Sinopharm Chemical Reagent (Shanghai, China). Acetonitrile (ACN, HPLC grade) was obtained from Tedia Co. (Fairfield, OH, USA). Ultra-pure water used throughout the study was purified with Milli-Q system (Milford, MA, USA).
Plant sample tissues (about 5 mg FW) were frozen with liquid nitrogen, transferred into a 1.5-mL centrifuge tube and then ground into powder. [2 H2 ]IAA (0.2 ng), [2 H6 ]ABA (0.2 ng), [2 H4 ]SA (2 ng) and stable isotope labeled CKs (0.01 ng) were added to the samples to serve as internal standards for the quantification. Then ACN (0.5 mL) was added into the mixture. After extraction at −20 ◦ C for overnight, the supernatant was collected upon centrifugation at 10,000 × g under 0 ◦ C for 20 min. Subsequently, SIPTE was carried out. Briefly, the supernatant was first mixed with 0.1% FA (v/v, 200 L) and then toluene (200 L) was added as inducing solvent. After mild mixing and standing, clear phase separation of the mixed solution was obtained. The aqueous phase (the lower phase) was collected and evaporated to dryness under mild nitrogen gas stream. Subsequently, the organic phase (the upper phase) was mixed with 50 mM NaHCO3 aqueous solution (400 L). After mild mixing and layering, the organic phase (the upper phase) was discarded. The aqueous phase (the lower phase) was acidified with
Fig. 3. The comparison of 0.1% FA and H2 O as extractant on recoveries of CKs (A). The comparison of different inducing solvents on recoveries of CKs using 0.1% FA as extractant (B). CK standards were spiked at 0.4 ng mL−1 . Internal standards (I.S.) were added into the enriched samples just before UPLC–MS/MS analysis.
Fig. 4. The percentage of acidic phytohormones in organic phase while CKs were extracted (A); the recoveries of acidic phytohormones extracted with 50 mM NaHCO3 (B).
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concentration at which signal-to-noise ratios were equal to 3 and 10, respectively. The method reproducibility was evaluated by the intraday and inter-day precisions. Both Intra- and inter-day relative standard deviations (RSDs) were calculated using plant samples spiked with phytohormone standards at three different concentrations. Five parallel extractions of sample solutions over 1 day gave the intraday RSDs, and the inter-day RSDs were determined by extracting sample solutions that was independently prepared for a continuous three days. The accuracy of the method was assessed by the recovery data of phytohormone standards spiked in plant samples at three different concentrations (Table 2). First, the endogenous concentrations of phytohormones were determined. And then, the spiked concentrations of phytohormones in plant samples were calculated by subtracting the endogenous concentration from the total concentration of the spiked sample. Finally, the recoveries were obtained by comparing the calculated concentration of phytohormones to the actual spiked values. Fig. 5. The effect of matrix on the signal response.
3. Results and discussion 3.1. Selection of inducing solvent
4 L formic acid and then mixed with butyl acetate (600 L). After layering, the organic phase (the upper phase) was collected and evaporated to dryness under mild nitrogen gas stream. The residues were redissolved in 30% MeOH (v/v, 50 L). 10 L was applied to UPLC–MS/MS analysis. 2.4. Instruments and analytical conditions Analysis of phytohormones was performed on a UPLC–ESI (+/−)–MS/MS system consisting of a AB SCIEX 4500 triple quadrupole mass spectrometer (Foster City, CA, USA) with an electrospray ionization source (Turbo Ionspray), a Shimadzu LC–30AD HPLC system (Tokyo, Japan) with two 30AD pumps, a SIL-30AC auto sampler, a CTO–30A thermostat column compartment, and a DGU–20A5R degasser. Data acquisition and processing were performed using AB SCIEX Analyst 1.6 software (Foster City, CA, USA). The HPLC separation was performed on a Shim-pack XR-ODS Ш column (75 mm × 2.0 mm i.d., 1.6 m) purchased from Shimadzu (Tokyo, Japan) at 40 ◦ C. A 27-min gradient of 0.05% FA (A) and ACN (B) was employed for the separation with a flow rate of 0.4 mL/min. A gradient programme of 2 min 5–5% B, 10 min 5–25% B, 8 min 25–80% B, 2 min 80–80% B, 2 min 80–5% and 3 min 5% B was used. Multiple reaction monitoring (MRM) and the appropriate product ions were chosen to quantify phytohormones (Table S1). The optimized conditions of MRM experiments were as follows: curtain gas, 40 psi; ion spray voltage, 5000 V for positive ion mode and −4500 V for negative ion mode; turbo heater temperature (TEM), 500 ◦ C; nebulizing gas (Gas 1), 55 psi; heated gas (Gas 2), 40 psi. All the optimal parameters of MS for each phytohormones were listed in Table S1. 2.5. Method validation To evaluate the linearity of the developed method, 11 samples with concentrations ranging from 0.01 to 10 ng/mL for CKs, 0.3–10 ng/mL for IAA, 0.1–10 ng/mL for ABA, JA, and 1.0–100 ng/mL for SA were analyzed in triplicate for establishing the calibration curves. The calibration curves were constructed by plotting the peak area ratios (analytes/I.S.) versus the phytohormones concentrations. Linearity was evaluated by the linear correlation coefficients (r) of the calibration curves. The limits of detection (LODs) and limits of quantifications (LOQs) were calculated as the
In the current study, the solvent induced phase separation of ACN–H2 O system was used for the pretreatment of plant tissue samples, in which highly effective inducing solvent is a crucial factor. Therefore, non-oxygenated hydrophobic solvent (toluene, chloroform, dichloromethane, n-hexane and cyclohexane) and oxygenated hydrophobic solvent (butyl acetate, ethyl acetate) were investigated as inducing solvent for ACN–H2 O system (500 L ACN/200 L H2 O). The results showed that the investigated solvents, except for ACN-miscible n-hexane and cyclohexane could trigger the phase separation, which was consistent with previous report [33]. But the required minimum volume of inducing solvent was different as listed in Table S2. The solvents that have greater Log P value required less volume to induce phase separation; more volume was required for oxygenated hydrophobic solvents than non-oxygenated solvents with similar Log P value. Therefore, in the current work, toluene, chloroform and butyl acetate were chosen as the model inducing solvents for the following experiments. 3.2. Optimization of SIPTE conditions 3.2.1. The effect of added amount of inducing solvent In the ACN–H2 O system (500 L ACN/200 L H2 O), the recovered volume of aqueous phase increased with the increase of added amount of inducing solvent until to maximum (data not shown). Here, CKs (iPR, iP9G, tZR, tZ9G, DHZR) in ACN were extracted with H2 O and the effect of added amount of inducing solvent (toluene) on the recoveries was investigated. As shown in Fig. 2, the recoveries increased with increasing amount of inducing solvent. This result may be ascribed to the increase of recovered volume of aqueous phase. However, the recoveries of CKs decreased with further increase of the inducing solvent volume after the volume of aqueous phase did not increase. The similar results were obtained by using chloroform and butyl acetate as inducing solvent (Fig. S1). Hence, 200 L of inducing solvent was used for the following experiment. 3.2.2. Comparison of CK recoveries by using different extractant and inducing solvent Adenine-derived CKs could be positively charged under acidic condition. Here, the extraction efficiencies of CKs with 0.1% FA (v/v) and H2 O as extractant were compared by using toluene as inducing solvent (Fig. 3A). The results showed that there was no obvious
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Table 2 Precisions and recoveries for the determination of phytohormones in O. sativa leaves. Analytes
Intra-day precision Low −1
(ng g iP iPR iP9G tZ tZR tZ9G DHZ DHZR IAA ABA JA SA
(R.S.D., %; n = 5)
Medium )
10.7 12.7 11.2 14.2 2.3 3.6 11.1 5.9 17.1 14.9 13.9 18.0
−1
(ng g
)
5.2 11.2 5.3 7.5 8.4 11.0 7.3 12.1 7.3 8.4 8.4 13.4
Inter-day precision
High
Low −1
(ng g 6.8 6.0 7.9 6.8 3.8 6.6 3.6 4.6 6.5 9.3 6.5 10.9
)
(R.S.D., %; n = 3)
Medium −1
(ng g
)
5.3 6.0 3.2 1.9 4.1 3.7 4.7 4.1 2.1 3.0 6.3 8.4
−1
(ng g 1.3 10.8 5.7 0.4 3.3 4.0 3.0 5.1 7.7 0.6 2.5 0.2
)
Recoveries (%)
High
Low −1
(ng g 3.6 4.2 5.0 0.5 3.5 1.2 1.4 3.1 5.1 7.3 3.1 5.4
)
Medium −1
(ng g
−1
)
(ng g
97.1 109.3 108.7 100.2 93.9 90.4 111.7 90.7 93.2 102.5 114.6 85.1
96.3 93.1 95.5 102.5 94.3 90.2 101.0 88.9 102.3 100.5 103.2 105.6
)
High (ng g−1 ) 96.5 96.4 96.1 113.9 98.8 94.9 106.9 95.3 91.0 97.4 107.8 88.8
Phytohormone standards were spiked in sample at three different concentrations (1, 5 and 25 ng g−1 FW for CKs; 5, 20 and 50 ng g−1 FW for IAA, ABA and JA; 100, 500 and 1000 ng g−1 for SA).
Table 3 Contents of detected endogenous phytohormones in Oryza sativa (unit, ng g−1 FW). Analytes
iP iPR iP9G tZ tZR tZ9G DHZ DHZR IAA ABA JA SA
Amount of Oryza sativa (mg)
RSD (%)
5
10
20
50
5.08 ± 0.07 2.92 ± 0.17 0.52 ± 0.02 0.24 ± 0.02 0.25 ± 0.01 N.Q. N.D. N.D. 80.25 ± 0.15 22.63 ± 0.20 6.25 ± 0.12 8571.43 ± 155.19
5.35 ± 0.19 3.30 ± 0.26 0.45 ± 0.01 0.18 ± 0.01 0.26 ± 0.03 N.Q. N.D. N.Q. 79.81 ± 0.24 22.86 ± 0.17 5.66 ± 0.02 7705.09 ± 219.57
5.31 ± 0.19 3.57 ± 0.04 0.40 ± 0.02 0.24 ± 0.02 0.28 ± 0.02 0.03 ± 0.00 N.D. N.Q. 79.55 ± 5.56 20.71 ± 0.38 5.39 ± 0.37 7490.62 ± 139.03
5.17 ± 3.60 ± 0.32 ± 0.22 ± 0.29 ± 0.02 ± N.D. 0.02 ± 78.99 ± 18.92 ± 5.09 ± 6178.12 ±
0.09 0.11 0.00 0.01 0.00 0.00 0.00 4.33 0.64 0.04 897.45
2.3 9.4 19.8 12.5 6.6 8.6 – – 0.7 8.7 8.8 13.2
N.D., not detected; N.Q., not quantified.
difference of the recoveries for more hydrophilic CKs (iP9G, tZR, tZ9G, DHZR) using 0.1% FA (v/v) or H2 O as extractant; while the CK with less hydrophilicity such as iPR exhibited different recoveries using the different extractants. The greater recovery of iPR in 0.1% FA (v/v)–ACN may be ascribed to the protonation of iPR at lower pH and thus increase of its hydrophilicity. Theoretically, the recovery of
iPR would increase when more extractant was used. However, more aqueous solution caused longer processing time for dryness. Hence, 200 L 0.1% FA was selected as extractant for the following experiment. Similar results were observed in the cases using chloroform and butyl acetate as inducing solvent. This results can be explained by the fact that the exocyclic amino group at the sixth position of
Fig. 6. The MRM chromatograms of phytohormones. 12 phytohormones standards were spiked in 30% MeOH solution (A) and endogenous phytohormones were detected in 5 mg O. sativa samples (B).
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Table 4 Contents of detected endogenous phytohormones in different organs (root, stem, leaf, flower and siliques) of Arabidopsis thaliana (unit, ng g−1 FW). Analytes
Root
Stem
Leaf
Flower
Silique
iPR iP9G tZR IAA ABA JA
0.91 ± 0.104 N.D. 0.44 ± 0.072 8.91 ± 0.393 N.D. 11.61 ± 0.788
2.41 ± 0.284 0.12 ± 0.011 2.32 ± 0.268 4.32 ± 0.464 7.33 ± 0.868 2.67 ± 0.028
0.54 ± 0.079 0.23 ± 0.013 0.45 ± 0.040 3.66 ± 0.628 18.29 ± 1.562 5.53 ± 0.466
0.92 ± 0.132 N.D. 2.30 ± 0.342 8.10 ± 1.406 21.16 ± 1.166 14.17 ± 0.480
0.48 ± 0.053 N.D. 2.74 ± 0.277 23.48 ± 0.650 28.97 ± 1.771 16.17 ± 0.994
N.D., not detected.
purine in the structure of CKs is all positively charged under acidic condition [34,35]. Therefore, 0.1% FA (v/v) was selected as extractant. For inducing solvent, as shown in Fig. 3B, greater recoveries of CKs could be achieved using toluene compared with chloroform and butyl acetate. In addition, toluene could drive the phase separation of acetonitrile aqueous mixture more effectively. Thus, toluene was selected as inducing solvent and the added amount was fixed to 200 L for the extraction of CKs. 3.2.3. Investigation of the extraction efficiencies of carboxylated phytohormones The extraction efficiencies of carboxylated phytohormones were also investigated in 0.1% FA–ACN system. When CKs were extracted, acidic phytohormones were mostly in organic phase (Fig. 4A). These carboxylated phytohormons at low pH presented at molecular form, leading to lower hydrophilicity. Next, 50 mM NaHCO3 aqueous solution was used as extractant for carboxylated phytohormomes extraction. As shown in Fig. 4B, the recovery of these acidic phytohormones reached nearly 100% due to their ionic forms at higher pH. To further purify the extracted acidic phytohormones, the aqueous phase after phase separation was acidified with FA and then extracted backward using butyl acetate with the recoveries ranged from 81.3 to 97.2% (Fig. S2). 3.3. Matrix effect Mass spectrometry is widely used for determination of phytohormones due to its high sensitivity and selectivity. However, the matrix effect commonly influences the accurate quantification of target analytes [36]. In previous work, it has been reported that matrix effect for drugs extracted from plasma could almost be eliminated after SIPTE [33]. Hereon, the isotope-labeled standards were spiked in pretreated sample and 30% MeOH (v/v) aqueous solution, respectively. The matrix effect of the proposed method was evaluated by comparing the signal response of labeled standards in pretreated sample and in 30% MeOH. As shown in Fig. 5, the signal responses of isotope-labeled compounds were similar in pretreated sample and 30% MeOH, except [4 H2 ]SA, indicating that the proposed strategy could effectively reduce the matrix effect. The signal of [4 H2 ]SA was suppressed because of high content of endogenous SA in plant. These results indicated that the matrix effect for analytes (CKs) in aqueous phase using SIPTE was hardly observed as similar as analytes (acidic phytohormones) in organic phase. 3.4. Method validation Under the optimal conditions, the performance of proposed method was evaluated. The MRM chromatograms of phytohormone standards were shown in Fig. 6A. The calibration curves were constructed by plotting the peak area ratio (analyte/I.S.) against the phytohormones concentrations. As listed in Table 1, good linearity was obtained with linear correlation coefficients (r) ranging from 0.9970 to 0.9993. The limits of detection (LODs) were calculated as the signal to noise ratios of 3:1. The results showed that LODs
for the 12 phytohormones ranged from 0.53 to 438.60 pg mL−1 , which could meet the requirement for the analysis of endogenous phytohormones in plant samples. The accuracy of the method was evaluated by spiking 12 phytohormone standards in the rice leaf matrices. The results showed that spiked phytohormones could be successfully determined with 85.1–114.6% recoveries (Table 2). Furthermore, the reproducibility of the proposed method was evaluated by investigating the intra- and inter-day precisions. Reproducibility of the proposed method was evaluated with the relative standard deviations (RSDs) between 0.2 and 18.0 % (Table 2).
3.5. Quantitative determination of endogenous phytohormones in plant samples Quantification of endogenous phytohormones is of great significance for knowing clearly the distribution of phytohormones in different plant tissues. It will be helpful for better understanding the interaction mechanism of phytohormones in plant. Rice is an important crop worldwide and is considered to be a model plant among monocots for biological research. A. thaliana is a small flowering plant that is widely used as a model organism in plant biology [37,38]. In the current study, phytohormones analysis was performed with 5–50 mg rice (O. sativa) for evaluating the performance of the proposed method. As listed in Table 3, phytohormones analysis could be completed with 5 mg rice sample and 9 phytohormoes were successfully detected, including 5 CKs, IAA, ABA, JA and SA. The MRM chromatograms of 9 detected endogenous phytohormones were shown in Fig. 6B. Furthermore, phytohormones in different plant tissues (root, stem, leaf, flower and siliques) of A. thaliana were also analyzed and the results were listed in Table 4. IAA and JA were detected with higher concentration in flower and siliques than other tissues. The distribution of phytohormones in rice and A. thaliana tissues could be obtained, demonstrating that the proposed method is sensitive and requires tiny amount of plant samples.
4. Conclusion In the current study, we described a simple and effective method for simultaneous determination of multiple endogenous phytohormones by the combination of sequential SIPTE and UPLC–MS/MS. The proposed sequential SIPTE was demonstrated to be a simple and effective approach to enrich and purify phytohormones from acetonitrile extract of plant tissues. The developed sequential SIPTE-UPLC–MS/MS method was successfully applied to analysis of multiple endogenous phytohormones in different issues of model plants (rice and A. thaliana) with small amount of sample size (5 mg, fresh weight). The developed method may be suitable for studying the distribution of endogenous phytohormones in different tissues of model plants.
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Acknowledgements This work was supported by the National Natural Science Foundation of China (21475098, 91217309) and the Fundamental Research Funds for the Central Universities. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jchromb.2015. 09.031. References [1] R. Bari, J.G. Jones, Plant Mol. Biol. 69 (2009) 473–488. [2] A. Santner, L.I.A. Calderon-Villalobos, M. Estelle, Nat. Chem. Biol. 5 (2009) 301–307. [3] R. Aloni, E. Aloni, M. Langhans, C.I. Ullrich, Ann. Bot-London 97 (2006) 883–893. [4] P. Reymond, E.E. Farmer, Curr. Opin. Plant Biol. 1 (1998) 404–411. [5] X.Q. Pan, X.M. Wang, J. Chromatogr. B 877 (2009) 2806–2813. [6] P. Tarkowski, L. Ge, J.W.H. Yong, S.N. Tan, Trends. Anal. Chem. 28 (2009) 323–335. [7] F. Du, G. Ruan, H. Liu, Anal. Bioanal. Chem. 403 (2012) 55–74. [8] R. Maldiney, B. Leroux, I. Sabbagh, B. Sotta, L. Sossountzov, E. Miginiac, J. Immunol. Methods 90 (1986) 151–158. [9] J. Engelberth, E.A. Schmelz, H.T. Alborn, Y.J. Cardoza, J. Huang, J.H. Tumlinson, Anal. Biochem. 312 (2003) 242–250. [10] E.A. Schmelz, J. Engelberth, H.T. Alborn, P. O’Donnell, M. Sammons, H. Toshima, J.H. Tumlinson, Proc. Natl. Acad. Sci. U. S. A. 100 (2003) 10552–10557. [11] E.A. Schmelz, J. Engelberth, J.H. Tumlinson, A. Block, H.T. Alborn, Plant J. 39 (2004) 790–808. [12] A. Fletcher, J. Mader, J. Plant Growth Regul. 26 (2007) 351–361. [13] G.U. Balcke, V. Handrick, N. Bergau, M. Fichtner, A. Henning, H. Stellmach, A. Tissier, B. Hause, A. Frolov, Plant Methods 8 (2012) 47. [14] Z. Liu, F. Wei, Y.-Q. Feng, Anal. Methods 2 (2010) 1676. [15] S.C. Farrow, R.J.N. Emery, Plant Methods 8 (2012) 42. [16] Z. Han, G. Liu, Q. Rao, B. Bai, Z. Zhao, H. Liu, A. Wu, J. Chromatogr. B 881–882 (2012) 83–89.
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Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Sequential solvent induced phase transition extraction for profiling of endogenous phytohormones in plants by liquid chromatography-mass spectrometry Bao-Dong Cai, Er-Cui Ye, Bi-Feng Yuan, Yu-Qi Feng ∗ Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), Department of Chemistry, Wuhan University, Wuhan 430072, China
a r t i c l e
i n f o
Article history: Received 5 May 2015 Received in revised form 10 September 2015 Accepted 20 September 2015 Available online 28 September 2015 Keywords: Phytohormones Sample pretreatment SIPTE UPLC–MS/MS
a b s t r a c t In the current study, a novel method for high-throughput and sensitive determination of 12 phytohormones in plants was developed by using sequential solvent induced phase transition extraction (SIPTE) coupled with ultra–performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS). In sequential SIPTE, 0.1% formic acid (v/v) and 50 mM NaHCO3 aqueous solution were used for enrichment and purification of alkaline and acidic phytohormones from the acetonitrile extract of plant tissues in sequence, in which hydrophobic solvent (toluene) was added to the acetonitrile aqueous mixture for driving the phase separation. Under optimized sequential SIPTE conditions, the phytohormones in acetonitrile extract of plant tissues could be effectively enriched and purified, which was in favor of the following UPLC–MS/MS analysis with less matrix effect. The phytohormones could be detected using the developed sequential SIPTE–UPLC–MS/MS method with the limits of the detection (LODs) ranging from 0.56 to 438.60 pg mL−1 and linear range over 2 orders of magnitude with correlation coefficients (r) > 0.9970. The relative recoveries of the detected phytohormones were in the range of 85.1–114.6%. Finally, the proposed method was applied to simultaneous determination of endogenous phytohormones in different tissues of model plants (Oryza sativa and Arabidopsis thaliana) with small amount of sample size (5 mg, fresh weight). The proposed method may be suitable for studying the distribution of phytohormones in model plants. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Phytohormones are a collection of small molecules which play vital roles in the regulation of plant growth, development and responses to biotic and abiotic stress [1,2]. They are generally grouped into several major classes including auxin, cytokinins (CKs), abscisic acid (ABA), jasmonates (JA), salicylic acid (SA), gibberellins (GAs), brassinasteroids (BRs) and ethylene (ET) according to their structure and physiological function. Although each class of phytohormones has characteristic biological effects, multiple phytohormones regulate physiological activities by synergistic or antagonistic actions referred to cross-talk [3,4]. To better understand molecular mechanisms and interactions of phytohormones, basic information about the concentration, diversification, spacial and temporal distribution of phytohormones at the organ
∗ Corresponding author. Fax: +86 27 68755595. E-mail address: [email protected] (Y.-Q. Feng). http://dx.doi.org/10.1016/j.jchromb.2015.09.031 1570-0232/© 2015 Elsevier B.V. All rights reserved.
, cellular and sub-cellular level is essential. Therefore, the simultaneous determination of multiple phytohormones would be greatly helpful for the progress. Many methods for determination of phytohormones have been developed and are summarized in several reviews [5–7]. Bioassay and immunoassay are the traditional method, but cross-reaction often results in reduced accuracy [8]. Gas chromatography–mass spectrometry (GC–MS) is endowed with high separation resolution capability and sensitivity, which plays a vital role in identification and quantification of phytohormones in early time [9–11]. On the other hand, it requires a chemical derivatization procedure before analysis and degradation of the thermally labile compounds may occur because of high temperature. In the past decade, liquid chromatography–tandem mass spectrometry (LC–MS/MS) is widely used for determination of multiple phytohormones with multiple reaction monitoring (MRM) mode because of its sensitivity and selectivity [12–16]. Typically, naturally occurring phytohormones are present at trace amounts in plants [7]. For routine analysis, organic solvent
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Fig. 1. The structures of 12 phytohormones.
is usually employed for extracting phytohormones from plant tissues [5]. However, the crude plant extract commonly contains large amounts of impurities such as lipids and hydrophobic matrix substances, which heavily affect the accurate quantification [17]. Therefore, sample preparation is essential before LC–MS analysis. Progresses in sample preparation for analysis of phytohormones have been made, including polymer monolith microextraction (PMME), solid-phase extraction (SPE), magnetic solid-phase extraction (MSPE), liquid–liquid extraction (LLE) [18–23]. However, most of them focused on the purification and enrichment of a single class, or a few classes of phytohormones (e.g. carboxylated phytohormones) [22,24,25]. Simultaneous enrichment of multiple phytohormones is still a challenging issue due to their chemical diversity. In this respect, multiple SPE was widely employed for selective purification and enrichment of acidic and alkaline phytohormones. Acidic and alkaline phytohormones were often isolated into different fractions and then were detected respectively [15,26,27] or together [28]. However, these multiple SPE based methods were tedious and time-consuming. Recently, Meulebroek et al. developed a generic extraction protocol and an UPLC–Orbitrap-MS based method for analysis of eight phytohormones, in which crude plant extract was just passed through a 30 kDa Amicon® Ultra centrifugal filter unit prior to LC–MS analysis [29]. Pan et al. used dichloromethane to extract and purify seven major classes phytohormones from plant extract before LC–MS analysis [5,19]. Although these proposed protocols are simple and have been applied for phytohormones analysis in plant, their efficiency of enrichment and purification should be further improved. Phase transition extraction is a separation technique derived from the fact that hydrophilic and water-miscible acetonitrile could be separated from water under induced conditions such as saltingout [30,31] and cooling at subzero temperatures [31,32]. It makes acetonitrile based LLE possible. More recently, Liu et al. proposed an improved phase transition extraction approach, hydrophobic solvent induced phase transition extraction (SIPTE), for extracting drugs from plasma, in which hydrophilic acetonitrile was used as extractant with high partition coefficient for analytes, while hydrophobic solvent such as chloroform could drive phase transition of acetonitrile aqueous mixture [33]. The proposed SIPTE exhibited high recovery rate, high pre-concentration ratio and less matrix effect. In the current work, we developed a sequential SIPTE for rapid and sensitive analysis of multiple phytohormones by
Table 1 Linearity, LODs and LOQs of phytohormones. Analytes
iP iPR iP9G tZ tZR tZ9G DHZ DHZR IAA ABA JA SA
Linear dynamic range
Regression line
(ng mL−1 )
Slope
Intercept
0.01–10 0.01–10 0.01–10 0.01–10 0.01–10 0.01–10 0.01–10 0.01–10 0.3–10 0.1–10 0.1–10 1.0–100
2.8066 3.8011 9.0771 4.7316 2.7343 29.3317 2.4921 4.3856 0.3512 0.7438 1.0856 0.0637
0.0020 −0.0075 −0.0185 −0.0014 −0.0043 −0.0715 −0.0121 −0.0108 0.0172 0.0249 0.0087 0.1097
LODs
LOQs
r Value
(pg mL−1 )
(pg mL−1 )
0.9993 0.9992 0.9987 0.9992 0.9970 0.9977 0.9990 0.9988 0.9990 0.9990 0.9971 0.9985
1.84 1.15 1.04 1.99 0.85 0.56 1.65 0.63 113.64 42.02 18.36 438.60
6.13 3.85 3.47 6.62 2.84 1.88 5.49 2.11 378.79 140.06 61.20 1461.99
Fig. 2. The effect of the added amount of toluene on recoveries of CKs. CK standards were spiked at 0.4 ng mL−1 . Internal standards (I.S.) were added into the enriched samples just before UPLC–MS/MS analysis.
UPLC–MS/MS. In our proposed sequential SIPTE, aqueous solutions at different pH were used for selective enrichment of alkaline CKs and acidic phythormones (Fig. 1), respectively, from plant
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acetonitrile extract. The phase separation of acetonitrile aqueous solution occurred with the assistance of toluene. Subsequently, the selectively enriched phytohormones were detected with UPLC–MS/MS in a single run. Using our proposed protocol, the quantification of endogenous phytohormones in different plant tissues (5 mg, fresh weight) was achieved.
25
2.2. Plant materials Oryza sativa was grown in green house at 30 ◦ C under 16 h light/8 h dark photoperiods for two weeks. Arabidopsis thaliana was kindly provided by Wuhan Botanical Garden, Chinese Academy of Sciences (Wuhan, China). All the plant materials were harvested, weighed, immediately frozen in liquid nitrogen, and stored at −80 ◦ C.
2. Experimental 2.3. Sample pretreatment 2.1. Chemicals and reagents Phytohormone standards: indole-3-acetic acid (IAA), abscisic acid (ABA), jasmonic acid (JA), salicylic acid (SA), N6 isopentenyladenine (iP), 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), and stable isotope-labeled standards: [2 H2 ]IAA, [2 H6 ]ABA, [2 H4 ]SA, [2 H6 ]iP, [2 H6 ]iPR, [2 H6 ]iP9G, [2 H5 ]tZ, [2 H5 ]tZR, [2 H5 ]tZ9G, [2 H3 ]DHZ, [2 H3 ]DHZR were purchased from Olchemim (Olomouc, Czech Republic). Formic acid (FA, 88%), sodium bicarbonate, toluene, chloroform, butyl acetate, ethyl acetate, n-octyl alcohol, dichloromethane, n-hexane and cyclohexane were all purchased from Sinopharm Chemical Reagent (Shanghai, China). Acetonitrile (ACN, HPLC grade) was obtained from Tedia Co. (Fairfield, OH, USA). Ultra-pure water used throughout the study was purified with Milli-Q system (Milford, MA, USA).
Plant sample tissues (about 5 mg FW) were frozen with liquid nitrogen, transferred into a 1.5-mL centrifuge tube and then ground into powder. [2 H2 ]IAA (0.2 ng), [2 H6 ]ABA (0.2 ng), [2 H4 ]SA (2 ng) and stable isotope labeled CKs (0.01 ng) were added to the samples to serve as internal standards for the quantification. Then ACN (0.5 mL) was added into the mixture. After extraction at −20 ◦ C for overnight, the supernatant was collected upon centrifugation at 10,000 × g under 0 ◦ C for 20 min. Subsequently, SIPTE was carried out. Briefly, the supernatant was first mixed with 0.1% FA (v/v, 200 L) and then toluene (200 L) was added as inducing solvent. After mild mixing and standing, clear phase separation of the mixed solution was obtained. The aqueous phase (the lower phase) was collected and evaporated to dryness under mild nitrogen gas stream. Subsequently, the organic phase (the upper phase) was mixed with 50 mM NaHCO3 aqueous solution (400 L). After mild mixing and layering, the organic phase (the upper phase) was discarded. The aqueous phase (the lower phase) was acidified with
Fig. 3. The comparison of 0.1% FA and H2 O as extractant on recoveries of CKs (A). The comparison of different inducing solvents on recoveries of CKs using 0.1% FA as extractant (B). CK standards were spiked at 0.4 ng mL−1 . Internal standards (I.S.) were added into the enriched samples just before UPLC–MS/MS analysis.
Fig. 4. The percentage of acidic phytohormones in organic phase while CKs were extracted (A); the recoveries of acidic phytohormones extracted with 50 mM NaHCO3 (B).
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concentration at which signal-to-noise ratios were equal to 3 and 10, respectively. The method reproducibility was evaluated by the intraday and inter-day precisions. Both Intra- and inter-day relative standard deviations (RSDs) were calculated using plant samples spiked with phytohormone standards at three different concentrations. Five parallel extractions of sample solutions over 1 day gave the intraday RSDs, and the inter-day RSDs were determined by extracting sample solutions that was independently prepared for a continuous three days. The accuracy of the method was assessed by the recovery data of phytohormone standards spiked in plant samples at three different concentrations (Table 2). First, the endogenous concentrations of phytohormones were determined. And then, the spiked concentrations of phytohormones in plant samples were calculated by subtracting the endogenous concentration from the total concentration of the spiked sample. Finally, the recoveries were obtained by comparing the calculated concentration of phytohormones to the actual spiked values. Fig. 5. The effect of matrix on the signal response.
3. Results and discussion 3.1. Selection of inducing solvent
4 L formic acid and then mixed with butyl acetate (600 L). After layering, the organic phase (the upper phase) was collected and evaporated to dryness under mild nitrogen gas stream. The residues were redissolved in 30% MeOH (v/v, 50 L). 10 L was applied to UPLC–MS/MS analysis. 2.4. Instruments and analytical conditions Analysis of phytohormones was performed on a UPLC–ESI (+/−)–MS/MS system consisting of a AB SCIEX 4500 triple quadrupole mass spectrometer (Foster City, CA, USA) with an electrospray ionization source (Turbo Ionspray), a Shimadzu LC–30AD HPLC system (Tokyo, Japan) with two 30AD pumps, a SIL-30AC auto sampler, a CTO–30A thermostat column compartment, and a DGU–20A5R degasser. Data acquisition and processing were performed using AB SCIEX Analyst 1.6 software (Foster City, CA, USA). The HPLC separation was performed on a Shim-pack XR-ODS Ш column (75 mm × 2.0 mm i.d., 1.6 m) purchased from Shimadzu (Tokyo, Japan) at 40 ◦ C. A 27-min gradient of 0.05% FA (A) and ACN (B) was employed for the separation with a flow rate of 0.4 mL/min. A gradient programme of 2 min 5–5% B, 10 min 5–25% B, 8 min 25–80% B, 2 min 80–80% B, 2 min 80–5% and 3 min 5% B was used. Multiple reaction monitoring (MRM) and the appropriate product ions were chosen to quantify phytohormones (Table S1). The optimized conditions of MRM experiments were as follows: curtain gas, 40 psi; ion spray voltage, 5000 V for positive ion mode and −4500 V for negative ion mode; turbo heater temperature (TEM), 500 ◦ C; nebulizing gas (Gas 1), 55 psi; heated gas (Gas 2), 40 psi. All the optimal parameters of MS for each phytohormones were listed in Table S1. 2.5. Method validation To evaluate the linearity of the developed method, 11 samples with concentrations ranging from 0.01 to 10 ng/mL for CKs, 0.3–10 ng/mL for IAA, 0.1–10 ng/mL for ABA, JA, and 1.0–100 ng/mL for SA were analyzed in triplicate for establishing the calibration curves. The calibration curves were constructed by plotting the peak area ratios (analytes/I.S.) versus the phytohormones concentrations. Linearity was evaluated by the linear correlation coefficients (r) of the calibration curves. The limits of detection (LODs) and limits of quantifications (LOQs) were calculated as the
In the current study, the solvent induced phase separation of ACN–H2 O system was used for the pretreatment of plant tissue samples, in which highly effective inducing solvent is a crucial factor. Therefore, non-oxygenated hydrophobic solvent (toluene, chloroform, dichloromethane, n-hexane and cyclohexane) and oxygenated hydrophobic solvent (butyl acetate, ethyl acetate) were investigated as inducing solvent for ACN–H2 O system (500 L ACN/200 L H2 O). The results showed that the investigated solvents, except for ACN-miscible n-hexane and cyclohexane could trigger the phase separation, which was consistent with previous report [33]. But the required minimum volume of inducing solvent was different as listed in Table S2. The solvents that have greater Log P value required less volume to induce phase separation; more volume was required for oxygenated hydrophobic solvents than non-oxygenated solvents with similar Log P value. Therefore, in the current work, toluene, chloroform and butyl acetate were chosen as the model inducing solvents for the following experiments. 3.2. Optimization of SIPTE conditions 3.2.1. The effect of added amount of inducing solvent In the ACN–H2 O system (500 L ACN/200 L H2 O), the recovered volume of aqueous phase increased with the increase of added amount of inducing solvent until to maximum (data not shown). Here, CKs (iPR, iP9G, tZR, tZ9G, DHZR) in ACN were extracted with H2 O and the effect of added amount of inducing solvent (toluene) on the recoveries was investigated. As shown in Fig. 2, the recoveries increased with increasing amount of inducing solvent. This result may be ascribed to the increase of recovered volume of aqueous phase. However, the recoveries of CKs decreased with further increase of the inducing solvent volume after the volume of aqueous phase did not increase. The similar results were obtained by using chloroform and butyl acetate as inducing solvent (Fig. S1). Hence, 200 L of inducing solvent was used for the following experiment. 3.2.2. Comparison of CK recoveries by using different extractant and inducing solvent Adenine-derived CKs could be positively charged under acidic condition. Here, the extraction efficiencies of CKs with 0.1% FA (v/v) and H2 O as extractant were compared by using toluene as inducing solvent (Fig. 3A). The results showed that there was no obvious
B.-D. Cai et al. / J. Chromatogr. B 1004 (2015) 23–29
27
Table 2 Precisions and recoveries for the determination of phytohormones in O. sativa leaves. Analytes
Intra-day precision Low −1
(ng g iP iPR iP9G tZ tZR tZ9G DHZ DHZR IAA ABA JA SA
(R.S.D., %; n = 5)
Medium )
10.7 12.7 11.2 14.2 2.3 3.6 11.1 5.9 17.1 14.9 13.9 18.0
−1
(ng g
)
5.2 11.2 5.3 7.5 8.4 11.0 7.3 12.1 7.3 8.4 8.4 13.4
Inter-day precision
High
Low −1
(ng g 6.8 6.0 7.9 6.8 3.8 6.6 3.6 4.6 6.5 9.3 6.5 10.9
)
(R.S.D., %; n = 3)
Medium −1
(ng g
)
5.3 6.0 3.2 1.9 4.1 3.7 4.7 4.1 2.1 3.0 6.3 8.4
−1
(ng g 1.3 10.8 5.7 0.4 3.3 4.0 3.0 5.1 7.7 0.6 2.5 0.2
)
Recoveries (%)
High
Low −1
(ng g 3.6 4.2 5.0 0.5 3.5 1.2 1.4 3.1 5.1 7.3 3.1 5.4
)
Medium −1
(ng g
−1
)
(ng g
97.1 109.3 108.7 100.2 93.9 90.4 111.7 90.7 93.2 102.5 114.6 85.1
96.3 93.1 95.5 102.5 94.3 90.2 101.0 88.9 102.3 100.5 103.2 105.6
)
High (ng g−1 ) 96.5 96.4 96.1 113.9 98.8 94.9 106.9 95.3 91.0 97.4 107.8 88.8
Phytohormone standards were spiked in sample at three different concentrations (1, 5 and 25 ng g−1 FW for CKs; 5, 20 and 50 ng g−1 FW for IAA, ABA and JA; 100, 500 and 1000 ng g−1 for SA).
Table 3 Contents of detected endogenous phytohormones in Oryza sativa (unit, ng g−1 FW). Analytes
iP iPR iP9G tZ tZR tZ9G DHZ DHZR IAA ABA JA SA
Amount of Oryza sativa (mg)
RSD (%)
5
10
20
50
5.08 ± 0.07 2.92 ± 0.17 0.52 ± 0.02 0.24 ± 0.02 0.25 ± 0.01 N.Q. N.D. N.D. 80.25 ± 0.15 22.63 ± 0.20 6.25 ± 0.12 8571.43 ± 155.19
5.35 ± 0.19 3.30 ± 0.26 0.45 ± 0.01 0.18 ± 0.01 0.26 ± 0.03 N.Q. N.D. N.Q. 79.81 ± 0.24 22.86 ± 0.17 5.66 ± 0.02 7705.09 ± 219.57
5.31 ± 0.19 3.57 ± 0.04 0.40 ± 0.02 0.24 ± 0.02 0.28 ± 0.02 0.03 ± 0.00 N.D. N.Q. 79.55 ± 5.56 20.71 ± 0.38 5.39 ± 0.37 7490.62 ± 139.03
5.17 ± 3.60 ± 0.32 ± 0.22 ± 0.29 ± 0.02 ± N.D. 0.02 ± 78.99 ± 18.92 ± 5.09 ± 6178.12 ±
0.09 0.11 0.00 0.01 0.00 0.00 0.00 4.33 0.64 0.04 897.45
2.3 9.4 19.8 12.5 6.6 8.6 – – 0.7 8.7 8.8 13.2
N.D., not detected; N.Q., not quantified.
difference of the recoveries for more hydrophilic CKs (iP9G, tZR, tZ9G, DHZR) using 0.1% FA (v/v) or H2 O as extractant; while the CK with less hydrophilicity such as iPR exhibited different recoveries using the different extractants. The greater recovery of iPR in 0.1% FA (v/v)–ACN may be ascribed to the protonation of iPR at lower pH and thus increase of its hydrophilicity. Theoretically, the recovery of
iPR would increase when more extractant was used. However, more aqueous solution caused longer processing time for dryness. Hence, 200 L 0.1% FA was selected as extractant for the following experiment. Similar results were observed in the cases using chloroform and butyl acetate as inducing solvent. This results can be explained by the fact that the exocyclic amino group at the sixth position of
Fig. 6. The MRM chromatograms of phytohormones. 12 phytohormones standards were spiked in 30% MeOH solution (A) and endogenous phytohormones were detected in 5 mg O. sativa samples (B).
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Table 4 Contents of detected endogenous phytohormones in different organs (root, stem, leaf, flower and siliques) of Arabidopsis thaliana (unit, ng g−1 FW). Analytes
Root
Stem
Leaf
Flower
Silique
iPR iP9G tZR IAA ABA JA
0.91 ± 0.104 N.D. 0.44 ± 0.072 8.91 ± 0.393 N.D. 11.61 ± 0.788
2.41 ± 0.284 0.12 ± 0.011 2.32 ± 0.268 4.32 ± 0.464 7.33 ± 0.868 2.67 ± 0.028
0.54 ± 0.079 0.23 ± 0.013 0.45 ± 0.040 3.66 ± 0.628 18.29 ± 1.562 5.53 ± 0.466
0.92 ± 0.132 N.D. 2.30 ± 0.342 8.10 ± 1.406 21.16 ± 1.166 14.17 ± 0.480
0.48 ± 0.053 N.D. 2.74 ± 0.277 23.48 ± 0.650 28.97 ± 1.771 16.17 ± 0.994
N.D., not detected.
purine in the structure of CKs is all positively charged under acidic condition [34,35]. Therefore, 0.1% FA (v/v) was selected as extractant. For inducing solvent, as shown in Fig. 3B, greater recoveries of CKs could be achieved using toluene compared with chloroform and butyl acetate. In addition, toluene could drive the phase separation of acetonitrile aqueous mixture more effectively. Thus, toluene was selected as inducing solvent and the added amount was fixed to 200 L for the extraction of CKs. 3.2.3. Investigation of the extraction efficiencies of carboxylated phytohormones The extraction efficiencies of carboxylated phytohormones were also investigated in 0.1% FA–ACN system. When CKs were extracted, acidic phytohormones were mostly in organic phase (Fig. 4A). These carboxylated phytohormons at low pH presented at molecular form, leading to lower hydrophilicity. Next, 50 mM NaHCO3 aqueous solution was used as extractant for carboxylated phytohormomes extraction. As shown in Fig. 4B, the recovery of these acidic phytohormones reached nearly 100% due to their ionic forms at higher pH. To further purify the extracted acidic phytohormones, the aqueous phase after phase separation was acidified with FA and then extracted backward using butyl acetate with the recoveries ranged from 81.3 to 97.2% (Fig. S2). 3.3. Matrix effect Mass spectrometry is widely used for determination of phytohormones due to its high sensitivity and selectivity. However, the matrix effect commonly influences the accurate quantification of target analytes [36]. In previous work, it has been reported that matrix effect for drugs extracted from plasma could almost be eliminated after SIPTE [33]. Hereon, the isotope-labeled standards were spiked in pretreated sample and 30% MeOH (v/v) aqueous solution, respectively. The matrix effect of the proposed method was evaluated by comparing the signal response of labeled standards in pretreated sample and in 30% MeOH. As shown in Fig. 5, the signal responses of isotope-labeled compounds were similar in pretreated sample and 30% MeOH, except [4 H2 ]SA, indicating that the proposed strategy could effectively reduce the matrix effect. The signal of [4 H2 ]SA was suppressed because of high content of endogenous SA in plant. These results indicated that the matrix effect for analytes (CKs) in aqueous phase using SIPTE was hardly observed as similar as analytes (acidic phytohormones) in organic phase. 3.4. Method validation Under the optimal conditions, the performance of proposed method was evaluated. The MRM chromatograms of phytohormone standards were shown in Fig. 6A. The calibration curves were constructed by plotting the peak area ratio (analyte/I.S.) against the phytohormones concentrations. As listed in Table 1, good linearity was obtained with linear correlation coefficients (r) ranging from 0.9970 to 0.9993. The limits of detection (LODs) were calculated as the signal to noise ratios of 3:1. The results showed that LODs
for the 12 phytohormones ranged from 0.53 to 438.60 pg mL−1 , which could meet the requirement for the analysis of endogenous phytohormones in plant samples. The accuracy of the method was evaluated by spiking 12 phytohormone standards in the rice leaf matrices. The results showed that spiked phytohormones could be successfully determined with 85.1–114.6% recoveries (Table 2). Furthermore, the reproducibility of the proposed method was evaluated by investigating the intra- and inter-day precisions. Reproducibility of the proposed method was evaluated with the relative standard deviations (RSDs) between 0.2 and 18.0 % (Table 2).
3.5. Quantitative determination of endogenous phytohormones in plant samples Quantification of endogenous phytohormones is of great significance for knowing clearly the distribution of phytohormones in different plant tissues. It will be helpful for better understanding the interaction mechanism of phytohormones in plant. Rice is an important crop worldwide and is considered to be a model plant among monocots for biological research. A. thaliana is a small flowering plant that is widely used as a model organism in plant biology [37,38]. In the current study, phytohormones analysis was performed with 5–50 mg rice (O. sativa) for evaluating the performance of the proposed method. As listed in Table 3, phytohormones analysis could be completed with 5 mg rice sample and 9 phytohormoes were successfully detected, including 5 CKs, IAA, ABA, JA and SA. The MRM chromatograms of 9 detected endogenous phytohormones were shown in Fig. 6B. Furthermore, phytohormones in different plant tissues (root, stem, leaf, flower and siliques) of A. thaliana were also analyzed and the results were listed in Table 4. IAA and JA were detected with higher concentration in flower and siliques than other tissues. The distribution of phytohormones in rice and A. thaliana tissues could be obtained, demonstrating that the proposed method is sensitive and requires tiny amount of plant samples.
4. Conclusion In the current study, we described a simple and effective method for simultaneous determination of multiple endogenous phytohormones by the combination of sequential SIPTE and UPLC–MS/MS. The proposed sequential SIPTE was demonstrated to be a simple and effective approach to enrich and purify phytohormones from acetonitrile extract of plant tissues. The developed sequential SIPTE-UPLC–MS/MS method was successfully applied to analysis of multiple endogenous phytohormones in different issues of model plants (rice and A. thaliana) with small amount of sample size (5 mg, fresh weight). The developed method may be suitable for studying the distribution of endogenous phytohormones in different tissues of model plants.
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