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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, Analytical technologies in the biomedical and life sciences (2015-10-07)
Bao-Dong Cai, Er-Cui Ye, Bi-Feng Yuan, Yu-Qi Feng
ABSTRACT

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 50mM 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 438.60pgmL(-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 (5mg, fresh weight). The proposed method may be suitable for studying the distribution of phytohormones in model plants.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Dichloromethane, anhydrous, ≥99.8%, contains 40-150 ppm amylene as stabilizer
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Toluene, anhydrous, 99.8%
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Ethyl acetate, ACS reagent, ≥99.5%
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Chloroform, contains 100-200 ppm amylenes as stabilizer, ≥99.5%
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Sodium bicarbonate, powder, BioReagent, for molecular biology, suitable for cell culture, suitable for insect cell culture
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Ethyl acetate, anhydrous, 99.8%
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Sodium bicarbonate, ACS reagent, ≥99.7%
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Dichloromethane, contains 40-150 ppm amylene as stabilizer, ACS reagent, ≥99.5%
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Formic acid, reagent grade, ≥95%
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Hexane, anhydrous, 95%
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Toluene, ACS reagent, ≥99.5%
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Chloroform, anhydrous, ≥99%, contains 0.5-1.0% ethanol as stabilizer
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Hexane, ReagentPlus®, ≥99%
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Cyclohexane, anhydrous, 99.5%
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Chloroform, anhydrous, contains amylenes as stabilizer, ≥99%
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Formic acid, puriss. p.a., ACS reagent, reag. Ph. Eur., ≥98%
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Dichloromethane, puriss. p.a., ACS reagent, reag. ISO, ≥99.9% (GC)
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Hexane, Laboratory Reagent, ≥95%
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Formic acid, ACS reagent, ≥96%
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Sodium bicarbonate, ReagentPlus®, ≥99.5%, powder
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Hexane, puriss. p.a., ACS reagent, reag. Ph. Eur., ≥99% (GC)
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Cyclohexane, ACS reagent, ≥99%
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Sodium bicarbonate, BioXtra, 99.5-100.5%
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Formic acid, puriss., meets analytical specifications of DAC, FCC, 98.0-100%
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Chloroform, contains ethanol as stabilizer, ACS reagent, ≥99.8%
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Ethyl acetate, puriss., meets analytical specification of Ph. Eur., BP, NF, ≥99.5% (GC)
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1-Octanol, anhydrous, ≥99%
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Butyl acetate, anhydrous, ≥99%
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Dichloromethane, suitable for HPLC, ≥99.9%, contains 40-150 ppm amylene as stabilizer
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Butyl acetate, ACS reagent, ≥99.5%