Determination of Acidic Plant Hormones by Derivative UPLC-MS

doi:10.11983/CBB22017

Abstract

Abstract: This study aims to establish a rapid and efficient method for qualitative and quantitative determination of plant acid hormones by ultra-high performance liquid chromatography/high resolution time-of-flight mass spectrometry (UPLC- TOF-MS). Several derivatization reagents, 2-bromoacetophenone (BP), 2-dimethylaminoethylamine (DMED), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and 3-bromoactonyltrimethylammonium bromide (BTA) were selected for derivatization with abscisic acid, gibberellic acid, indoleacetic acid, jasmonic acid and salicylic acid, respectively. The effect of derivatization reagent was evaluated by comparing the response value of the derivative hormones. The plant hormone standards were diluted in gradient and reacted with the derivatization reagent, and the detection limit and quantitation limit of the derivative hormones were determined. Reagent with the best derivatization effect was reacted with the plant crude extract to test its application effect. All the four derivatized reagents could improve the MS response value of acidic hormone, among which the MS sensitivity of hormone derived by DMED is the best. Reaction with DMED was stable and reproducible. The quantitation limits of ABA-DMED, GA₃-DMED, IAA-DMED, JA-DMED and SA-DMED were 0.05, 0.2, 0.1, 0.1 and 0.5 ng∙mL^-1, respectively. Compared with non-derivatized hormones, the quantitative limits of ABA, GA₃, IAA, JA and SA were reduced by 100, 25, 50, 50 and 10 times, respectively. So, DMED derivatization increased the mass spectrometry sensitivity of several hormones by 10-100 times. The method was applied to the determination of endogenous acidic hormones in rice, wheat and broad bean, and the sensitivity was significantly higher than that before derivation. A simple, rapid and reproducible UPLC-TOF-MS method based on derivatization was established, which greatly improved the sensitivity of the determination of acidic hormones in plants.

Key words: derivatization, high resolution mass spectrometry, acidic plant hormones, quantitative determination

Dai Chen, Wang Jin, Lu Yaping. Determination of Acidic Plant Hormones by Derivative UPLC-MS[J]. Chinese Bulletin of Botany, 2022, 57(4): 500-507.

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URL: https://www.chinbullbotany.com/EN/10.11983/CBB22017

https://www.chinbullbotany.com/EN/Y2022/V57/I4/500

Figures/Tables 7

References 20

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Analyte	Formula	Ion mode	m·z^-1	RT (min)
ABA	C₁₅H₂₀O₄	[M-H]^-	263.1283	7.02
GA₃	C₁₉H₂₂O₆	[M-H]^-	345.1338	5.52
IAA	C₁₀H₉NO₂	[M+H]⁺	176.0712	6.06
JA	C₁₂H₁₈O₃	[M-H]^-	209.1178	7.61
SA	C₇H₆O₃	[M-H]^-	137.0239	6.19

Analyte	Formula	Ion mode	m·z^-1	RT (min)
ABA	C₁₅H₂₀O₄	[M-H]^-	263.1283	7.02
GA₃	C₁₉H₂₂O₆	[M-H]^-	345.1338	5.52
IAA	C₁₀H₉NO₂	[M+H]⁺	176.0712	6.06
JA	C₁₂H₁₈O₃	[M-H]^-	209.1178	7.61
SA	C₇H₆O₃	[M-H]^-	137.0239	6.19

Analyte	Linear equation	R²	RSD (%)	LOD (ng∙mL^-1)	LOQ (ng∙mL^-1)
BA	y=23.75x+54.37	0.999	5.1	2	5
GA₃	y=31.31x+72.43	0.999	3.7	1	5
IAA	y=21.22x-3.499	0.998	3.9	5	10
JA	y=27.11x+118.7	0.996	4.1	2	5
SA	y=33.63x+141.8	0.997	2.8	2	5

Analyte	Linear equation	R²	RSD (%)	LOD (ng∙mL^-1)	LOQ (ng∙mL^-1)
BA	y=23.75x+54.37	0.999	5.1	2	5
GA₃	y=31.31x+72.43	0.999	3.7	1	5
IAA	y=21.22x-3.499	0.998	3.9	5	10
JA	y=27.11x+118.7	0.996	4.1	2	5
SA	y=33.63x+141.8	0.997	2.8	2	5

Analyte	Formula	Ion mode	m·z^-1	RT (min)	Response^a	Response^b	Response^c
ABA-BP	C₂₃H₂₆O₅	[M+H]⁺	383.1858	8.93	2.4e⁶	4.5e⁵	2.5e⁴
GA₃-BP	C₂₇H₂₈O₇	[M+H]⁺	465.1913	7.62	1.76e⁴	2.7e³	ND
IAA-BP	C₁₈H₁₅NO₃	[M+H]⁺	294.113	8.69	5.6e⁶	8.7e⁵	5.6e⁴
JA-BP	C₂₀H₂₄O₄	[M+H]⁺	329.1753	9.55	1.4e⁶	2.6e⁵	9.8e³
SA-BP	C₁₅H₁₂O₄	[M+H]⁺	257.0814	7.65	9.8e³	4.4e³	ND
ABA-DMED	C₁₉H₃₀N₂O₃	[M+H]⁺	335.2335	4.89	1.7e⁷	1.6e⁶	1.45e⁵
GA₃-DMED	C₂₃H₃₂N₂O₅	[M+H]⁺	417.2389	4.67	9.1e⁶	1.2e⁶	8.8e⁴
IAA-DMED	C₁₄H₁₉N₃O	[M+H]⁺	246.1606	3.79	7.3e⁶	5.9e⁵	3.5e⁴
JA-DMED	C₁₆H₂₈N₂O₂	[M+H]⁺	281.2229	5.38	8.9e⁶	1.1e⁶	7.6e⁴
SA-DMED	C₁₁H₁₆N₂O₂	[M+H]⁺	209.1290	3.43	3.1e⁵	4.0e⁴	9.2e³
ABA-EDC	C₂₃H₃₇N₃O₄	[M+H]⁺	420.2862	6.01	1.1e⁷	1.2e⁶	1.8e⁵
GA₃-EDC	C₂₇H₃₉N₃O₆	[M+H]⁺	502.2917	5.14	4.0e⁶	4.4e⁵	6.2e⁴
IAA-EDC	C₁₈H₂₆N₄O₂	[M+H]⁺	331.2134	5.89	9.8e⁶	2.6e⁶	3.2e⁵
JA-EDC	C₂₀H₃₅N₃O₃	[M+H]⁺	366.2757	5.40	8.8e⁶	1.2e⁶	2.3e⁵
SA-EDC	C₁₅H₂₃N₃O₃	[M+H]⁺	294.1818	ND	ND	ND	ND
ABA-BTA	C₂₁H₃₁NO₅	[M+H]⁺	378.228	5.82	4.0e⁶	5.37e⁵	5.4e⁴
GA₃-BTA	C₂₅H₃₃NO₇	[M+H]⁺	460.2335	4.87	1.3e⁶	1.6e⁵	2.1e⁴
IAA-BTA	C₁₆H₂₀N₂O₃	[M+H]⁺	289.1552	4.96	8.0e⁵	8.5e⁴	1.7e⁴
JA-BTA	C₁₈H₂₉NO₄	[M+H]⁺	324.2175	6.40	1.2e⁶	7.4e⁴	9.6e³
SA-BTA	C₁₃H₁₇NO₄	[M+H]⁺	252.1236	4.70	4.9e⁴	4.8e³	ND