Determination of plant hormones in plant tissues by UPLC-MS/MS

ObjectivePlant hormones have pivotal roles in plant growth, development and response to biotic and abiotic stress. Quantitative analysis is required for studying the chemical synthesis, transportation, metabolism and molecular regulation of plant hormones. However, hormones are trace level and unstable in most tissues of plants. Moreover, the analysis of plant hormones is seriously interfered by co-existed secondary metabolites. In addition, some plant materials are rare and their amounts are low. To address above-mentioned questions, a method was developed for simultaneously determining multiple plant hormones in Populus tomentosa leaves, including indole-3-acetic acid (IAA), abscisic acid (ABA), gibberellins (GA₃), jasmonic acid (JA), and salicylic acid (SA) using ultra-highperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), which could provide technical support for better studying plant hormones.

MethodLeaching solution of 2-propanol, water, and hydrochloric acid (2∶1∶0.002, v/v/v) was used to extract IAA, ABA, GA₃, JA, and SA, and five internal standards (²H₅-IAA/²H₆-ABA/2H-JA/²H₅-SA, 10 ng per each, ²H₂-GA₃ 20 ng) were simultaneously introduced into the samples at 4 ℃. Crude extract was re-extracted by dichloromethane and then analyzed by UPLC-MS/MS. The target analytes were separated by Agilent SB-C₁₈ column, with gradient elution using acetonitrile and 0.1% acetic acid in water as mobile phases. The flow rate was 0.8 mL/min and the column temperature was 30 ℃. Negative electrospray ionization with multiple reaction monitoring (MRM) mode was used for detection. Internal standard curve method was used for quantitative analysis.

ResultFive plant hormones were separated thoroughly and detected within 5 mins. The limits of detection were 0.01−0.05 pg/g, and the limits of quantitation were 0.05−0.15 pg/g. The correlation coefficients (r) of internal calibration curves ranged from 0.999 4 to 0.999 9. The relative standard deviation (RSD) of peak areas was 1.1%−3.9%. The results showed that the contents of IAA, ABA, GA₃, JA, and SA in Populus tomentosa leaves were (74.38 ± 4.62) ng/g, (193.80 ± 6.04) ng/g, (674.67 ± 18.08) ng/g, (286.62 ± 7.48) ng/g, and (746.02 ± 13.93) ng/g, respectively.

ConclusionThe established method is easy-to-operate, high sensitivity and time-saving, which is suitable for the simultaneous identification and quantification of multiple hormones in plant tissues.