Establishment and validation of UPLC-MS/MS method for simultaneous determination of 19 phytohormones in soil

Objectives The purpose of this study is to establish an effective method for simultaneously extracting a total of 19 of phytohormones in rhizosphere soil by using ultra performance liquid chromatography tandem mass spectrometry (UPLS-MS/MS). These phytohormones include ABA, GA3, JA, IAA, tZ, tZR, IPR, IBA, IP, SL, cZ, DZR, IPA, MeIAA, SA, DZ, ICA, MeJA, and BL.

Methods The rhizosphere soil were extracted by the solution of isopropyl alcohol∶ water∶ formic acid (80∶19∶1, V/V/V). After ultrasonic treatment and centrifugation, the phytohormones extracts were concentrated by drying at room temperature, and redissolved with methanol. The Waters ACQUITYUPLC ® HSST3 chromatographic column was used for separation of 19 phytohormones, 0.30 mmol/L of ammonium formate aqueous solution (containing 0.01% formic acid) was used as mobile phase A and 0.30 mmol/L of ammonium formate acetonitrile (containing 0.01% formic acid) was used as mobile phase B, accompanying a gradient elution, the flow rate was 0.3 mL/min and the column temperature was maintained at 30℃. Then the phytohormones were identified and quantified with ultra performance liquid chromatography tandem mass spectrometry through a multiple reaction-monitoring mode. The JA, IAA, tZ, tZR, IPR, IBA, IP, SL, cZ, DZR, IPA, MeIAA, DZ, ICA, MeJA, and BL were scanned with the positive ion mode, while the ABA, GA3, SA were scanned by the negative ion mode. External standard method was used to quantify the recovery.

Results The concentration and corresponding peak area correlation coefficients (r) of 19 kinds of phytohormones such as ABA, GA3, JA, IAA, tZ, tZR, IPR, IBA, IP, SL, cZ, DZR, IPA, MeIAA, SA, DZ, ICA, MeJA, and BL were more than 0.99 in the concentration range of this experiment. The detection limits were ranged from 0.02 ng/g to 1.06 ng/g, the recoveries of 19 phytohormones were between 70.2%–117%, and the relative standard deviations were ranged from 0.2% to 7.3%. The phytohormones in rhizosphere soil were determined by the optimized experimental method, and the detected five phytohormones (IAA, ICA, MeIAA, SA, SL) were ranged from 0.55 to 5.79 ng/g in rhizosphere soil.

Conclusions In the modified procedure, the phytohormones do not require overnight extraction, they can be effectively extracted using dichloromethane through a series of centrifugation, concentration and resolution. The modified procedure could produce better selectivity and higher sensitivity for the 19 main phytohormones in soil.