Oasis max solid phase extract cartridge

For analysis of ABA content in soybean seeds, the previously protocol we used (Shu et al., 2013 (link)) were employed in this study. Firstly, the seeds were ground to powder in liquid nitrogen, and 300 mg of seeds powder was homogenized and extracted for 24 h in methanol containing D6-ABA (OIChemIm Co. Ltd.) as an internal standard. Purification was performed with an Oasis Max solid phase extract cartridge (Waters) and eluted with 5% formic acid in methanol. Subsequently, the elution was dried and reconstituted, and it was then injected into a liquid chromatography–tandem mass spectrometry system consisting of an Acquity ultra performance liquid chromatograph (Acquity UPLC; Waters) and a triple quadruple tandem mass spectrometer (Quattro Premier XE; Waters). Three biological replications were performed.

Quantification of ABA and GA were performed according to the methods described in our previous studies (Chen et al., 2011 ; Shu et al., 2013 (link), 2016a (link)).
For ABA, dry and imbibed (6 h after sowing, 400 mg) soybean seeds were ground in liquid nitrogen and extracted for 24 h in methanol containing D6-ABA (OIChemIm Co. Ltd.) as an internal standard. Purification was performed using an Oasis Max solid-phase extract cartridge (Waters) and eluted with 5% formic acid in methanol. The elution was then dried and reconstituted, and injected into a LC–tandem MS system consisting of an Acquity ultra-performance LC (Acquity UPLC; Waters) and a triple-quadrupole tandem MS (Quattro Premier XE; Waters). Three biological replications were performed.
For GA, dry and imbibed (6 h after sowing, 400 mg) soybean seeds were ground in liquid nitrogen and extracted with 80% (v/v) methanol. GA d2 isotope standards were added to the samples before grinding. The crude extracts were purified by reversed-phase solid-phase extraction, ethyl-ether extraction, and derivatization. The resulting mixture was injected into a capillary electrophoresis-MS system (Agilent Technologies) for quantitative analysis. Three biological replications were performed.

IAA was quantified as previously described [6 (link)]. Briefly, 200 mg (fresh weight) of whole root or shoot for each treatment was quickly frozen in liquid nitrogen and ground into a fine powder, and then, tissues were homogenized and extracted for 24 h in methanol containing ²H-IAA (CDN isotopes) as an internal standard. Purification was performed using an Oasis Max solid phase extract cartridge (Waters) after centrifugation. IAA measurement was carried out with a liquid chromatography–tandem mass spectrometry system consisting of Acquity Ultra Performance Liquid Chromatography (Acquity UPLC; Waters) and a triple quadruple tandem mass spectrometer (QTRAP 5500; AB SCIEX).