A Simple Purification of Indole-3-Acetic Acid and Abscisic Acid for GC-SIM-MS Analysis by Microfiltration of Aqueous Samples through Nylon

Abstract

A simple procedure was developed for the partial purification of plant tissue samples to be analyzed simultaneously for indole-3-acetic acid (IAA) and abscisic acid (ABA). The procedure relies on removal of contaminants by filtration through nylon and partitioning into dichloromethane. This procedure successfully purified both IAA and ABA from muskmelon, cotton, and broccoli tissue. Twenty individual samples can be purified and methylated in 8 h for analysis of free IAA and ABA with gas chromatographyselected ion monitoring-mass spectrometry. The use of microfiltration of aqueous samples through nylon offers new opportunities for improving the efficiency of existing sample purification procedures.with molecular fragments unique to the hormone of interest are monitored (3,15,16). The high degree of detector selectivity coupled with the efficiency of capillary gas chromatography requires less sample purity than normally required for analysis of IAA and ABA from plant tissue.Nylon preparations, including nylon 66, have been used to inactivate and remove phenolic compounds from plant samples (7, 9). Microfiltration through nylon 66 syringe filters was recently reported to be an effective step in the preparation of plant samples for the immunoassay of ABA (13, 21). Consequently, experiments were conducted to assess the potential value of using nylon 66 microfiltration in the preparation of plant samples for the analysis of IAA and ABA by GC-SIM-MS. IAA and ABA (4,8,12,17). The GC-MS analysis is particularly sensitive when only a few selected ions associated 'Abbreviations: GC-SIM-MS, gas chromatography-selected ion monitoring-mass spectrometry; BHT, butylated hydroxytoluene; MATERIALS AND METHODSMeABA, methyl ester of ABA; MeIAA, methyl ester of IAA. Sample PreparationCotton floral buds (squares) and fruiting branches were harvested from field plots prior to anthesis. Muskmelon flowers were harvested from greenhouse-grown plants at anthesis. Broccoli heads including floral buds were harvested at commercial maturity from field plots. Each plant tissue was lyophilized, homogenized with a Polytron PT 10/35 (Brinkman Instruments, Westbury, NY)2 in 70% acetone (40 mL g-' dry weight that contained 200 mg of BHT and 100 mg of Na ascorbate L-' (6).

Stomatal Responses in Rainfed Lowland Rice to Partial Soil Drying; Comparison of Two Lines

“…The leaf blades were placed in aluminum foil then immediately immersed in liquid N and stored at -80 o C until analysis. Extraction and purification were done following the protocol of Dunlap and Guinn (1989). Samples were then fed into an HPLC system consisting of an LC 10AT HPLC pump, SIL-10AD auto injector, a CTO-10AVP column oven and an SPD 10 AVP (Shimadzu Corporation, Kyoto, Japan).…”

Stomatal Responses in Rainfed Lowland Rice to Partial Soil Drying ; Evidence for Root Signals

“…For ABA analysis, leaves were sampled at 1000 h, placed in aluminum foil, then immediately dipped in liquid N and stored at −80 o C until analyzed. Extraction and purification were performed following the protocol of Dunlap and Guinn (1989). Samples were then analyzed using an HPLC consisting of an LC 10AT HPLC pump, SIL-10AD auto-injector, a CTO-10Avp column oven and an SPD-10 Avp detector (Shimadzu Corporation, Kyoto, Japan).…”

A Microscale Technique for Gas Chromatography-Mass Spectrometry Measurements of Picogram Amounts of Indole-3-Acetic Acid in Plant Tissues

“…GC-MS provides precise measurements of small amounts of IAA, with the accuracy of each estimate being checked by comparing response ratios of several characteristic fragments (Cohen et al, 1986). However, as with other methods, several purification steps are needed if accurate quantitative estimates of IAA in plant extracts are to be obtained (Rivier, 1986;Vine et al, 1987;Chen et al, 1988;Dunlap and Guinn, 1989). By using a double-focusing HR mass spectrometer, the selectivity of the analysis increases and overcomes some of the problems caused by interfering substances.…”