Simultaneous Quantitation of Indole 3-Acetic Acid and Abscisic Acid in Small Samples of Plant Tissue by Gas Chromatography/Mass Spectrometry/Selected Ion Monitoring

Abstract

Indole 3-acetic acid (IAA) was analyzed in apple, orange, and prune tissue by GC-MS by monitoring the protonated molecular ion of its methyl ester at mass to charge ratio (m/z) 190 together with the major fragment ion at m/z 130 and the corresponding ions from the methyl esters of either 12H4JIAA (m/z 194, 134) or I2HS5IAA (m/z 195, 135).Abscisic acid (ABA) was analyzed by monitoring the major fragment ions of its methyl ester at m/z 261 and m/z 247 and the corresponding ions from the methyl ester of rH31ABA (m/z 264, 250 The method to be described here, for simultaneous quantitation by GC-MS-SIM of free IAA and free ABA in small samples (0.1-0.5 g dry weight), arose from research on morphogenesis in tissue cultures and intact plants of apple (9, 13), prune (1), and 'Abbreviations: GC-MS-SIM, gas chromatography-mass spectrometry-selection ion monitoring; Me-IAA, methyl ester of IAA; Me-ABA, methyl ester of ABA; MH+, protonated molecular ion; m/z, mass to charge ratio. citrus (10). Of the few methods available for simultaneous measurement of IAA and ABA (2, 4-6, 8, 11) none was suitable for our purposes. Those based on chromatographic procedures alone (2, 5, 8, 11) require extensive purification of extracts and lack the specificity of GC-MS. Published methods involving GC-MS (4, 6) are primarily qualitative in nature and do not include use of deuterated internal standards and calibration curves. These methods lack the necessary precision for quantitative work. Accordingly, it was decided to modify a currently used quantitative GC-MS assay for ABA (12) to enable IAA to be measured in the same extract. MATERIALS AND METHODSPlant Materials, Extraction, and Derivatization. The plant materials were leafy microcuttings from aseptic proliferating shoot cultures of apple (cv Jonathan) and prune (cv D'Ente), 1-year-old extension shoots of Jonathan apple, and fully expanded mature leaves from adult cropping orange trees (cv Valencia).All glassware was soaked in a 5% solution of Surfasil (Pierce Chemical Co., Rockford, IL) in hexane for 5 min, rinsed with methanol and oven-dried for 1 h at 100°C. All the sample manipulations were carried out under dim light to minimize isomerization and photodecomposition. Samples and extracts were stored in the dark at -20°C when not in use. All solvents were of analytical reagent grade and were distilled before use.The tissues were harvested, frozen by immersion in liquid N2, and freeze-dried. Although the possibility of losses of IAA and ABA during freeze drying cannot be completely ruled out, care was taken to ensure that the same technique and conditions were used for the freeze drying of all tissues. Samples of the freezedried material (100-500 mg) were ground in a mortar and extracted overnight (4°C) with 70% (v/v) IAA (purchased from MSD Isotopes, Montreal, Canada). The slurry was centrifuged (2 min) and the supernatant was retained.The pellet was reextracted with 70% acetone and centrifuged and the combined supernatants were then evaporated to the aqueous phase under a ...

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

“…The ABA was determined from traces at m/z 162, 166, 190, and 194 using the isotope dilution equation described for IAA (3). Radiolabeled internal standards were used to estimate recoveries of the target compounds (1) and confirmed according to procedures presented by Vine et al (19) (19). The aqueous residue left after rotary flash evaporation as described in the section on sample preparation was adjusted to a pH of 8.5 with a saturated solution of NaHCO3 and partitioned 3 times against equal volumes of petroleum ether followed by 3 equal volumes of diethyl ether.…”

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.…”

Hydrolysis of Indole-3-Acetic Acid Esters Exposed to Mild Alkaline Conditions

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ABSTRACTEster conjugates of indole-3-acetic 16,18,22,23) following various protocols suggested that a more systematic evaluation of this process was needed. In this report we present data on the kinetics of hydrolysis of reagent IAA-GLU2 at selected pH values and compare the lability of IAA-GLU to that of IAA-MI and several other ester conjugates of IAA.

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