Identification of Abscisic Acid by LC/MS System and Rapid Determination of the Abscisic Acid Concentration in Soybean Seeds Using High Performance Liquid Chromatography

Abscisic acid (ABA) is one of the photohormones related to plant metabolism, development and senescence. For determination of ABA, tritium- or deuterium-labeled ABA has been used as an internal standard, because incomplete recovery was expected in the extraction and purification procedures. The simple purification method was applied for the determination of ABA concentration in soybean seeds without any internal standard. The soybean seeds were extracted with methanol including 2.6-di-tert-butyl-p-crezol as an antioxidant. After methanol was evaporated to dryness, the residue was redissolved in water, and the impurities in the solution were removed by mini column of octadecylsilane (C 18). The ABA was quantitatively recovered from the column and ABA gave a single UV peak by high performance liquid chromatography with a SIL 5-B column. The identification of ABA was done by a liquid chromatography/mass spectrometer (LC/MS) system. The mass spectrum of standard ABA and the peak with the same retention time from soybean seeds gave the similar fragmentation patterns with various voltages of ionization. Also, the values obtained by mass spectrum with 263 m/z, a major peak of ABA and those by UV absorbancy, showed the same concentrations. UV spectrum of standard ABA and isolated ABA fraction from LC showed the same curve. From these results, the ABA concentration in soybean seeds can be analyzed simply by partial purification with a C 18 mini-column and analyzed by HPLC with UV detecter without an internal standard. The LC/MS system is very useful to identify the LC peak and to detectimpurities in the ABA fraction. ABA concentration change in seeds was compared in two isogenic lines, nodulated T 202 and non-nodulated T 201, grown in a rotated paddy field of Niigata Agricultural Station during seed development. In the early stage of seeds development (September 1, 1993), the ABA concentration of T202 seeds was initially higher and then decreased, while that of T201 seeds remained high in the middle stage of seeds development (September 22, 1993). The differential changes in the ABA concentration of seeds may influence nitrogen nutrition of plants or seed storage protein accumulation