Sains Malaysiana 33(2):21-34 (2004)                                                                                 Sains Fizis dan Gunaan /

Physical and Applied Sciences

Pengembangan Kaedah Analisis bagi Tiga Jenis Fitohormon

Secara Kromatografi Gas ­Spektroskopi Jisim

(Development of Analytical Method for Three Types of Phytohormones

Using the gas Chromatography-mass Spectroscopy)

Jamaluddin Mohd Daud & Nor Azlina Zainal Abidin

Pusat Pengajian Sains Kimia dan Teknologi Makanan

Fakulti Sains dan Teknologi

Universiti Kebangsaan Malaysia

43600 UKM Bangi, Selangor, Malaysia

ABSTRAK

Satu kaedah penyediaan sampel dan analisis kualitatif yang pantas bagi fitohormon iaitu asid absisik (ABA), asid indol-3-asetik (IAA) dan asid 1­-aminosiklopropana-l-karboksilik (ACC) dilaporkan. Ekstrak daripada tumbuhan Umbellifereae sp. dilakukan pengekstrakan fasa pepejal dengan menggunakan katrij C₁₈ dan efluen yang dipungut ditindakbalaskan bagi membentuk terbitan pentafluorobenzil bagi campuran ABA, IAA dan ACC. Campuran terbitan tersebut dipisahkan dan dilakukan pengionan kimia ion negatif dengan menggunakan metana di dalam instrumen kromatografi gas-spektrometri jisim (GC-MS). Spektrum jisim tiga hormon tersebut agak mudah dan oleh itu adalah sesuai bagi pemonitoran ion terpilih (S/M) pada kepekaan tinggi bersama dengan piawai dalaman masing-masing yang berlabel deuterium. Walau bagaimanapun, penentuan kuantitatif hanya dapat dilakukan untuk IAA menggunakan kaedah GC-MS melalui penisbahan luas puncak kromatogram S/M bagi m/z 179 dengan m/z 174. Ini membolehkan pembetulan kehilangan IAA endogen semasa pengekstrakan berbanding piawai dalaman tanpa perlu pensampelan yang teliti, kecuali semasa penambahan piawai dalaman. Had pengesanan bagi IAA melalui kaedah ini ialah 5 femtomol/g berat basah tumbuhan.

ABSTRACT

A faster method for sample preparation and qualitative analylysis of phytohormones, i.e. abscisic acid (ABA), indole-3-acetic acid (IAA) and 1­-aminocyclopropane-l-carboxylic acid (ACC) is reported. Solid phase extrac­tion of Umbellifereae sp. plant extract was performed using C₁₈ catridge ana the effluent collected was reacted to form the pentafluorobenzyl derivatives of ABA, IAA and ACC mixture. The derivatives mixture was separated and negative ion chemical ionization was carried out by using methane in a gas chromatograph-mass spectrometric instrument (GC-MS). Mass spectra of the three phytohormones are quite simple and therefore suitable for selective ion monitoring (S/M) at high sensitivity, together with their respective internal standards labeled with deuterium.  However, only IAA can be determined  quantitatively by GC-MS technique through the peak area ratio of SIM chromatograms for m/z 179 with m/z 174.  This method allows the correction for the loss of IAA endogen during extraction when compared to the internal standard without the need for precise sampling, except during addition of the internal standard.  The detection limit of IAA by this method is  5 femtomole/g wet weight of the plant.

RUJUKAN/REFERENCES

Cohen, J.D., Baldi, B.G. & Slovin, J.P. 1986. ¹³C₆-[Benzene Ring]-Indole-3-Acetic Acid. A New Internal Standard for Quantitative Mass Spectral Analysis of Indole-3-Acetic Acid in Plants. Plant Physiol., 80: 14-19.

Hedden, P. 1993. Modern Methods for the Quantitative Analysis of Plant Hormones. Ann. Rev. Plant Physiol. Plant Mol. Biol., 44: 107-129.

IIIic, N., Buta, N.J. & Cohen, J.D. 1992. Synthesis of  ¹³C₆-abscisic acid for use as an Internal Standard for GC-MS Quantification. Plant Physiol., 99: 65 (Suppl.) Magnus.

V., Bandurski, R.S. & Schulze A. 1980. Synthesis of 4,5,6,7 and 2,4,5,6,7 Deuterium-labeled Indole-3-Acetic Acid for Use in Mass Spectromeric Assays. Plant Physiol., 66: 775-781.

Milborrow, B.V. 1971. Abscisic Acid. Dalam : T.W. Goodwin (ed), Aspects of Terpenoid Chemsitry and Biochemistry, Acad. Press, London, ms. 137-151.

Netting, A.G. & Duftield, A.M. 1985. Positive and Negative Ion Methane Chemical Ionization Mass Spectrometry of Amino Acid Pentatluorobenzyl Derivatives. Biomed. Mass Spectrum., 12: 668-672.

Netting, A.G. & Milborrow, B.V. 1988. Methane Chemical Ionization Mass Spectrometry of the Pentatluorobenzyl Derivatives of Abscisic Acid, its Metabolites and Others Plant Growth Regulators. Biomed. Environ. Mass Spec­trum., 17: 281-286.

Prinsen, E., Rudelsheim, P. & Van Onckelen, H. 1991. Extraction, Purification and Analysis od Endogenous Indole-3-Acetic Acid and Abscisic Acid. Dalam : I. Negrutiu dan F. Fharti-Chhetri (eds). A. Laboratory Fuide for Cellular and Molecular Plant Biology, Birkhauser Verlag. Basel, ms. 198-209.