title: Analysis of neem oils by LC-MS and degradation kinetics of azadirachtin-A in a controlled environment. Characterization of degradation products by HPLC-MS-MS.
abstract: Since it was first isolated, the oil extracted from seeds of neem (Azadirachtin indica A juss) has been extensively studied in terms of its efficacy as an insecticide. Several industrial formulations are produced as emulsifiable solutions containing a stated titer of the active ingredient azadirachtin-A (AZ-A). The work reported here is the characterization of a formulation of this insecticide marketed under the name of Neem-azal T/S and kinetic studies of the major active ingredient of this formulation. We initially performed liquid-liquid extraction to isolate the neem oil from other ingredients in the commercial mixture. This was followed by a purification using flash chromatography and semi-preparative chromatography, leading to (13)C NMR identification of structures such as azadirachtin-A, azadirachtin-B, and azadirachtin-H. The neem extract was also characterized by HPLC-MS using two ionization sources, APCI (atmospheric pressure chemical ionization) and ESI (electrospray ionization) in positive and negative ion modes of detection. This led to the identification of other compounds present in the extract-azadirachtin-D, azadirachtin-I, deacetylnimbin, deacetylsalannin, nimbin, and salannin. The comparative study of data gathered by use of the two ionization sources is discussed and shows that the ESI source enables the largest number of structures to be identified. In a second part, kinetic changes in the main product (AZ-A) were studied under precise conditions of pH (2, 4, 6, and 8), temperature (40 to 70 degrees C), and light (UV, dark room and in daylight). This enabled us to determine the degradation kinetics of the product (AZ-A) over time. The activation energy of the molecule (75+/-9 kJ mol(-1)) was determined by examining thermal stability in the range 40 to 70 degrees C. The degradation products of this compound were identified by use of HPLC-MS and HPLC-MS-MS. The results enabled proposal of a chemical degradation reaction route for AZ-A under different conditions of pH and temperature. The data show that at room temperature and pH between 4 and 5 the product degrades into two preferential forms that are hydrolyzed to a single product over time and as a function of pH change.
authors.affiliation: Service Central d'Analyse, USR 059, CNRS, BP 22, 69390 Vernaison, France.
authors.forename: Sami
authors.initials: S
authors.lastname: Barrek
authors.forename: Olivier
authors.initials: O
authors.lastname: Paisse
authors.forename: Marie-Florence
authors.initials: MF
authors.lastname: Grenier-Loustalot
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005989:Glycerides / Q000032:analysis*; D007700:Kinetics; D008027:Light; D036701:Limonins / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry / Q000379:methods*; D013696:Temperature; D013729:Terpenes / Q000032:analysis*
chemical_list: D005989:Glycerides; D036701:Limonins; D013729:Terpenes; C002443:neem oil; C010329:azadirachtin