An Efficient Method for the Quantification of Hydroxamic Acids From Wheat by Thin Layer Chromatography–Densitometry C. Garcı ´a, 1 S. Garcı ´a, 1 H. Heinzen, 1 P. Moyna 2 and H. M. Niemeyer 2 * 1 Facultad de Quı ´mica, Universidad Mayor de la Repu ´blica, General Flores 2124, Montevideo, Uruguay 2 Departamento de Ciencias Ecolo ´gicas, Facultad de Ciencias, Universidad de Chile, Casilla 653, Santiago, Chile A new method is described for the quantification of 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA) and 2,4-dihydroxy-1,4-benzoxazin-3-one (DIBOA), the main hydroxamic acids in wheat and rye extracts, respectively, in cereal extracts based on densitometry of scanned thin layer chromatographic plates. The method allows the simultaneous quantification of up to five samples, and is linear between 0.5– 7 mg and 10–30 mg for DIMBOA and between 0.5–3.0 mg and 10–30 mg for DIBOA. Quantification of DIMBOA by this method generates a linear correlation with results obtained following analysis by high performance liquid chromatography. The possibility of applying this methodology to mixtures of DIBOA and DIMBOA is discussed. # 1998 John Wiley & Sons, Ltd. Keywords: hydroxamic acids; benzoxazinones; DIMBOA; DIBOA; thin layer chromatography–densitometry. INTRODUCTION Hydroxamic acids present in cereal extracts confer resistance to the plant towards insects such as the European corn borer and several species of aphids (Niemeyer and Pe ´rez, 1995). Hydroxamic acids are present in the plant as 2-b-O-D-glucopyranosides which are hydrolysed to the corresponding aglucones when the tissue is damaged (Hofman and Hofmanova, 1971). Three groups of methods have been described in order to quantify hydroxamic acids in cereal extracts. In the first, the tissue is ground and then either frozen, thawed and heated or simply heated to promote hydrolysis of the glucosides to agluconic benzoxazinones and their further decomposition to benzoxazolinones. These latter deriva- tives are then separated chromatographically and quanti- fied by UV-VIS spectroscopy (Beck et al., 1957; Molot and Anglade, 1968), by spectrofluorimetry (Bowman et al., 1968) or by infrared spectroscopy (Scism et al., 1974), or are quantified directly by isotopic dilution (Klun and Brindley, 1966), by gas chromatography (GC) (Pessi and Scalorbi, 1979; Malan et al., 1986), or by visual determination after thin layer chromatographic (TLC) separation (Robinson et al., 1982). These methods assume that decomposition of hydroxamic acid aglucones to benzoxazolinones is quantitative, or at least that a single correction factor may be applied to all samples. These asumptions have not been validated by experi- ments. The decomposition reaction is not quantitative, the yield of benzoxazolinones being not only a function of pH (Niemeyer et al., 1982) and temperature (Wood- ward et al., 1978a; Bravo and Niemeyer, 1986a), but also of the composition of the reaction medium (Woodward et al., 1978b; Argandon ˜a et al., 1981; Bravo and Niemeyer, 1986b). A second group of methods is based on the quanti- fication of hydroxamic acids as Fe (III) complexes (Hamilton, 1964; Long et al., 1974; Sullivan et al., 1974). These methods suffer from uncertainties owing to the non-specific nature of the colourimetric reaction of Fe(III) and also to their inability to quantify different hydroxamic acids that may be present in the samples. However, the method has been applied successfully to series of related cereals (Woodward et al., 1979a). The third group of methods allows for the formation of aglucones and the quantification of these compounds directly by GC (Woodward et al., 1979b) or by high performance liquid chromatography (HPLC) on silica columns (Gutie ´rrez et al., 1982) or on reverse-phase columns (Lyons et al., 1988; Niemeyer et al., 1989; Xie et al., 1990; Mayoral et al., 1994). This has become the method of choice for hydroxamic acid analysis. Direct TLC–densitometric quantitation has been applied to a number of natural products in extracts with high accuracy and repeatability, e.g. cuticular waxes (Davyt et al., 1995), carbohydrates (Cesio et al., 1996), steroidal alkaloids (Ferreira et al., 1993), and cineole from the essential oil of Eucalyptus (Rossini et al., 1995). Moreover, the UV spectra taken directly from a TLC plate was useful in identifying a series of flavonoids (Garcı ´a et al., 1993). In this paper, we describe a new method to quantify 2,4-dihydroxy-7-methoxy-1,4-ben- zoxazin-3-one (DIMBOA), the main hydroxamic acid in wheat extracts, and/or 2,4-dihydroxy-1,4-benzoxazin-3- one (DIBOA), the main hydroxamic acid in rye extracts, based on the TLC separation from plant extracts of these PHYTOCHEMICAL ANALYSIS Phytochem. Anal. 9, 278–282, (1998) CCC 0958–0344/98/060278–05 $17.50 # 1998 John Wiley & Sons, Ltd. * Correspondence to: H. M. Niemeyer, Departamento de Ciencias Ecolo ´gicas, Facultad de Ciencias, Universidad de Chile, Casilla 653, Santiago, Chile. (E-mail: niemeyer@abulafia.ciencias.uchile.cl) Contract/grant sponsor: PEDECIBA Program; Contract/grant number: PNUD URU84/002. Contract/grant sponsor: International Program in the Chemical Sciences. Received 7 May 1997 Revised 17 October 1997 Accepted 3 November 1997