J. Agric. Food Chem. 1994,42,2029-2034
2029
Gas Chromatography of Allelochemicals Produced during Glucosinolate Degradation in Soil Paul D. Brown, Matthew J. Morra,* and Vladimir Borek Division of Soil Science, University of Idaho, Moscow, Idaho 83844-2339
Glucosinolate degradation products from Brassica spp. may control soil-borne plant pests; however, a comprehensive method for the analysis of the responsible glucosinolate-derived allelochemicals in soils is lacking. Splitless injection and a 5% phenyl-substituted methylpolysiloxane fused silica capillary column in a gas-liquid chromatograph equipped with a flame ionization detector were used to separate various isothiocyanates, nitriles, and oxazolidinethione in less than 22 min. A linear relationship between detector response for isothiocyanates and molecular weight was observed, allowing estimation of isothiocyanate concentrations for which standards are not readily available. Detection limits for seven of nine compounds were less than 0.1 pg/mL for 1 pL injections. The efficacy of various solvents for extraction was examined, as was the effect of salt and extraction time. Quantities of isothiocyanate and nitrile in CH2C12 extracts of five soils exceeded 94% of the added spikes, and oxazolidinethione recovery exceeded 84%. Maximum extraction of glucosinolate degradation products from soil amended with crushed seed meal of Brassica napus was obtained in 15 min when 2 mL of 0.20 M CaC12 was included with the CHzClz extractant.
Keywords: Glucosinolates; isothiocyanates; allelochemicals; Brassica spp. INTRODUCTION Glucosinolates are organic anions containing B-Dthioglucose and sulfonated oxime moieties. Nearly 100 glucosinolates, differentiated by the specific aliphatic or aromatic side group, have been isolated or identified from degradative products (Chew, 1988). Produced exclusively in dicotyledonous plants, highest concentrations of glucosinolates are found in plants from the families Resedaceae, Capparidaceae, and Brassicaceae (Fenwick et al., 1983). Enzymatic degradation of glucosinolates by thioglucoside glucohydrolase (EC 3.2.3.1) results in the formation of biologically active products. These allelochemicals have the potential to suppress a wide variety of soil-borne plant pests and thus reduce the need for application of synthetic organic pesticides. Analysis of glucosinolate degradation products in soil is necessary t o evaluate the mechanisms by which glucosinolate-containing Brassica spp. participate in biological pest control strategies. Packed column gas chromatography (GC) of isothiocyanates produced in plant tissue extracts as a result of glucosinolate degradation was initiated by Youngs and Wetter (1967). Improvements and modifications of this method have facilitated the analysis of additional glucosinolate degradation products (Daxenbichler et al., 1970; Daun and Hougen, 1977; Daxenbichler and VanEtten, 1977). Similar GC methods continue to be used (Cole, 1976; Louda and Rodman, 1983; Itoh et al., 1984; Al-Shehbaz and Al-Shammary, 1987; Finnigan et al., 1989). These methods were developed primarily as indirect measures of glucosinolate content in Brassica tissues. Because methodology for direct quantitation of glucosinolates is now possible, much of the impetus for further development of methods to quantify degradation products has been eliminated. A comprehensive method to measure the numerous glucosinolate degra-
* Author t o whom correspondence should be addressed [telephone (208) 885-6315; fax (208) 885-7760; e-mail
[email protected]. 0021-8561/94/1442-2029$04.50/0
dation products, such as isothiocyanate (ITC), nitriles, and 5-vinyl-2-oxazolidinethione (OZT), is lacking: Previous soil analysis has been limited mainly to methyl ITC (not common in natural sources) because of its direct application in soil fumigants or indirect application through a methyl dithiocarbamate precursor. In addition, capillary GC was used to quantify benzyl ITC in root exudates from papaya (Tang and Takenaka, 1983) and allyl ITC extracted from a simplified solid matrix (Choesin and Boerner, 1991) as well as soil (Williams et al., 1993). Our goal was to develop an improved and comprehensive method for capillary GC of glucosinolate degradation products in soils amended with Brassica tissues. MATERIALS AND METHODS Soils. Prior to the addition of any amendments,soils (Table 1)were air-dried, crushed to pass through a 2-mm sieve, and characterized accordmg to methods previously described (Brown and Morra, 1993). Particle size for Avonville and Trout Creek soils was determined using the pipet method (Gee and Bauder, 1986). Instrumentation. Sample analysis was conducted using a Hewlett-Packard 5890 Series I1 gas chromatograph, He as a carrier gas, flame ionization detector (FID), and flame
photometric detector (FPD) having a sulfur filter. Columns (30 m x 0.32 mm; J&W Scientific, Folsom, CA) included a DBWax [poly(ethylene glycol); 0.25 pm film], DB-1701 (14% cyanopropylphenyl-substituted methylpolysiloxane; 1.0 ym film), DB-5 (5%phenyl-substitutedmethylpolysiloxane; 0.25 pm film), and DB-5MS (5%phenyl; 0.50 ym film). A similar GC system with a DB-5MS column (0.33ym film), coupled to a Hewlett-Packard598941 quadrupole mass detector,was used for identification of the compounds. Parameters for electron impact sample ionization were as follows: interface temperature, 280 "C; repeller, 7 V, emission, 300 V, electron energy, 70 eV; source temperature, 200 "C. Product Identification. Identification was based on retention time, presence of sulfur as measured by GC-FPD, prediction from glucosinolate precursors present in the plant tissues (Brown et al., 1991), and mass spectral data. Mass spectral data were in general agreement with previously published information (G1035A Wiley Database, Hewlett0 1994 American Chemical Society
2030 J. Agric. Food Chem., Vol. 42, No. 9, 1994
Brown et al.
Table 1. Characteristics of Soils Used in Extraction Experiments soil series Portneuf si1 Latahco si1 Andisol sila Avonville si1 Trout Creek cla a
subgroup Durixerollic Calciorthid Argiaquic Xeric Argialboll Typic Vitricryand Andic Xerumbrept Andic Paleoboralf
pH 7.55 6.10 6.25 5.65 5.90
organic C,gkg 11.6 41.0 15.0 39.0 2.2
total C,@g 15.6 41.0 15.0 39.0 2.2
total N,g/kg 1.7 3.9 1.8 3.9 0.3
clay, gkg 157 159 117 96 698
sand, gkg 48 122 190 265 35
water at -0.033MPa,% 27.5 39.2 58.0 46.8 36.2
Not an official series name
Table 2. Potential Glucosinolate Degradation Products, or Analogues, Examined in Study name abbrev compd source allyl cyanide ACN CHz=CHCHzCN Aldrich, Milwaukee, WI allyl isothiocyanate AITC CHz=CHCHa=C=S Aldrich benzyl isothiocyanate BITC CsH&HzN=C-S Aldrich 3-butenyl isothiocyanate 3BuITC CHz=CHCHzCHzN=C=S plant tissue 5-vinyl-2-oxazolidinethione(goitrin) OZT Lancaser Synthesis, Winham, NH
=
CH?
indole-3-acetonitrile
IAN
methyl isothiocyanate 4-pentenyl isothiocyanate phenethyl isothiocyanate phenyl isothiocyanate propyl isothiocyanate
MITC 4RITC PeITC PITC PrITC
CH
