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Sample handling and analysis of allelochemical compounds in plants
E. Eljarrat*
and D. Barceló
I.I.Q.A.B., C.S.I.C., Environmental
Chemistry Department, Jordi Girona 18¯26, 08034 Barcelona, Spain
Available online 22 October 2001.
An overview is presented of current methodologies for the determination of allelochemical compounds, and especially the chemical family of benzoxazinones. These compounds are relevant since they are potential substitutes for pesticides in crop protection. Analysis of allelochemicals is usually carried out by LC, LC¯MS, LC¯MS¯MS, CE, GC¯MS or GC¯MS¯MS. The advantages and disadvantages of the various chromatographic techniques will be discussed. Since allelochemicals are generally found in plants, foliage and roots, sample preparation strategies are reviewed. This is a critical step since some of these compounds can be hydrolysed during sample preparation and consequently relevant environmental information can be lost.
Author Keywords: Allelochemical; Benzoxazinone;
Benzoxazolinone
Allelopathy has been defined by the International Allelopathy Society as `any process involving secondary metabolites (allelochemicals) produced by plants, micro-organisms, viruses and fungi that influence the growth and development of agricultural and biological systems (excluding animals), including positive and negative effects' [1]. Allelochemicals from plants are released into the environment by exudation from roots, leaching from stems and leaves, or when the plant material is decomposed. In recent years there has been an increasing focus on the prospects of exploiting allelopathy as an alternative strategy for controlling weeds in particular, but also insects and diseases [2, 3]. Weeds can be controlled either by growing a crop with the ability to exude allelochemicals or by incorporating plant residues with a high content of allelochemicals into the soil.
A number of chemical classes, such as tannins, cyanogenic glycosides, several
flavonoids, and phenolic acids present allelopathic activity. However, the
chemicals identified as the most active allelopathic compounds in different
crops such as wheat, rye or maize are of the same chemical family, the
benzoxazinones [4].
The first discovery of benzoxazolin-2(3H)-one (BOA) in plants was in rye,
reported in 1955 [5].
Shortly after, the methoxy derivative, 6-methoxybenzoxazolin-2(3H)-one
(MBOA), was isolated from wheat and maize [6].
In 1959 it was shown that these compounds do not occur in plants but are
degradation products of 2,4-dihydroxy-1,4-benzoxazin-3(4H)-one, its
7-methoxy derivative, and their corresponding glucosides [7,
8].
Today, a broad variety of benzoxazinones is known. The main structures are shown
in Fig.
1. Hydroxamic acids are found as 
-glucosides [7].
When plant tissues are damaged, -glucosides are enzymatically hydrolysed
to their corresponding aglucones [9].
The aglucones are converted into their corresponding benzoxazolinones, MBOA and
BOA, when heated in aqueous solutions [10,
11]
(Fig.
2).
Fig. 1.
Benzoxazinone derivatives present in plants.
(2K)
Fig. 2.
Formation of benzoxazolinones from the
The qualitative and quantitative analysis of the various allelochemicals is of interest in relation to several problems in plant physiology and pathology. Also, the analysis of these compounds is of interest since discussions are taking place on their possible use as substitutes for pesticides in crop protection. An overview of the current analytical methods for the determination of benzoxazinones is presented here, and various aspects such as sample preparation, extraction, purification, and final determination will be reviewed.
As none of the hydroxamic acids or aglucones is commercially available it is
necessary to isolate all reference substances from natural sources. The
methoxylated benzoxazinoids, DIMBOA¯Glc, HDMBOA¯Glc,
HMBOA¯Glc, DIMBOA and HMBOA could be isolated from young corn (Zea
mays) plants [7],
while DIBOA¯Glc, DIBOA and HBOA could be obtained from Aphelandra
tetragona roots [12].
MBOA and BOA could be obtained by heating aqueous solutions of DIMBOA and DIBOA,
respectively, at 70°C for 1 h [13].
An overview of the chemical synthesis of these compounds was published recently
[14].
Basically, the isolation procedure involves the following steps: extraction with
ethyl acetate, separation of the organic phase, and further purification with a
silica gel column to obtain aglucones, extraction of the aqueous phase with
n-BuOH, and further purification on a Sephadex column to obtain -glucosides.
Finally, heat treatment of the aglucones produces the benzoxazolinones (see Fig.
3).
Fig. 3.
Isolation of aglucones,
Allelochemicals can be present in several parts of plants including roots, rhizomes, leaves, stems, pollen, seeds and flowers. Most of the published studies of benzoxazinones and related compounds have been carried out on roots, seeds, and leaves of different plants.
As the hydroxamic acid glucosides are very sensitive to enzymatic deglucosylation followed by chemical degradation, careful sample preparation is of major importance. Several research papers describe the decomposition of the hydroxamic acids by enzymatic and/or thermal degradation prior to the extraction [15, 16]. This does not allow any differentiation between glucosides and aglucones. In order to avoid these problems, Baumeler et al. [12] froze the samples under liquid nitrogen immediately after harvesting and performed all further manipulations (storing, maceration, weighing) at 0°C. The frozen powder (an amount of 0.1¯0.5 g) is extracted with methanol at 20°C. The extracts are evaporated and diluted with water.
When the sample preparation is performed carefully the direct analysis of the hydroxamic acids and their glucosides should allow an insight into the distribution of the benzoxazinoids in plants. The results recently obtained by Cambier et al. [17] showed that in uninjured plants only the glucosylated forms of DIMBOA and related compounds were present, and aglucones were only detected when enzymatic deglucosylation occurred during sample work-up.
As an example of the importance of the direct analysis of the glucosylated forms, Grambow et al. [18] have shown that HDMBOA degraded very rapidly into MBOA, the same degradation product as from DIMBOA. Nevertheless, some researchers consider MBOA to result exclusively from the DIMBOA degradation and continue to quantify DIMBOA¯Glc by the sum of the measured quantities of DIMBOA and MBOA. This method can lead to significant errors when the concentration of HDMBOA¯Glc in the plant is high.
In the raw extracts of plants, the broad variety of substances (salts, lipids, glycosides, phosphates, peptides, macromolecules, chlorophyll) can influence the quantifications. This fact was neglected in all cited publications where the raw extracts were analysed directly. However, Baumeler et al. [12] observed that some peaks (according to their UV spectra, probably flavonoids or flavonoid glycosides) eluted with retention times identical to the benzoxazinoids. Thus, a purification of the samples prior to instrumental analysis is recommended. A simple pre-purification procedure consists in the use of a Sep-Pak® C18 solid-phase extraction cartridge.
Several methods for the qualitative and quantitative analysis of benzoxazinones and related compounds have been developed. The simplest method is to measure the total hydroxamic acid content by measuring the absorbance of the blue complex formed between hydroxamic acids and FeCl3 [19, 20], but this can only estimate total cyclic hydroxamic acids because FeCl3 does not react with benzoxazolinones. Another method is based on the assumption that 1 mol of the hydroxamic acid (DIMBOA) produces 1 mol of the benzoxazolinone (MBOA) after degradation, and measures the hydroxamic acid content as benzoxazolinones. The benzoxazolinones were quantified by isotopic dilution [21], infrared spectrophotometry [22], fluorometry [23], gas chromatography (GC) [24] and thin-layer chromatography [25]. However, it has been found that the amount of MBOA formed from DIMBOA varies with temperature, pH and composition of the reaction medium and always yields less than 75% MBOA from DIMBOA [13]. Therefore, these methods can lead to erroneous estimates of DIMBOA content in plant extracts. In order to obtain a more complete picture on the distribution and exact composition of all benzoxazinoids, new methods for their separation and quantification were sought. An overview of the various chromatographic methods for determination of allelochemicals is shown in Table 1.
Table 1.
Chromatographic analysis of allelochemicals in plants
(17K)
During the 1980s and 1990s, several procedures were developed for the
separation and quantification of cyclic hydroxamic acids and their degradation
products in plant extracts using liquid chromatography (LC) [15,
16,
17,
26,
27,
28,
29,
30,
31,
32].
UV detection at 288, 280, 263 or 254 nm was most commonly used. It should be
pointed out that the identification is less sensitive when samples are analysed
at 263 nm because MBOA has a minimum at this absorbance. DIM2BOA,
HMBOA and DIMBOA have similar but distinct UV spectra, with absorbance maxima at
wavelengths of 264, 261 (shoulder 289 nm) and 264 nm (shoulder 292 nm),
respectively. MBOA has two absorbance peaks with maxima of 230 and 292 nm.
Mayoral et al. [15]
used a LC¯DAD since some confirmation is possible through the UV
spectrum. Because of the differences in the UV absorption spectra of the
analytes, a wavelength of 288 nm was chosen to allow their joint determination
with the best quantitative results. Xie et al. [16]
also used a DAD with scanning range between 190 and 400 nm, and the limits of
detection of their method were 1 
g/ml for DIM2BOA, DIMBOA and
MBOA, and 0.5 g/ml for HMBOA. These detection limits are considered acceptable when
compared with the levels normally found in the literature. The relative levels
of benzoxazinones and related compounds vary between species (Table
2), but a range between 10 g/g and 100 mg/g is usually found.
Table 2.
Levels of some allelochemicals in different plants
The LC¯UV method can lead to an erroneous determination of allelochemical contents in plant extracts because of possible co-elution of several compounds and it can only be used when pure reference compounds are available. To overcome the LC¯UV limitations, the use of LC¯MS has clear advantages. Various interfaces are currently available for LC¯MS experiments. The main advantages of atmospheric pressure interfaces are the higher sensitivity (especially when using atmospheric pressure chemical ionisation, APCI), robustness, and ease of use. This approach, which allowed the unequivocal identification of allelochemical compounds, was recently used by Cambier et al. [31] for determining DIMBOA and related compounds in maize. Spectra were acquired in the negative-ion mode. LC¯MS allowed the determination of, e.g., DIMBOA¯Glc and DIM2BOA¯Glc. The peak of deprotonated DIMBOA¯Glc (m/z=372) and the peak of deprotonated DIM2BOA¯Glc (m/z=402) were the base peaks of these two compounds. Cambier et al. [17] also developed a LC¯APCI¯tandem mass spectrometry (APCI¯MS¯MS) method for the confirmation of several rarely described benzoxazinones (i.e., HM2BOA¯Glc, DIM2BOA¯Glc and HDMBOA¯Glc) and for the identification of a new compound, HDM2BOA¯Glc. These compounds are probably present in the majority of plants studied, but were not characterised by the analytical methods used. Fig. 4 shows the mass spectrum of an unknown chromatographic peak, with two predominant ions (m/z=386 and 476), as well as the further fragmentation spectrum of the peak at m/z=386. The information obtained from these spectra allowed the identification of the unknown peak as HDM2BOA¯Glc.
Fig. 4.
(a) Mass spectrum of HDM2BOA¯Glc, and (b) fragmentation spectrum of the peak at m/z=386. Reprinted from [17] with permission from John Wiley and Sons.
For the chromatographic separation of the analytes, various chromatographic
columns and conditions have been reported. The first studies were carried out
using silica-based columns [26,
30].
Mayoral et al. [15]
used a poly(styrene¯divinylbenzene) resin because this stationary
phase is more stable at any pH than the silica-based packing materials.
Recently, Baumeler et al. [12]
developed a method that allows good separation of the hydroxamic acids, DIBOA
and DIMBOA, their glucosides DIBOA¯Glc and DIMBOA¯Glc,
their precursors HBOA and HMBOA, HMBOA¯Glc and the benzoxazolinones
BOA and MBOA, using a reversed-phase Ultrasphere ODS 5-m column
(C18) (Fig.
5). A number of authors [17,
28,
29,
31,
33,
34]
also used reversed-phase 5-m columns (C18) for
chromatographic separation. They worked with a two-solvent system to generate
the mobile phase: solvent A was acetic acid or H3PO4 in
water and solvent B was 100% methanol. However, Baumeler et al. [12]
found that the solvent system that gave the best results was H2O as
solvent A, and MeOH/iso-PrOH (95/5)+0.025% HOAc as solvent B. The main
differences from previously published methods are the replacement of methanol by
a higher alcohol in solvent B, and the addition of the acid to the alcoholic
solvent B.
Fig. 5.
HPLC separation of hydroxamic acids, their glucosides, their precursors and benzoxazolinones. Key to peak identity: 1, DIBOA¯Glc; 2, DIMBOA¯Glc; 3, DIBOA; 4, DIMBOA; 5, BOA; 7, HDMBOA¯Glc; 10, HMBOA¯Glc; 11, HBOA; 12, HMBOA. Reprinted from [12] with permission from Elsevier Science.
Lippmann et al. [35]
and Thunecke et al. [36]
studied the enantiomeric separation of benzoxazinones. Lippmann et al. [35]
developed a LC method for the separation of enantiomers of some cyclic
hydroxamic acids on a -cyclodextrin-modified chiral stationary phase. However, the system
does not allow the separation of DIBOA and DIMBOA because of a rapid
oxo¯cyclo-tautomerisation of these compounds. Thunecke et al. [36]
used capillary electrophoresis (CE) for the enantioseparation of
DIBOA¯Glc and DIMBOA¯Glc using borate buffers (pH
9¯10 range) and cyclodextrins as chiral additives. The CE proved to
be superior to LC, providing greater separation efficiency.
Gas chromatography coupled with mass spectrometry (GC¯MS) is regarded as a powerful analytical tool for the characterisation of complex organic mixtures. This technique has also been reported by some authors for the determination of allelochemical compounds [37, 38, 39]. This approach has the advantage of high sensitivity and selectivity, and the existence of mass spectrum libraries for screening of unknown samples, but derivatisation is required prior to analysis. Various derivatisation reagents have been reported to convert analytes into volatile compounds, e.g., bis(trimethylsilyl)trifluoroacetamide with 1% trimethylchlorosilane [38], or N-methyl-N-(trimethylsilyl)trifluoroacetamide [39]. Thus, the trimethylsilyl derivatives are analysed. The MS detection is performed in the electron ionisation mode.
Another group of chemicals that present allelopathic activity is the family of phenolic acids. p-Hydroxybenzoic, vanillic, p-coumaric, syringic and ferulic acids have been regarded as some of the major phenolic acids predominantly identified in wheat [40, 41]. These compounds were also identified in vulpia (Vulpia myuros) [42]. Although phenolic acids and cyclic hydroxamic acids have been reported in relation to allelopathies, no attempt has been made to simultaneously determine these two distinct groups of compounds. Recently, Wu et al. [39] reported a procedure using GC and tandem mass spectrometry (GC¯MS¯MS) for the simultaneous determination of some phenolic acids and one of the hydroxamic acids (DIMBOA) in wheat samples. They used an ion-trap detector to perform MS¯MS analyses. Compared to single-stage MS, MS¯MS enhances instrumental selectivity and sensitivity. Because of the complexity of the sample matrix studied, GC¯MS analysis shows the co-elution of some phenolic acids and DIMBOA with background substances. The tandem mass spectrometry technique was introduced in order to filter out this unwanted chemical background. The signal to noise ratios for all the analytes were much lower in GC¯MS than in GC¯MS¯MS, i.e., the ratio for p-chlorobenzoic acid was 50 times higher under MS¯MS conditions than under MS conditions.
The literature shows that the type of detected benzoxazinoids depends
strongly on the working-up procedure of the plant material. The isolation of the
glucosides involves the inactivation of the hydrolytic enzymes present in the
plant tissues before extraction, since in damaged plant tissues a -glucosidase
converts them into aglucones. Therefore a main source of error appeared in the
sample preparation procedure used.
Two different approaches for the separation of different hydroxamic acids and related compounds, GC and LC, have been reported. However, most of the work reported in the literature used the LC system because this procedure does not require the derivatisation step that is needed prior to the GC analyses. Recently, some authors have used LC¯APCI¯MS, because of its high sensitivity.
It should be pointed out that less purification of the raw extracts is
required when MS detectors are used. When analyses are carried out by
LC¯UV systems, the clean-up step is highly recommended to avoid
overlapping between analytes and interferences.
This work was financially supported by the programme `Quality of life and
management of living resources (1998 to 2002)' of the European Union,
FATEALLCHEM contract no. QLRT-2000-01967.
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*Corresponding
author. Tel.: +34 (93) 400-6100; Fax: +34 (93) 204-5904; E-mail: eeeqam@cid.csic.es
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