Elsevier

Talanta

Volume 144, 1 November 2015, Pages 375-381
Talanta

Simultaneous enantiomeric determinations of acid and ester imidazolinone herbicides in a soil sample by two-dimensional direct chiral liquid chromatography

https://doi.org/10.1016/j.talanta.2015.06.062Get rights and content

Highlights

  • A two-dimensional LC–LC chiral method allows acid and ester IMI enantiomeric determination.

  • Multiple response factorial design makes it easy to get optimum values of the principal variables.

  • The mobile phase in the achiral column and the mobile phase in the chiral column are compatibilized.

  • The direct chiral method allows determination of IMIs and their enantiomeric ratios in a soil sample.

Abstract

A two-dimensional HPLC method for the simultaneous direct chiral enantiomeric determination of acid and ester IMI herbicides has been described. Difficulties arising from differences in polarity were overcome. Firstly, the imazaphyr, imazethapyr and imazamethabenz methyl herbicides were separated in a C18 achiral column. Then, their respective enantiomers were separated using a protein chiral AGPTM column; a heart-cut mode was used. Mobile phases of the two systems were compatibilized, after optimizing by factorial design using multiple response analysis. The proposed method has been validated by recovery studies from an enriched soil sample. Important enantiomer parameters such as enantioresolution higher than 1.12, enantiomeric ratio (ER) close to 1 and enantiomeric fraction (EF) around 0.5 were obtained for standards, confirming that herbicides are present as racemates.

Introduction

At present, about 25% of pesticides are chiral; i.e., they exist as enantiomers. It is known that the chemical properties of enantiomers are alike but they often differ in their toxicity and biological activity [1]; the active enantiomer should have the desired effects on a target species, while the inactive one may have adverse effects on some non-target species. Imazapyr (IM), imazethapyr (IMP) and imazamethabenz-methyl (IMBM) herbicides are chiral members of the imidazolinone family (IMIs), which are used in modern agriculture and are applied both via foliage and through the soil. The imidazolinone structure shows a stereogenic center in the imidazolinone ring, which is a ramified chain amino acid inhibitor [2], [3]. The inhibitor power of the R(−)-IMP enantiomer to acetohydroxy acid synthase was found to be 10-fold that of the S(+)-enantiomer [4], [5], [6] and their persistence in agricultural soil is high, affecting crop rotations [4], [7], [8], [9]. Therefore, in order to reduce the amount of herbicides used and prevent unnecessary enantiomer waste, which has an adverse impact, several European countries have suggested that only the active enantiomer should be employed [10], [11]; Sweden has implemented a tax on agrochemicals based on the weight of the active ingredient and Netherlands and Switzerland have revoked registration for racemic mixtures of chiral phenoxyalkanoic acids, while approving the registration of single-isomer products; however, few single-enantiomer pesticides are synthesized or produced [12].

Degradation of IMIs in soil depends on this pH; wet and alkaline media allow a significant biological breakdown of IMIs, while dry and acid ones bind them strongly to the soil, limiting their mobility and slowing down their biological degradation; for example, the p-IMBM enantiomers are more stable in the environment than the m-IMBMs and, therefore, they are present in greater proportion [8]. Consequently, there is an urgent need to develop analytical methods to determine the stereoselectivity, bioactivity and environmental behavior of these chiral pesticides [10].

HPLC is one of the most powerful techniques for enantiomer analysis; historically, it has been the standard technique and the first choice for chiral analysis because a wide variety of chiral stationary phases (CSPs) are available. The separation of enantiomers by HPLC using CSPs is based on the formation of transient diastereomeric complexes between the enantiomorphs of the solute and a chiral selector which is an integral part of the stationary phase. The difference in stability between these complexes leads to a difference in retention time; the enantiomer that forms the less stable complex will be eluted first. These diastereomeric adsorbates must, therefore, differ adequately in free energy for enantiomer separation to be observed [13].

In general terms, LC stationary phases are quite specific, making analysis difficult when herbicides of the same family have a different polarity, as is the case when acid and neutral groups are present.

For acid IMP and IM enantiomer separation, some stationary phases have been used, the normal phase mode was successfully applied for chiral analysis of these herbicides in spiked soil samples; however, there is no data regarding simultaneous acid and ester IMI enantiomer separation (Table 1). Furthermore, available information about IMBM enantiomer analysis refers only to the separation of the p- and m- isomers [5], [6], [8], [15] or to their corresponding four enantiomers, which has been achieved recently with acceptable resolution using a protein-based α-acid glycoprotein (AGP) stationary phase [21] by HPLC in reverse phase., The mechanism of chiral recognition by proteins is largely unknown because of their complex structures. The solute is retained on this type of chiral stationary phase mainly by combinations of ionic bonding, hydrophobic interaction, hydrogen bonding, and charge transfer interaction. In fact, the general advantages of protein-based CSPs include the use of an aqueous mobile phase [23].

Given that AGP is able to bind protein for neutral, acid and basic drugs as well as binding a variety of hydrophobic compounds due to interactions with an apolar cavity formed by the folding of the secondary structure of AGP [24], it should be possible for AGP to allow the simultaneous separation of both neutral and acid IMIs.

Direct chiral analysis of more than one enantiomer pair with very different polarities continues to present limitations. The low load capacity and the high cost of chiral columns [25] should ideally require the injection of a very clean sample or pure enantiomer pairs, which can be done by two-dimensional HPLC using a C18 column to separate individual enantiomer pairs in the first dimension and a chiral column in the second dimension. Careful selection of the experimental conditions is required for this technique; advantages include online sample clean-up and increased selectivity. Different variables are involved in optimization, so experimental design may be useful [26].

Taking into account the need for a clean sample in direct chiral analysis and the difficulty in simultaneously separating nonpolar and polar IMI enantiomers, a two- dimensional LC–LC chiral method has been described in this paper, which allows the simultaneous determination of three IMI enantiomer pairs; two acids and one ester. Firstly, the IM, IMP and IMBM herbicides were separated in a C18 achiral column. The herbicide peaks were transferred to the chiral system through a valve by the “heart-cut” mode, and then, their respective enantiomers were separated using a protein chiral AGPTM column. The most critical variables for achieving enantiomeric separation were optimized by multiple response analysis in both achiral and chiral chromatographic systems in order to determine the enantiomeric ratios of the IMIs in a soil sample. Particular attention was paid to the compatibilization of the mobile phase for achiral analysis with the mobile phase for chiral analysis.

Section snippets

Standards and reagents

Imazapyr, Imazethapyr and Imazamethabenz-methyl (99%) herbicides were purchased from Cymit Chemica (Germany). All organic solvents were HPLC grade; methanol (MeOH) and diethyl ether (Et2O) from Romil (Teknokroma, Spain) and 2-propanol (2-PrOH), acetonitrile (ACN) and dichloromethane (DCM) from Scharlau (Spain) were purchased. Anhydrous acetic acid (HAc) (99%) and ammonium acetate (NH4Ac) were obtained from Sigma-Aldrich (Germany), formic acid (HCOOH) (98–100%) from Scharlau (Spain) and ammonium

Results and discussions

The simultaneous separation of the three IMI enantiomer pairs was attempted using the AGP column but it was not possible using ACN or using MeOH or 2-PrOH as modifiers in isocratic mode in a great variety of experimental conditions. The two enantiomer pairs of the first two herbicides (IM and IMP) were resolved in isocratic mode when a small concentration of modifier, about 2% of MeOH, was used. However, the enantiomer pair from the third neutral herbicide was strongly retained due to their

Conclusions

The AGP chiral column has the advantage that it allows the separation of the three IMI enantiomer pairs; this has not been achieved according to the literature.

The two-dimensional method allows the simultaneous determination of three enantiomer pairs, two acids and one ester, which have quite different polarities. This was not possible directly on the protein αAGP chiral column using neither ACN nor MeOH nor 2-PrOH as modifiers. However, the use of an LC–LC two-dimensional achiral–chiral system

Acknowledgments

The present work has received financial support from Project 152GR10581, supported by Complutense University of Madrid (Spain).

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