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    Journal of Chromatography A, 1216 (2009) 45394552

    Contents lists available at ScienceDirect

    Journal of Chromatography A

    journa l homepage: www.e lsev ier .com/ locate /chroma

    ethod validation and comparison of acetonitrile and acetone extraction forhe analysis of 169 pesticides in soya grain by liquid chromatographytandem

    ass spectrometry

    onara R. Pizzutti a,, Andr de Kokb, Maurice Hiemstrab, Cristine Wickertc, Osmar D. Prestesc

    Federal University of Santa Maria, Chemistry Department, Center of Research and Analysis of Residues and Contaminants (CEPARC), Santa Maria, RS, BrazilVWA Food and Consumer Product Safety Authority, Chemistry Laboratory, R&D Group, National Reference Laboratory (NRL) for Pesticide and Mycotoxin Analysis in Food,msterdam, The NetherlandsFederal University of Santa Maria, Chemistry Department, Laboratory of Pesticide Residue Analysis (LARP), Santa Maria, RS, Brazil

    r t i c l e i n f o

    rticle history:eceived 20 October 2008eceived in revised form 23 March 2009ccepted 24 March 2009vailable online 28 March 2009

    eywords:esticidesoya grainulti-residue method


    a b s t r a c t

    An acetonitrile-based extraction method for the analysis of 169 pesticides in soya grain, using liquidchromatographytandem mass spectrometry (LCMS/MS) in the positive and negative electrospray ion-ization (ESI) mode, has been optimized and validated. This method has been compared with our earlierpublished acetone-based extraction method, as part of a comprehensive study of both extraction meth-ods, in combination with various gas chromatography(tandem) mass spectrometry [GCMS(/MS)] andLCMS/MS techniques, using different detection modes. Linearity of calibration curves, instrument lim-its of detection (LODs) and matrix effects were evaluated by preparing standards in solvent and in thetwo soya matrix extracts from acetone and acetonitrile extractions, at seven levels, with six replicateinjections per level. Limits of detection were calculated based on practically realized repeatability rela-tive standard deviations (RSDs), rather than based on (extrapolated) signal/noise ratios. Accuracies (as% recoveries), precision (as repeatability of recovery experiments) and method limits of quantification(LOQs) were compared. The acetonitrile method consists of the extraction of a 2-g sample with 20 mL ofacetonitrile (containing 1% acetic acid), followed by a partitioning step with magnesium sulphate and asubsequent buffering step with sodium acetate. After mixing an aliquot with methanol, the extract can beinjected directly into the LCMS/MS system, without any cleanup. Cleanup hardly improved selectivityand appeared to have minor changes of the matrix effect, as was earlier noticed for the acetone method.Good linearity of the calibration curves was obtained over the range from 0.1 or 0.25 to 10 ng mL1, withr2 0.99. Instrument LOD values generally varied from 0.1 to 0.25 ng mL1, for both methods. Matrix

    effects were not significant or negligible for nearly all pesticides. Recoveries were in the range 70120%,with RSD 20%. If not, they were still mostly in the 5070% range, with good precision (RSD 20%). Themethod LOQ values were most often 10 g kg1 for almost all pesticides, with good repeatability RSDs.Apart from some minor pros and cons, both compared methods are fast, efficient and robust, with good

    he twd 20method performances. Tduring surveys in 2007 an

    . Introduction

    Nowadays, consumers tend to change their food consumptionabits towards a preference for healthy fruits and vegetables pro-iding also the right nutritional value. The benefits of soybean

    Glycine max (L.) Merrill] and the importance to include this legumend its products in the daily diet have been very well known by thehinese people since more than 2000 years B.C. In the beginningf the 20th century, the main properties of soya, the high content

    Corresponding author. Tel.: +5555 3220 9458; fax: +5555 3220 9458.E-mail address: (I.R. Pizzutti).

    021-9673/$ see front matter 2009 Elsevier B.V. All rights reserved.oi:10.1016/j.chroma.2009.03.064o methods were applied successfully in a routine analysis environment,08.

    2009 Elsevier B.V. All rights reserved.

    of oils and proteins, called the attention of the occidental peoplethat started to use this grain not just for consumption, but also forindustrial and technical applications, such as amongst others, in theareas of cosmetics, textiles, inks, disinfectants, fertilizers and bio-fuel. However, the applications of soybean for food and feed are stillthe most important [1].

    Soybean belongs, together with wheat, rice, corn and potatoes,to the five most important food and feed crops in the world. Brazil

    is the second biggest producer of soya grain, but the first exporter.Seventy-five percent of the total Brazilian production of soybean isexported, mainly to China and the European Union [2]. The Nether-lands is the second biggest importer of soya grain in the world, afterChina.

  • 4 atogr. A 1216 (2009) 45394552












    540 I.R. Pizzutti et al. / J. Chrom

    Taking into account the continuous increase in consumption ofll products originating from soybean and other grains, legislationas established recently [3] in order to keep pesticide residues at

    he lowest possible levels, and guarantee food and feed safety. SinceSeptember 2008, a new European Union regulation became into

    orce setting around 145,000 harmonised maximum residue levelsMRLs) for the tens of thousands commoditypesticide combina-ions [4]. Compliance with these MRLs requires an efficient controlia regulatory monitoring programs and enforcement, analysisf raw and processed food and feed [5], risk assessment, field-pplication trials, organic food verification [6] and border controlor international trade of food and feeding stuffs [7].

    The development of sensitive, selective and reproducible analyt-cal methods and techniques has always been a prerequisite for thechievement of high-quality results in enforcement and monitor-ng programmes. Nowadays, other characteristics of an analyticaluantitative method are also requested, such as usage of the small-st possible amount of sample and chemicals, more environmentalriendly approaches, safer and less hazardous chemicals to the ana-ysts, cost-effective, less laborious and faster methods [810].

    Until recently, most improvements in multi-residue methodsor pesticides have been achieved for crops with a high water con-ent, like fruits and vegetables. In the last years, research has nowlso been directed towards analysis of pesticide residues in moreomplex matrices, such as grains and its derived products, andatrices with a high fat and sugar content. These latter matrices

    re now gradually becoming less difficult, thanks to the newest,ore advanced chromatographic detection techniques [1115].Classical analytical methods based on acetone [16], acetonitrile

    17] and ethyl acetate [18] extraction have been continuously opti-ized and modified in such a way to improve considerably their

    erformances and applications for pesticide residues analysis, alsoor the more complex matrices mentioned above.

    Recent versions of the acetone [12,13] and acetonitrile [19]ethods and its many modifications and applications [2024] and

    lso methanol-based [25] and ethyl acetate-based [26] methodsave achieved the state-of-the-art stage of multi-residue methods

    or pesticide residues.As follow-up of our previous study [12] on the development of

    n acetone-based extraction method, this study aimed at the opti-ization and validation of an acetonitrile-based extraction method

    27] for the analysis of 169 pesticides in soya grain by LCMS/MSsing both the positive and negative ESI ionization mode.

    The results for the validation parameters, such as matrix effect,ccuracy, precision, linearity range, detection and quantitation lim-ts, sensitivity and selectivity, were compared for the two methods.

    . Experimental

    In this study, soya extract 1 means the extract obtained byhe acetone extraction [1,12] method (Fig. 1) and soya extract 2

    eans the extract obtained by the acetonitrile extraction methodescribed in Section 2.5 of this paper and also shown schematically

    n Fig. 2. For both methods cited, no clean up procedure was appliedor analysis by LCMS/MS.

    .1. Chemicals and reagents

    Methanol, toluene, acetone, acetonitrile (pesticide grade), andnhydrous magnesium sulphate and sodium acetate (analyticaleagent grade) were purchased from Merck (Darmstadt, Germany).

    esticides reference standards (purity > 97%) were obtained fromr. Ehrenstorfer (Augsburg, Germany); Riedel de Han (Seelze,ermany); Hayashi (Osaka, Japan), Bayer (Leverkusen, Germany);eneca (Wilmington, DE, USA); Janssen (Beerse, Belgium), NovartisBasel, Switzerland), Agrevo (Frankfurt, Germany), Rohm and HaasFig. 1. Scheme of the acetone extraction method for the determination of pesticidesin soya grain.

    (Philadelphia, PA, USA), Cyanamid (Princeton, NJ, USA) and DuPont(Wilmington, DE, USA).

    2.2. Standard solutions

    Standard stock solutions were prepared at 1.00 mg mL1 mainlyin toluene. However, for those compounds with solubility problemsin toluene, methanol or acetone were used. A stock standard mix-ture solution containing all 169 pesticides studied, was preparedat 1 g mL1 of each pe


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