Pesticides, HPLC analysis

Shibata Y, Oyama M, Sato H, et al. 1998. Simultaneous cleanup method for multi pesticide residue analysis by GC and HPLC. J Food Hyg Soc Jpn 39(4) 241-250. 

Acetone, n-hexane, acetonitrile, ethyl acetate, pesticide residue analysis grade Aluminum oxide, Aluminumoxid 90, activity 11-111, 70-230 mesh MSTM (Merck) Anhydrous sodium sulfate, sodium chloride, special grade Distilled water, HPLC grade... 

The most common and diverse approach to cleanup (and extraction of water samples) in pesticide residue analysis is SPE. Over the last 20 years, improvements and diversifications in SPE formats, sorbent types, and apparatus have made SPE a widely used approach for a variety of applications, including the analysis of pesticide residues. SPE cartridges or disks can be likened to low-resolution HPLC columns in that similar stationary and mobile phases are used. A typical particle size in SPE is 40 pm, and the plastic cartridges are generally packed with 0.1-1 g of sorbent in plastic tubes. The choice of reversed-phase, normal-phase, and ion-exchange media in SPE is very diverse, and Table 2 lists some of the more popular SPE applications for the cleanup of pesticides. 

Acetone, acetonitrile, dichloromethane, ethyl toluene pesticide residue analysis grade Distilled water HPLC grade Sodium chloride special grade... 

You have the task of purchasing some n-hexane for use in three different applications (i) pesticide analysis by gas chromatography, (ii) as a solvent to extract some non-polar high-boiling (200-300°C) oils from a soil sample, and (iii) as a mobile phase for HPLC analysis with UV detection. List and contrast the performance characteristics you need to take into account for purchasing the appropriate grade of hexane in each case. n-Hexane boils at about 70°C. Will any of your choices of hexane be suitable for use for HPLC analysis with fluorescence detection Explain your decision. 

Tables 8 and 9 summarize the separation conditions used in HPLC analysis of carbamate and urea pesticides.

Carbamate pesticides can be determined using different detectors in GC or HPLC analysis. A characteristic feature of a carbamate molecule is the nitrogen atom, which can form the bases for quantitation and some carbamates also contain chlorine, sulfur, or other heteroatoms in the molecule. This allows the use of various detection techniques for their determination (139,140), such as electrical conductivity (165), alkali flame (141) photometry, and mass spectrometry (44,166). 

Direct determination of urea pesticides by high-performance liquid chromatography has been widely reported in the literature (10,32-36,127-130). Ultraviolet detection has often been used (32,33,35,36,60,127) with usually acceptable sensitivity, although this detector is nonspecific and the sensibility is, in general, low. To overcome this problem, several techniques have been assayed, such as precolumn enrichment (60), postcolumn derivatization (34,10), and the use of other detection techniques such as the electrochemical (129), photoconductivity (128,130), and fluorescence detectors (9,10,34). Table 9 summarizes representative papers using these techniques in HPLC analysis. 

High-performance LC determination is also compatible with the extraction of environmental water with an organic solvent such as methylene chloride or ethyl acetate (31,32) and solid-phase extraction (SPE) (33,34). Solid-phase extraction and liquid-liquid extraction (LLE) have been compared with respect to their ability to preconcentrate pesticides prior to HPLC analysis. The reproducibility of the method is better when C,8 cartridges are used than with conventional LLE, but LLE sometimes gives better recoveries, for example, for dimethoate, chlor-pyriphos ethyl, and carbofenothion (35). 

High-performance LC methods for pesticide residue analysis were first developed for nonvolatile or thermally labile compounds, such as carbamate insecticides. Because HPLC offers a simpler and/or faster approach to analysis for a wide range of other compounds, it is becoming more and more widely accepted, and its applications are steadily increasing in number. Although HPLC has been used in the analysis of OCPs and OPPs, the literature on its application in food is scarce. The methods reported have been summarized in Table 4. 

The most commonly used detector for pesticide residue analysis by LC is UV-VIS. It includes fixed wavelength and variable wavelength. Most OCPs and OPPs absorb appreciably only at wavelengths below 250 nm, the same spectral region where many solvents, solvent impurities, and matrix-derived interferences absorb. Analysis of these compounds by HPLC is still possible with very clean environmental substrates such as water. 

E. A. Hogendoorn, C. E. Goewie and P. van Zoonen, Application of HPLC column switching in pesticide residue analysis, Fresenius , J. Anal. Chem. 339 348-356 (1991). 

On-Line Systems Flowing MMLLE systems have been established in different layouts with automation and on-line hyphenation to GC and HPLC analysis. An automated on-line FS-MMLLE-GC system with a loop-type interface compatible with LVI was used for the extraction of pesticides and PAHs in surface waters.86 In another study, pressurized hot water extraction (PH WE) was coupled on-line to a FS-MMLLE-GC-FID system and applied to the analysis of PAHs in soil, where MMLLE was used as a cleanup and concentration step of the PH WE extract prior to final GC analysis.87 In addition, an HF-MMLLE setup was incorporated in PHWE and GC, resulting in an online PHWE-HF-MMLLE-GC system, where the HF membrane module contained 10-100 HFs. The system served for the extraction and analysis of PAHs in soil and sediments ... 

Aulakh, J.S., A.K. Mailk, V. Kaur, et al. 2005. A review on solid phase micro extraction—High performance liquid chromatography (SPME-HPLC) analysis of pesticides. Crit. Rev. Anal. Chem. 35 71-85. 

A Critical Comparison of Pre-Column and Post-Column Fluorogenic Labeling for the HPLC Analysis of Pesticide Residues... 

Yao S, Meyer A, Henze G. 1991. Comparison of amperometric and UV-spectrophotometric monitoring in the HPLC analysis of pesticides. Fresenius J Anal Chem 339 207-211. 

High-pressure pumps are required to force solvent through a tightly packed HPLC column, and electronic detectors are used for monitoring the appearance of material eluting from the column. Figure 12.17 shows the results of HPLC analysis of a mixture of 14 common pesticides, using coated silica microspheres as the stationary phase and acetonitrile/watei as the mobile phase. 

The HPLC analysis of a mixture of 14 agricultural pesticides. The structures of the pesticides can be found in the Merck Index. 

Each laboratory prepared separate calibrants according with their own laboratory procedure. These were used for calibrating the detector within its dynamic range. Calibrants were prepared, avoiding serial dilution. A minimum of five calibration points were required. The coordinator supplied the participants with pure pesticides samples with certified purity. The participants were requested to use these calibrants or to check their own calibrants. For GC and HPLC analysis at least one internal standard was used for the final determination. 

Figure 7.10. HPLC analysis of pesticides using photodiode array detection. Levels of pesticides are 100-2,000 parts-per-trillion preconcentrated by solid-phase extraction. Chromatogram courtesy of PerkinElmer, Inc.

This table illustrates one of the major impediments to the rapid assimilation of immunochemical technology into pesticide residue analysis labs. Because of the amount and variety of work involved, new method development costs may be high when compared to routine chromatographic methods. However, the low cost per run allows for rapid recovery of the initial investment with sufficiently high sample loads. For example, the cost of reagents and supplies for an ELISA for diflubenzuron was estimated to be 0.20/sample as compared with 4 for HPLC or 11 for GC (35). In addition to the lower reagent and supply costs, the major economic advantage of immunoassay is the dramatic decrease in labor costs. 

Most of the CMs are phenyl A -methyl esters of car-bamic acid. On hydroly.sis or metabolism, CMs produce phenols. Phenols are polar compounds and thus have to be alkylated before GC determination with either ECD or NPD. HPLC analysis combined with the appropriate detector appears to be the better choice. Aprea et al. (2002) reviewed various methods for unchanged CMs in blood and urine, as well methods for specific CM metabolites, such as benomyl metabolites earbcndazlm and methyl-5-hydroxy-2-benzaimidazolecarbamate 1-napthoI 2-isopropoxyphenoI (metabolite of propoxur) and carbo-furan phenol, which is a metabolite of several pesticides (carbofuran, benfuracarb, carbosulfan, and furathiocarb). 

In addition to laboratoiy glassware and equipment necessary fOT cleanup of the extract, traditional pesticide residue methods require expensive chromatogrsqihic instrumentation for identification and quantitation of residues. EIA methods require minimal amounts of glassware, disposable plasticware, or other supplies. Quantitative EIAs often make use of 96-well microtiter plates fOT multiple simultaneous assays of about a dozen extracts and associated reference standard. Major equipment consists of a plate reader, which automatically measures the absorbance of each well. Plate readers can be used alone or in conjunction with a personal computer, which can correlate replicate measurements, construct the calibration curve, calculate results, and provide a complete statistical analysis. Such an EIA workstation can be obtained for roughly half the cost of the GC or HPLC system typically used for pesticide residue analysis. 

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