Residue analysis Solid Phase Extraction

In our laboratory, we also prefer to omit derivatization when confirming the results of analysis by HPLC/DAD. For the the latter method of analysis, solid phase extraction as described in Chapter 7 is carried out and the residue is taken up in 100 pi methanol. Of the remainder of the methanol extract for HPLC analysis, 1 pi is injected into the GC/MS. 

Analytical methods for parent chloroacetanilide herbicides in soil typically involve extraction of the soil with solvent, followed by solid-phase extraction (SPE), and analysis by gas chromatography/electron capture detection (GC/ECD) or gas chromatog-raphy/mass spectrometry (GC/MS). Analytical methods for parent chloroacetanilides in water are similarly based on extraction followed by GC with various detection techniques. Many of the water methods, such as the Environmental Protection Agency (EPA) official methods, are multi-residue methods that include other compound classes in addition to chloroacetanilides. While liquid-liquid partitioning was used initially to extract acetanilides from water samples, SPE using... 

Prior to analysis of -lactam residues in liquid foods such as milk, a pretreatment step for fat removal, accomplished by centrifugation (69-71), is usually required. In instances where milk is to be submitted to ultrafiltration, dilution with water/acetonitrile (72-76) or water/acetonitrile/methanol (77-79) is often needed. Milk filtration (80) or dilution with acetate (81, 82) or phosphate buffers (83) is sometimes essential prior to solid-phase extraction. Unlike milk, semisolid food samples such as muscle, kidney, and liver require normally more intensive sample pretreatment. Tissue break-up is mostly carried out by the combined use of a mincing apparatus and a tissue homogenizer. 

Using mentioned extraction/deproteinization procedures, the obtained aqueous or organic extracts often represent very dilute solutions of the analyte(s). These extracts may also contain coextractives that, if not efficiently separated prior to analysis of the final extract, will increase the background noise of the detector making it impossible to determine the analyte(s) at the trace residue levels likely to occur in the analyzed samples. Hence, to reduce potential interferences and concentrate the analyte(s), the primary sample extracts are often subjected to some kind of additional sample cleanup such as liquid-liquid partitioning, solid-phase extraction, or online trace enrichment and liquid chromatography. In many instances, more than one of these cleanup procedures may be applied in combination to allow higher purification of the analyte(s). 

In some instances, combinations of normal- and reversed-phase columns can also be used for better purification of the crude extract. Combinations of Ci8 and alumina or Cig and silica solid-phase extraction columns have been successfully employed in the analysis of ivermectin residues in animal tissues (346) and bovine plasma (348), respectively. Elimination of coextracted materials and concentration of the analytes has also been accomplished using mixed-phase extraction columns. Such a copolymeric bonded silica column with both hydro-phobic and cationic functions has been employed in the analysis of hygromycin B in plasma, serum and milk (326). 

In contrast to liquid-liquid partitioning cleanup, which is particularly suitable for individual drugs or groups of drugs with similar chemical properties, solid-phase extraction is more appropriate for multiresidue analysis. On that account, solid-phase extraction in combination with liquid-liquid partitioning has become the method of choice in many laboratories for the purification of residues of sedatives and -blockers that may occur in biological matrices. Purification is usually accomplished on reversed-phase solid-phase extraction columns. Optimum retention of seven sedatives and carazolol on a reversed-phase solid-phase extraction column was reported when 10% sodium chloride solution was added to the acetonitrile hssue extract prior to its solid-phase extrachon cleanup (523, 524). A silica-based diol solid-phase extraction column was further suggested for efficient isolation of sedative and -blocker residues from food extracts (526). 

During recent years, however, the distinction between these two types of methods has become less clear because of the improved methods of sample cleanup that allow selective isolation of groups of compounds. Mixed-mechanism solid-phase extraction procedures and multi-immunoaffinity techniques are clear examples of liquid chromatographic developments that have contributed greatly to the current state of the art within residue analysis. 

G Font, J Manes, JC Molt6, Y Pico. Solid-phase extraction in multi-residue pesticide analysis of water. J Chromatogr 642 135-161, 1993. 

To obtain clear solutions, the extracts are either filtered or centrifuged and the residues reextracted with the same solvents (108). Another possibility is to transfer the chlorophylls from the acetone phase into an ether phase by adding sodium chloride (114) or sodium sulfate (93). More recently acetone extracts were purified and fractionated with C18 solid-phase extraction cartridges, and dephytylated pigments were eluted with 70% acetone and phytylated pigments with 90% acetone (115). Often a concentrating of the pigment solution with a rota-evaporator under vacuum is necessary before analysis. Chlorophyll extracts should be analyzed as soon as possible, but if they have to be stored they must be kept in the cold and dark (107). 

Albanis, T.A. and D.G. Hela (1995). Multi-residue pesticide analysis in environmental water samples using solid-phase extraction discs and gas chromatography with flame thermionic and mass-selective detection. J. of Chromatogr. A, 707 283-292. 

Recovery and Analysis of Abamectin Residues. Sample preparation was a modification of the initial steps of Merck Co. Method 8001 (9). Cg solid-phase extraction columns (Fisher Prep-Sep, 300 mg resin) were conditioned by consecutive washes with hexane, ethyl acetate, methanol, acetonitrile, and water (20 ml. each). Samples (100 ml of glass-distilled water) were spiked with various amounts of abamectin, adjusted to 25% (v/v) acetonitrile, and applied to the columns. The columns were washed with 10 ml of water, and then the abamectin was eluted in 12 ml of acetonitrile. The eluates were evaporated to 1.0 ml at 70s under nitrogen. Dilutions of these samples were made in PBS-Tween-20% acetonitrile, and mixed with equal volumes of MAb B11C2.1 (1 200 in the same buffer) in sealed 1.4 ml polypropylene tubes. After incubation for 2 hr to about 14 hr (overnight) at room temperature, replicate aliquots (100 pi) were transferred to Immukm 2 EIA wells coated with 25 ng ivermectin 4"-hemisuccinate-CON for the standard competition EIA. Standard curves consisted of 8 dilutions of abamectin, from 0.01 ppb to 500 ppb, in triplicate. 

A simple first strategy to recover residues for ELA is to try existing multiresidue methods, or to use solvents and solid-phase extraction media that have proven satisfactory in the first steps of more involved instrumental analyses. This approach makes the transition from instrumental methods to ELA easier for technical personnel, and it facilitates confirmatory instrumental analysis during method validation. The uncommon structure of avermectin antibiotics makes it very unlikely that the ELA would detect compounds other than an avermectin in multi-residue extracts, though this remains to be tested. The greater concerns are completeness of the extractions, efficiency and reproducibility of recoveries, and freedom from non-specific matrix effects and interference by solvents. 

Wells, M. J. M. and Michael, J. L. 1987. Reversed-phase solid-phase extraction for aqueous environmental sample preparation in herbicide residue analysis, J. Chromatog. Sci., 25 345-350. 

Buiarelli et al. (2004) extended the above analytical approach to many more related steroids when they published a method for the direct analysis of 15 urinary anabolic steroids in a single run, namely T, epitestosterone, dehydroepiandrosterone (DHEA), androsterone, etiocholanolone, their sulfates and their glucuronides (Figure 2,2), They extracted 2 mL of human urine by solid-phase extraction with methanol elution and reconstituted the residue in aqueous methanol in the presence of deuterated internal standards (da-epitestosterone glucuronide, [16,16,17-"H3 testosterone sulfate and [16,16,17-2H3]testosterone), then monitored, for example, mJz. 289-97 and 109 for T and epitestosterone, miz 367-97 for their sulfates, and m/z 463-113 and 287 for their glucuronides. The method does not achieve quantitation, but it allows the estimation of ratios, which makes it possible to monitor the urinary steroid profile, which is useful for monitoring the abuse of anabolic steroids. 

The analysis of food contaminants, in particular any toxic or biologically active residue, is important for public health or quality control reasons.19 Examples are mycotoxins (aflatoxins) and pesticide and drug residues. Sample preparation is typically elaborate and might involve deproteinization, solvent extraction, and clean-up via solid-phase extraction (SPE).The use of highly sensitive and specific LC/MS/MS is increasing and has simplified some of the sample preparation procedures. 

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