Liquid-solid chromatographic cleanups

The quantification of PAHs on a routine basis is often conducted using liquid chromatography (LC) in conjunction with a luminescence technique since they exhibit fluorescent properties. In general, the algorithms of analysis include extraction, cleanup procedure, concentration, chromatographic separation, and determination. Liquid-liquid, solid-phase and cloud point extractions have been interfaced with flow-based methodologies. 

A cleanup procedure is usually carried out to remove co-extracted matrix components that may interfere in the chromatographic analysis or be detrimental to the analytical instrument. The cleanup procedure is dependent on the nature of the analyte, the type of sample to be analyzed, and the selectivity and sensitivity of the analytical instrument used in the analysis. Preliminary purification of the sample extracts prior to chromatographic separation involves liquid-liquid partitioning and/or solid-phase extraction (SPE) using charcoal/Celite, Elorisil, carbon black, silica, or aminopropyl-silica based adsorbents or gel permeation chromatography (GPC). 

Diethylstilbestrol is particularly difficult to quantitate below 1.0 ppb in bovine tissues, especially in liver, which is among the last tissues to contain diethystilbestrol after cattle are withdrawn from receiving tire drug (101, 102). Interferences from tissue matrix constitute a major problem that might be due to nonspecific interference of lipids and fatty compounds (103, 104). In addition, problems with false-positive results often appear in urine analysis unless a chromatographic step such as a solid-phase extraction cleanup (105, 106) is introduced. Simple sample preparation procedures such as those based on solvent extraction and liquid-liquid partitioning do not usually give satisfactory results (107, 108). 

Detection in liquid chromatography is mostly performed by fluorescence and/or ultraviolet absorption. In a few instances, electrochemical detection has also been employed (357, 368). For compounds that exhibit inherent intense fluorescence such as albendazole and metabolites (319, 320, 338, 355), closantel (344), and thiabendazole and metabolites (378), fluorometric detection is the preferred detection mode since it allows higher sensitivity. Compounds that do not fluoresce such as eprinomectin, moxidectin, and ivermectin, are usually converted to fluorescent derivatives prior to their injection into the liquid chromatographic analytical column. The derivatization procedure commonly applied for this group of compounds includes reaction with trifluoroacetic anhydride in presence of A-methylimidazole as a base catalyst in acetonitrile (346, 347, 351, 352, 366, 369, 372-374). The formation of the fluorophore is achieved in 30 s at 25 C and results in a very stable derivative of ivermectin and moxidectin (353) but a relatively unstable derivative of eprinomectin (365). However, the derivatized extracts are not pure enough, so that their injection dramatically shortens the life of the liquid chromatographic column unless a silica solid-phase extraction cleanup is finally applied. 

Following extraction/cleanup, quinoxaline-2-carboxylic acid can be detected by electron capture, or mass spectrometric techniques, after gas chromatographic separation on capillary or conventional columns. A prerequisite of quin-oxaline-2-carboxylic acid analysis by gas chromatography is the derivatization of the molecule by means of esterification. Esterification has been accomplished with methanol (419, 420, 422), ethanol (421), or propanol (423) under sulfuric acid catalysis. Further purification of the alkyl ester derivative with solid-phase extraction on a silica gel column (422), thin-layer chromatography on silica gel plate (420), or liquid chromatography on Hypersil-ODS, 3 m, column (423), has been reported. 

Solid-phase extraction columns offer a rough cleanup of the crude extract, which might nevertheless not be sufficient for some detection systems such as mass spectrometry. Some authors have proposed a combination of solid-phase extraction and liquid chromatography columns for extract cleanup (440). Other methods appeal to liquid chromatography on Cig columns with automated fraction collection. Fractions containing the analyte of interest were evaporated to dryness, yielding a residue that in most cases was suitable for gas chromatographic detection after suitable derivatization (445, 437). 

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. 

Liquid-liquid extractions (13) permit the elimination of slightly polar molecules (phospholipids, fatty acids, etc) that may interfere in the HPLC determination of carbohydrates. Hence, for solid foodstuffs, some form of extraction will be required prior to the chromatographic procedure, and even in liquid food samples it may be necessary to modify the solvent composition of the liquid phase to make it compatible with the HPLC eluent. The major role of the extracting solvent is to obtain all of the carbohydrate present in the food sample dissolved in a liquid phase, be it for direct injection into the chromatograph or for subsequent cleanup stages prior to HPLC. 

M Hiemstra, JA Joosten, A De Kok. Fully automated solid-phase extraction cleanup on-line liquid chromatographic determination of benzimidazole fungicides in fruit and vegetables. J AOAC Int 78 1267-1274, 1995. 

J Tarbin, WH Farrington, G Shearer. Determination of penicillins in animal tissues at trace residue concentrations. 1. Determination of benzylpenicillin in milk by reversed-phase liquid chromatography with solid-phase extraction and liquid-chromatographic fractionation cleanup. Anal Chim Acta 318 95-101, 1995. 

A de Kok, M Hiemstra. Optimization, automation, and validation of the solid-phase extraction cleanup and on-line liquid chromatographic determination of V-methylcarbamate pesticides in fruits and vegetables. J Assoc Off Anal Chem 75 1063-1072, 1992. 

Another major drawback of classical extractions is that additional clean-up procedures are frequently required before chromatographic analyses. Solid phase extraction (SPE) avoids the emulsion problems often encountered in liquid-liquid extraction. A wide range of adsorbents are commercially available and may be divided into three classes polar, ion-exchange, and nonpolar adsorbents. Solid-supported liquid-liquid extraction on Extrelut columns is frequently reported for efficient cleanup of crude tropane alkaloid mixtures. Basifled aqueous solutions of alkaloids maybe transferred to Extrelut columns and the bases recovered in dichloromethane-isopropanol mixture [13]. 

Goldade, D.A., Primus, T.M., Johnston, J.J., Zapien, D.C. (1998). Reversed-phase ion-pair high-performance liquid chromatographic quantitation of difethialone residues in whole-body rodents with solid-phase extraction cleanup. J. Agric. Food Chem. 46(2) 504-8. 

Used as preservatives in many topical pharmaceutical products, paraben esters have been found to exhibit estrogenic effects that have been linked to breast cancer. This has led to requiranents for their rapid and quantitative detection. Traditional methods of analysis are slow and tedious. For gas chromatographic methods, derivatization as silyl- or fluoroacetyl derivatives followed by extraction and sample cleanup complicate the process, while liquid chromatographic methods require extraction and long chromatographic run times. To increase speed and decrease complexity, solid phase microextraction with ion mobility spectrometry (SPME-IMS) was used for the determination of parabens in pharmaceutical formulations. ... 

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