Bioanalytical Applications Solidphase Extraction

The organic phase containing the extracted analyte is isolated, evaporated to dryness, and reconstituted in liquid chromatographic mobile phase for analysis. One of the benefits of LLE is that with proper selection of solvent and pH, very clean extracts can be obtained with good selectivity for the target analytes. However, the disadvantages of LLE are that

• it is a very labour intensive procedure,

• it requires large volumes of organic solvents which can be expensive to purchase and dispose,

• exposure of personnel to these solvents can be hazardous to health,

• it cannot easily be automated,

• emulsions have been demonstrated to occur,

• evaporative losses can occur upon dry-down with volatile analytes.

Despite its drawbacks, LLE continues to be used for drug bioanalysis when there is adequate labour and its associated costs are not prohibitive, and the sample throughput can be adequately met.

Protein precipitation

A fast and simple method of sample preparation is protein precipitation, also referred to as 'dilute and shoot'. This nonselective technique involves adding a water-miscible organic solvent (e.g. acetonitrile) or inorganic acid (e.g. trichloroacetic acid, 10%) to the biological matrix (usually in a 3 : 1 or 4 : 1 ratio, v/v), centrifuging or filtering to remove precipitated proteins and injecting an aliquot of the diluted supernatant. This technique is often performed in pharmaceutical drug discovery laboratories as the first attempt to prepare samples for bioanalysis. Satisfactory analyses have been demonstrated with this rapid sample preparation approach, but it has disadvantages. This technique dilutes the sample by a factor of four or five, so it is useful only when sample levels are relatively high, typically in the low IgmL-1 range. Also, matrix components are not efficiently removed, and thus may co-elute with the analyte in the isolated supernatant or filtrate. When present, these contaminants have been shown to interfere with detection techniques and lower the signal for the analyte of interest. This approach thus lacks selectivity and problems can arise from column fouling since the precipitation efficiency is not complete.

Solid-phase extraction

Solid-phase extraction has, during the last 18 years, become recognized as a preferred technique for extracting drug analytes from complex biofluids using adsorption chemistry. The attraction of the analyte for a solid phase adsorbent ('sorbent') is exploited to the exclusion of other compounds through a selective wash step. Elution of the analyte is achieved with an organic solvent that disrupts the attraction to the solid sorbent. Solvent exchange is followed by analysis on a chromatographic system. The traditional format for SPE has been single disposable columns and cartridges. Its advantages are that multiple samples can be prepared in parallel, low volumes of solvents are used and procedures can be readily automated. The technology has been improved in recent years with the introduction of more selective solid sorbent chemistries, disc-based SPE devices, smaller bed mass sorbent loading, on-line SPE techniques and introduction of the 96-well plate format for improved productivity.

An on-line SPE technique has recently shown great utility. A commercial device (Prospekt™ (Spark Holland)) combines an autosampler and a solvent delivery unit to aliquot liquid samples into the flow path of solvent. An SPE cartridge is preconditioned and is in-line with the solvent flow. The cartridge retains target analytes while a weak solvent elutes unretained salts and polar matrix components. An optimized sequence of solvents, each with increasing solvent strength, is used to wash out weakly retained components. A final elution with LC mobile phase elutes the analytes of interest from the SPE cartridge and onto an analytical column for chromatographic separation followed by detection.

The autosampler within this device can be preloaded with up to 160 samples and the entire tray can be analysed in an automated procedure. Its advantages are: unattended sample prep and analysis, minimized adsorptive losses, since sample transfers are not performed as in off-line techniques, and trace enrichment of analyte occurs. Some disadvantages are that analysis is serial (although a sample is always being analysed while another is being extracted and prepared for injection) and sample stability may be an issue for some drugs as a result of extended storage times in the autosampler.

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Solar Panel Basics

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