Solid phase extraction with 384-well plates

The study concluded that Once wash steps are optimized, samples prepared by solid phase extraction are cleaner than those prepared by protein precipitation. Samples prepared by extraction with a Multi-SPE plate resulted in lower LOQs than samples prepared by solvent precipitation. Drug recoveries were acceptable (>80%) for both the SPE and the solvent precipitation methods. Well-to-well reproducibility of samples was slightly better with extraction with a Multi-SPE plate. Evaporation and reconstitution, while more time-consuming, yield better chromatographic performance, allow analysis of lower concentration samples, and require optimization for good analyte recovery. 

In our laboratories, a cycle time of 90 sec can be achieved with a dilution factor of 1 25 for a given sample concentration, allowing the purity and identity control of two and a half 384-well microtiter plates per day. The online dilution eliminated an external step in the workflow and reduced the risks of decomposition of samples in the solvent mixture (weakly acidic aqueous solvent) required for analysis. Mao et al.23 described an example in which parallel sample preparation reduced steps in the workflow. They described a 2-min cycle time for the analysis of nefazodone and its metabolites for pharmacokinetic studies. The cycle time included complete solid phase extraction of neat samples, chromatographic separation, and LC/MS/MS analysis. The method was fully validated and proved rugged for high-throughput analysis of more than 5000 human plasma samples. Many papers published about this topic describe different methods of sample preparation. Hyotylainen24 has written a recent review. 

Zimmer D. et al., 1999. Comparison of turbulent-flow chromatography with automated solid-phase extraction in 96-well plates and liquid-liquid extraction used as plasma sample preparation techniques for liquid chromatography-tandem mass spectrometry. J Chromatogr A 854 1999. 

Products/technologies Porvair sells the Microlute Solid Phase Extraction in a Microplate system that provides 96 solid phase extractions in one compact unit (using any brand of sorbent). It can be automated using most standard liquid handling and robotic systems. Porvair s 384-well plate is compatible with most automated liquid handling instruments, readers for EIA, fluorescence, luminescence, and scintillation assays as well as robotic-handling devices. 

Biological matrices are not directly compatible with LC-MS analysis, since these samples tend to block LC columns and contaminate the ion source. Extraction of compounds of interest from biological fluids is required prior to LC-MS/MS analysis [20]. Sample extraction can be achieved off-line with protein precipitation (PP), liquid-liquid extraction (LLE), or solid-phase extraction (SPE) [21]. With the ease of use and sophistication of automated liquid-handling systems, sample extraction procedures in a 96-well format can handle microliter volumes with multiple sorbents per plate and can simplify and expedite SPE method development [22,23]. The technique can be used to routinely develop methods for multiple analytes and examine a set of eluent compositions for each analyte [16]. 

Zimmer, D. Pickard, V. Czembor, W. Muller, C. Comparison of Turbulent Flow Chromatography with Automated Solid-Phase Extraction in 96-Well Plates and Liquid-Liquid Extraction used as Plasma Separation Techniques for Liquid Chromatography-Tandem Mass Spectrometry, J. Chromatogr. A 854, 23-35 (1999). 

Many different sample preparation techniques are available to the drug discovery scientist. Off-line sample preparation procedures include protein precipitation, filtration, dilution followed by injection, liquid-liquid extraction (LLE), and solid-phase extraction (SPE). Typically, these procedures are performed in an automated, high-throughput mode that features a 96-well plate format. Online sample preparation procedures include SPE and turbulent flow chromatography (TFC) with conventional chromatographic media or restricted access media (RAM). These online approaches are often simple and easy to automate. 

These reagents were utilized in the usual fashion to scavenge thiol as well as amines in the synthesis of a library of sulfides and amides with a high degree of purities and yields (Scheme 8.24). A significant feature of this approach was the purification utilizing plate-to-plate fluorous SPE as well as an automated solid phase extraction on a RapidTrace system, thus demonstrating the capacity of fluorous platforms for automation. 

Solid-phase extraction systems have been designed in a 96-well microtiter plate format, so they can be processed automatically. Single-block plates with 96 wells contain either packed beds or disks of sorbent particles, in an 8-row X... 

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