Solid phase extraction 96-well format

Solid phase extraction (SPE) is a very simple, rapid and reproducible cleanup technique that is now widely accepted as an alternative to the time-consuming liquid-liquid extractions. Additionally, SPE uses relatively small volumes of solvents, and is easy to automate. It is available in a number of different formats, including cartridges, disks, loose material, well plates or SPME using film-coated capillaries. SPE can be considered as an extraction technique when used for isolation and concentration or a cleanup technique when used to remove co-extractives from solvent extracts. The use of SPE for cleanup is discussed later. 

SP refers to a family of solid/liquid handling techniques to extract or to enrich analytes from sample matrices into an analyzable format, namely, the final analyte solution. While SP techniques are well documented, " few publications address the specific requirements for drug product preparations, most of which tend to employ the simple dilute and shoot approach. A more elaborate SP is often needed for complex sample matrices (e.g., lotions and creams). Many newer SP technologies such as solid-phase extraction (SPE), " supercritical fluid extraction (SFE), "i° pressurized fluid extraction or accelerated solvent extraction (ASE)ii"i and robotics " " are topics of numerous research papers, symposia and commercial promotion. However, for reasons discussed later, these newer developments have had little impact on the way pharmaceutical laboratories conduct their SP for drug products today. 

T. K. Nevanen, H. Simolin, T. Suortti, A. Koivula, and H. Soderlund, Development of a High-Throughput Format for Solid-Phase Extraction of Enantiomers Using an Immunosorbent in 384-Well Plates, Anal. Chem. 2005, 77, 3038. 

Allanson, J. P. Biddlecombe, R. A. Jones, A. E. Pleasance, S. 1996. The use of automated solid phase extraction in the 96 well format for high throughput bioanalysis using liquid chromatography coupled to tandem mass spectrometry. Rapid Commun. Mass Spectrom., 10,811-816. 

Certain techniques are very good at particular tasks (such as protein precipitation for removal of proteins and cellular components), but perhaps not as good in general. With the emphasis on greater efficiency, many of these approaches have become automated or semiautomated. Thus, there has been greater emphasis on the 96-well formats an approach that can lend itself more readily to automated workstations. This format has been used successfully for protein precipitation, liquid—liquid extraction, and solid-phase extraction [8-10]. Ultrafiltration (UF) in a 96-well format is also being evaluated and shows some potential, but products and applications are not yet fully developed. Automated techniques for sample preparation and each of the sample preparation techniques listed in Table 1 are described below. 

Figure 12 Typical shallow-well 96-well format solid-phase extraction vacuum manifold adapted for use with liquid-handling workstations.

Examples of this approach has been reported by Janiszewski [10] and others [53] by utilizing extraction disks in a 96-well format to perform quick, automated solid-phase extractions under very simple wash and elution conditions. In one approach [10], a Tomtec Quadra 96-well workstation was used to perform the semiautomated solid-phase extraction with Empore C extraction disks. The advantage of this piece of equipment is that it allows liquid to be transferred to or from all 96-wells simultaneously, thus giving the greatest throughput advantage. 

RS Plumb, RD Gray, AJ Harker, SJ Taylor. Use of reduced sorbent bed and disk membrane solid-phase extraction for the analysis of pharmaceutical compounds in biological fluids, with applications in the 96-well format. J Chromatogr B 687 457 161, 1996. 

Products/technologies These include solid phase extraction products in 96-well format and Mercury , which is a small, low-cost NMR spectrometer. The acquisition of Rainin allows Varian to compete in the preparative HPLC market and gives it a lower cost product line to complement its research grade LC systems. 

Biddlecombe, R.A. Benevides, C. Pleasance, S. A Clinical Trial on a Plate The Potential of 384-Well Format Solid Phase Extraction for High-Throughput Bioanalysis Using Liquid Chromatography/Tandem Mass Spectrometry, Rapid Commun. Mass Spectrom. 15, 33 0 (2001). 

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]. 

Ronan, M.C. Buffet, C. Masson, L. Marfd, R Humbert, H. Maurer, G. "Practice of Solid-Phase Extraction and Protein Precipitation in the 96-Well Format Combined with High-Performance Liquid Chromatography-Ultraviolet for the Analysis of Drugs in Plasma and Brain, J. Chromatogr. B 754,45-55 (2001). 

Plumb, R.S. Gray, R.D.M. Jones. C.M. "Use of Reduced Sorbent Bed and Disk Membrane Solid-Phase Extraction for the Analysis of Pharmaceutical Compounds in Biological Fluids, with Application in the 96-Well Format, J. Chromatogr. B 694, 123-133 (1997). 

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. 

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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