February 1, 2006 (Vol. 26, No. 3)

Providing the Highest Level of Cleanliness for Drug Discovery and Drug Development

Solid-phase extraction (SPE) can be used to capture almost any compound of interest for further analysis or to capture an impurity to maintain the quality of that which remains in the sample. First used in environmental applications, SPE has found its way into a variety of biological analyses.

John Siira, product manager at Caliper Life Sciences (www.caliperls.com) tells the anecdote of a sultan of a Middle Eastern country, convinced that he owned the worlds fastest camel and that his fellow racers were giving performance-enhancing drugs to their camels. The sultan purchased an automated instrument to do solid-phase extraction because the positive pressure capabilities of the system could handle the viscous camel specimen samples.

SPE is used for sample preparation steps before mass spectrometry or liquid chromatography applications, Siira says. Often, it has been used for method development to determine what levels of a drug might leach into an animals liver or determine the analyte of a drug of abuse.

Customers can define what they want to do with SPE, which refers to immobilizing a compound of interest on a bed and eluting it off, according to Greg Robinson, liquid-handling product manager at Gilson (www.gilson.com). The beds can be made of silica, CaC18, filter papers, or polymers. Essentially, SPE is a preconcentration step that enables a large volume of water to go through a cartridge, which just holds onto an analyte of interest.

SPE involves a material that needs to be extracted and a resin medium in a cartridge format that interacts with the sample, explains Srikanth Srinvasan, product support specialist at Polymer Laboratories (www.polymerlabs.com).

In drug discovery, polymeric or silica-based media in cartridge formats are used to remove trifluoroacetate (TFA), acetic acid, and formic acid in the process of developing drug libraries. In life science applications, there are ionic interactions with the SPE media, Srinvasan adds.

According to Jeff Mazzeo, Ph.D., director of applied technology at Waters (www.waters.com), SPE can be one of the simplest, most cost-effective and versatile methods of sample preparation. Utilizing low-cost, prepackaged, disposable cartridges containing chromatographic packing, a sample analyte of interest is separated from other species in the sample matrix by loading the sample onto the device and selectively eluting the desired compounds using different solvents.

SPE assures assay robustness and sensitivity for LC/MS/MS studies of drug metabolism studies in plasma, Dr. Mazzeo adds. Endogenous compounds in plasma can lead to assay variability and reduce sensitivity in these analyses. Using SPE to clean up the sample gives you a better quality sample while keeping the instrument running a lot longer.

In 1996, Waters introduced Oasis sample extraction products, which incorporated hydrophilic-lipophilic-balanced water-wettable copolymers. It enabled sample cleanup for a combination of reversed-phase and ion exchange chromatography.

Library Degradation

In medicinal chemistry repository collections, the degradation of compound libraries and the subsequent false positives (combined with the price of library resynthesis and rescreening), cost the pharmaceutical industry somewhere between $600 and $900 million annually, according to Srinvasan.

A significant cause of library degradation is when compounds are stored as TFA salts in solutions. Polymer Laboratories StratoSpheres SPE products help to generate freebase compounds to preserve the lifetimes of the libraries. Peptide chemists also need to work with TFA and remove it at the end of the process. The cartridges filled with polymeric media interact with the TFA.

Dietary Supplements

Currently, there is an initiative to establish industry-wide standards to ensure that dietary supplements are manufactured consistently.

HPLC analysis and solid-phase extraction can be used to determine the identity, purity, quality, strength, and composition of triterpene glycosides in dietary supplement formulations, according to Laura Watson, director of international marketing at Polymer Laboratories.

Nutraceuticals or phytoceuticals, natural products that are used to supplement the diet by increasing the total dietary intake of important nutrients, may include nutritional supplements such as vitamins, minerals, herbal extracts, antioxidants, amino acids, and proteins. Ephedra is a naturally occurring substance derived from the Chinese herbal Ma Huang. Its principal active ingredient is ephedrine, which, when chemically synthesized, is regulated as a drug.

The FDA has made a proposal to prohibit marketing of dietary supplements containing 8 mg or more of ephedrine per serving; therefore it is imperative to be able to determine the quantity of ephedra found in various foods and dietary supplements.

An automated solid-phase extraction method coupled with HPLC can determine the various levels of ephedra and ephedra-like compounds in dietary supplements more easily and accurately than manual solid-phase extraction, according to Joan Stevens, Ph.D., applications manager at Gilson.

Natural products are a complex matrix, such that interfering peaks may compromise the identification and quantitation of components and trace components. The automated solid phase extraction procedure removes much of the interfering matrix that would otherwise interfere with analysis at 210 nmthe necessary wavelength for detection of ephedra alkaloids.

An automated SPE system plays an integral part in the sample preparation of natural products and provides a higher extraction rate (relative recovery) than manual SPE.

Gilson products automate SPE applications from smaller sample processing to higher sample throughput. The full range of column dimensions (16 mL), including the 96-well format, is accommodated within the layout of the instruments. Graphically designed, drag-and-drop software simplifies method development, sample processing, and 21CFR Part 11 compliance, according to the company.

Gilsons SPE 215 System offers high-throughput solid-phase extraction and sample purification through 96-well extraction plates and 1- and 3-mL SPE cartridges. Positive-pressure elution is designed for precise liquid transfers.

The ASPEC XL4 uses positive pressure elution to automate solid-phase extraction for both routine and method development work. The system processes four samples in parallel and reportedly has the capability to process up to 50 samples per hour and up to 108 samples in a single batch. The ASPEC XLi offers automated sample pretreatment with on-line injection.

The SPE 215 System allows the user to automate SPE and liquid-handling applications from the initial wash to the final transfer, Robinson adds. This instrument uses positive pressure for SPE applications. In the case of the SPE 215, the positive pressure elutions are achieved via Gilsons integrated sealing foot design. The SPE 215 also has the ability to perform parallel injectionsinjecting up to 8 samples simultaneously.

Calipers AutoTrace SPE is a high-throughput workstation dedicated specifically for automating SPE, says Siira. The AutoTrace automates the cartridge conditioning, sample loading, and elution steps for large volumes aqueous solution extractions (up to 2,000 mL).

The unit can process up to six samples in parallel, automatically conditioning, washing, and eluting SPE cartridges with a choice of up to five reagents, Siira says. The unit also segregates aqueous and solvent waste using separate waste lines for efficient, cost-effective waste handling.

The RapidTrace SPE Workstation is a modular, scalable, automated, high-throughput solid-phase extraction platform, according to Siira. Utilizing 1-mL or 3-mL industry-standard SPE cartridges, the RapidTrace can process up to 100 samples in less than two hours, unattended. Each module can be loaded with 10 cartridges, and up to 10 modules can be connected together and controlled through a software package.

The RapidTrace specializes in automating low-volume solid-phase extractions. It is currently being used in a wide variety of industry applications primarily for bio-extractions, and especially within the regulated pharmaceutical, clinical, and forensic environment.

DNA Purity in Biobanking

Another SPE application is maintaining the purity of DNA in biobanking. By storing dried blood, we will achieve significant savings in terms of both cost and storage space, says Tim Peakman, executive director of the UK Biobank.

By providing for room temperature storage, Whatmans (www.whatman.com) FTA and FTA Elute eliminate the freeze/ thaw sample degradation of stored cellular fractions inherent in traditional ultra-low temperature storage approaches, Peakman explains.

Through a heat and water elution step, FTA Elute releases the DNA, reducing contamination from the matrix and minimizing the number of PCR amplification cycles needed to reach a saturation endpoint, according to Peakman. FTA Elute binds proteins, hemoglobin, and other PCR inhibitors to create a stable environment that allows DNA to be stored at room temperature, enabling the release of stored DNA into liquid for multiple amplification reactions from a single DNA sample.

Peptide sequencing labs frequently use detergents to solubilize peptides and proteins from electrophoresis gels, according to Amos Heckendorf, Ph.D., of the Nest Group (www.nestgrp.com). Detergent buildup can ruin subsequent runs by reversed-phase HPLC, leading to bubble formation that blocks further sample intake in automated peptide sequencers, and accumulates on the PVDF membranes used for sequencing.

The Nest Group offers a range of small-volume, microSPE products designed for desalting, concentration, detergent removal, or ionic fractionation of biological molecules. The detergent removal products use a hydrophilic interaction mechanism to remove nonpolar impurities from peptides or proteins. Elution is in low organic solvent so that the samples are available for direct analysis by reversed phase LC/MS.

These minispin columns, unlike traditional SPE, are spun to dryness in a micro centrifuge, eliminating wasted residual bed volume of liquid remaining in the tub, according to the company. The bed of the microSPE chemistry is large to enable repeated cycling, and the elution volume is small enough to use for MS sample prep directly.

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