Technology Forum: Sample Prep

This month's Technology Forum looks at the topic of Sample Prep and the trends and issues surrounding it. Joining us for this discussion is LCGC columnist Ron Majors of Agilent Technologies, Willam Ciccone of Mircosolv Technology Corporation, and and Yibai Chen of Fox Chase Cancer Research Center.

What trends do you see emerging in Sample Prep?

Majors: I see a trend in the development of SPE sorbents that are more selective. As samples get more complex, oftentimes only a single analyte or maybe a class of analytes is of interest to the analyst. If the SPE phase can selectively remove and concentrate these analytes, it makes the analytical job easier. Just as in HPLC, many analysts choose a C18 phase for their solid-phase extraction. In many cases, this is the wrong approach since this phase tends to sorb everything in the sample that has a carbon atom; in other words, it is non-selective. One is much better off looking at the chemistry of the analyte and then choosing an SPE phase that is optimized for its functionality, hopefully differentiating it from the sample matrix. For example, recently, I have seen a number of mixed mode SPE phases that have come onto the market. A mixed-mode phase is one that contains multiple points of interaction, such as a hydrocarbon portion (e.g. C8, phenyl) and an ion exchange portion (e.g. SCX, strong cation exchanger). Such phases display an added level of selectivity compared to a typical C18 or C8 SPE phase.

The ultimate selectivity is achieved with a molecular imprinted polymeric (MIP) phase. These phases are very selective since they are synthesized for one or a single class of analytes. The polymer is constructed in the presence of a template which happens to be the analyte of interest or one very closely matched. After the polymer is completed, the template is extracted leaving behind an imprint of the template molecule. The separation mechanism is purported to be similar to a “lock and key” principle where only the template molecule can fit into the imprint much better than similar or other molecules. Thus, it is very selective similar to an affinity phase in bioseparations. The MIPs have been very popular in Europe but are now being seen in the U.S. The MIPs that may prove to be the most useful are those designed for certain popular classes of compounds such as beta agonists or tobacco-specific nitrosamines. Making a MIP for a single analyte may prove to be too costly but could be justified for a compound that would be analyzed for years to come.

Example of other selective phases that have become available are those for the depletion of high abundance protein for proteomics studies, restricted-access media for drugs in biological fluids, titania phases that are selective for phosphorylated peptides, scavengers for metal species, silver-bound phases for fatty acid methyl ester isolation and various environmental phases.

Another trend I’ve noted is the wide variety of formats of SPE devices that are available. The SPE cartridges and disks have been available for sometime but in the last few years, 96-well SPE plates, pipette tips with sorbents coated or bonded to the inner walls, in-syringe SPE devices, SPE mega-columns, and spin columns to name a few. Thus, the analyst can now match the SPE format to the sample size or throughput needs in his/her laboratory.

Many of the SPE formats are well suited to automation which is a continuing trend. I was surprised to see a number of new SPE automation products at this year’s Pittcon in Chicago. It seemed like for years that SPE automation had taken a backseat to other high throughput automation instrumentation. A useful accessory for SPE automation is the method development 96-well plate. Instead of having a single SPE phase in each well, there will be various phases (e.g. C18, C8, SCX, SAX, mixed mode and polymeric) packed into the wells. Thus, using an automated SPE instrument, an entire array of stationary phases and washing/elution conditions can be automatically and rather quickly performed making method development an easier task.

Ciccone:I see sample prep developing more and more toward faster and faster ways to accomplish the preparation.

Chen: People will begin to prepare samples in an on-line setting for LC-MS high throughput experiments. Manual preparation is labor intensive and hard to keep reproducible.

What is the future of Sample Prep?

Majors:Actually I see three application areas of increased focus: 1) high abundance protein depletion; 2) food safety; and 3) environmental. With the increasing role of proteomics in investigating of new drug targets and biomarkers of disease, there is hope that this field of human research can help in improving the health of millions. However, most of these proteins that are indicative of disease states are buried deep in the proteome in minute concentrations. The tiny amounts of protein are masked by the massive amounts of high abundance plasma proteins, such as human serum albumin and IgG, so that they cannot be sufficiently uncovered. The development of high abundance protein depletion sample preparation columns is a step forward in helping biochemists and molecular biologist to uncover the trace proteins that could be identified and characterized to help in the ongoing battle to defeat cancer and other life threatening illnesses. I see more of these products that will deplete more and more of these high abundance proteins.

Food safety and food contamination is on everybody’s mind. Recently, there have been major scares-the melamine tainted pet food from China, the e coli contaminated spinach, chloramphenicol in honey and shrimp, Malachite Green in fish, and acrylamide in baked and fried food to name a few. Our nation’s food supply is no longer home grown; we import food products from countries that don’t have the best food safety standards and inspection systems. New sample preparation products are needed to tackle these complex matrices that can range from simple (lettuce) to complex samples (animal parts). New selective SPE phases can help analysts more quickly check out these food samples for potential contamination.

The environment is an area of continued concern. Although our environmental laws are rigid, regulatory agencies cannot be everywhere and methods for the analysis of some trace toxins are not readily available. Since SPE is a technique that can concentrate trace organics from large volumes of water or can help in the final stages of analyte extraction from solid wastes, I forsee continued development in the area of environmental sample prep. Combined with on-line assays using multidimensional and comprehensive separation techniques like GCXGC, fully automated systems could continually monitor complex environments.

Ciccone: Sample prep is and will continue to be the time limiting factor for many analytical and biochemical methods and tests. Therefore sample prep strategies and technologies will continue be developed and improved.

Chen: For most of my work, we focus on working with small sample quantities. It is difficult to prepare samples in the nano-gram range for MS analysis and be confident in the results.

What is the Sample Prep application area that you see growing thefastest?

Majors: With analyte quantities getting smaller and matrices getting more complex, improvements in sample prep, including SPE, will continue to be made. Fortunately, instrumentation improvements continue to help in the process. More sensitive and selective analytical techniques such as LC/MS-MS require smaller amounts of sample and thus go hand in hand with smaller analyte quantities. The continuing trend in more selective phases such as the protein immunodepletion phases will result in cleaner extracts as well as allowing for more efficient concentration. The MIPs and restricted-access media (RAM) coupled on-line via switching valves will become more useful, as reported at the recent HPLC 2007 meeting in Belgium. On-line SPE has become quite fashionable once again and instrumentation to make such experiments more convenient can now be interfaced to and controlled by modern gas and liquid chromatographs.

With the trend to make sample preparation safer and more environmentally friendly, SPE with pipette tips and 96-well plates use greatly reduced amounts of solvent which reduces both purchase and disposal costs. Solvent-less technique like SPME or reduced solvent techniques like matrix-assisted sorbent extraction will also reduce solvent usage. If less solvent is used for sample prep, then the will be less solvent impurities that may show up during the concentration step.

Besides reducing the amount of solvent, simpler sample preparation techniques are in development. Already, QuEChERS (standing for quick, easy, cheap, effective, and safe and is pronounced "catchers") developed for the sample preparation of pesticides in foods and agricultural samples could be expanded beyond these application areas. The technique only uses small amounts of solvent and salt and is coupled with SPE to make a fairly efficient sample prep protocol to handle complex food matrices. An extension of SPME, stir-bar sorbent extraction (SBSE) can isolate trace amounts of analyte from liquid or semi-solid matrices. When coupled to on-line thermal desorption-GC/MS systems, sample prep problems from many different application areas can be solved using very simple approaches.

The drivers in laboratory analysis will also drive the need for better, faster, more efficient sample preparation. For example, every company wants to get to market faster and thus laboratory productivity is increasingly important. With fewer skilled chemists available and sample loads increasing, everybody wants to do more with less. This has a direct impact on sample preparation which is often viewed upon as the slowest part of the analytical cycle. Automation for sample prep, in general, and SPE, in particular, will still continue to be developed and optimized. With the automated SPE devices such as the 96-well plates and the micropipette tips ever expanding (and perhaps new formats on the horizon), the high throughput user will have a variety of choices. On-line SPE devices like the Prospekt (Spark Holland) and a number of 96-well plate autosamplers already available, SPE-HPLC is already being practiced and manufacturers should continue to promote this multidimensional technology.

Ciccone: Proteins and Peptides for desalting and fractionation.

Chen: Removing abundant proteins and extracting targeted proteins from a "dirty" environment are in high demand.

What obstacles stand in the way of Sample Prep development?

Majors: I don’t believe that there are major obstacles to further SPE development. The SPE market is already there and growing, especially in the automation area. The need for better, faster, more efficient sample prep protocols is there. The driver for isolating trace analytes from samples of ever increasing complexity is there.

I would say the biggest obstacle in sample preparation (and SPE) development is its stature in the analytical laboratory. Sample preparation has always been viewed as a manual, tedious technology that was oftentimes given to the lower skilled workers to perform. It has been relatively unattractive from an academic viewpoint and unrecognized as a field of analytical chemist. Hence, sample preparation technology gets very little recognition and study in the universities. In addition, most analysts consider themselves as above sample preparation as well and view their job as running sophisticated instruments, evaluating and implementing new analytical technologies and generating and interpreting data. Even the large instrument companies have avoided large investments in sample prep instrumentation leaving the market to smaller companies who address sample prep automation.

One of perceived difficulties to the acceptance of SPE is the time/experience in developing a method. With a wide range of SPE chemistries, a multitude of choices for manipulating solvent and pH conditions, various loading and flow rate studies required, and the multiple steps in applying the technique sometimes makes SPE more difficult to grasp than other sample prep techniques. For example, initially 96-well SPE plates were used for drugs in biological fluid assays but have been giving way to the use of 96-well “crash” plates where protein is precipitated and the supernatant analyzed by LC/MS. Even though the extracts of the protein precipitation techniques are admittedly dirtier, the time invested in developing a successful SPE method was considered an obstacle. Much of SPE method development has been “trial and error”; there needs to be more time spent on educating sample prep technologists on developing rugged and robust SPE methods, which is not that much different than developing rugged and robust HPLC methods.

Ciccone: For R&D purposes, the sample sizes are becoming increasingly small and difficult to handle. Also, many analytical methods are changing. For example as more and more methods become LCMS, the matrices and samples must be compatible with MS. Completing the sample prep becomes more and more of a challenge.

Chen:There are not many good sample prep products for some specific needs such as the extraction and enrichment of phosphopeptides on the nanogram scale. The products currently available are unreliable and their protocols are not robust. The development of this product is so slow. Possibly, the chemistry is difficult or the profit is not high enough.