Sample Preparation

Innovations in sample preparation have continued to proliferate in recent years, enhancing and revolutionizing separation science. Here, our panel of experts discuss the latest developments in this rapidly advancing field.

Joining us for this discussion are Michael Halvorson of Gilson, Inc.; Don Shelly of UCT; Michael Campognone of Phenomenex, Inc.; Joni Stevens of Agilent Technologies; Jack Cochran of Restek Corporation; Tom Hall of FMS; and Vivek Joshi and Estelle Riche of Millipore.

What new developments in sample preparation have you the most intrigued?

Halvorson:I am intrigued with the advances that have been made in the developmentof molecularly imprinted polymers (MIPs) so that they are a morereliable, specific, and robust tool for sample preparation.

Shelly:Several factors have converged to create new approaches to sample preparation. Economic realities, improved sensitivity in instrumentation, the green chemistry movement, and the need for faster results have encouraged innovators to develop sample preparation techniques that use less solvent, avoid the use of chlorinated solvents, allow a smaller sample size, and reduce sample preparation time. An example is the QuEChERS approach. I recall using the Luke method to extract agricultural products back in the 1980s. Some matrices would take days for extraction and clean-up, not to mention liters of solvent and expensive glassware. Now, we can attain the same or better results in 20 min using QuEChERS.

Campognone:The desire to spend less time, effort, and resources on sample preparation has created a trend for more selective sample preparation procedures that achieve better cleanup and improved analysis at lower concentrations. This trend has led to adsorptive media and format solutions that resulted in the emergence of more analyte selective media that is more rugged and robust in formats that allow for less labor-intensive usage and higher throughput capabilities such as 96-well plates and online cartridges.

Stevens:I am impressed with the “just enough” sample preparation approach that is emerging with the advent of highly selective and sensitive detection systems, for instance, LC–MS-MS and GC–MS-MS. These analytical tools allow us to approach sample preparation from an entirely different angle. In the past, sample preparation required extensive/exhaustive procedures involving many steps in order to achieve required levels of detection. Not only was sample preparation time consuming and expensive, but usually required a high level of expertise. Implementing these sensitive and selective detection systems offers an opportunity for sample preparation to be faster, cheaper, and greener than current techniques by employing a “just enough” approach. This approach, although in its infancy, is being used with great success in the food safety and homeland security market via QuEChERS sample extraction technique. The QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe, pronounce “catchers”) procedure has only two simple additional steps associated to the extraction, unlike SPE, which usually requires at least four flow rate monitored additions. Steps (1) extraction/partitioning and (2) dispersive-SPE in the QuEChERS sample preparation procedure are easily performed manually, requiring minimal sample preparation experience and common laboratory instruments, e.g. centrifuge, vortexer, and pipettes. Although QuEChERS is an extraction procedure versus an extraction/concentration procedure like SPE, it has produced remarkable results. For example, from a simple QuEChERS extraction procedure, over 400+ pesticides can be detected within a single analysis method, unprecedented via SPE. As stated by many, the driving force behind sample preparation techniques now and in the future is balancing cost of implementation, consistent and repeatable results, while delivering the lowest possible failure rate and fastest throughput. I believe the QuEChERS procedure delivers on all points and we are and will be seeing its advancement beyond food products and biological matrices.

Cochran:The fast batch sample preparation approach, QuEChERS, has been the most exciting development to come along in quite a while for the pesticide-residues-in-food analysis people. One method produces 6, 12, or more extracts for gas and liquid chromatography in about 30 min. And the analyte and commodity types (originally fruits and vegetables) for QuEChERS continue to be expanded with only slight adjustments to the original method. It’s such an adaptable approach that it is crossing over into forensic, pharmaceutical, and environmental application areas too.

Hall:Some of the new techniques that use smaller sample volumes and reduce solvent usage. Micro extractions, QuEChERS, and better solid phase chemistries are proving to be excellent high throughput, cost effective screening tools.

Joshi and Riche:The use of solid phase separation for sample preparation has improved recovery and reproducibility, and presents a great opportunity for further improvements in this technology. The most recent advances in this area include paramagnetization of the solid phase, enabling efficient isolation from the liquid phase, and affinity purification using solid phases that selectively retain analytes of interest.

Has the importance of sample preparation increased in light of the recent melamine and food safety scares? Has there been an increased interest in the use of QuEChERS for safety testing?

Halvorson:The use of mass spectrometry (MS) has increased significantly as ananalytical tool for detecting melamine as well as a number of otherunwanted residues that can be found in foods. As a result, the need forsample preparation has increased as well, since it is important toremove any compounds in foods that may interfere with MS analysis.

Yes, I have observed an increased interest in the use of QuEChERS as asample preparation tool. I have also seen an interest in automatingQuEChERS in labs that are currently processing large numbers of samplesusing this method.

Shelly:There has definitely been an increase in the awareness that food safety is an issue that requires immediate attention. I have seen a huge surge in the food testing industry internationally. While QuEChERS has been rapidly gaining in popularity, many analysts have been led to believe that the “dilute and shoot” approach to sample preparation will decrease analysis time. They quickly learn that instrument down time for maintenance and column replacement costs rapidly abate any short term time savings.

Campognone:Each time there is a public scare it underscores the need for further improvements in food safety, environmental analysis, and toxicology; each of which carry their own sample prep challenges based on cleanup, concentration needs, and matrix constituents. If you can dilute your sample or simply inject it, great; but if you cannot, which is usually the case, the sample (fruits and vegetables, water, blood/plasma/serum, urine, cooking oil, milk, etc.) will require sample prep. We have found in our studies that selectivity is the key to providing a viable solution to the challenges of sample prep.

QuEChERS is a technique that uses the selectivity of a solid phase extraction media, but in a dispersive form with some salts. In many ways sample prep, using QuEChERS, is very similar to SPE in technique but at the same time it can seem different and complicated. One can use an SPE sorbent to retain an analyte while washing off contaminants that are present in their sample, or one can use an SPE sorbent to retain contaminants and allow target analytes to be washed and captured for analysis. Both are great ways to utilize SPE media but it simply depends on what the goals of the analyst are and how we can help them achieve those goals. One approach provides the ability to cleanup and concentrate, the other allows for cleanup only, but may be a faster procedure.

Stevens:Yes, the need for sample preparation has increased relative to food safety issues, however food adulterations like melamine are extremely difficult to decipher. Not only are their matrix issues, but it’s synonymous with trying to find a needle in a haystack where we are looking for “something” but not sure what that “something” is. SPE will still play a very important role in food adulteration especially if samples require selective extraction and concentration prior to analysis. Additional sample preparation techniques are MIP (molecular imprinted polymers) for fungal contamination and pathogen screening through DNA-based PCR, Immunoassays (ELISA). Chromatographic analysis will allow evaluation of the results against the compound libraries especially in association with deconvolution reporting software available for GC–MS+, offering identification within a day not a week.

Cochran:Certainly the importance of sample preparation has increased in light of the recent food safety scares, mainly in the area of needing faster sample preparation approaches to handle increased numbers of samples and/or to obtain results more quickly. Again, that is where QuEChERS (the Qu for Quick!) fits nicely. And, because QuEChERS is a multi-residue approach, i.e., it extracts a wide range of compound types, the chances of isolating an unknown (and potentially hazardous) food adulterant for screening purposes is good.

Hall:Yes, particularly in determining persistent organic pollutants in final products and products used in producing a final product that are imported and exported to or from global locations.

Yes, although a manual process its’ popularity is growing throughout the world as a cost effective, quick, and easy way to do food testing.

Joshi and Riche:Indeed, food adulteration and food safety issues have become a serious concern worldwide. Sample preparation is of paramount importance when working with food matrices, and QuEChERS is increasingly being used, not only for pesticide analysis, but also for detection of other compounds such as veterinary drugs in animal products. Food safety issues have also led to the development of higher resolution analytical methods (such as LC-MS or GC-MS). As these analytical methods become more sensitive, the solvents used for sample preparation, including water, need to be of increasingly high purity in order to avoid contaminating the samples and to keep background noise low.

What sample preparation application area do you see growing the fastest?

Halvorson:The use of solid phase extraction (SPE) as a sample preparation toolwill continue to grow, especially in the areas of pharmaceuticals andfood and beverage analysis. There is a high interest in the automation ofSPE prior to analysis by LC–MS and other tandem mass spectrometrymethods. Sample preparation using ASE/PSE will also continue to grow inimportance.

Shelly:Food safety testing is still seeing rapid growth. The demand for QuEChERS products is increasing because of the international concern for food safety. This measurement is based on the wide variation in requests that I have received for QuEChERS applications and products.

Campognone:Most definitely food safety and environmental applications because new requirements and methods are promulgated from regulatory agencies as the need is uncovered (like with melamine). The challenges set forth are unique in each case. Each matrix is so very different, much different than understanding a regulated biological matrix and the recurring pattern in concepts of selective adsorption and associated benefits, etc. These two industries and their needs are just in their infancy, especially in the countries that will lead the world in global growth for the foreseeable future.

Stevens:There’s a growing interest in food safety and homeland security. Recent issues like milk and pet food adulteration, worldwide use of pesticides, terrorism via intentional contamination, and counterfeiting for profit will be a driving force for reliable sample preparation in order to analyze food stuffs. QuEChERS, dispersive-SPE, and MSPD (matrix solid phase dispersion) are examples of sample preparation techniques, which are simple and effective for the analysis of pesticides and other adulterants from food stuffs. Since this technique employs small amounts of solvent it is considered a “green” sample preparation approach. Over the last couple of years QuEChERS has expanded into a variety of food stuffs and other matrices. We might say it’s “catching on.”

Cochran:Food safety.

Hall:The largest growth for applications has been in applied markets such as food, beverage, and clinical.

Joshi and Riche:As mass spectrometry (MS) becomes more and more routine, moving out of core facilities into individual laboratories, there is an increasing demand for sample preparation technologies that are geared for MS analysis. Because MS is incredibly sensitive, signals are easily confounded by the presence of contaminating molecules; therefore, samples must be extremely pure. One way to help ensure optimum sample purity is to use freshly-drawn ultrapure water during sample prep and LC-MS analysis. The need for dedicated, MS-specific sample preparation tools that are compatible with a broad range of sample compositions is driving rapid growth in this area of sample preparation.

What was the greatest advancement in the sample preparation field during the past year?

Halvorson:The use of biosensors as well as the use sample preparation products inthe "field" rather than the lab.

Shelly:The greatest advancement that I have seen is the willingness of many regulators and lab managers to accept or at least try new technologies. Labs that rejected QuEChERS or solid-phase extraction a few years ago are now eagerly embracing the chemistry.

Campognone:Specialty solutions, using the same general chemistry and retention mechanisms but in formats that provide ease-of-use, rugged and robust low-level analysis, and simplified method development. There are a few that I can think of, but most importantly the application of current technologies has taken leaps forward this past year. We see savvy analysts using relatively new technology from the past 5-10 years in ways that are far more powerful and efficient than ever.

Stevens:The greatest advances have been in the development of new rapid sample preparation products and techniques that are required to address the complex matrices ranging from simple (apples) to complex (animal food stuffs). Food safety and contamination is a very real concern- melamine tainted milk and pet food, veterinary drug residue in fish, antibacterial agents in honey/shrimp, and acrylamide in fried foods are just the tip of the iceberg. World food demand is at an all time high, requiring food products being imported and exported all over the world from countries that may not abide by strict food safety standards or inspection processes. Automation will play an integral role in sample preparation because it facilitates the processing of large numbers of samples while minimizing human interactions with possible unknown or identified toxins. Sample preparation techniques offering very quick turnaround time for food products will in-turn increase productivity, safety, and reduce the cost to market.

Cochran:I think the greatest advancement was the continued expansion of the QuEChERS approach to different analytes and sample types. This was very obvious at the 4th International Symposium on Recent Advances in Food Analysis that I just attended in Prague, where QuEChERS was used for dietary supplements, cereals and grains, animal feeds, coffees, wines, and juices. Targeted analytes were not only pesticides, but also mycotoxins and veterinary drugs. And, as stated above, I see QuEChERS being used in the forensics and pharmaceuticals analysis worlds.

Hall:Automated one-step sample prep, sample prep systems that combine and merge several sample prep processes into one seamless process. Put the sample into the system, get the final prepped sample out and inject it.

Joshi and Riche:The past year has shown a dramatic trend toward using smaller pore sizes in membrane microfiltration of samples used for extremely sensitive downstream analysis. Specifically, the increased popularity of ultra-high pressure liquid chromatography (UHPLC) demands that samples be free of even the smallest particulates, and leads users to prepare samples by filtration through membranes with 0.2 micron pores.

What obstacles stand in the way of further advances in sample preparation development?

Halvorson:One obstacle is the need to develop more standardized, robust, andreliable sample preparation methods that are readily transferable fromlaboratory-to-laboratory around the world.

Another challenge is the development of faster and robust methods forthe removal of lipids from complex matrices. The current methods thatexist are either very efficient, but slow or fast, but lack the abilityto remove large amounts of lipid from a complex sample matrix orcompromise sample integrity. It would be nice to have a samplepreparation method that is both efficient in removal of lipids andrapid.

Shelly:Our only obstacle is our natural fear of change. We must open our minds, innovate, and accept the reality that there may be a better way.

Campognone:Unfortunately, many analysts will sacrifice analytical performance in any attempt to avoid adequate sample preparation. It is a common misnomer to assume that sample prep will increase cost and slow results. Few investigators actually interpolate the cost and time spent on instrument downtime and sample reanalysis. It is unfortunate that the powerful and selective technique of sample preparation is often an underrated and underutilized solution for improving analytical throughput, reproducibility, and lower-level concentration analysis. The only obstacle inside the laboratory to furthering advances in sample preparation is adoption.

Stevens:Sample preparation has always been viewed by researchers as labor-intensive, time consuming, and boring. Many popular sample preparation techniques have been around for a very long time and work quite well (SPE, LLE), although they may be slow and labor-intensive or require automation, in some cases. Alternatives to existing sample preparation techniques like QuEChERS, SLE (supported liquid extraction), PPT (protein precipitation), dispersive-SPE, and MSPD (matrix solid phase dispersion) offer sample extraction with minimal sample preparation time and may actually be the key to unlocking obstacles associated with sample preparation.

Cochran:One of the biggest obstacles that I see is efficiently dealing with sample extracts (e.g., fish, beef, poultry, some dietary supplements, etc.) that contain macromolecules like lipids, which sabotage gas chromatographic liners and columns. If we want to avoid compound degradation in comprehensive monitoring efforts, we need to use gel permeation chromatography (GPC) to remove these compounds from extracts, which can be slow and is solvent-intensive. There are some nice automated GPC systems out there now though that not only do the cleanup, but concentrate the sample for analysis as well.

Preparing fatty samples for analysis in general is problematic, including for QuEChERS, where the polar solvent used, acetonitrile, can be poor at extracting hydrophobic residues (e.g., organochlorine pesticides).

Hall:Cost is a big concern for many looking to automate sample prep processes. Once the cost of reducing labor, solvent consumption, solvent disposal, and reruns due to human error is understood, in most cases it is the right move for a lab. Maintaining properly trained staff, many sample prep processes are tedious, labor intensive and prone to error, in many sample prep labs technician turnover is high. Slow acceptance/adoption of new sample prep techniques.

Joshi and Riche:The industry should be aiming toward the automated, seamless integration of sample preparation with its corresponding downstream analytical application, built into a single, dedicated instrument. Achieving this goal will require close collaboration between analytical equipment and sample preparation technology developers.

What do you see in the future for the field of sample preparation?

Halvorson:There will always be a need for sample preparation. New samplepreparation methods will be more environmentally friendly and use lesssolvents. They will become more standardized from lab-to-lab. Samplepreparation methods will also become more automated, especially whenmass spectrometry is used for analysis.

Shelly:I see the continuing trend towards faster, greener, less expensive, and smaller-scale applications with the ability to capture more analytes in a single extraction. We are already well on the way to accomplishing this goal with the development of new SPE technologies in all areas of analysis.

Campognone:The future will be centered around unlocking the versatile uses and selectivity advantages of many of the solutions that currently exist in smaller dimensions and easier to use formats. The demands of the analytical scientist have become more intensive and require better, more reproducible cleanup and/or concentration to push the limits of the existing chromatography systems and detectors. The challenges are far greater today than yesterday and will be greater tomorrow. We have worked with requests for applications to extract femtogram levels of disease markers in biological samples with subsequent analysis in less than one min. This is a perfect example of an application that will require and benefit tremendously from sample prep, specifically ion-exchange which is the most selective form of SPE.

Stevens:Sample preparation is going to be absolutely necessary for certain samples because they are in a complex matrix, analyte concentration is so low that even sophisticated analytical methodologies like LC–MS-MS or GC–MS-MS cannot provide adequate sensitivity and/or selectivity. Food safety, which has been cited several times above, is such a case. Food toxins are often presented in low concentrations and matrix interferences cause ion suppression in the mass spectrometer requiring sample cleanup. This is also observed in environmental samples. Biological samples, particularly in the area of proteomics can be extremely challenging since biomarkers can be a million-fold or more, lower in concentration than “house-keeping” proteins. These trace proteins must be isolated and concentrated for further study and identification. Only presenting a few fields in this technology forum we can see that there is a very vast and wide requirement for sample preparation as long as the process is faster, better, cheaper, and greener than existing techniques.

Cochran:Even faster sample preparation methods with better residue recoveries and cleaner extracts. Isn’t that what everyone else sees? Or is that just what everyone wants?! On a serious note, I would hope that sample preparation methods, including QuEChERS, become more “green.” We should continue to reduce our solvent usage and plastic waste wherever possible.

Hall:Making sample prep simpler. Combining more of the individual sample prep processes into one seamless step with a high level of integration into analytical instrumentation for direct injection increasing sample throughput by eliminating the manual sample handling bottleneck.

Joshi and Riche:The future promises continuation of current trends toward higher throughput and automation compatibility. More efficient preparation of small volume samples or complex samples (such as biological solutions or complicated food matrices) containing multiple analytes will be required. Technologies that will improve the signal-to-noise ratios in analysis of complex mixtures will include advancements in enriching specific analytes of interest, either by affinity purification or by depletion of molecules other than the desired analyte. As these new technologies are developed, high purity water will undoubtedly continue to play an important role in reducing potential contamination of samples and instruments.

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