Sample Preparation

LCGC: What trends do you see emerging in sample preparation?

Paul H. Roberts: Simplifying workflows by combining or eliminating steps and reducing sample preparation time alongside improved automation and throughput are major trends. Reduction in sample volumes, with consequent reduction in solvent use and evaporation time is also a trend. Matrix scavenging techniques instead of analyte‑targeted sample preparation is a growing area.

Alicia Douglas Stell: Some of the fastest growing trends in sample preparation include the need for faster and easier‑to‑use systems that yield repeatable results without a technician dedicating significant time to their setup and use. As analysis techniques become more sophisticated and can report lower detection limits the need for rapid and consistent sample preparation is the ideal for accurate analyses.

Peter Dawes: The need for automation of sample preparation processes is obvious and is often stated as necessary, but broad adoption has been very slow with automation seen as expensive, requiring skilled operators to implement, and most automation platforms are intended for high-throughput applications. The broader need is for systems that are flexible and easy to set up for many different jobs in a laboratory’s day. Typically, sample runs are between 10 and 100 samples. Systems designed with this in mind are now becoming available.

The drive for development of automated sample preparation systems is extreme ease-of-use with a reduction in cost for sample preparation and a greater reliability in the results.

Oliver Lerch: The trend towards miniaturization and automation of sample preparation that we have seen recently will continue. This means that less sample and less solvent is required for the analysis and less waste is produced. Virtually all core sample preparation techniques are influenced by this trend. As more companies develop innovative sample preparation equipment, more users will start adopting this equipment for their daily laboratory routine.

Nonselective sample preparation techniques are also needed for the emerging trend of nontargeted analysis, but selective workflows are necessary for target compound analysis. The trend towards multitarget methods with more than 100 analytes will continue.

The rising interest in extraction of polar compounds from water matrices, as well as nonpolar compounds from fatty matrices, poses a challenge in sample preparation.

Matt Brusius: While the technologies that feed into sample preparation have generally remained unchanged, certain applications and types of equipment are becoming more popular, which has ultimately changed the overall landscape of “sample preparation”.

Sample preparation of large molecules has increased and the need for a more streamlined workflow at the commercial level is helping to drive innovation in this space as scientists diversify beyond small molecule. In addition, further adoption of automated sample processing platforms (robotics) along with devices like positive pressure manifolds have provided a better, more effective, and reliable way to process samples providing a quicker route to the instrument and downstream analysis.

Danielle Mackowsky: In the past year, sample preparation techniques traditionally used in a specific industry have begun to have broader appeal across multiple disciplines. For example, QuEChERS was developed in 2003 for pesticide residue testing in food‑based matrices. With the emergence of the cannabis industry worldwide, QuEChERS is now being introduced to a new subsection of scientists. In addition, this technology has expanded into use on samples that are not of an agricultural realm. Numerous post-mortem forensic toxicology laboratories are starting to incorporate this methodology for the universal extraction of drugs of abuse from a variety of sample types.

LCGC: In your opinion, what is the future of sample preparation?

Paul H. Roberts: Sample preparation often follows analytical instrumentation developments. Although liquid chromatography–mass spectrometry (LC–MS) systems are becoming more sensitive, and detection limits may be met by using simple “dilute and shoot”- type sample preparation, this can also lead to problems with the analytical system as matrix components accumulate over time. So, sample preparation needs to effectively clean-up samples, but be balanced with simplicity and ease of workflow.

Alicia Douglas Stell: The future of sample preparation frees technicians up from manual and time-consuming setup and extraction techniques and yields repeatable results in a fraction of the time when compared to existing techniques.

Peter Dawes: The future of sample preparation must be first to enable a move away from the large amount of manual handling with repetitive liquid handling. The liquid handling aspect of sample preparation is the easiest to automate and enables the introduction of a much greater degree of precision and reliability. However, too often we are asked to simply automate legacy sample preparation methods, which can be done, but it is grossly inefficient. The future lies with a whole raft of new tools and techniques designed for efficient automation providing faster and more reliable sample preparation than the current manual processes. These include, but are not limited to, special solid-phase extraction (SPE) cartridges, enzymatic reactors, affinity chromatography, derivatization, filtering, extreme accuracy of liquid handling, and better controlled reaction kinetics.

Oliver Lerch: Sample preparation will have its place in the world of chromatographic mass spectrometric analysis. The key point is to find the right balance between the effort spent on sample preparation and the robustness of the analysis method. In this sense, the “just enough” approach will replace the “dilute and shoot” approach. This will lead to many simple sample preparation techniques being implemented, including dilution, centrifugation, filtration, protein precipitation, and various combinations of these techniques.

Comprehensively optimized and sophisticated sample preparation workflows combined with the most sensitive gas chromatography (GC)–MS and LC–MS equipment will be needed to meet the requirements of certain legislation, for example, for the analysis of baby food or in the context of the EU water framework directive.

The acceptance of automated sample preparation will increase both in dedicated systems that integrate sample preparation and GC–MS or LC–MS analysis, and in off‑line workstations on the laboratory bench that prepare samples for multiple instrument types.

Matt Brusius: This depends on the application. In general, I ascribe to the notion that the future of sample preparation looks like a “quick-and-dirty” clean-up directly in front of a very powerful high resolution mass spectrometer. It is also possible that things like MS prefilters, that is, ion mobility, could eventually eliminate the need for sample preparation and chromatography entirely. For a more conservative prediction, as instruments become more powerful, I think that more matrix-specific filters and streamlined SPE procedures will be implemented to reduce total amount of time spent performing sample preparation.

Danielle Mackowsky: The future of this discipline will allow scientists to use minimal sample volume to achieve their analysis goals. As instrument detectors become more sensitive and less cost prohibitive, less sample is required for each extraction. In pain management laboratories, it is not uncommon to use as little as 100 µL for each patient sample and this could possibly become the norm across multiple disciplines. Reducing sample volume also cuts down the overall amount of organic solvent needed, making sample preparation a greener and more cost-effective choice for laboratories as a whole. To keep up with this trend, sample preparation consumables will be adjusted accordingly to accommodate smaller sample volumes.

LCGC: What one recent development in the area of sample preparation would you say is the most important?

Paul H. Roberts: Simple, user-friendly automation that is tailored specifically for techniques, such as SPE or supported‑liquid extraction (SLE), rather than “bolted on” to liquid handling devices.

Alicia Douglas Stell: Rapid automated sample preparation is the most significant development realized in more than 15 years. With traditional sample preparation taking 62% of the time spent on typical chromatographic analysis, sample preparation is the bottleneck to rapid LC and GC analysis. Systems that can cut typical sample preparation times down from over 30 min for each sample to 5 min or less are well positioned to eliminate the bottleneck.

Peter Dawes: Apart from the rapidly growing range of automatable processes in sample preparation, the increasing availability of ion mobility mass spectrometry systems is important in my opinion. It is understood that there are no universal truths in analytical chemistry that cover all sample types, but there are many situations where ion mobility mass spectrometry indirectly streamlines the requirements of sample preparation workflows where difficult matrices are involved. Along the same lines as the huge power that can be achieved through orthogonal multidimensional chromatography separations, the application of the same principles of orthogonal mechanisms to sample preparation or detection can reduce the need for high performance and critical‑to‑operate analytical separation systems.

Oliver Lerch: This is difficult to say; in my view no single groundbreaking new development has been made recently. The QuEChERS method in its multiple variations for all kinds of matrices is definitely one of the main achievements over the last decade.

Matt Brusius: I would say that generally it has been more about the increase in instrument capability than one vital breakthrough in sample preparation. Most of the commercially available developments have been incremental and are mostly still based on technology that is 10- to 15-years‑old. For example, polymeric‑based SPE media has undergone some improvements in that some products provide additional application-based benefits, such as in-well hydrolysis, or phases that are truly water wettable, which improves overall processing time. Ultimately, I think the most interesting developments are the ones that combine some type of clean-up device directly with ambient ionization mass spectrometry to bypass chromatography entirely.

Danielle Mackowsky: In the United States, opioid addiction has claimed the lives of millions of Americans. Forensic toxicologists need to stay on top of what analogues are being abused in their region in an accurate and rapid way. Development of fast and efficient extraction methodologies has allowed them to distinguish between the emerging compounds and more traditional drugs of abuse. In the environmental sector, per- and polyfluoroalkyl substances (PFASs) are a diverse group of synthetic organofluorine compounds that have been widely used in industrial applications and consumer products. PFASs are bioaccumulative in wildlife and humans and can now be tested for accurately in drinking water and other available matrices thanks to sample preparation technologies.

LCGC: What obstacles do you think stand in the way of sample preparation development?

Paul H. Roberts: The focus on analytical instrumentation developments can lead to loss of expertise and innovation within the “chemistry” of sample preparation.

Alicia Douglas Stell: The greatest obstacle that stands in the way of dramatic sample preparation development is fear of change. In order to make a dramatic step forward, sample preparation will have to abandon the “tried and true” methods of the past 50 years and design, from the ground up, technologies that meet the demands of today’s busy laboratories, including: speed, simplicity, and reproducibility in an effective platform with a small footprint.

Peter Dawes: As with the introduction of any new technology there is the burden of personal and financial investment in existing methods that hold back the adoption of better and more robust methods. For example, the new automated sample preparation techniques work better with smaller sample sizes achieving better limits of detection and limits of quantitation, but so many regulated analytical methods define that large volumes be analyzed. It is obviously important to ensure a sample being analyzed is representative, but automation does not lend itself well to using a litre of sample when a few millilitres is more than adequate, which we recently had to deal with for fire retardant analysis (PFAS) in water.

Oliver Lerch: From the users' perspective, sample preparation method development is increasingly a challenge. Laboratory resources are limited and qualified laboratory personnel is increasingly hard to find. This means that we may see method development being shifted from the end user to suppliers of analysis equipment in the future.

Manufacturers of sample preparation tools and systems have invented several new techniques over the past years, but customer acceptance is lower than expected and this may slow down further innovations. Many users take a lot of convincing and stick to tried and trusted products and concepts. Generally, sample preparation only gets limited attention in analytical chemistry research and this may also be a factor that slows down new development.

Matt Brusius: The cost for each sample is always something that limits the new types of sample preparation technology. I think to justify significant up-front cost and time in sample preparation the technology must provide some type of intangible benefit, such as peace of mind that your system will not fail overnight in the middle of a run. I think it is the other components of the workflow, such as the column and the MS system, that drive sample preparation development, and not the other way around.

Danielle Mackowsky: Laboratories are constantly under pressure to reduce their turnaround times. Because of this, the development of universal sample preparation materials and extraction methodologies is critical. It can be challenging from a product and method development standpoint to create an all‑encompassing sample preparation solution without compromising any results from a specific analyte class. In short, there is a definite struggle at times to find a “one‑size-fits-all” solution that is amendable to a variety of compounds and settings.

LCGC: What was the biggest accomplishment or news in 2017/2018 for sample preparation?

Paul H. Roberts: Simplified workflows and matrix scavenging techniques for urine samples.

Alicia Douglas Stell: The biggest accomplishment for sample preparation in 2017/2018 was the introduction of a new technology for rapid automated solvent extraction of samples for analysis by GC and LC that included built-in methods optimized for a wide variety
of sample types. This takes the guesswork and much of the method development time out of the sample preparation equation and produces repeatable results in a “hands-off” automated fashion.

Peter Dawes: I have been very impressed with the clinical analyzers that are now on the market (with more coming) that automate the entire workflow from sample preparation to LC and MS detection. It will be even more impressive when the LC part of the process, which after all in many analyses is just sample clean-up for the mass spectrometer, can be eliminated. This will be possible as a result of better sample preparation processes, such as small particles (less than 3 µm instead of 50 µm) and high‑resolution SPE cartridges that make the kinetics of the process less critical and more effectively remove matrix and fractionate the sample. If we can already do it reliably with difficult matrices like whole blood and urine for targeted analysis directly into a mass spectrometer, it should be much simpler for targeted environmental analysis.

Oliver Lerch: As I mentioned previously, there are some obstacles to new approaches being adopted. The implementation of nanomaterial with unique physical and chemical properties will definitely boost sample preparation techniques going forwards. Applications of nanomaterial for dispersive µ-SPE in the form of (magnetic) molecularly imprinted polymers, magnetic beads, and coatings were reported in 2017 and 2018.

Matt Brusius: I don’t know if you can necessarily pinpoint one single accomplishment, but overall, sample preparation is always changing and improving. Product advances are making it even easier to get cleaner samples in less time. Coupled with the other advances in LC-, GC-, and MS instruments, this has been an exciting year for sample preparation and many advances have been developed to bridge the gap into full workflow solutions. I think the immediate future holds further incremental improvements while the instrumentation platform will most likely drive the true innovation.

Danielle Mackowsky: Sample preparation purists are constantly up against the enemy that is “dilute and shoot”. Within the past year, the tide has begun to turn away from this methodology and back to sample preparation solutions because the required detection limits are becoming lower and lower. Constant replacement of guard and high performance liquid chromatography (HPLC) columns can quickly become a large financial setback for laboratories that have multiple
LC–MS/MS instruments. In addition, the time it can take to clean a source that has been exposed to diluted matrices, such as urine or even plant material, can be detrimental to laboratories up against tight deadlines. Investing in sample preparation on the front end of your sample workflow can pay off dramatically downstream, and many scientists are now starting to re-embrace this mentality.

Paul H. Roberts is the Global Product Manager, Analytical Consumables & Systems at Biotage.

Alicia Douglas Stell is a Senior Scientist, Molecular Sample Preparation Division, at CEM Corporation.

Peter Dawes is the President of Eprep Pty Ltd.

Oliver Lerch is a Senior Application Scientist at Gerstel GmbH & Co. KG.

Matt Brusius is a Product Manager, Sample Preparation at Phenomenex.

Danielle Mackowsky is a Forensic Technical Specialist at UCT.