Trends in Sample Preparation

Taking a look at the main trends in sample prep
Taking a look at the main trends in sample prep

An Updated Perspective on Today’s Needs and Tomorrow’s Opportunities.

“Sample Preparation” is a broad term, one that can be used in many contexts and applications. In fact, at a recent seminar, which focused on “sample preparation”, it was clear that there were nearly as many definitions of sample preparation as there were presenters and attendees.

For the sake of this article, then, sample preparation is defined as the preparation of samples containing known or unknown analytes for analysis using gas (GC) or liquid chromatography (LC) with detection by mass spectrometry (MS), tandem mass spectrometry (MS-MS), conventional detectors, or a combination of these.

In addition, the focus will be on sample preparation techniques designed for applications covering “small molecules,” leaving for future discussion those opportunities that lie within the fields of protein and peptide analysis.

Sample Preparation Fundamentals for Chromatography1 is an excellent reference for the broader range of sample preparation techniques and application areas that has been recently released. This new handbook provides an extensive discussion on options, use, and principles of sample preparation for chromatography, and is a valuable guide for today’s scientists facing many of the challenges encountered when dealing with sample preparation, based on a survey of LCGC readers conducted in early January 2013.2

There are four main contributors to today’s sample preparation trends:

  1. Instrument evolution
  2. Sample Preparation Miniaturization
  3. Changing Regulatory Environment
  4. Ongoing economic pressure

Each of these will be discussed in the context of sample preparation, with examples provided to highlight how sample preparation approaches adapt to each challenge and change.

Instrument Evolution

Today’s analytical instrumentation offers chemists exciting performance advantages in terms of specificity, acquisition rate, and sensitivity. Gas and liquid chromatography columns and systems support short separation cycles, considerably reducing analysis times and increasing throughput. Narrow-bore GC columns and small-particle HPLC columns offer high resolution, narrow peak widths and fast run times.

Detectors have evolved with these improved capabilities, becoming faster to ensure characterization of peaks with increasing selectivity, allowing for deconvolution of signals that may overlap or co-elute in the shorter analytical run time. These changes in instrumentation also require changes in the sample preparation approaches in order to support these capabilities.

One consideration is the analytical run time. With short runs, the instrumental analysis is no longer the bottleneck in reporting a sample. Instead, the time needed for sample preparation may be the largest contributor to the overall time it takes to complete a sample analysis. This results in a need for sample prep approaches that are shorter, less labour-intensive and more streamlined. An example of this type of adaptation is the QuEChERS technique. QuEChERS, which stands for Quick, Easy, Cheap, Effective, Rugged, and Safe, is an approach to sample preparation that incorporates a salting-out partitioning step plus a cleanup step using dispersive solid phase extraction (dSPE).

This provides an extract that is sufficiently clean for instrumental analysis, particularly in conjunction with tandem mass spectrometry, which delivers a higher level of selectivity. While QuEChERS was originally developed in 2003 for the extraction of pesticide residues from fruits and vegetables,3 the approach has since been found to be amenable to a wider range of target compounds and sample types than originally intended. As such, QuEChERS approaches are applied to applications and compound classes in a wider range of markets.

This trend in sample preparation is highlighted by several recent publications. In one study, the QuEChERS approach was used to prepare whole blood samples for the analysis of pharmaceuticals.4 Whole blood samples were prepared following the QuEChERS extraction/ partition and dSPE cleanup methods, evaluating the effects of different salts and cleanup sorbents, as well as the selection of an appropriate acidification step during the extraction. The study results, including comments on sample cleanliness, are summarized in Table 1.

Table 1 Summary of the method development study for a mini-QuEChERS approach to pharmaceutical analysis in whole blood.
Table 1 Summary of the method development study for a mini-QuEChERS approach to pharmaceutical analysis in whole blood

Following extraction and cleanup, the combination of salts and dSPE selection were compared for final extract cleanliness from a visible perspective, and all combinations resulted in a final extract that was clear and suitable for LC-MS/MS analysis (Figure 1). Good recoveries and precision were demonstrated, and the chromatography shows a fast run time with good peak shape and lack of interference at the 10 ng/mL concentration (Figure 2).

Figure 1 Final extract cleanliness, using a combination of extraction salts and dSPE cleanup steps
Figure 1 Final extract cleanliness, using a combination of extraction salts and dSPE cleanup steps

Figure 2 LC-MS/MS chromatogram of a 10 ng/mL spiked whole blood sample, following extraction with AOAC acetate salts and cleanup using 50 mg PSA sorbent plus 150 mg MgSO 4
Figure 2 LC-MS/MS chromatogram of a 10 ng/mL spiked whole blood sample, following extraction with AOAC acetate salts and cleanup using 50 mg PSA sorbent plus 150 mg MgSO 4

The QuEChERS approach and protocol has also been applied to other food types, such as meats, teas, and fish.5,6,7 Work continues to be published using QuEChERS as scientists see the value of this methodology because it addresses important pain points in the lab in terms of cost, throughput and simplicity. By providing a final extract that is cleaner than simple dilution would yield, while minimizing steps and reducing the use of more toxic reagents and solvents, QuEChERS is expected to continue to grow, particularly as a complement to today’s analytical instruments.

Sample Preparation Miniaturization

Just as the added selectivity of tandem MS instrumentation supports simplified sample preparation approaches such as QuEChERS and dilute-and-shoot, the increasing sensitivity of today’s instruments also support a trend toward miniaturization of the entire sample preparation workflow. This includes collection of smaller sample sizes at the beginning and extends to techniques that are tailored to this small sample size. These include online solid phase extraction (online SPE), single-drop microextraction (SDME), and functionalized magnetic particles, among other miniaturized techniques. Nanoparticles are also an intriguing area of development, offering an approach to sample preparation that allows for a high level of selectivity while being compatible with a smaller sample size.8

An example of how instrument sensitivity and selectivity allows for reduction in sample size can be seen in the analysis of organic contaminants in environmental water samples. Traditional analysis of contaminants in water requires collection and extraction of 1 L of water.9 For the environmental lab, this sample size results in additional costs for logistics, storage, and preparation. Using online SPE, or even direct injection, the collected sample size can be reduced by 90 per cent or more.10 Similar reductions in sample size can be achieved in bio- analysis when using dried matrix or dried blood spots in combination with microextraction techniques.11

A study of herbicides in surface water using online SPE coupled with liquid chromatography and tandem mass spectrometry (LC-MS/ MS) demonstrates the power of pairing reduced sample size with more sensitive instrumentation.10 Selected herbicides were analyzed at low ppt (ng/L) levels in a variety of water sources, using a 900 μL injection volume and online SPE. For online SPE, the sample is applied to a cartridge containing sorbent, in this case PLRPs polymeric SPE, allowing the sample to pass through to waste while collecting analytes on the sorbent. The online SPE cartridge is positioned in the system such that the valve controls can direct sample flow through the cartridge. Following sample application, flow is reversed through the SPE cartridge to elute the sample, focused as a tight band, onto the LC analytical column, where separation occurs. Detection using multiple reaction monitoring (MRM) with two or more transitions acquired per target compound enables sensitive, selective detection, with resulting limits of detection determined to be 0.1 ppt or lower for the compounds tested. Figure 3 shows the reconstructed ion chromatogram for the target herbicides at 0.1 ppt using the MRM method.

Figure 3 Chromatogram of a water sample spiked at 0.1 ppt with selected herbicides, analyzed using online sPe and lc-ms/ms
Figure 3 Chromatogram of a water sample spiked at 0.1 ppt with selected herbicides, analyzed using online sPe and lc-ms/ms

Changing Regulatory Requirements

As the instrumentation available to scientists becomes more sensitive, regulations in countries and regions adapt accordingly, particularly as more becomes known about the effects on biological systems of even very low concentrations of active compounds. Keeping up with these changing regulations requires scientists working in environmental, food, and similar types of industries to adapt by deploying new instrumentation, incorporating more selective sample preparation techniques, or a combination thereof. In food analysis, another complication may be the need to analyze according to a range of reporting limits, based on differences in import and export regulations.

An example of this is in the regulation of hormones in meats and meat products. Different countries and regions have different regulatory limits and exclusions on hormones that may be used in the production of meat. Being able to address these changing requirements requires an analytical method that offers the flexibility to prepare samples for a wide range of compounds, combined with an instrument capable of delivering the required sensitivity.

Solid phase extraction (SPE) is a sample preparation method with a long history of use, one which continues to be relevant even as sample prep trends expand into other techniques. Because of the effectiveness at sample cleanup, ease of use, and ability to have a generic methodology, SPE remains a relevant and viable option for sample preparation.2

Even as trends in sample preparation turn to novel sorbents, formats, or alternate techniques, SPE continues to grow. A recent application demonstrated the utility of SPE for the analysis of hormone residues in pork.12 This application is a good example of the challenge of meeting regulatory requirements that change or vary from region to region, and the sample preparation method using SPE offers a simple, effective cleanup step that is amenable to an analytical method that can meet a broad range of requirements. This flexibility is an important consideration when working in regulated environments.

Continued Economic Pressure

An important macroeconomic trend in the analytical world is the continued economic challenge faced in many regions. This challenge to continue to deliver high-quality analytical results while also carefully balancing the level of investment into the workflow puts considerable pressure on the sample preparation steps. These steps in the workflow are typically the largest contributors to the overall cost of analysis, in terms of time, labour and materials.1 Looking for opportunities to reduce this cost component results in changing sample preparation methods, deploying alternate techniques, and in some cases cases, completely changing the analytical approach. An example of this may be converting a method that uses GCMS with extraction and sample derivatization over to an LC-MS method that uses a simple “dilute-and-shoot” approach without derivatization.

A recent review by Deventer, Pozo, Verstraete, and Van Eenoo evaluated dilute-and-shoot LC-MS/MS methods, or DS-LC-MS, as the authors termed this process, in the context of testing urine for sports doping applications.13 Of the studies included in the review, less than 40 per cent of the methods used were simply dilute-and-shoot.

For a large majority of the studies reviewed, an additional cleanup step was incorporated, such as centrifugation, filtration, or a combination of centrifugation plus filtration. The authors note specific challenges that result from the dilute-and-shoot approach, namely issues with particulates, ion suppression or enhancement, and elution of salts affecting reproducibility of early eluting compounds, depending on the LC conditions.

Clearly, while dilute-and-shoot with LC-MS is an option for reducing costs, this route needs to be evaluated for unintended consequences that may affect method ruggedness or overall cost of analysis. Increased consumption of LC columns due to blockage or contamination, increased frequency of maintenance to the mass spectrometer or HPLC or UHPLC system, and or a higher rate of sample repeats due to matrix can all increase costs and decrease productivity, working against the intended goal.

Conclusions

There are many factors that affect sample preparation in today’s analytical labs. This review focused on just of a few of those factors. This area within our workflows lays the foundation for success, and as such, scientists continue to develop new approaches, to apply new techniques to current products, and to figure out how to be successful within their area of research. Exploiting the exciting developments in analytical instrumentation to reach lower detection limits, achieve higher throughput and make new discoveries leads to opportunities to evaluate sample preparation approaches. Taking even more advantage of increased instrument capabilities allows miniaturization of the sample prep approaches, including collecting, transporting and using less sample. Keeping up with regulations may put pressure on the entire workflow, from validation to instrument selection to sample preparation methodologies. Finally, meeting the day-to-day challenges of working in the lab while also trying to do more with less provides opportunities to streamline or completely change sample preparation workflows to reduce cost, while maintaining the quality of results. Finding the right balance may require trial and error, but there are resources available to help find the best approach.

Trisa Robarge, product manager for Agilent Technologies sample preparation products has over 20 years of experience with sample preparation, gas chromatography, and mass spectrometry. She has a Bachelor of Science in Biology with an emphasis on pre-medicine and biochemistry and an MBA in Marketing.

References

1. Majors, R. Sample Preparation Fundamentals for Chromatography. www.agilent.com/chem/SamplePrepBook.

2. Majors, R. LCGC North America. Volume 31, Issue 3, pp. 120-203. (2013).

3. M. Anastassiades, S.J. Lehotay, D. Štajnbaher, and F.J. Schenk, J. AOAC Int. 86, 4121 (2003).

4. Stevens, J. http://www.chem.agilent.com/Library/applications/5990-8789EN.pdf, retrieved December 30, 2013.

5. Wozniak, B., Zuchowska, I.M., Zmudzki, J. Journal of Chromatography B, Volume 940, 1 December 2013, pp. 15-23.

6. Zhai, C.H., http://www.chem.agilent.com/Library/applications/5990-6400EN.pdf, retrieved January 7, 2014

7. Chang, M., https://www.chem.agilent.com/Library/applications/5991-2408EN.pdf, retrieved January 6, 2014.

8. Tian, J. Xu, J., Zhu, F., Lu, T, Su, C, Ouyang, G. Journal of Chromatography A, Vol. 1300, pp. 2–16 (2013).

9. United States EPA Method 524.3. Determination of Semivolatile organic chemicals in drinking water by solid phase extraction and capillary column gas chromatography/mass spectrometry (GC/MS). EPA Document #: EPA/600/R-12/010. Version 1.0. February, 2012.

10. Mohsin, S.B., Woodman, M. http://www.chem.agilent.com/Library/applications/5991-2731EN.pdf. Retrieved January 2, 2014.

11. Arora, R. Hudson, W., Boguszewski, P. http://www.chem.agilent.com/Library/applications/5990-9929EN.pdf. Retrieved January 2, 2014

12. Zhai, C.H. http://www.chem.agilent.com/Library/applications/5991-3660EN.pdf. Retrieved January 6, 2014.

13. Deventer, K., Pozo, O.J., Verstraete, A.G., Van Eenoo, P. Trends in Analytical Chemistry, Vol. 55, pp. 1-13 (2014).

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