Solid-Phase Microextraction in Clinical Diagnostics

July 24, 2015
Bethany Degg

LCGC Associate Editor

The Column

The Column, The Column-07-24-2015, Volume 11, Issue 13
Pages: 12–14

Researchers at the University of Waterloo in Canada are collaborating with clinicians at Toronto General Hospital to develop preclinical and clinical applications of solid-phase microextraction (SPME). Bethany Degg of The Column spoke to Barbara Bojko from the team to find out more.

Researchers at the University of Waterloo in Canada are collaborating with clinicians at Toronto General Hospital to develop preclinical and clinical applications of solid-phase microextraction (SPME). Bethany Degg of The Column spoke to Barbara Bojko from the team to find out more.

 

Q. What are your main research interests at present?
A:
My professional background is in clinical laboratory diagnostics, and so my research interests have always centred on clinical applications. I am currently a member of Professor Pawliszyn’s research group, the inventor of solid-phase microextraction (SPME). My main investigations are focused on new clinical application of this technology and the one that caught my attention was in vivo tissue analysis.

Q. What are the advantages of solid-phase microextraction (SPME) over other sampling methods in bioanalysis?
A:
First of all, we need to remember that a single technique cannot be a panacea and solve all the problems we face in bioanalysis. As Professor Pawliszyn always emphasizes: “SPME is another tool in the toolbox” and we should know where and how to use it.

From my perspective, the most important feature of SPME that makes it a superior technique for clinical and especially in vivo analysis is its minimal invasiveness. As mentioned a number of times in our articles, there are a very limited number of analytical approaches that permit such studies and standard methods based on biopsy are too invasive to be used in applications requiring repeated analysis.

The other advantage is that we are very flexible in terms of the chemistry of the coatings, so we can cover a very wide range of analytes or we can use a selective coating to target specific analytes (for example, a drug or biomarker). We can also provide fully quantitative results, which allow us to use SPME for applications such as pharmacokinetics, or we can evaluate compound distribution in the organ(s) when using several probes simultaneously without causing damage to the tissues. In addition, when coupling to mass spectrometry (MS) we very rarely see the matrix effect.

Q. You recently developed a low invasive in vivo tissue sampling method for monitoring biomarkers and drugs during surgery. How did this come about?

A: We have been developing low invasive simple tools (SPME devices) based on approximately 100 micrometre diameter flexible wires coated by a thin layer of sorbent material with biocompatible morphology and applying it to a range of applications, including environmental, food, and pharmaceutical analysis. We have determined that this approach could also be effective in the clinical environment because this wire can be placed into living tissue to sample small molecules that can be removed and transferred to analytical instrument for characterization. Historically, the platform of choice for SPME was gas chromatography (GC) or gas chromatography coupled with mass spectrometry (GC–MS) because the method was mainly used for sampling volatile compounds. Nowadays, especially for the clinical and pharmaceutical applications, we use liquid chromatography–mass spectrometry (LC–MS) systems to analyze non- and semi-volatiles in biological matrices.

We’ve been collaborating with a number of clinicians from different specializations for the past few years, which has resulted in many interesting applications of SPME. Tissue analysis is particularly important because standard sample preparation protocols are very tedious and require collection of the biopsy, which makes them highly invasive when it comes to analysis of living systems. This particular application of SPME was used to evaluate organs for transplantation, and was a perfect case for us to demonstrate unique features of the method, such as low invasiveness and the possibility of repetitive analysis, thus its applicability to monitoring changes of biochemical profile of grafts in time.1 Our collaborators from Toronto General Hospital work on novel methods for organ preservation, which permits extending their lifetime and improves their performance, and aims to increase the pool of organs available for transplantation. Our joint research gives us a chance to better understand changes occurring in the organs during their preservation and to select the ones with the lowest risk of rejection.

Surprisingly, current evaluation of the organs, which could potentially be used for transplantation, is very limited, based mainly on general and non-specific tests and visual assessment. Therefore, information about the entire biochemistry of the grafts will definitely improve this process and will allow specific biomarkers to be selected; these could be determined within a few minutes, and allow clinicians to make an immediate decision on the usability of the organ, or its condition and level of damage.

Q. What were your main conclusions from this study?
A:
Initially, part of our work was focused on optimizing the sampling procedure and validating the practical aspects of the intervention because, although it is minimally invasive, it is still direct sampling of a living system. However, after receiving positive feedback from the surgeons regarding the sampling step itself we were ready to evaluate the analytical part of the studies. In this instance this meant determining what compounds we are able to extract, what type and size of the probes we should use, what time of exposure is optimum for our studies, and so on.2 In the end, we used an optimized protocol for analysis of a few pig models. We were able to extract analytes with a wide polarity range, starting from amino acids, sugars up to lipids, which are involved in many metabolic pathways. We have already characterized the molecules we may capture with the coating we used here in more detail in several other matrices, and the information can be found in our previous papers.3–7

Our results demonstrated that we are able to monitor differences between “healthy” and “injured” organs as well as identify progressing changes during the time of preservation of the organs and, subsequently, after their transplantation and reperfusion. For example, we found the difference in the profile of some compounds involved in Krebs cycle when we compared liver subjected to warm ischemia and control liver. The changes suggested impairment of enzymes in the injured livers. The “nice” finding was that monitoring the metabolism of one of the drugs routinely used during this medical procedure showed inhibition of the metabolism of the parent drug, which confirmed the assumption regarding the enzyme function made based on the metabolomics study. Of course, finding the selective biomarkers and drawing the clinically relevant conclusions requires performing studies on a large number of individuals - we have to first learn how to walk before we start running!

 

Q. What developments are needed for the sampling technique to be used in the clinic?
A:
We have demonstrated that an “acupuncture-sized” flexible wire coated with appropriate sorbent is a suitable medium to extract biomarkers from the living tissue. Now we need to make an easy-to-handle tool for medical personnel. The more applications we develop and the more experience we gain in this field the more ideas we will have on what should be improved to meet the expectations of clinicians: the future users of the method. Currently, we are working on a new tissue sampler, which could be easily used by the surgeon during the medical intervention.

Also, we still push the limits of the size of the device to minimize the invasiveness of the sampling. Of course, such miniaturization of the devices results in a lower recovery of the extracted compounds, thus more sensitive instruments needs to be used for the detection. However, today’s technologies meet this expectation, making the microextraction methods great alternatives to standard approaches. The manufacturers of the analytical platforms tune the sensitivity and decrease the amount of sample needed for the analysis and this is all in favour of “micro” methods. Moreover, current trends lead towards direct introduction of samples/extracts to detectors such as mass spectrometers with no prior chromatographic separation and SPME is a perfect tool to assist in such a coupling. Such a hyphenated rapid diagnostic tool can provide immediate, precise, accurate and reproducible results.8Q. How do you see this technology developing?
A:
I’m just hoping that our work can be translated to the use of this technology globally. It is difficult to introduce a novel analytical approach for daily use because it not only involves practical issues, such as going through the regulatory processes and financial involvement of medical institutions to set up a new method in their facilities, but also overcoming some mental barriers of the everyday users: the physicians or technicians who simply get used to standard approaches and most of them don’t see the need of replacing “good enough with better”. But on the other hand, we are looking for the niche in clinical analysis where SPME could fit and so far the response we have received from our collaborators is promising, so maybe we can make a case and SPME will become an established technology in the clinical environment.

References

  1. B. Bojko et al., Laboratory Investigation94, 586–594 (2014).
  2. B. Bojko et al., Anal Chim Acta 803, 75– 81(2013).
  3. B. Bojko et al., Trends in Analytical Chemistry 61, 168–180 (2014).
  4. D. Vuckovic et al., Angewandte Chemie International Edition50, 5618–5628 (2011).
  5. D. Vuckovic et al., Angewandte Chemie International Edition 123, 5456 –5460 (2011).
  6. V. Bessonneau et al., Bioanalysis 5, 783–792 (2013).
  7. B. Bojko et al., Journal of Pharmaceutical Analysis 4, 6–13 (2014).
  8. G. Gomez-Rios and J. Pawliszyn, Chem. Commun. 50, 12937–12940 (2014)

 

Dr. Barbara Bojko is an Associate Professor at the Department of Pharmacodynamics and Molecular Pharmacology, Collegium Medicum Nicolaus Copernicus University (Poland) and Research Associate in Prof. Pawliszyn’s group at the Department of Chemistry, University of Waterloo (Canada). Previously she was an Assistant Professor at the Medical University of Silesia, Poland. In 2001 she obtained her medical diagnostician diploma and masters degree; in 2005 her PhD (Hon) in pharmaceutical sciences; and in 2014 she obtained her Doctor of Science degree. She is an author of over 60 publications in peer-reviewed journals and two book chapters. She was awarded for her scientific achievements by the Minister of Health (2004, 2007, and 2011), the President of Medical University of Silesia (2009, 2010, and 2012), and the President of Nicolaus Copernicus University (2014).

E-mail:    bbojko@uwaterloo.ca
Website:    https://uwaterloo.ca/pawliszyn-group/about/people/bbojko

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