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What areas are key to providing a solution to these requirements?
TS: Good methods are the starting point, which starts from the sample preparation to the separation science and then the detector. We have focused very much on doing the appropriate sample preparation and apply a lot of Solid Phase Extraction (SPE) to give us the best possible sample clean up and then follow that up by using HPLC with high levels of separation efficiency. We employ a lot of new technology including SOLA SPE and Solid Core Columns.
MO: Tim has made some excellent points. The research we have conducted at Thermo Scientific has demonstrated that most bioanalytical methods are designed around the isolation and quantification of the test analytes, and with little consideration of the matrix components and what effect they can have on the performance of the assay. This work has also shown that inappropriate sample preparation techniques can actually result in build up of residual matrix on the analytical system, which affects not just one sample, but subsequent samples within the analytical batch. This often results in situations where the assay appears to perform well most of the time but falls over in an unpredictable manner.
Within bioanalysis why is sample preparation required?
TS: The biologic samples that we are working with be it blood, plasma, brain or any other biological matrix are a very complex mixture of components. Generally we are looking to analyse extremely low levels of one or more analytes of interest. Therefore, we have to simplify the sample, while retaining the analytes, to allow robust analysis. In many cases we are looking to selectively extract the analyte and concentrate it while removing the rest of the matrix components to allow extremely low level detection. The remove of the unwanted matrix components reduces the background interferences in the assay and also reduces the potential for these interferences to cause significant issues especially when using Mass Spectrometric detection.
MO: The introduction of LCMS to bioanalytical laboratories was seen as a huge advance in detection capability and selectivity, sensitivity and specificity of the technique was seen as a panacea. For the first few years of its routine use in bioanalytical laboratories there was a belief that complex sample preparation and chromatography were not required. This was shown not always to be the case when dealing with low level analysis from complex matrices due to the effects of ion suppression, which is prevalent in atmospheric ionization sources. For these reasons the need for good sample preparation and chromatography to ensure good quality data became apparent.
What does a bioanalyst want or require from their SP technique?
TS: Ease of use is a critical parameter and over the past 15 years the introduction of automated SPE and the use of polymeric phases have been instrumental in the robustness of SPE. Reproducibility is now the key and guaranteeing a product is reproducible well to well, plate to plate and batch to batch guarantees day to day, month to month and year to year analytical robustness. On top of this your sample preparation technique should allow you to selectively capture the analytes of interest whilst actively removing the interferences.
MO: SPE hardware has not changed significantly over the last 20 years (loose packed material positioned between two frits). As a result analysts have had to cope with analytical variability due to bed inconsistencies caused by packing discrepancies and bed instability (caused during production or transportation) which can lead to voiding, channelling and even loss of stationary phase, which ultimately results in a lack of robustness. Solving these issues has focused our research activities when developing our innovative SPE technology, SOLA which overcomes these problems and results in greater robustness and ease of use. Coupled to this is the availability of a range of different chemistries which allows the analyst choice when optimizing the extraction process to best remove interferences and selectively capturing the analyte of interest.
What technologies are currently employed to facilitate sample preparation within bioanalysis?
TS: The main techniques of sample preparation routinely employed for bioanalysis are; Solid Phase Extraction (SPE), Liquid Liquid Extraction (LLE), and Protein Precipitation (PPT). The are a few others that are not as main stream but are continuing to gain popularity including, supported liquid extraction (SLE), online techniques which includes turbulent flow and online SPE and recently the use of immunoaffinity techniques are starting to become more common either using columns or bead based techniques.
MO: The points that Tim has made are very good. Although SPE does have a wide range of selectivity’s it is important for the bioanalyst to have a wide range of analytical techniques at their disposal, since SPE may not be applicable in all cases and indeed the research we have been performing at Thermo Scientific, has demonstrated that for certain compounds different approaches other than SPE provide optimal performance either due to compound stability and solubility issues.
How do these technologies currently meet the requirement of the bioanalyst?
TS: PPT is a great technique if you can afford to live with the limitations in sample clean up, but for a lot of early stage work this is more than acceptable. Implementation of automation using PPT plates which have additional functionality to selectively remove specific interferences is a step forward and also allows easier automation. However, they still have limitations on the separation the can be achieved. LLE can be a very good technique if the assays are appropriately set up but it is very labour intensive and time consuming. I remember working 10 hour days doing a single batch of LLE samples and it was hard work. SLE has allowed for miniaturization and automation of this technique making it far easier to implement. SPE is in my view the best sample clean up in terms of automation and separation but it comes at a cost. The technique can be significantly more expensive when compared to PPT or LLE and the optimisation of the methods can take longer (although probably should not!). SPE gives the best opportunity for sample clean up and concentration and if done correctly will give the most robust assays that achieve the lowest limits of quantification.
MO: Tim again makes some good points. There are a number of options for sample clean up and choice of the one that fits the analysis best is critical to the success of the assay. In some cases, whilst clean up is not necessarily optimal PPT can be the best choice, for its ease of use and speed. Where, for an assay requiring ultimate sensitivity and reproducibility SPE would be the most appropriate technique.
Why do these technologies currently fail to meet the desired standard for the bioanalyst?
TS: Normally assays fall down due to the unknown, for example we get a matrix effect in samples that was not observed during the assay development or validation. This is commonly observed when using PPT, but can often be avoided when using SPE. We anticipate that a certain percentage of batches will not meet acceptance criteria and we put this down to the difficulty of the samples we are handling. However, working with more robust technology enables us to drive this percentage down and improving the overall robustness of the assay. Automation is still a major limitation and while we can semi-automate the extraction, especially with SPE, this often presents a major challenge. These issues are normally linked to the hardware rather than the technique, for example opening tubes, reading labels and handling poor samples, for example dealing with blood clots. As we are moving to new analyte classes especially peptides and proteins, the problems we face are very different to small molecule analytes and we are being challenged to develop new, more appropriate sample preparation techniques.
MO: Anticipating where the next analytical challenges will be is a major focus for us. We see the step change in reproducibility requirements and the move towards lower sample volumes, lower limits of detection and more complex molecules as the major areas of interest over the next few years. SOLA’s unique technology is already providing considerable benefits with higher levels of reproducibility combined with the ability to provide lower elution volumes.
Why are these factors so critical to the workflow and are these demands likely to become more important in the future?
TS: Timelines are becoming more important every day, everyone wants to go faster and this puts stress on the bioanalytical workflow. Improvements in robustness allow us to be more accurate in our planning as we need less contingency and we can produce data faster to meet customer demands. The current drive to reduce sample volume has lead to a move towards dried blood spots (DBS) and capillary microsampling (CMS) which gives the bioanalyst much smaller sample from which to generate the same high quality data. The use of more rigorous sample clean up and high sensitivity methods are crucial in allowing this to happen. Data quality is another major driver and the introduction of the Incurred Sample Reanalysis (ISR) has lead to greater focus on this area. The only way to reduce risk when you analyze your study samples and guarantee that you are not going to have an issue with the reproducibility is to do as much separation as possible (sample preparation and chromatography) to remove as many unknowns as possible.
MO: Tim makes 2 very salient points relating to assay robustness and sample volume. These are important issues that the modern bioanalyst faces on a day to day basis. These can be addressed by using robust technologies which are applicable to smaller sample volumes. Solid phase extraction technologies have moved some way to providing a solution, however issues with the inherent design of loose packed SPE media can result in variability in reproducibility of the assay.
Is there anything currently available which addresses these needs?
TS: Sample preparation is always moving forward, be it from the introduction of polymeric materials for SPE to the newer materials which are giving better separation and control of the sample clean up process e.g. SOLA. Chromatography is also an ever changing landscape and over the last 10 years we have seen the advent of ultra high pressure liquid chromatography, small particle sizes and more recently the solid core particles e.g. Accucore.
MO: The technology associated with SOLA SPE ensures that sample to sample robustness is assured, while also providing lower elution volumes and thus enabling the use of lower sample volumes. This is achieved by combining the polymeric frit material and stationary phase into a single entity, rather than an assembled form consistent of powder and separate frits as in conventional loose packed SPE. The conventional SPE format is prone to voiding, channelling and inconsistencies in packing, which is accentuated at lower bed weights. The manufacturing process associated with SOLA’s unique design ensures that consistent bed weight, bed size are achieved even when subjected with physical stresses. This results in providing a solution which addresses the issues that Tim mentions.
What impact has this new technology on the bioanalytical workflow?
TS: We have been able to develop more robust methods in a shorter time. The analysis is also done in shorter time but also with less problems. We can increase sensitivity using new SPE, HPLC and detector technology and this allows us to implement both DBS and CMS samples within our company enabling us to refine and reduce our animal use.
MO: The comments that Tim makes are very encouraging, in developing this new technology we have conducted extensive in house testing which supports Tim’s experiences within a leading high throughput bioanalytical laboratory. From feedback from our customers they are seeing a step chance in performance, robustness and quality of their results since implementing this novel technology.
Do you ever envisage a time when advancements in LCMS instrumentation will remove the need for SP? For example direct analysis, especially in conjugation with MS and tissue bioanalysis, shows promising potential, but, in your opinion, will some sort of sample preparation/extraction always be required?
TS: While in some situations the use of direct analysis is feasible, bioanalysis will always be subject to the limitations of matrix effects. So for quantification in a regulated environment the use of some form of sample clean up will be required for the foreseeable future. In qualitative work the use of high resolution instrumentation (or other developments) may allow the analysis with limited or no separation but the risks of slight changes in the sample matrix impacting any quantification will for the meantime result in us continuing to do significant amounts of separation chemistry. During the advances over the past 20 years there have been a couple of times when the death of sample preparation has been heralded only for us to then find many reasons why we should continue to do good, fit for purpose sample clean up prior to analysis.
MO: I agree with Tim’s statements. Sample preparation not only provides reduced interferences within the Mass Spectrometer, but also reduces the amount of maintenance associated with a particular assay. Finally the use of sample preparation allows for pre-concentration of the sample which provides increased sensitivity. Whilst a radical new design in the source of an MS may reduce the amount of ion suppression this will not address other issues associated with contamination of the sampling device and the requirement for the development of more sensitive assays.
What do you see as the future for SP within bioanalysis, new innovations are continually being developed, but what sample preparation/extraction technology would you like to see in the future?
TS: Continued developments in handling smaller sample volumes in well plate or tip format are important. These techniques need to be backed up by consumables for handling the sample from collection to analysis in a 96 well or higher format. I believe these have not become main stream techniques due to limitations in the format and ease of use issues. Automation and software that can handle the whole process from sample arrival to reported sample results, encompassing automated sample preparation, sample analysis and data reporting is crucial. Large molecules are a ‘relatively’ new area for chromatographic based regulated bioanalyitcal methods, especially with a mass spectrometry end point. Developments in this area to support the process would be nice to see. Easier integration of immunoaffinity analysis into the workflow so that we can utilise this more easily and cost effectively would also be a nice to have.
MO: There are 3 key areas for future development of sample preparation. As Tim has alluded to more selective phases will ensure less matrix effects are observed, providing the bioanalyst with more robust assays. The development of the bio-pharmaceutical industry has resulted in many new challenges in the field of bioanalysis. Large bio-molecules are incredibly complex and ensuring that the sample preparation is selective will be a major challenge for the industry. Finally there is the question of dried blood spots, and the haematocrit issue which requires a solution.
Of growing importance is ‘green’ bioanalysis and bioanalytical chemistry, but what are the main implications of this in sample preparation and extraction?
TS: Reduction of solvent usage is probably the biggest driver and this can be achieved by using low bed volume SPE. This also then follows through to the HPLC, if you run at 100ul/min instead of 1ml/min you can reduce solvent use by 10 fold. I remember when the first dedicated online SPE systems were released and we were told that you could re-use the cartridge 20 times! Can we come up with an SPE plate we can reuse to reduce wastage and decrease costs but without any risks?
MO: With the advent of new sample preparation technologies there has been a reduction in the requirements for organic solvent. As the industry drives towards even lower sample volumes manufactures will be tasked to provide solutions which will address these challenges. This will result in greener assays and analysis.