60 May / June 2019
With Great Power Comes Great Responsibility
One of the challenges of an analytical scientist is to ensure that the developed assays are robust, and so analysing the same sample should give the same result. In this article we will look at how the use of electrospray can result in a large variability in the detector response, and how the root cause of this variability is associated with the sample preparation and the chromatography.
The advent of atmospheric pressure ionizsation (API) sources allows the coupling of high performance liquid chromatography (HPLC) to mass spectrometry (MS) [1], which has resulted in assays having lower detection limits and a greater degree of specificity. Other detector types also use an electrospray type interface such as Evaporative Light Scattering Detector (ELSD) and Charged Aerosol Detector (CAD). Electrospray ionisation (ESI) is one of the most widely used LC/MS interfaces and has applications in the analyses of a variety of different analytes such as characterisation of peptides and proteins [2,3,4], screening of drugs and steroids [5,6,7] and detection and quantification of residual pesticides in food [6,8,9,10]. The broad range of applications is due to the compatibility of the technology to a wide range of organic molecules.
Within the field of bioanalysis the use of mass spectrometry coupled to liquid chromatography has become mainstream. The initial introduction of this technology was seen as resolving many, if not all of the issues that were associated with the analysis of compounds from a biological fluid. Approaches to sample preparation were significantly simplified, with terminology such as “‘dilute and shoot” ’ being introduced into the analytical scientist vocabulary. However it was soon found that this approach also came with its own challenges which need to be addressed, notably;
• Robustness of the system Blocked columns Blocked syringes
Precipitation of the sample causing blockages
• Carryover • Ion suppression • Sensitivity issues
The addition of sample preparation and a chromatographic separation to the work flow ensures that all of these issues are addressed to a greater or lesser extent depending on the quality of the separation and also the sample preparation that is utilised.
The sample preparation is usually the most important aspect of the whole workflow, however typically it will involve the least amount of effort to optimise this part of the process. Sample preparation is often seen as something where generic approaches are employed and does not require the level of skill that is associated with the operation of the detection and chromatographic part of the process. However, since the analytical technique is dependent on the quality of the sample that is being analysed, any deficiencies that occur within the
sample preparation will be transferred into the final analysis.
The sample preparation starts with the collection of the sample to ensure that the sample is representative of what is being measured. In most cases within a bioanalytical laboratory, the sample will be a biological fluid, which means that any sampling is highly likely to be representative due to the nature of the fluid flow within a biological system.
Subsequent to the sampling, the sample is then stored, and the nature of the storage container needs to be considered carefully to ensure that sample is not lost or modified. This is particularly important for larger molecules where the possible interactions is substantially increased compared to smaller molecules where the possible interactions may be limited due to the size of the molecule. It should also be noted that the configurational arrangement of a large molecule can be influenced by the storage vessel, thus proteins can denature in the presence of a hydrophobic surface and so consequently it is important to understand the nature of the analyte and also perform a series of simple experiments to determine if there are any issues at this stage of the sample preparation. These experiments would be as simple as monitoring the concentration levels over a period of time and also monitoring in different storage vessels to determine if there is any temporal differences in the observed concentration.
Once the sample storage has been addressed, the next aspect of the sample preparation to consider is how to remove the matrix components. Typically for a biological fluid this will focus on the removal of proteinaceous components and also looking at endogenous salts etc.
The final stage of the workflow is the combination of the chromatography and detection techniques. This article will focus on the LC-MS as matrix effects do cause significant effects using this technology. The impact that the initial sample preparation has on the sample analysis can be very subtle and in certain situations will not be easily identified.
Typically, the separation is optimised for the analyte molecules and this can result in some interesting issues that would not occur when using less selective detection technology, where co-eluting peaks can be clearly seen, and late eluting peaks can also be readily seen. This results in a degree of variability in the detector response due to suppression issues and varying retention times of matrix components potentially impacting the signal observed from the same sample.
In LC-MS there is an assumption that the injected sample is fully eluted from the column, or is irreversibly retained on the column, however when dealing with very complex samples this assumption
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