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Chromatography Today Help Desk Challenges with Sample Preparation
The introduction of liquid chromatography mass spectrometry (LC-MS) into the analytical laboratory has transformed the ability to identify and quantify compounds at low concentrations. Initially scientists had thought that the use of this technology, which allowed for much greater specificity, would eliminate the need for any sample preparation, and the concept of dilute and shoot was readily applied to a range of samples. It was very evident that this approach has limited applicability in disciplines which require quantitive analysis as the detected levels for the same concentration of sample could vary substantially depending on the nature of the matrix components. The explanation for the variability is due to the ionisation process, which can be greatly affected by co-eluting components, or indeed the analyte itself since mass spectrometers have a limited concentration range over which they give a linear response as a function of analyte concentration. The use of sample preparation can reduce or even eliminate co-eluting species derived from the matrix which will also reduce the suppression effects caused by matrix components.
However, even when some form of sample preparation is performed, the matrix can still affect the ionisation efficiency and the performance of an assay. So called ‘matrix effects’ [1-3] are well recognised for their potential to distort the analytical data, the use of appropriate sample preparation or chromatography, however where the sample matrix varies the analyst can never be truly confident, and in this scenario the use of isotopically labelled internal standards can provide greater levels of assurance to the assay. These matrix effects arise because of the complexity of the matrix, which for a biological fluid, can contain several tens of thousands of different compounds with a very wide range (>107
) of concentrations [4]. Each
of the endogenous compounds can, and does, vary from sample to sample [5]. Many of these compounds will interfere with the analyte ionisation process which results in them either;
• competing for the available charge in the ion source of the mass spectrometer [6]
• enhancement of the ionisation capabilities of other compounds [7] • reduction in solvent evaporation [8]
There are also other processes, including space charge effects, micelle formation and gas phase interactions [9] that exist and can also cause variable responses from the mass spectrometer.
The variability in matrix composition potentially means that the degree of ionisation will vary from one sample to another with possible adverse effects on the analysis of target analytes. Therefore; it is critical that the compound is resolved from any endogenous materials that produce matrix effects in order to reduce or eliminate ion suppression within the mass spectrometer source. This can be achieved either through the initial sample preparation or by the final chromatographic separation to eliminate co-elution of the matrix component and the analyte. It should be noted that in biological samples which contain tens of thousands of matrix components this will be challenging to say the least.
An interesting observation is the variability of analyte response that
can be observed with the same sample and the helpdesk will look at what can cause this issue. The introduction of Incurred Sample Reanalysis (ISR) [10] as part of the validation criteria in 2009 has resulted in this issue having much greater significance and as such is a necessary component of bioanalytical method validation. ISR is intended to verify the reliability of the reported subject sample analyte concentrations and is conducted by repeating the analysis of a subset of subject samples from a given study in separate runs on different days to critically support the precision and accuracy measurements established with spiked QCs; the original and repeat analysis is conducted using the same bioanalytical method procedures.
Repeating the analysis on the same sample can potentially highlight when there is an issue with the assay. There are a variety of reasons that could cause the assay not to give the same result, some pertaining to the sample stability and some relating to the performance of the assay. If the sample deteriorates over a period of time, then the assay performance should pick this up. This article will, however, focus on sample preparation issues that can affect the assay stability.
Sample Preparation
Within many bioanalytical laboratories, the typical workflow will be to perform some form of sample preparation followed by a LC-MS/ MS based analysis. There are a range of different sample preparation techniques that can be employed including dilution, protein precipitation, liquid-liquid extraction, and solid phase extraction. Optimisation of each of these approaches can require some effort, making method development quite daunting. In general, the less selective the extraction technique the more economical will be the process and the quicker will be the sample preparation approach. However, the disadvantage is that there will be substantially more matrix components that reaches the chromatographic system and ultimately this will have a detrimental effect on the performance of the system.
Two common approaches of sample preparation that are often employed are protein precipitation and solid phase extraction. Protein precipitation has been successfully applied to the analysis of a wide range of compounds within a variety of biological matrices. It relies on altering the solubility of the protein by changing the configuration of the protein using a variety of chaotropic reagents, with the most common being acetonitrile and acids such as trichloroacetic acid (TCA). Different chaotropic reagents will preferentially affect different bonding mechanisms within the protein structure. Proteins commonly cause significant issues, either due to irreversible adsorption to active surface sites on the column, co- elution or causing MS ion suppression. The removal of these matrix components increases column lifetime and also significantly reduces ion suppression effects within the detector. However, this approach does not remove all of the matrix components, and one particular classification of compounds, phospholipids, which are present in high concentrations within a biological matrix can cause high levels of ion suppression.
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