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52 February / March 2016


Chromatography Today Help Desk An interesting problem came to the helpdesk some time ago, and is one that has been seen by a few experienced bioanalysts.


When developing a method for the analysis of a compound in plasma, a simple protein precipitation method was used. This approach is often used in bioanalysis labs where Pharmacokinetics is being sought. Pharmacokinetics is a study of the effect that the biological system has on the concentration of a compound in the biological system, and is used to determine how quickly compounds are eliminated. Analysis of the blood, or plasma samples aids the drug development process as it allows some of the performance characteristics of the drug to be observed. During the method development the method was providing some very good data. The accuracy and precision measurements were very tight and there was good linearity between the actual response and the expected response. The drug was then administered to the first test subject during an iv (intravenous) study. The data was reported back and the concentration profile observed after the iv administration is shown below in red, compared to the expected blue, iv dose trace.


that this would rectify the issue and that as a consequence of this diligent new procedures would be introduced into the animal houses to ensure that the samples were correctly labelled.


Imagine his surprise when after an expensive re-run of the trial exactly the same data was obtained. By now the project to launch the drug was running behind schedule and the rate determining step was the PK analysis. Everybody in the team was put under immense pressure to determine the cause of the peculiar data.


Eventually a question was asked that is not often asked since most bioanalysts know what the answer is and are frightened of the consequences. The question posed was “Is the sample the same as the QC’s?” The implication being that there is a significant difference between the sample that was taken from the test subject compared to the sample that was prepared in the laboratory. The answer is obvious that the test subject samples are different from the spiked samples, but the assumption is often made in a bioanalytical laboratory that the samples are close enough in nature not to have an effect on the nature of the analysis. Indeed, when analysing for endogenous compounds it is quite common to use surrogate matrices, such as stripped plasma or even PBS.


Figure 1


Concentration profile of a drug given by iv and that observed by a reader which more matches an oral dose


For those familiar with PK (pharmacokinetic) curves it is very evident that this is not the response that would be expected for an iv dose, instead it is the response that would be expected for an oral dose. Initially the analytical scientists involved assumed that there had been a mix up in the labelling and asked that the samples were reanalysed, with more care being taken on the labelling to ensure that the correct profile was achieved. The results came back and to the surprise of the analytical scientist the data was exactly the same. There was still at no point during the problem solving process any thought that it was the assay that was causing the issue only that it must have been something associated with the sampling. The analytical scientist then asked that the study was repeated as it was clearly an issue with the technicians who took the original samples mislabelling. So at some expense the study was repeated. The analytical scientist was confident


There are also known issues associated with the source of the sample, as certain ethnicities are known to have a different composition of the constituents that make up plasma. An example would be that the western population tends to have a more fatty diet than that observed in Asia, and this will be reflected in the composition of certain lipids within the bloodstream and consequently within the plasma sample. A suitable sample preparation technique will ensure that these differences do not affect the final assay, however in most organisations there is ever increasing pressure to reduce costs which will invariably mean that a less effective approach to matrix removal is employed. A common example of this is when laboratories employ protein precipitation in preference to a solid phase or liquid-liquid extraction methodology. Although the protein precipitation will remove a large amount of the matrix components and indeed in many cases this approach may be suitable, or ‘fit for purpose’ there are examples where this can cause issues. In most cases this results in some form of ion suppression or instrumentation issues which if this was consistent would not present a major challenge. However, not only is the amount of ion suppression not consistent due to the sample but also due to memory effects on the analytical system. This will also be affected by the injection sequence. This has been demonstrated on many occasions.


However, this does not explain the current situation where the profile obtained is dramatically different to what would be expected and so further questions of what were the differences between the sample and the spiked solutions were posed. It was not immediately obvious but eventually the team that were assigned to investigate the problem suggested that the dosing vehicle may be causing problems. In this


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