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case in order to aid solubility of the drug into the blood stream a dosing vehicle had been added to the original compound. Dosing vehicles are commonly used within the Pharmaceutical industry as it allows for dissolution of the compound into a suitable part of the biological system. These are chosen to primarily aid the solubility of the drug, and also to have negligible effect on the toxicity of the overall drug dose. They are often surfactants molecules, comprising of a polar moiety and a non-polar moiety. These can be polymeric in nature or a single molecule although polymers are in general favoured due to their greater diversity in terms of solubility. In this case the dosing vehicle was a PEG 400 polymer.
Another issue associated with bioanalysis is that only the compound of interest is typically monitored when using SRM for quantification. This is facilitated by the generic use of mass spectrometry within the industry, and the detuning of all compounds apart from the analyte of interest. Thus, in general it is not feasible to determine if there are any matrix components that are still left in the sample after the sample preparation step and also if there is any co-elution of the matrix components with the analyte. Many laboratories overcome these issues by the use of an isotopically labelled sample, where the internal standard will mimic very closely the response of the analyte, however even this approach did not work in this situation.
Further investigation revealed that the PEG 400 did indeed co-elute with the analyte and that it did cause some ion suppression. However this still left a mystery as to why the ion suppression should vary in such a consistent manner. To get a better understanding of why this is the case it is necessary to understand some of the pharmacokinetics process of a drug and the other constituents that make up a final product pharmaceutical formulation. Pharmacokinetics is a study of the effect that the biological system has on the concentration of a compound in the biological system, so in this case it is used to determine how quickly compounds are eliminated. Different compounds can be eliminated at different rates. In this scenario the elimination rates of the PEG 400 and the compound and consequently
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the amount of ion suppression is variable. Figure 2 shows the actual concentration of the dosing vehicle and the compound, and also the measured concentration which is lower than the true concentration due to the PEG 400.
Figure 2 Concentration profiles of drug, dosing vehicle and observed drug concentration
Once the team had determined this, efforts were focused on an approach which would remove the PEG from the sample to be injected onto the chromatographic system. The use of SPE here was essential as it allowed a much greater degree of selectivity, between the compound and the dosing vehicle, than could be obtained using a protein precipitation methodology and resulted in complete removal of the ion suppressant from the injection sample. Subsequent to this change in the analytical procedure the assay performed well, however the cost to the business was significant, substantially more than the perceived cost of an expensive sample preparation technique. It should be noted that altering the chromatography could have also resulted in the same result as it would have separated the PEG envelope from the analyte.
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