In this technical article you will learn about which properties are necessary when choosing a compound to use as an internal standard.
It is very important that the internal standard never be found in the sample. This should be pretty obvious – if the internal standard (IS) is normally, or possibly present in the sample and you add a known amount of IS to each sample, how can you tell if the peak you observe is attributed to the IS added or the IS normally in the sample? There are certain cases for LC-MS when it is impossible to completely avoid this. It should also be obvious that the internal standard must be well-resolved from the other peaks in the chromatogram. If the IS overlaps with another peak, how are you going to assign an area or peak height to the IS peak? There is one exception to the requirement of chromatographic resolution, and this is for stable-label (isotopic label) internal standards with LC-MS. Because the MS detector can usually differentiate between the IS and analyte if they co-elute, they don’t have to be chromatographically resolved.
By reading the full article you will discover that if you select an internal standard with the properties outlined, it is more likely that you will have a dependable internal standardization method than if you were to skip some of these requirements.
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