Chromatography Forum

Hello,

I am getting into to quantitation of small compounds using LC-MS. I know an isotopically labeled internal standard is the best way to go, but I only have the sample in it's native state as an internal standard.

By my reckoning I should be able to add a known amount of internal standard to each sample (say 20ug) and then use the increase in peak area compared to an otherwise identical sample, to determine the recovery of the standard. Is this sound logic, or have I missed something?

Am I able to simply add the same compound and use it as an internal standard in this manner, or do I need to have an isotopically labeled standard?

One other question, once I have the standard how do I use the information? If it's down by 10%, do I just increase my sample value by 10%. This just doesn't seem right. So how do I make use of the standard for quantitation? Is there some paper or book that someone could refer me to.

Thank for your help,

Walter

Be careful not to confuse the terminology. An internal standard is a molecule different from your analyte, be it an isotope or another chemical entity. When you add your analyte to your unknown sample, as you described, it is called the Standard Addition Method. Look it up in this forum or on the web and you'll find lots of pages that explain it.

If you search this forum for internal standards, you will find that some have had success in MS detection without using them. In my experience, it depends on your sample matrix and the day-to-day performance of your MS. Spike your sample matrix with known concentrations. Run an external standard curve and compare the response to ensure that your sample matrix is not causing ion suppression. Also, if you run a sequence, you should intersperse standards throughout your sequence to ensure that your MS response is consistent. If you have a realtively clean sample matrix, you may find that you don't need an internal standard. Even if you can't get an isotopically-labeled species to use as one, you could probably find something else that has the same functional groups and elutes closely.