Missing Internal Standard

[hannahbrodeen]

Hello,

We have encountered an issue that we are unable to resolve. Our analyst has prepared the internal standard as stated by the SOP, however, when the samples are analyzed, we noticed that some of the samples do not have 6-MAM internal standard. It seems to be random; some samples have 6-MAM, but the response is low, and some samples are missing the internal standard altogether. It is the only one that is missing. We have prepared new internal standard and have prepared new samples, however we are still encountering this issue. Please let me know if you have seen this before, and if so what can be done to resolve this issue. Thank you very much for your time!

[Rndirk]

We need more information. You can't assume we know your SOP.

- What are the samples

- What is the sample preparation procedure

- What are the chromatographic conditions

-..

[tom jupille]

As a complete "shot in the dark", I've encountered a similar situation in the past. It turned out the analyst neglected to vortex the tubes afer adding the internal standard.

[lmh]

... if Tom's guess is correct, then you may find that the internal standard is much more variable than the actual analyte.

As a general approach, if you find that you're losing an unacceptable amount of internal standard, then you can try adding it at different stages of the procedure, to narrow down where things are going wrong. For example, in an LC-MS method, adding the internal standard immediately before measurement differentiates between problems in the extraction procedure and problems during measurement (e.g. cosuppression etc.).

You're quite right to investigate. If your internal standard is getting lost, then one of two situations has happened: (a) you're losing only the internal standard, but not the analyte; in this case your measurements will be drastically overestimated, and as variable as the loss of internal standard; (b) you're losing both the internal standard and the analyte, which is fine provided both are lost equally and you still have plenty of signal. It goes wrong, however, if you're losing 99% of both, with the result that you've hardly got any signal left, and if you happen to lose 99% of one and 98% of the other, you have a 2-fold change in the final measurement.