Have you ever seen a LC-MS/MS method uses both internal standards and external standards (in matrix matching purpose) but the concentrations of internal standards are outside the calibration curve gained by external standards with matrix matching?
LC-MS/MS methods typically use internal standards for correction and external standards for calibration, with internal standard concentrations usually within or near the calibration range. Having internal standard concentrations outside the external standard calibration curve is unusual and potentially problematic, as it may lead to quantification inaccuracies and suggests a need for method adjustment. This situation warrants a review of the method development process, possibly requiring changes to the calibration range or internal standard concentrations.
The best way is to use deuterated internal standard of you molecule of interest in the matrix. It compensate the effects of the matrix in the ionization of the molecule and in the Tr.
Best regards
A properly selected Internal Standard (IS) should show a similar response as the actual sample(s) under analysis. The concentration used for the IS should be at or near the average sample response. Use of an IS that is outside the range of the calibration does not follow good chromatography practices and may be reason to declare the method invalid.
If a paper for testing of illegal dye in foods, use 50 ppb internal standards but they claim the linearity of method is 1 to 20 ppb. Should this be a big mistake?
In the meantime, the paper did not provide recovery of every dye in the test, if it is acceptable?
If there is evidence that the author intentionally hides the information of misusage of internal standards, is it a serious problem?
Jien Wu: Sadly, many journal articles (and application notes, white papers, even some regulatory applications, etc) may have poorly developed, outdated or even completely invalid methods. This is true of the newer "open-access" as well as established printed journals. It is rare today that any paper is reviewed by one or more individuals with the needed training and knowledge to evaluate the article for scientific completeness. *Journals do not pay reviewers anything for their time or efforts. When reviewing submitted articles, editors often ask the reviewers to essentially re-write the entire article for the author(s). Journal editors have a difficult time finding anyone to volunteer to review papers. The result is that some papers simply share misinformation or invalid methods. This helps spread misinformation and poor quality methods.
As a past lead reviewer for many top scientific journals, I routinely saw 20-30% of the new submissions that came across my desk showed the authors had absolutely no knowledge of what they were doing. Many Journals rely on publication acceptance to generate income. Doing good science is not a priority for many of them. They are "publishing houses".
My advice offered to students is; "Never use other 'papers' as examples of what to do. Instead, review them with someone knowledgeable in the topic to see if they follow good practices first. Always question, keep learning and hone your skills researching answers.
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