Internal Standard positive correlation to [Analyte]

[toomunchsam]

Hello,

I was wondering if I could get some help understanding internal standards. I am trying to develop a method to quantify psilocin in whole blood. I am using d10-psilocin as my internal standard. In my LC-MS/MS method, both psilocin and d10-psilocin elute at the 2.2 mins mark. As I was starting to estimate my linear range, I noticed that as my analyte concentration increased, my internal standard (ISTD) also showed an increase in signal, especially with the higher calibrators. This could be seen even if I prepared my whole calibration line in MeOH, and not in the whole blood matrix. It would have made sense if my internal standard response may have started showing slight decreases in signal due to ion suppression by higher analyte concentrations, but I am unable to explain this positive correlation. Theoretically, if I am spiking the same amount of ISTD, I should expect the same response all throughout the calibration line right? Might any of you help me understand this?

[copperfoils]

What transitions are you monitoring? Any possible isobaric overlaps?

[toomunchsam]

I am monitoring the following transitions:

Psilocin: 205.1 -> 58 (Quantifier transition)
Qualifier transitions:
205.1 -> 160.0
205.1 -> 132.0
205.1 -> 117
205.1 -> 115
205.1 -> 89.0
205.1 -> 77.0
205.1 -> 63.0

d10-psilocin: 215 -> 66.1 (Quantifier transition)
Qualifier transitions:
215.1 -> 164
215.1 ->136
215.1 ->118
215.1 -> 117
215.1 -> 90.6

[bcd_GCLCMSMS]

I am curious is all of your ISTD qualifier transitions show the same trend as the quantifier transition?

Commonly, the most intense (or the most stable) ISTD quantifier transition provides good stability, when all samples are spiked and treated the same.

In this case, the chosen quantifier fragments are very low molecular weight, possibly a secondary fragment. If there is a larger mass fragment in good abundance, that may be more stable for your quantifying transitions.

I am monitoring the following transitions:

Psilocin: 205.1 -> 58 (Quantifier transition)
Qualifier transitions:
205.1 -> 160.0
205.1 -> 132.0
205.1 -> 117
205.1 -> 115
205.1 -> 89.0
205.1 -> 77.0
205.1 -> 63.0

d10-psilocin: 215 -> 66.1 (Quantifier transition)
Qualifier transitions:
215.1 -> 164
215.1 ->136
215.1 ->118
215.1 -> 117
215.1 -> 90.6

[toomunchsam]

When I checked the response of all my qualifier transitions, they all exhibit the same behavior, in that the ISTD response increases with higher analyte concentration. I noticed too that its response becomes "more stable" with lesser differences between calibrators at these higher concentrations too.

[millerk8561]

When I checked the response of all my qualifier transitions, they all exhibit the same behavior, in that the ISTD response increases with higher analyte concentration. I noticed too that its response becomes "more stable" with lesser differences between calibrators at these higher concentrations too.

Is it possible that there are active sites along the flow path of the instrument that become occupied with analyte instead of ISTD as the concentration increases, thereby freeing up more ISTD to then make it to the detector? That would partially explain why the ISTD concentration rises and then levels off at higher concentrations of analyte.

[toomunchsam]

Hello everyone,

Thank you so much for all your help. Looks like I got it resolved. I noticed that the fluctuations started decreasing whenever I decreased the concentration of my ISTD. I then noticed that with this decreased [ISTD], one of my qualifier transitions each for d10-psilocin and psilocin were much less variable at my set linear range. Although it got resolved, I would appreciate if anyone might be able to explain why this was so, I honestly stumbled upon this resolution by some gut intuition!