Urinary steroid hormone extraction - unusual IS abundance

[Tones]

Hi all,

I am perplexed by the internal standard values I am getting for a couple of my urine samples. For example, I am only detecting 15% of the internal standard that I expect in my QC 12 samples. Why is this urine leading to such low internal standard abundance?

The LC-MS/MS method I am using is already validated by several others in my department. The QC urine samples are generated by individuals with healthy kidney function. I store all urine samples in the same -80C freezer. I have observed the same low ISTD abundance in QC 12 over the past two extractions.

I add the same amount of internal standard to all samples at the same time, using a positive-displacement pipette. I do a liq-liq extraction using Ethyl acetate. I evaporate under nitrogen gas while in a 30C water bath. And I reconstitute with 50/50 MeOH/H2O. I run this in ESI-negative.

Anyone know why I may be getting particularly low ISTD abundance on a single urine sample? Any ideas are greatly appreciated. Thanks!

Here is a clip of my numbers:

(A-D are various steroid compounds I am extracting. Each urine is extracted in duplicate.)

[Don_Hilton]

One possibility is the recovery of the IS from the urine. Is the IS pH sensitive and have you buffered the urine? Are you sure nothing happens to give a retention time shift in this particular sample, leaving you looking at the wrong peak?

[zokitano]

So you're saying that you've observed a consistent lower recovery only for QC 12 samples.
Are these samples prepared in a different glassware or plasticware than the other QC samples (different glass type, manufacturer etc)? From your results I can notice a consistent generally lower values for your analytes as well (for QC 12).
Can you repeat the QC 12 urine samples with the glassware used previously for some "good" QC samples. Just to exclude possible wall adsorption effects, maybe?

[Tones]

I am not sure whether the IS is pH sensitive. I am using the same synthetic glucocorticoid compound as others before me have used (but I don't know what troubles they may have run into). The urine I am using isn't buffered. They are collected (from the same male and female each time, different days), vortexed, aliquoted out, and then stored at -80C.

It seems as though there isn't anything overtly wrong with the chromatograms -- I believe I am looking at the right peaks.


Here are the chromatograms of QC 12 (urine) vs. Standard H (my controls that contain only IS and nothing else, in dH2O):









The RTs are very similar and there aren't any other peaks to confound the integrations.


All the samples are prepared in the same glassware at every step. QC 12 is treated the same as other QC urines as well as the standards.

There is one change though. The protocol calls for 15 mL plastic falcon tubes for the liq-liq extraction. I ran out of those, so I've been using (plastic rubber-lined) screw-cap glass tubes. This couldn't have caused this variability, right?

[zokitano]

There is one change though. The protocol calls for 15 mL plastic falcon tubes for the liq-liq extraction. I ran out of those, so I've been using (plastic rubber-lined) screw-cap glass tubes. This couldn't have caused this variability, right?

This could be a possible cause for your analyte/IS loss during the QC 12 analysis. Why don't you try preparing the QC 12 samples in plastic tubes instead of glass and report us back what is the outcome

Good luck

[Tones]

Sorry, I guess I wasn't clear when I said that. All the samples were prepared using the same type of glass tubes. I did not mean to imply that QC 12 was prepared using a different type of tubing compared to the other QCs.

[zokitano]

Ok, sorry for my misinterpretation of your previous post

Have you tested the LC-ESI/MS procedure for possible matrix effects, particularly for the problematic QC 12 samples?

If you run the same LC-ESI/MS method only by using some scanning MS acquisition mode (like Q1 scan or Q3 scan, lets say in range m/z 50-1000) instead of MRM, for your QC 12 samples, do you observe additional peaks near your analyte/IS Rt in the scan MS chromatogram? And if you repeat the same procedure for the good QC samples, do they look "cleaner" in scan mode?

Or if you syringe infuse your IS through a tee piece together with the column effluent in to the ESI source and your again analyze your QC 12 samples (without IS added) in MRM, do you see any dip in the MRM transition signal for your IS near its retention time?