Hello,
I am analyzing steroids with LC-MS/MS.
while developing sample extraction method, I noticed substantial signal reduction after nitrogen evaporation & reconstitution process.
When I prepare 2 samples as follows,
1. X ng/ml in 50% MeOH
2. 10X ng/ml in 50% MeOH + 1/10 dilution with 100% MeOH >> nitrogen evaporation >> reconstitute with 50% MeOH (final conc. of X ng/ml as 1.)
The number 1 sample always has higher signal (~twice higher).
What do you think is the cause?
The evaporation step takes less than 3 hours, and nitrogen flow is quite gentle.
Would it be better to sonicate in water bath in reconstitution step?
Any advice will be a great help, thanks.
If the sample matrix is hard to dissolve after dry down, sonication in a water bath should always be added to the reconstitution step. It seems that you have a doubt about the solubility, can you get a clean and homogenous solution after reconstitution? Try to add more organic to complete solubilization as an alternative. Afterward, compare the 1X and concentrated 10x signals by bringing the 1x to the same organic composition as concentrated 10x.
Assuming your nitrogen source is pure enough and thereby there is not any oxidation effect. 3 hours is a relatively long procedure for nitrogen evaporation. You may incubate your 1x sample at the same conditions without nitrogen application and you can compare the responses before and after the incubation period. Within this test, you may verify whether it is originated from the evaporation process or the time of the process.
This sounds like a solubility issue to me. Are you sure your analytes are soluble in methanol at your final concentration (the evaporated sample)? If there are any doubts then ultrasonication will definitely help.
I would suggest that your 10X solution wasn't homogen that's why you get less concentrated sample therefore less signal
If it's not a solubility issue then it's decomposition or evaporative loss. If you repeat the evaporation what happens to the signal?
Thank you all.
Sonicating in water bath for 3 min definitely recovered the signal.
- Badges
- Science topic
- Similar topics
- Geoscience
- Asia
- India