Chromatography Forum

I have a Quattro Micro which is being used to determine basic drugs in
plasma in electrospray positive ion mode by reverse phase HPLC.


Some compounds are linear from 0.5 to 2000 ng/ml (5 ul injected
on-column), but others from 0.5 ng/ml to 1000.


Is there anyway to increase the linearity such as varying the
electorspray voltage, nebulizing gas temperature (desolvation gas),
nebulizing gas temperature, water added post-column (analytes and
metabolites elute at fairly high methanol content), etc??


Thanks for the help.

Is 0.5 ng/ml your LOQ or just the lowest concentration you need to measure?

Regards Bert

Just the lowest concentration I need to measure. The analysis also has the hydroxy and demethylated metabolites present. I have standards for all of them, and the response for all three metabolites tend to flatten some above 1250 ng/ml.

I didn't reconstitute and inject directly from above the protein pellet. However, the samples in water matrix show the same effect. The actual concentrations noted are somewhat less than apparent since the sample is diluted about 2.5 to 1 during protein precipitation.

The study can have a wide range of responses from 1 ng/ml to 1700. Would like to avoid diluting the samples if possible, since I am somewhat lazy.

Hoping to understand if way to increase dynamic range without decreasing lower end sensitivity significantly.

If the sensitivity of your method is good enough, why don't you just lower your injection volume? If the sensitivity is good, I think your accuracy and precision should remain fine at a 0.5 - 1 ng/ml concentration level.

the sensitivity on the low end is adequate, but don't have a lot of extra sensitivity.

Guess I could inject different amounts for different samples?

But would probably have to make multiple analyses for samples.

Was hoping to determine a way to analyze all the same way, just increase the linearity of the analysis above 1000 ng/ml and keep the sensitivity at 0.5 ng/ml.

I had tried fitting a quadratic, but wasn't pleased with the resduals calculated afterwards.

Thanks for the suggestion

James,

I surely think that electorspray voltage, nebulizing gas temperature etc. influence linearity, but in my experience it is trial and error. Changing the organic in your mobile phase to fi acetonitril might also help. We always tune the MS with rather low concentration of analyte, and infuse at 10 to 20 µl/min. Did you try to tune your instrument with high analyte concentrations, with the actual flow of the LC-analysis?

regards Bert

always try to tune at low concentration with the flow and the eluent similar to that used in the analysis. Usually T in post column about 200 ng/ml solution of component.

THis particular analysis uses methanol since (see section on matrix interferences on my web page) it elutes the phospolipids in plasma samples quicker from the column.

methanol use at:

http://users.chartertn.net/slittle/