MS Quantitation

[Karen01]

I have not done quantitation by LC/MS before...

I have to quantitate 4 polar compounds (2 water soluble vitamins and two other small polar molecules) from a really messy matrix at concentrations too low to do by UV... I have an LC/QToF to work with so I can look at parent or fragments so just seeing the analytes should not be a problem unless the concentrations are too low.

I can handle the chromatography development i'll need to do ... but I'm not sure about the quantitation part...

My understanding is that one should use internal standards that are isotopically labeled forms of the analytes to be able to deal with variable ionization .. particularly if there is stuff that co-elutes with the analytes that competes for ionization.

But I don't have isotopically labeled standards... So can it be done without them? I suppose if I get lucky with the chromatography and separate all interferences, i might be able to use a 'different' internal standard, or even external standards ... but that is not likely.

So without isotopically labeled analytes for standards, would standard addition be the way to go?

Thanks,
- karen

[Jetjamnong]

I think most of labelled internal standard is very expensive but its quite similar chemical properties with the analytes. I think non-labelled IS can be use in quantitative analysis if such compound that you selected as IS have similar properties such as polarity and ionization efficiency with MS (+ive or -ive)
For example, if you are analysis 3 benzodiazepines and 2 tricyclicantidepressant drus diazepam, nordiazepam, alprazolam amitriptyline and nortriptyline, you can use another compoud as IS but it must the same properties with your analytes, in this case use of Trimipramine for example.

Another case for Buspirone you can use Reserpine as internal standard, just example its depend on your sample matrix and your requirements.

The purpose of use isotropic labelled internal standard is mattrix effect correction becuase most of labelled IS have the same retention time with analyte for example, analysis of Vitamin D2 and Vitamin D3 by using D6-Vitamin D3 as their internal standard all compouds were present at the same retention time with same mattrix effect range

[Don_Hilton]

Labeled internal standards have a benefit beyond that noted for ion supression. They have the same chemical characteristics as the unlabeled compunds and thus suffer the same losses in sample preparation steps. Thus small changes in matrix or sample hadiling may resut in slight losses of analytes - but the internal standard is lost in the same way, maintaining the ratio of analyte to standard. This can be expected to benefit accuracy and precisision of the method.

[Peter Apps]

IFyou can get matrix with no analyte in it, you can use straightforward external calibration. If you can ony get matrix with analytes then you have to calibrate by increase in peak area vs added quantity.

Peter

[Karen01]

IFyou can get matrix with no analyte in it, you can use straightforward external calibration. If you can ony get matrix with analytes then you have to calibrate by increase in peak area vs added quantity.

Peter

So standard addition is a viable approach.

Thanks,
- Karen

[Peter Apps]

IFyou can get matrix with no analyte in it, you can use straightforward external calibration. If you can ony get matrix with analytes then you have to calibrate by increase in peak area vs added quantity.

Peter

So standard addition is a viable approach. Beware that if your analyte concentration is already high your additions will soon run off the top of the linear part of the calibration. Also, with additions to matrix that already has analyte in it, your results are strictly an extrapolation (or at best are right at the bottom of the curve). There are two ways of calibrating by addition; either you do one calibration series using spiked matrix and just run the samples as they are, or you spike different levels into each sample so that every sample has its own calibration.

Peter

Thanks,
- Karen