Chromatography Forum

Hello,

I am attempting to follow an older quatitation method, which uses an internal standard spiked into every sample,

They also ran a calibration curve,

I simply cannot understand how quanlynx is working out concentrations, based upon the calibration or internal standard.

If anyone has any knowledge of quantification with internal standards I would be very much appreciative

Thank you

I've not worked with Quanlynx, but in general internal standardization also requires a calibration plot. The computations involved for "external" and "internal" standardization are almost the same.

The only difference is that whereas in "external standardization" the calibration plot is area (Y) vs. amount (X), in "internal standardization", the calibration plot is area ratio (Y) vs. amount (X). "Area ratio" is just what it sounds like: the ratio of analyte peak area to internal standard peak area (this assumes that the amount of internal standard is held constant).

QuanLynx is not very self-explanatory or straightforward...
Have a look into this tutorial. If this does not help you further, come back and I'll try to do my best to help you.

Regards,
Jörg

Thanks both, very much.

Yes I had found this tutorial on my quest to find some answers. Thank you for the clarification regarding internal standard and response factors, I have since further read the method and they claim to use standard isotope dilution method

I am not overly convinced that you can simply use the ratio of peak area of internal and actual unknown to provide concentration of unknown.

They basically spike the unknow with an isotopically labelled standard and use that work out unknown quantity - is this analytically sound ?

I'm not 100% sure ....back to my books.

Quanlynx certainly doesn't appear to calculate without some sort of calibration, I certainly cannot get it to work on just peak areas of standards

They basically spike the unknow with an isotopically labelled standard and use that work out unknown quantity - is this analytically sound ?

This sounds somewhat equivalent to standard addition method. You would need at least three different amounts of labeled standard added to each of your samples; I guess using a simple calibration using response factor is much less work when analysing >4 samples.

Quanlynx certainly doesn't appear to calculate without some sort of calibration, I certainly cannot get it to work on just peak areas of standards

Sorry I can't help you at this point, I never used quanlynx this way.

If it were done by isotope dilution/standard addition, then you wouldn't actually need a calibration curve, so you can easily tell if Quanlynx is really doing that (which I doubt).

I strongly suspect it's just badly described. I use TargetLynx, and my understanding is that QuanLynx is just a cut-down version. In TargetLynx, any compound where you don't specify an internal reference compound (see page 297 of bunnahabhain's helpful link), is treated as external: it's quantified by external calibration, and if you specify how much of it is in the standards, the software will make an external calibration curve Area versus amount.

If you specify an internal reference, then the software uses internal standard quantification and prepares a calibration curve of Area(target) divided by Area(Internal reference) * amount or concentration of internal reference, versus amount of target compound. This is entirely typical internal standard calibration. TargetLynx actually indicates this on the axis of the calibration curve plot when you review the data.

You do need to specify the amounts of the target compounds and internal reference compounds (either in the method if they're constant, or in columns of the batch table if they vary; confusingly you can also multiply the concentrations in the columns by factors in the method, but that's another story, and it has a very practical use...).