My analysis requires a extremely wide concentration range from 1-5000 ng/mL. As expected, the calibration curve flattens out at the higher concentrations. I have ruled out detector saturation (see my last question).
I have noticed that there is an in-source fragment (lets call it X) of my parent compound (X-glucuronide) that I can quantify with its deuterium labeled internal standard. This has a perfect linear calibration curve from 25-5000 ng/mL. Before I perform exerpiments to force more in-source fragmentation to reach a lower LLOQ, I want to know if this is actually a good approach to solve my issue. I haven't found any literature that does the same thing.
You need see before this, if you have analytical signal to quantificate your compound. Linearity is not only the factor you need to quantify a target compound, your linear model couldn't be apropriate for singnal and you should weigh your calibration curve.
I think you know your equipament and analytical method to chose sorce fragmentation instead of Q2 fragmentation.
Using the deuterated aglycon of your analyte as an internal standard would probably work but it seems to be a rather disadvantegous choice: You have the price/effort of a proper deuterated standard but not the benefit because you are using a somewhat different compound as internal standard rather than an isotopomere of your analyte.
I agree with Marcos in that weighing the calibration model and/or using a non-linear model should improve the results of your external calibration.
Maybe cleaving the glucuronic acid during sample preparation would be an alternative? In that case you could use the deuterated standard.
Marcos Steola There is indeed an actual signal with S/N>5 (linear with 1/(x*x), all points are
Asal Asali I don't know if this is possible in your experiment, but you could dilute samples with high concentration and work a shorter range, another approach is mass software quantification could estimate signal saturation ( I know Sciex Multiquant ® has this option avaible and work well for estimate quantification).
Marcos Steola Unfortunaltely, it is not possible to dilute te sample in this case. In the biological mixture, there are also other analytes to be analyzed that require highest sensitivity. Thus, diluting the samples will make them undetectable. The challenge in this assay is that I have analytes that are very high in concentration and analytes that are very low in concentration. I am still trying to find out how to overcome this matter.
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