Hi,
Could anyone explain how Sciex OS Analytics software internally handles IS peak area calculations to account for Solid Phase Extraction recovery of a particular analyte in a targeted analysis of XIC peaks (i.e. MRM IDA EPI scans)?
My understanding is that the software is able to automatically account for the amount of analyte of interest that is lost during solid phase extraction (SPE), by calculating some sort of "peak area ratio", but I'm not sure which sample type the software is using to do this.
To elaborate further: my typical SPE and HPLC-MS/MS workflow (for charged lipids extraction) consists of first precipitating (and discarding) proteins out of either solid or liquid biological matrices (with HPLC-grade MeOH at -80C), followed by spiking a mix of known amounts of deuterated internal standards into each sample to be SPE-processed, right before running samples through a SPE column for charged lipid fraction enrichment. Simultaneously, I add said internal standard mix into all my Standard Curve sample vials, as well as a separate vial that I name "100% IS recovery", which just has the mix of known amounts of each IS analyte. Note that neither standard curve samples nor the "100% IS recovery" samples are passed through SPE columns (hence the "100% recovery" in my vial name) , they are just a mix of purified synthetic standards that are injected into the HPLC-MS system.
The manual, non-software automated way of calculating SPE efficiency for each "unknown" analyte of interest (being measured in biological, "unknown", samples) would be to simply divide the peak area of the deuterated IS analyte in the biological sample (that has undergone SPE) by the peak area of the same deuterated IS analyte in the "100% IS recovery" sample (NOT SPE-processed). This ratio (quotient of the division) is then applied (multiplied to) to the peak area of the analyte of interest in that same "unknown" biological sample.
Supposedly, Sciex OS automatically calculates SPE recovery efficiency for each analyte (as long as you assign an IS to a "Quantifier" analyte of interest), but I'm not sure how it is doing these calculations, i.e. which peak areas of which analyte and/or sample type is using to derive this simple ratio, that is then used for adjusting the final "Actual Concentration" of the "Quantifier" analyte that it reports.
Any pointers to any kind of literature source (software manuals, training material, webinars, etc.) would be much appreciated!
Sciex software is not smart than you think. It cannot know if you spike the IS or standards nor passing SPE. The software just calculate the integrated peak area for both peaks of analyte and IS, then plot them by analyte concentration / IS concentration for X axis and analyte peak area / IS peak area for Y axia. Finally, apply the "unknown" sample peak area / IS peak area on the calibration curve to calculate the "unknown" concentration. If the sample type is "Quality Control" or "Standard", and there is concentration keyed in under "Analyte Concentration", then the "Accuracy" would show the number which is from "Calculated concentration / analyte concentration" times 100%, and this is called "recovery". Unfortunately, it could not be a "recovery" for SPE.
To evaluate the SPE recovery, you have to prepare matrix matched calibration curve, blank, pre-spike (spike standard before SPE), post-spike (spike standard after SPE) and QC samples with the same concentration of IS (spike IS with sample preparation". The recovery will be (post-spike concentration / pre-spike concentration) times 100%. You can also calculate the IS intensity or peak area for both pre-spike and post-spike sample to evaluate the SPE recovery by loss rate of IS.
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