I am going to run GC-MS for headspace fecal sample's VOCs analysis. I will have about 300 putative compounds (non-targeted analysis) in each run. For compound quantification analysis, I need to use an internal standard. I am not sure if I can use one chemical such as toluene-d8 as internal standard. How can it help me for quantification of each compound?
Hi Mohsen,
I have used an internal standard (n-Tetradecane) to quantitate compounds in GC-MS of essential oils, and I am guessing it will be the same process for your work, though i can't tell you if toluene-d8 is the right standard to use or not.
Essentially each of your 300 samples needs to include a known amount of the internal standard. In my case we mixed 0.25mg of Tetradecane per litre of ethanol solvent that was placed in the vials. That way, when a sample is run on the GC-MS there will be a peak for the internal standard, and if you integrate the area under that peak then that area represents the amount of 0.25mg/L. The area of the standard becomes a reference for calculating the quantity of all the other compounds.
Simply integrate the other compounds' peaks and you then compare the area of those peaks to the area of your Internal Standard's peak. Since you know the true amount of the Internal Standard in the sample (because you put it there in a measured way!) you can then estimate an actual amount for each compound relative to the internal standard's amount.
e.g. if the standard peak has an area of 200,000 units, and another compound peak has an area of 600,000 units, then it's quantity = (600,000/200,000) x Weight_of_standard.
Hello, Mohsen. Just as David said, at a first moment it is not possible to say if toluene-d8 is the best Internal Standard (IS) in your application. I have successfuly used it in the analysis of VOCs in water, also by HS-GC-MS.
Some observations:
- The IS must be a substance with chemical properties similar to those of your analyte(s) and, for this reason, the IS and the analyte(s) are expected to have similar - but different - retention times. For a wide range of analytes in a single run (i.e. wide range of chemical properties, and so a wide range of retention times...) the use of more than one IS is common. The method must provide good peak resolution for the IS, which is not so hard to achieve with GC-MS.
- The use of an IS minimizes the error (improves accuracy) because the same amount of such compound (toluene-d8, for example) will be added to all of your samples, so if there is any variation in the recovery of your analytes (for many reasons, including variations in the injected volume), such positive or negative deviation will also affect the IS peak area. This is why, when using IS method, the peak area of the analyte is replaced by the so-called "area ratio" (analyte peak area/IS peak area), and such area ratio is now the response considered. The analyte concentration will be calculated according to the respective calibration curve, which must be constructed also by using the same IS method, with the resulting graph "area ratio" vs.analyte concentration. The SAME amount (concentration) of IS must be added to all the replicates of the calibration curve and to your samples.
The analyte concentration can not be calculated (at least accurately) as a "multiple" of the IS concentration, based only in the area ratio. For this to work, the analysis should provide exactly the same sensitivity to both the analyte and the IS, which is not likely to occur.
Kind Regards,
Allen Barros
.
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