Retention times internal standards, sample matrix effect

Figure 3. Sample matrix effect on reproducibility of internal standard retention times , standards in sample A, standards alone. (Reproduced with permission from reference 26. Copyright 1985 Lewis Publishers.)...

The internal standard method of qualitative analysis is used to help identify components in a mixture when the reproducibility of retention times of the components in the sample versus the standard mixture is inadequate. The change in retention times from the standard mixture to the sample mixture is usually due to sample matrix effects that are difficult to mimic in the standard mixture. Thus, a small amount of a compound that is known to be absent from the sample is added to both the standard mixture and the sample prior to analysis. 

Different compounds have variable ionization intensities, which are further affected by factors such as chromatographic retention times and suppression by compounds present in the analyte fluid and sample matrix, so it is essential to use isotope-labeled internal standards, which are physicochemically identical to the molecule of interest rather than structural analogs, to normalize for effects that can lead to erroneous quantification. Isotope-labeled standards also account for any differences arising out of sample preparation and absorptive loss due to selective binding on surfaces during chromatography (32). 

The abbreviated multiple mass chromatogram shows sample values for each selected ion printed every three seconds. The masses of 58, 60 and 64 are the pre-selected ions for the endogenous, undeuterated Dq choline, the labelled tracer choline, and the internal standard Dg choline. Mass 71, an abundant ion for unlabelled choline, is continuously sampled as a validity check. The relative abundances of the selected ions are estimated from the regression coefficients (FLT) of the data fitted to an idealized curve. The estimated retention times, T, are shown. After each control injection, a summary spectrum for all the control samples is included. The values from this matrix are used to estimate quantities in subsequent samples. The variant signal/quantity ratios are proportionality values that account for overall isotope effect manifested in unequal observed base peak signal attributable to each variant in an equimolar mixture. 

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