Optimizing LC-MS/MS Conditions
We are interested in accurately quantifying small oligonucleotide therapeutics in bodily fluids such as plasma. Here we randomly chose an 18-mer phosphorothioate oligonucleotide TL0901, with molecular weight of 5731.6, as a model compound. As mock metabolites we created shortened versions of TL0901—TL0901 (n-1) is the corresponding 17-mer, TL0901 (n-2) the corresponding 16-mer, and so forth.
Using ion-pair reverse-phase HPLC, TL0901 could be fully separated chromatographically from its n-6 metabolite (12-mer), but the chromatogram shows TL0901’s peak retention time slightly overlapping with that of the n-4 metabolite (14-mer).
Thus HPLC alone is inadequate when separating this oligo from its longer metabolites. As such the selection of an appropriate MRM transition for this oligonucleotide is needed to distinguish it from these metabolites. This can be done by analyzing corresponding neat oligonucleotide solutions on a mass spectrometer equipped with an appropriate HPLC system under optimized LC conditions.
Figure 1 shows a typical Q1 scan mass spectrum of TL0901 on an API5000 with a turbo ion spray ion source. The m/z of the various peaks allows the ionization state of the compound to be calculated based on its molecular weight. For example, the peak at 715.6 is TL0901 losing eight protons (M-8H), while that at 818.0 is TL0901 losing seven protons (M-7H).
From Q1 the ionized compounds undergo fragmentation in Q2, yielding a variety of smaller products, and the fragments are then analyzed in Q3. Many of these fragments (for example, that with m/z of 319) are seen to be shared among the products of the different parent ions (for example, M-8H and M-7H). Therefore, multiple signature MRM transitions can be used for monitoring TL0901.
For quantitative determination of TL0901 in pure neat solution samples, any of these signature MRM transitions can be used. However, for complex samples that contain metabolites and components from a biological matrix, the key, then, is to choose which transition(s) will accurately quantify full-length TL0901. It is especially important to ensure that the chosen transition does not suffer interferences from the compound’s metabolites that have retention times overlapping with those of the parent compound.
Figure 2 is an example of the process of selecting the appropriate MRM transitions. We injected a neat solution containing n-1 and n-2 metabolite mixture (2,500 ng/mL, the highest testing concentration) onto a LC column under optimized conditions (column: 2x50 mm C18 column; mobile phases: HFIP and TEA buffered water and methanol).
In addition to monitoring the corresponding n-1 and n-2 metabolite MRM transitions, we also monitor up to 10 different MRM transitions identified for TL0901. The top trace of Figure 2 shows the chromatogram of TL0901 (n-1) and TL0901 (n-2). The bottom three traces show the chromatograms monitored with three MRM transitions of TL0901.
The expected peak retention time of TL0901 is approximately 1.32’ in this experimental condition. The major peaks in both the 645.9 → 319 and the 817.7 → 319 transition traces represent the contribution from TL0901 (n-1) and TL0901 (n-2), making them inappropriate for quantitative analysis of TL0901.
However, neither of these two metabolites contributes to a detectable peak of the 715.4 → 319 transition at the expected TL0901 retention time (~1.32').Therefore, the quantitation of TL0901 will not be affected by the n-1 and n-2 metabolites by monitoring the 715.4 → 319 transition during LC-MS/MS analysis.