7 Table 5: MRM conditions of Mipomersen analogues (QTOF-LC-MS).
Mipomersen -2’-deoxy was detected with the highest sensitivity. A linear calibration curve was obtained in the concentration range of 2 - 1000 ng/mL with a limit of quantification of 2 ng/mL (Figure 4).
Figure 4: Quantitative analysis of Mipomersen-2’-deoxy using QTOF-LC-MS: MRM chromatogram (left) and calibration curve (right).
The calibration curve range and sensitivity of the analogues are shown in Table 6.
Table 6: MRM conditions of Mipomersen analogues (TQ-LC-MS).
Figure 3: Results of charge state deconvolution.
High-resolution MRM for quantitative determination of Mipomersen and related compounds
In Mipomersen, phosphorothioate is introduced into all phosphate- ester linkages between nucleotide bases, and a signal amplification effect in MS detection can be expected by multiple generations of a PSO2-fragment through a single MS/MS fragmentation. Using fragment ions derived from the phosphorothioate oligonucleotide backbone of Mipomersen (PSO2 -: m/z 94.9362) as a monitored ion, a high-resolution MRM using Q-TOF was developed for the determination of Mipomersen and the analogues. As the precursor ion, an octavalent ion with the highest valence was selected (Table 5), which was expected to generate PSO2-fragment more frequently.
Table 4: Results of molecular weight confirmation for Mipomersen analogues.
Quantitative analysis of Mipomersen and related compounds by TQ-LC-MS
MRM measurement of TQ-LC-MS was performed using fragment ions (PSO2 -: m/z 94.9362) derived from the phosphorothioate oligonucleotide backbone of Mipomersen in the same manner as high-resolution MRM. Detailed optimisation of the MRM conditions was executed using the parameter optimisation software LabSolutions. The optimised parameters are shown in Table 6.
Table 7: Results of molecular weight confirmation for Mipomersen analogues.
The limit of quantification for Mipomersen -2’-deoxy was 1 ng/mL, and good linearity was obtained in the calibration curve concentration range of 1 - 1000 ng/mL (Figure 5).
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