(T1130-09-53) Sensitivity Improvement for the Quantitation of Antisense Oligonucleotides in Plasma Using Hybridization Microflow LC-MS/MS

Purpose: Oligonucleotide therapeutics has been rapidly growing over the past decades. Quantitative analysis of antisense oligonucleotides (ASOs) in biological matrices is crucial to study their pharmacokinetic and pharmacodynamic properties. LC-MS/MS has been reported as an orthogonal technology to routinely support bioanalytical studies of ASOs. Higher sensitivity of a bioanalytical method is always desired to quantify the ultra-low concentration analytes in biological matrices. Here, in the present work, our purpose is to evaluate sensitivity improvement of ASOs in plasma on microflow LC-MS/MS.

Methods: Quantitation of ASO was conducted on a SCIEX QTRAP 6500+ mass spectrometer with multiple reaction monitoring (MRM) in negative mode. The mass spectrometer was couple with either an ExionLC system and Ion Drive Turbo V ion source, or an M3 MicroLC system and an OptiFlow Turbo V ion source. Reversed-phase ultra-high-performance liquid chromatography (UHPLC) with ion pairing agents 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) and N,N-dimethylcyclohexylamine (DMCHA) in water and acetonitrile were used to separate ASOs. For analytical flow, a Clarity 1.7 µm Oligo-TX 100 Å LC column 50 × 2.1 mm (Phenomenex, Terrace, CA) was used at the flow rate of 0.5 ml/min with a gradient from 0 % B to 30 % B within 3 min. For microflow, two micro-columns Gemini 3 µm C18 110 Å LC column 50 × 0.5 mm and Gemini 3 µm C18 110 Å LC column 50 × 0.3 mm (Phenomenex, Terrace, CA) were used at the flow rates of 15 ul/min and 10 ul/min with a gradient from 10 % B to 40 % B within 5 min. A synthetic ASO composed of 20 bases were used as a test compound. Calibration standards and quality control (QC)samples were prepared in neat solution and plasma. The ASO in 200 µL plasma was extracted by hybridization method and reconstituted in 500 µL reconstitution solution. Same volume of sample extract (5 µL) was injected for both the analytical flow and microflow methods.

Results: MS/MS conditions were optimized to achieve chromatograms with good sensitivity and selectivity. Figure 1 showed the extracted ion chromatograms of blank, 0.25 ng/ml and 1 ng/ml ASO in rat plasma after hybridization method with analytical flow LC with 50 × 2.1 mm column (top panel), microflow LC with 50 × 0.5 mm column (middle panel) and microflow LC with 50 × 0.3 mm column (bottom panel). Clean background was observed in the blank samples. The signal-to-noise ratio of 0.25 ng/mL ASO was 2.8 with analytical flow LC 50 × 2.1 mm column; 9.1 with 50 × 0.5 mm column and 11.4 with 50 × 0.3 mm column, which indicated a 4-fold S/N improvement was achieved using microflow. The LLOQ was 0.25 ng/mL with microflow LC and 1 ng/mL with analytical LC. Precision and accuracy of the ASO in plasma with the microflow were assessed by spiked QC samples (n = 6) at four concentration levels (LLOQ, QCL, QCM, and QCH). As shown in Table 1, the coefficient of variation (CV) was within 6.7 % and accuracy of all QC and LLOQ samples was above 87 %. Similar sensitivity improvement was achieved with ASO in neat solution; also, similar sensitivity improvement was achieved with two other ASOs.

Conclusion: An ultra-sensitive microflow LC-MS/MS method for quantifying ASOs in plasma was developed, a 4-fold sensitivity improvement is achieved compared to the analytical flow LC method. This method has the potential to support pharmacokinetic and pharmacodynamic studies of ASOs.

Figure 1. XICs of ASO in blank, 0.25 ng/mL, 1 ng/mL in rat plasma with analytical flow 2.1 × 50 mm column (Top), microflow 0.5 × 50 mm column (Middle) and microflow 0.3 × 50 mm column (Bottom)



Table 1. Precision and accuracy of ASO quantitation in rat plasma (0.25-250 ng/mL) with 0.3 × 50 mm column