(T0930-06-38) Quantitative Analysis of GalNAc-siRNA Conjugates in Tissue Homogenate Using Two-Dimensional Liquid Chromatography Coupled with Tandem Mass Spectrometry

Purpose: Metabolism of GalNAc-siRNA conjugates is mainly catalyzed by exonucleases and endonucleases, resulting in various sizes of shortened oligonucleotides, which could inference the quantitation of GalNAc-siRNA conjugates. Therefore, bioanalytical scientists have to use a long injection time to separate analyte from metabolites when using LC-MS/MS for quantitation. Ion-pair chromatography is the recommended method for determining oligonucleotides at reverse chromatography, but its disadvantages is too hard to rebalance (return column to initial condition) the column system within short time before next injection. How to determine oligonucleotides, or separate them within a short time is another problem to solve. To overcome those challenges, we applied a Two-Dimensional(2D) LC system coupled with tandem mass spectrometry to quantitatively analyze GalNAc-siRNA conjugates in tissue homogenate. The injection time can be shortened from 5.5 to 3.5 and the accuracy and precision not be affected. It is well-known that the GalNAc-siRNA conjugates contains large number of phosphate groups which make the molecules negatively charged. This characterize lets the analyte easily to adsorb to material surfaces such as polypropylene vials, pipette tips and the column solid phase. Therefore, the significant challenges of oligonucleotide bioanalysis are a bad peak shape, low recovery at lower concentration, bad linearity relationship, and enormous carryover when using LC-MS/MS for determination in complex bio samples. In this study, we found an additive which could solve these problems effectively.

Methods: Ion-pair reagents (dibutylamine (DBA) and hexafluoroisopropanol (HFIP)) coupled with Waters ACQUITYTM Premier Peptide BEH C18 column were used as chromatography condition. Tissue homogenate samples (liver and stomach as representative matrices in this study) were added with an additive to improve peak tailing and increase recovery before purified and concentrated by SPE processing. The extracts were analyzed by liquid chromatograph (Waters ACQUITY UPLC system and Waters ACQUITY UPLC Premier 2D system) coupled with triple quadrupole mass spectrometry (Sciex 6500+ and Waters Xevo TQ Absolute).

Results: The 2D LC system have two binary solvent managers (BSM) and a column manager with two six-way 2-position valves, so sample analysis alternates between two identical columns with the same flow path. When samples are injected, separated, and regenerated on one column, the other column is re-balanced to allow more samples to be analyzed in a given time. By using the 2D LC system instead of the traditional UHPLC, the injection time of a GalNAc-siRNA conjugate could be shortened from 5.5 to 3.5 mins without affecting the separation efficiency, which can highly increase the yield of the instrument at the unit time. The within-run accuracy was 0.8% to 4.9% and precision was 3.5% to 8.9%. The commonly used method such as increasing the column temperature (this will shorten column life) or adding more EDTA in mobile phase (this can inhibit analyte ionization) cannot improve peak shape and minimize carryover for quantitation of GalNAc-siRNA conjugates using LC-MS/MS. We found an additive, which is extracted from animals’ excrement could effectively improve peak shape and minimize carryover. By adding the additive into tissue homogenate before SPE, the peak shape was significantly improved (as shown in Fig.2). In addition, the linearity relationship got well (the linearity correlation factor “r” is improved from 0.9846 to 0.9968) and carryover was reduced from 3.1% to 0.5% of ULOQ area. This additive had been used to 20 tissues homogenate for several GalNAc-siRNA conjugates and they have the similar efficiency. Maybe there are some small peptides and lipids which can be adsorbed to the column solid phase and protect the GalNAc-siRNA conjugates to be adsorbed severely.

Conclusion: A high-throughput and robust method was developed and used to accurately quantify multiple GalNAc-siRNA conjugates in bio matrices.

Fig.1 The gradient method of those two UPLC systems
The retention time of Analyte-01 is about 2.8mins. Those two UPLC systems had the similar gradient before 3.5mins. But the difference was that the 2D LC system has two pumps and it needs two columns to work together. These two columns were used alternatively by a switching valve. One was used to separate analytes by the Alpha pump while the other was rebalanced by regeneration pump at the same time. So, the run time could be cut to 3.5mins.

Fig.2 The chromtograpy of Analyte-02 for liver homogenate after SPE
(a) Without additive. (b) With additive.