A fully automated novel solid-phase extraction method for siRNA drug extraction and quantitation across multiple...

Approaches such as liquid-liquid extraction and two-step liquid-liquid extraction combined with SPE has been widely used for extraction of oligonucleotide therapeutics. It has been more challenging to develop a generic one-step SPE method that can be used across multiple tissue types.

An SPE method was developed for extraction of oligonucleotide therapeutics from tissue homogenates utilizing an ASO internal standard and a siRNA analyte. The method was fully automated using a liquid handler and an automated SPE station. Quantification of the siRNA antisense was performed using LC-MS, with calibration samples prepared in a surrogate matrix.

The fully automated SPE method enabled quantification of the siRNA antisense across nine different tissues from three different species. The SPE method utilizing surrogate matrix calibration was compared to liquid-liquid extraction by analyzing in-vivo samples using both workflows, showing good agreement between the two different extraction methods.

This manuscript presents a one-step SPE method greatly reducing blank tissue requirements and sample preparation workload by enabling use of surrogate matrix calibration and full automation. The presented SPE method simplifies and reduces analytical time in bioanalysis of tissue biodistribution studies and adheres to the principles of 3R.