In drug development, quantitative analysis of oligonucleotides in biological samples is crucial for pharmacokinetic (PK), pharmacodynamic (PD), and toxicological safety evaluation. Due to the complex chemical properties of oligonucleotides and the special sample matrix, sample pretreatment techniques are also correspondingly complex. Liquid liquid extraction (LLE) and solid phase extraction (SPE) are commonly used pretreatment methods, among which SPE is more suitable for low LLOQ requirements and complex matrix situations [2]. However, traditional manual SPE methods are time-consuming and susceptible to human error.

Liquid chromatography-mass spectrometry (LC-MS/MS) has high sensitivity, low detection limit, high selectivity, and specificity, and can quickly and accurately detect and quantify low concentration target compounds in complex matrices without the need for special reagents and probes. It is widely used in oligonucleotide biological analysis [3].

In order to solve the problems in sample pretreatment, we have developed an automated SPE workstation based on the Bravo platform, combined with the new generation triple quadrupole liquid chromatography-mass spectrometry 6495D LC-MS/MS, providing a complete solution from sample pretreatment, sample analysis to data processing. The 6495D LC-MS/MS system exhibits excellent performance in high sensitivity, low detection limit, and resistance to matrix interference, while the automated SPE workstation has significant advantages in reducing manual intervention time, improving operational consistency, and reliability. This complete automated SPE biological analysis workflow significantly improves the efficiency and reliability of quantitative biological analysis of oligonucleotide drugs.