Methods and systems for hydrophilic-phase extraction
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Publication Number
US-12405253-B2
Publication Date
2025-09-02
Expiration Date
Abstract
Disclosed herein are embodiments of methods for oligonucleotide analysis using a novel solid-phase extraction and hydrophilic-interaction liquid chromatography. The unique polar-based retention methods provided herein provide a high-recovery extraction. The methods improve assay reliability and reproducibility and reach picomolar sensitivity with the demonstrably beneficial accurate mass platform. Also disclosed herein are systems and computer program products for performing these methods.
Core Innovation
The invention provides a method for determining an amount and/or a presence of a compound of interest in a sample from a subject, where the sample is a biological sample and the compound of interest is an RNA or a DNA oligonucleotide. The method includes conducting a hydrophilic-phase extraction on a sorbent bed comprising an aminopropyl phase on a silica base, to extract the RNA or DNA oligonucleotide from the biological sample. The extracted compound of interest is then isolated from other components via liquid chromatography and analyzed via a mass spectrometer.
In the described extraction workflow, aqueous acid and an internal standard are added to the sample prior to loading the sample onto the aminopropyl-phase/silica sorbent bed. The sorbent bed is conditioned with a solution comprising 9:1 v:v acetonitrile: 2.0% formic acid, followed by washing the sorbent bed and eluting the compound of interest with a solution comprising ammonium hydroxide and acetonitrile. The workflow is characterized in the claims by operating under ion-pairing-free conditions.
The system aspects of the invention include a hydrophilic-phase extraction system with the aminopropyl phase on a silica base, together with a liquid chromatography system and a mass spectrometer system, for determining an amount and/or a presence of the RNA or DNA oligonucleotide in a biological sample. The claims also include a tangible, non-transitory computer readable medium storing a computer-program product that causes one or more data processors to perform actions directing the extraction, isolation via liquid chromatography, and analysis via a mass spectrometer.
Claims Coverage
The document includes three independent claims covering a method, a system, and a computer-readable medium. Across the independent claims, five main inventive features repeat: hydrophilic-phase extraction on an aminopropyl-phase silica sorbent bed with defined sample loading, conditioning, and elution; isolation by liquid chromatography; analysis by a mass spectrometer; and ion-pairing-free conditions in the claim set.
Hydrophilic-phase extraction on aminopropyl-on-silica sorbent bed
Conducting a hydrophilic-phase extraction on a sorbent bed comprising an aminopropyl phase on a silica base on a biological sample containing an RNA or a DNA oligonucleotide.
Defined sample loading reagents: aqueous acid and internal standard
Adding an aqueous acid and an internal standard to the sample prior to loading the sample onto the aminopropyl-phase/silica sorbent bed.
Conditioning with acetonitrile/formic acid and elution with ammonium hydroxide/acetonitrile
Conditioning the sorbent bed with a solution comprising 9:1 v:v acetonitrile: 2.0% formic acid and eluting the compound of interest with a solution comprising ammonium hydroxide and acetonitrile.
Isolation via liquid chromatography and analysis via mass spectrometer
Isolating the compound of interest from other components of the sample via liquid chromatography and analyzing the compound of interest via a mass spectrometer.
Ion-pairing-free conditions
The method is carried out under ion-pairing-free conditions.
Overall, the independent claims cover a workflow for determining an amount and/or a presence of an RNA or DNA oligonucleotide in a biological sample using hydrophilic-phase extraction on an aminopropyl-on-silica sorbent bed, followed by liquid chromatography isolation and mass spectrometry analysis, with ion-pairing-free conditions specified in the claim set.
Stated Advantages
Documented Applications
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