Hydrophilic-Phase Extraction for Ion-Pairing-Free Oligonucleotide Analysis

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Solution Overview

Problem

Existing bioanalytical platforms face challenges in the quantitative analysis of oligonucleotides due to the use of ion-pairing reagents and extraction methods designed for hydrophobic small molecules, leading to issues like excessive instrumental downtime, reduced column lifetimes, signal drift, and sensitivity loss.

Innovation Solution

A novel method employing hydrophilic-phase extraction and hydrophilic-interaction liquid chromatography, utilizing a modified sorbent bed with an aminopropyl phase on silica, followed by mass spectral analysis without ion-pairing reagents, to isolate and analyze oligonucleotides.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Quantity of substance

If ion-pairing reagents are used for oligonucleotide extraction, then extraction efficiency is improved, but instrumental downtime increases and column lifetime decreases

Engineering Contradiction:

Improveextraction efficiencyVSAvoidinstrumental downtime

Core Design Contradiction:

Quantity of substanceVSLoss of time

Solution Approach 1:

The patent removes ion-pairing reagents from the extraction process entirely, using instead a hydrophilic-phase extraction system with a polar stationary phase that selectively retains oligonucleotides through hydrophilic interactions. This extraction approach achieves high recovery of oligonucleotides without requiring ion-pairing reagents, thereby eliminating the need for extensive instrument cleaning and column maintenance associated with ion-pairing reagent removal.

Inventive Principle:

Principle #2Taking out (Extraction)

Solution Approach 2:

The patent changes the fundamental parameter of the extraction mechanism from ion-pairing based on charged reagents to hydrophilic interaction based on polarity. By using a polar stationary phase and adjusting mobile phase composition to favor hydrophilic interactions, the system achieves selective retention of oligonucleotides without ion-pairing reagents, resolving the contradiction between extraction efficiency and instrumental maintenance requirements.

Inventive Principle:

Principle #35Parameter changes

2Quantity of substance

If ion-pairing reagents are used for oligonucleotide extraction, then extraction efficiency is improved, but column lifetime decreases

Engineering Contradiction:

Improveextraction efficiencyVSAvoidcolumn lifetime

Core Design Contradiction:

Quantity of substanceVSDuration of action of stationary object

Solution Approach 1:

The patent eliminates ion-pairing reagents from the system, using a hydrophilic-phase extraction approach where a polar stationary phase selectively interacts with oligonucleotides through hydrophilic forces. This removes the harmful chemical interactions that degrade column lifetime, allowing for extended column operation without the need for frequent replacement or extensive maintenance.

Inventive Principle:

Principle #2Taking out (Extraction)

Solution Approach 2:

The patent employs a disposable 96-well plate format for the extraction process, replacing the need for durable, long-lasting columns that are susceptible to ion-pairing reagent damage. This approach trades the investment in expensive, long-lived columns for a system using inexpensive, single-use extraction plates, thereby eliminating column replacement costs and maintenance requirements.

Inventive Principle:

Principle #27Cheap short-living objects (Disposable)

3Ease of operation

If traditional extraction methods for hydrophobic small molecules are used, then extraction simplicity is maintained, but sensitivity and resolution are reduced for polar and ionic biologics

Engineering Contradiction:

Improveextraction simplicityVSAvoidsensitivity

Core Design Contradiction:

Ease of operationVSMeasurement precision

Solution Approach 1:

The patent fundamentally changes the extraction parameter from hydrophobicity-based (non-polar) to hydrophilicity-based (polar) interactions. By using a polar stationary phase and adjusting mobile phase composition to emphasize hydrophilic interactions, the system achieves high sensitivity and resolution for polar and ionic oligonucleotides while maintaining operational simplicity through an automated 96-well plate format.

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The patent inverts the traditional extraction approach by using a polar stationary phase instead of a non-polar one. This inversion allows polar and ionic oligonucleotides to be retained and separated effectively, achieving high measurement precision for biologics that were previously difficult to analyze using conventional hydrophobic extraction methods.

Inventive Principle:

Principle #13The other way round (Inversion)

4Quantity of substance

If ion-pairing reagents are used, then extraction capability is improved, but signal drift occurs and sensitivity is lost

Engineering Contradiction:

Improveextraction capabilityVSAvoidsignal stability

Core Design Contradiction:

Quantity of substanceVSMeasurement precision

Solution Approach 1:

The patent removes ion-pairing reagents from the extraction process, using instead a hydrophilic-phase extraction system that relies on polar interactions between the stationary phase and oligonucleotides. This eliminates the source of signal drift and sensitivity loss associated with ion-pairing reagents, producing stable signals and high measurement precision throughout the analysis.

Inventive Principle:

Principle #2Taking out (Extraction)

Solution Approach 2:

The patent converts the natural polarity of oligonucleotides, which previously caused incompatibility with conventional hydrophobic extraction methods, into a beneficial feature. By using a polar stationary phase, the system exploits the hydrophilic character of oligonucleotides to achieve selective retention and high sensitivity detection, transforming what was a hindrance into a key separation mechanism.

Inventive Principle:

Principle #22Blessing in disguise (Convert harm into benefit)

Applied Scientific Principles

This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.

Function Achieved in This Case

This approach achieves high-recovery, ion-pairing free extraction with picomolar sensitivity, improving separation resolution and reducing instrumental downtime, while maintaining column longevity and sensitivity.

Implementation Method 1

conducting an extraction of the compound of interest, wherein the extraction is a hydrophilic-phase extraction

Methodology Applied

Scientific EffectHydrophilic interaction: Hydrophile

Implementation Method 2

isolating the compound of interest from other components of the sample via liquid chromatography

Methodology Applied

Scientific EffectChromatography: Chromatography

Implementation Method 3

analyzing the compound of interest via a mass spectrometer

Methodology Applied

Scientific EffectIonization: Ionisation

Data Source

PatentUS12405253B2Methods and systems for hydrophilic-phase extraction

Publication Date: 2025.09.02 LABORATORY CORPORATION OF AMERICA HOLDINGS INC

AI summary

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.