DECAF: Deconvoluted Extracted Ion Chromatogram-Based Quantification of Therapeutic Oligonucleotides

Area of Science:

• Analytical Chemistry
• Mass Spectrometry
• Biochemistry

Background:

• Quantification in LC-MS is hindered by co-eluting analytes with overlapping isotope patterns.
• Conventional methods can lead to biased results due to double-counting shared signals, especially for oligonucleotides.
• Degradation products and impurities complicate relative quantification of oligonucleotide therapeutics.

Purpose of the Study:

• To develop a method for deconvoluting overlapping isotope patterns in MS1 data.
• To enable accurate quantification of individual components in complex mixtures.
• To provide a transparent and efficient alternative to existing quantification software.

Main Methods:

• Developed DECAF, a procedure for deconvoluting overlapping isotope patterns from MS1 data.
• Modeled experimental isotope distributions as mixtures of theoretical templates across retention time.
• Generated deconvoluted ion chromatograms to reflect individual component contributions.

Main Results:

• DECAF reliably estimated mixing proportions in two oligonucleotide mixture datasets.
• Enabled visualization of component-specific elution profiles.
• Analysis of a typical sample was completed in 1-2 minutes, demonstrating computational efficiency.

Conclusions:

• DECAF accurately quantifies components in mixtures with overlapping isotope patterns.
• The method offers a practical and efficient solution for pharmaceutical analysis and quality control.
• DECAF provides a transparent and accessible alternative to commercial deconvolution software.