• Application Note

LC-MS Analysis of Synthetic Oligonucleotides

LC-MS Analysis of Synthetic Oligonucleotides
  • Waters Corporation
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Abstract

This application note demonstrates LC-MS analysis of synthetic oligonucleotides.

Introduction

LC-MS – The method of choice for characterization of oligonucleotides

Quality control and characterization is an important requirement for therapeutic oligonucleotides. Liquid chromatography with mass spectrometry detection (LC-MS) is the most suitable method for this analysis. Liquid chromatography using XTerra MS C18 Columns provides good oligonucleotide resolution up to 60mer using a mobile phase compatible with electrospray mass spectrometry (ESI-MS). The methods were developed for sensitive LC-MS analysis of native and modified oligonucleotides using a 1.0 x 50 mm XTerra MS C18 Column with a Capillary HPLC System and ESI (Tof) mass spectrometer.

Experimental

Preparation of 16.3 mM TEA - 400 mM HFIP buffer

Dissolve 41.5 mL of HFIP in ~950 mL of water. While mixing vigorously add 2.3 mL of TEA. Adjust volume to 1L with water. The pH of solution should be close to 7.9.

Preparation of 8.6 mM TEA - 100 mM HFIP buffer

Dissolve 10.5 mL of HFIP in ~950 mL of water. While mixing vigorously add 1.2 mL of TEA. Adjust volume to 1L with water. The pH of solution should be close to 8.3.

Results and Discussion

XTerra Columns for Sensitive LC-MS analysis and HPLC conditions

XTerra MS C18 Columns are packed with porous 2.5 μm hybrid particles. The sorbent has extended stability at temperatures and pH’s typically used for oligonucleotide separations (50–60 °C; pH 7–9). The 1.0 x 50 mm column is operated at the mobile phase flow rate compatible with direct sensitive MS detection. Mobile phases consist from aqueous triethylamine (TEA) and hexafluoroisopropanol (HFIP) solutions (ion-pairing buffer) and methanol. Oligonucleotide resolution achieved with this system was greater than with traditional triethylammonium acetate (TEAA) ion-pairing buffer. Contrary to TEAA based mobile phases, little or no ion suppression was observed with TEA-HFIP buffers.

Figure 1. LC-MS analysis of 25mer phosphorothioate oligonucleotides.

Principles of Oligonucleotide Identification by MS

The comparison of theoretical and measured mass was used for oligonucleotide identification. The Tof mass spectrometer routinely achieves mass accuracy ±1Da for oligonucleotides <50mer or even longer. Figure 2 shows oligonucleotides generated by digesting 25mer with 3’ exonuclease. They were positively identified by their molecular mass. The difference in mass for 24/25mer pair was 329.2 Da, indicating the loss of G mononucleotide. Similarly, the difference in mass for 23/24, 22/23, and 20/21 shows a loss of A, T and C mononucleotide, respectively (313.2, 304.2 304.2, and 289.2 Da). This method can be used for sequence verification and failure products identification of therapeutic and diagnostic oligonucleotides (Gilar, Anal. Biochem. 298 (2001) 196–206). Figure 3 shows analysis of TaqMan oligonucleotide. Accurate mass measurement was used for identification of singly-labeled failure products from “one-pot” synthesis. First elute nonlabeled oligonucleotides, followed with 5’FAM products, 3’TAMRA labeled oligonucleotides and the dually-labeled target product. Later eluting peaks are 1-4mer TAMRA labeled products and un-conjugated dye.

Figure 2. LC-MS identification of oligonucleotides.
Figure 3. LC-MS identification of 21mer TaqMan and failure by-products generated by one-pot Probe synthesis.

WA20773, June 2003

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In-depth Insights: LC-MS Profiling of Synthetic Oligonucleotides

LC-MS (Liquid Chromatography-Mass Spectrometry) analysis of synthetic oligonucleotides is a powerful and sophisticated technique used in molecular biology, pharmaceuticals, and various research applications. This analytical method plays a crucial role in characterizing and validating synthetic oligonucleotides, providing valuable information about their purity, composition, and structural integrity.

The process begins with sample preparation, where the synthetic oligonucleotides are purified and extracted, ensuring that the analyzed sample is representative of the intended product. LC-MS is then employed for the separation and identification of oligonucleotide molecules based on their mass-to-charge ratio.

Liquid chromatography is utilized to separate the oligonucleotide mixture into its individual components. This separation is achieved through a stationary phase that interacts differently with each oligonucleotide, leading to distinct elution times. The separated oligonucleotides are subsequently introduced into the mass spectrometer.

Mass spectrometry is a key component of the analysis, providing accurate mass measurements of the oligonucleotides. It involves ionizing the separated molecules, generating charged ions that can be accelerated and separated based on their mass-to-charge ratio. The resulting mass spectra offer insights into the molecular weight, sequence, and presence of any modifications in the synthetic oligonucleotides.

LC-MS analysis allows for the detection of impurities, sequence variants, or modifications, which is crucial for quality control in oligonucleotide synthesis. The technique can identify and quantify different oligonucleotide species, providing a comprehensive understanding of the sample composition.

Moreover, LC-MS is particularly valuable for confirming the success of post-synthetic modifications, such as labeling or conjugation, ensuring the accuracy of the intended modifications. It also aids in assessing the overall quality and homogeneity of the synthetic oligonucleotide batch.

The LC-MS analysis of synthetic oligonucleotides is a versatile tool, offering high sensitivity and resolution. It is instrumental in research areas like antisense oligonucleotide therapeutics , gene editing, and diagnostics, where the precise characterization of synthetic oligonucleotides is crucial for their biological activity and therapeutic efficacy.

In summary, LC-MS analysis of synthetic oligonucleotides is a sophisticated and integral technique in molecular biology and pharmaceutical research. It provides detailed insights into the purity, composition, and structural characteristics of synthetic oligonucleotides, ensuring their reliability and suitability for various applications.