Development of a Versatile High-through-put Oligonucleotide LC-MS Method to Accelerate Drug Discovery.

Animals Rats Chromatography, Liquid / methods Tandem Mass Spectrometry / methods Drug Discovery / methods Oligonucleotides, Antisense / administration & dosage Liquid-Liquid Extraction / methods Oligonucleotides High-Throughput Screening Assays / methods RNA, Long Noncoding Liquid Chromatography-Mass Spectrometry
LC–MS crosstalk-free internal standard high-through-put oligonucleotide (Oligo) sensitivity improvement

Journal

AAPS PharmSciTech
ISSN: 1530-9932
Titre abrégé: AAPS PharmSciTech
Pays: United States
ID NLM: 100960111

Informations de publication

Date de publication:
10 Oct 2024
Historique:
received: 01 07 2024
accepted: 03 09 2024
medline: 11 10 2024
pubmed: 11 10 2024
entrez: 10 10 2024
Statut: epublish

Résumé

Liquid chromatography-mass spectrometry (LC-MS) is an effective tool for high-throughput quantification of oligonucleotides that is crucial for understanding their biological roles and developing diagnostic tests. This paper presents a high-throughput LC-MS/MS method that may be versatilely applied for a wide range of oligonucleotides, making it a valuable tool for rapid screening and discovery. The method is demonstrated using an in-house synthesized MALAT-1 Antisense oligonucleotide (ASO) as a test case. Biological samples were purified using a reversed liquid-liquid extraction process automated by a liquid handling workstation and analyzed with ion-pairing LC-MS/MS. The assay was evaluated for sensitivity (LLOQ = 2 nM), specificity, precision, accuracy, recovery, matrix effect, and stability in rat cerebrospinal fluid (CSF) and plasma. Besides some existing considerations such as column selection, ion-pairing reagent, and sample purification, our work focused on the following four subtopics: 1) selecting the appropriate Multiple Reaction Monitoring (MRM) transition to maximize sensitivity for trace-level ASO in biological samples; 2) utilizing a generic risk-free internal standard (tenofovir) to avoid crosstalk interference from the oligo internal standard commonly utilized in the LC-MS assay; 3) automating the sample preparation process to increase precision and throughput; and 4) comparing liquid-liquid extraction (LLE) and solid-phase extraction (SPE) as sample purification methods in oligo method development. The study quantified the concentration of MALAT-1 ASO in rat CSF and plasma after intrathecal injection and used the difference between the two matrices to evaluate the injection technique. The results provide a solid foundation for further internal oligonucleotide discovery and development.

Identifiants

DOI: 10.1208/s12249-024-02934-3 PMID: 39390148
pubmed: 39390148
doi: 10.1208/s12249-024-02934-3
pii: 10.1208/s12249-024-02934-3
doi:

Substances chimiques

Oligonucleotides, Antisense 0
Oligonucleotides 0
RNA, Long Noncoding 0

Types de publication

Journal Article

Langues

eng

Sous-ensembles de citation

IM

Pagination

239

Informations de copyright

© 2024. Merck & Co., Inc., Rahway, NJ, USA and its affiliates, under exclusive licence to American Association of Pharmaceutical Scientists.

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Auteurs

Changhong Yun (C)

Pharmacokinetics, Dynamics, Metabolism, and Bioanalytics, Merck & Co., Inc., Boston, Massachusetts, 02115, USA.
ORCID: 0000-0002-9432-6735

Hyun Chong Woo (HC)

Pharmacokinetics, Dynamics, Metabolism, and Bioanalytics, Merck & Co., Inc., Boston, Massachusetts, 02115, USA.

Ditte Lovatt (D)

Biology-Discovery, Merck & Co., Inc., West Point, Pennsylvania, 19446, USA.

Craig A Parish (CA)

Discovery Chemistry, Merck & Co., Inc., Rahway, New Jersey, 07065, USA.

Daniel S Spellman (DS)

Pharmacokinetics, Dynamics, Metabolism, and Bioanalytics, Merck & Co., Inc., West Point, Pennsylvania, 19446, USA.

Honglue Shen (H)

Pharmacokinetics, Dynamics, Metabolism, and Bioanalytics, Merck & Co., Inc., Boston, Massachusetts, 02115, USA. honglue.shen@merck.com.

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Classifications MeSH

questionsmedicales.fr › Eucaryotes › Animaux › Development of a Versatile High-through-put...
questionsmedicales.fr › Eucaryotes › Animaux › Chordés › Vertébrés › Mammifères › Eutheria › Rodentia › Muridae › Murinae › Rats › Development of a Versatile High-through-put...
questionsmedicales.fr › Techniques d'investigation › Techniques de chimie analytique › Chromatographie › Chromatographie en phase liquide › Development of a Versatile High-through-put...
questionsmedicales.fr › Techniques d'investigation › Techniques de chimie analytique › Spectrométrie de masse › Spectrométrie de masse en tandem › Development of a Versatile High-through-put...
questionsmedicales.fr › Techniques d'investigation › Développement de médicament › Découverte de médicament › Development of a Versatile High-through-put...
questionsmedicales.fr › Actions chimiques et utilisations › Utilisations spécialisées de produits chimiques › Produits chimiques de laboratoire › Sondes moléculaires › Sondes d'acide nucléique › Éléments antisens (génétique) › Oligonucléotides antisens › Development of a Versatile High-through-put...
questionsmedicales.fr › Techniques d'investigation › Techniques de chimie analytique › Fractionnement chimique › Extraction liquide-liquide › Development of a Versatile High-through-put...
questionsmedicales.fr › Acides nucléiques, nucléotides et nucléosides › Nucléotides › Polynucléotides › Oligonucléotides › Development of a Versatile High-through-put...
questionsmedicales.fr › Techniques d'investigation › Tests de criblage à haut débit › Development of a Versatile High-through-put...
questionsmedicales.fr › Acides nucléiques, nucléotides et nucléosides › Acides nucléiques › ARN › ARN non traduit › ARN long non codant › Development of a Versatile High-through-put...