How to scale up a method from low throughput to high throughput for the quantification of 16 nucleosides? Lesson from experience.

Quentin Vandoolaeghe (1, 2, 3) , Valérie Bouchart (3, 1, 4) , Isabelle Vaudorne (4) , Raphaël Delépée (1, 4)

1 ANTICIPE - Unité de recherche interdisciplinaire pour la prévention et le traitement des cancers
2 UNICANCER/CRLC - Centre Régional de Lutte contre le Cancer François Baclesse [Caen]
3 LABÉO - LABÉO, Pôle d’analyses et de recherche de Normandie
4 PRISMM - Plateforme de Recherche et d'Innovation en Spectrométrie de Masse et Métabolomique

Biological plausibility of the increase of cancer incidence related to agricultural occupational exposures need to be more documented. Pesticides are suspected to belong to the causes of this increase of cancer incidence in farmers. Oxidative stress and epigenetic alterations are possible mechanism suspected for pesticides cancerogenicity. Aldehydes are produced during lipid peroxidation associated with oxidative stress. These highly hydrophilic compounds can react with DNA to form DNA adducts. These biomarkers have been identified as crucial in the exposome study. Quantified in a specific population, these biomarkers could be linked with professional exposure effects. This imply the analysis of several hundred to several thousand of samples from cohort. Starting from an HPLC-HRMS/MS method developed in the laboratory [1], we decide to develop a high throughput method for the quantification of epigenetic biomarkers and DNA adducts from human white blood cells. The original method, able to quantify 9 exocyclic DNA adducts has been improved by adding 5 new epigenetic biomarkers (i.e. 5-Methylcytidine, 5-Hydroxymethylcytidine, 5-Formylcytidine, 5-Hydroxymethyluridine, 5-Carboxylcytidine) and 2 new DNA adducts (i.e. ethenodeoxyadenosine and ethenodeoxycytidine). The previous method was time consuming and adapted for the preparation of only 20 samples per week. Samples preparation steps are: reduction, precipitation, internal standards addition and enzymatic hydrolysis of DNA. Redesigning of all these steps was needed to increase the throughput of the method. A high throughput improvement as been made thanks to the automatization of most of the steps. A pipetting robot Integra Assit Plus as been used for this automatization. This pipetting Robot was used for the reduction, the precipitation and hydrolysis. The use of individual tube was no longer possible and 96 well plates have been used instead. As an example of optimization, the reduction step was shortened from 2 hours to 30 minutes. In its current state, the preparation method allows the preparation of more than 300 samples each week. The modification applied on the original method have been tested for the preparation of 700 samples obtained from an agricultural cohort. Obtained results are robust and human error are limited thanks to the use of a pipetting robot.

Domaines: Chimie

Quentin Vandoolaeghe, Valérie Bouchart, Isabelle Vaudorne, Raphaël Delépée. How to scale up a method from low throughput to high throughput for the quantification of 16 nucleosides? Lesson from experience.. Congrès ANALYTICS 2022, Sep 2022, Nantes, France. ⟨hal-03846446⟩