Sensitive GC-MS/MS Method to Measure Deuterium Labeled Deoxyadenosine in DNA from Limited Mouse Cell Populations

Abstract

A rapid and sensitive gas chromatography-tandem mass spectrometry (GC-MS/MS) method was developed to quantitatively measure low levels of DNA base deoxyadenosine (dA) and its isotopologues (e.g., dA M+1) from limited mouse cell populations. Mice undergoing allogeneic hematopoietic transplantation (AHSCT) received deuterated water at biologically relevant time intervals post AHSCT, allowing labeling of DNA upon cell division, which was detected as the dA M+1 isotopologue. Targeted mouse cell populations were isolated from lymphoid organs and purified by multiparameter fluorescence activated cell sorting. Cell lysis, DNA extraction, and hydrolysis were accomplished using available commercial procedures. The novel analytical method utilized a hydrophilic-lipophilic balanced sample preparation, rapid online hot GC inlet gas phase sample derivatization, fast GC low thermal mass technology, and a recently marketed GC-MS/MS system. Calibration standards containing dA and fortified with relevant levels of dA M+1 (0.25-20%) and dA M+5 (internal standard) were used for sample quantitation. The method employed a quadratic fit for calibration of dA M+1 (0.25-20%) and dA, demonstrated excellent accuracy and precision, and had limits of detection of 100 fg on-column for the dA isotopologues. The method was validated and required only 20 000 cells to characterize population dynamics of cells involved in the biology of chronic graft-versus-host disease, the main cause of late morbidity and nonrelapse-mortality following AHSCT. The high sensitivity and specificity of the method makes it useful for investigating in vivo kinetics on limited and important cell populations (e.g., T regulatory cells) from disease conditions or in disease models that are immune-mediated, such as diabetes, human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS), arthritis, inflammatory bowel disease, and multiple sclerosis.