title: Quantitation of isocitrate dehydrogenase (IDH)-induced D and L enantiomers of 2-hydroxyglutaric acid in biological fluids by a fully validated liquid tandem mass spectrometry method, suitable for clinical applications.
abstract: A recent update of the hallmarks of cancer includes metabolism with deregulating cellular energetics. Activating mutations in isocitrate dehydrogenase (IDH) metabolic enzymes leading to the abnormal accumulation of 2-hydroxyglutaric acid (2-HGA) have been described in hematologic malignancies and solid tumours. The diagnostic value of 2-HGA levels in blood to identify IDH mutations and its prognostic significance have been reported. We developed a liquid chromatography tandem mass spectrometry method allowing a rapid, accurate and precise simultaneous quantification of both L and D enantiomers of 2-HGA in blood samples from acute myeloid leukaemia (AML) patients, suitable for clinical applications. The method was also develop for preclinical applications from cellular and tissues samples. Deuterated (R,S)-2-hydroxyglutaric acid, disodium salt was used as internal standard and added to samples before a solid phase extraction on Phenomenex STRATA™-XL-A (200mg-3mL) 33μm cartridges. A derivatization step with (+)- o,o'-diacetyl-l-tartaric anhydride permitted to separate the two resulting diastereoisomers without chiral stationary phase, on a C18 column combined to a Xevo TQ-MS Waters mass spectrometer with an electrospray ionization (ESI) source. This method allows standard curves to be linear over the range 0.34-135.04μM with r(2) values>0.999 and low matrix effects (<11.7%). This method, which was validated according to current EMA guidelines, is accurate between-run (<3.1%) and within-run (<7.9%) and precise between-run (<5.3CV%) and within-run (<6.2CV%), and is suitable for clinical and preclinical applications.
authors.affiliation: Gustave Roussy Cancer Campus Grand Paris, Service de Pharmacologie, Département de Biologie et Pathologie Médicales, Villejuif 94805, France.
authors.forename: Vianney
authors.initials: V
authors.lastname: Poinsignon
authors.affiliation: Gustave Roussy Cancer Campus Grand Paris, Service de Pharmacologie, Département de Biologie et Pathologie Médicales, Villejuif 94805, France.
authors.forename: Lionel
authors.initials: L
authors.lastname: Mercier
authors.affiliation: INSERM U 1170, Institut Gustave Roussy, Villejuif 94805, France.
authors.forename: Koïchi
authors.initials: K
authors.lastname: Nakabayashi
authors.affiliation: INSERM U 1170, Institut Gustave Roussy, Villejuif 94805, France.
authors.forename: Muriel D
authors.initials: MD
authors.lastname: David
authors.affiliation: Gustave Roussy Cancer Campus Grand Paris, Service de Pharmacologie, Département de Biologie et Pathologie Médicales, Villejuif 94805, France.
authors.forename: Alexandre
authors.initials: A
authors.lastname: Lalli
authors.affiliation: INSERM U 1170, Institut Gustave Roussy, Villejuif 94805, France.
authors.forename: Virginie
authors.initials: V
authors.lastname: Penard-Lacronique
authors.affiliation: INSERM U 1170, Institut Gustave Roussy, Villejuif 94805, France.
authors.forename: Cyril
authors.initials: C
authors.lastname: Quivoron
authors.affiliation: INSERM U 1170, Institut Gustave Roussy, Villejuif 94805, France.
authors.forename: Véronique
authors.initials: V
authors.lastname: Saada
authors.affiliation: INSERM U 1170, Institut Gustave Roussy, Villejuif 94805, France.
authors.forename: Stéphane
authors.initials: S
authors.lastname: De Botton
authors.affiliation: Gustave Roussy Cancer Campus Grand Paris, Service de Pharmacologie, Département de Biologie et Pathologie Médicales, Villejuif 94805, France.
authors.forename: Sophie
authors.initials: S
authors.lastname: Broutin
authors.affiliation: Gustave Roussy Cancer Campus Grand Paris, Service de Pharmacologie, Département de Biologie et Pathologie Médicales, Villejuif 94805, France; Université Paris-Sud, Laboratoire de Vectorologie et Thérapeutiques Anticancéreuses, UMR 8203, Villejuif 94805, France; Centre National de la Recherche Scientifique (CNRS), Laboratoire de Vectorologie et Thérapeutiques Anticancéreuses, UMR 8203, Villejuif 94805, France; Gustave Roussy Cancer Campus Grand Paris, Laboratoire de Vectorologie et Thérapeutiques Anticancéreuses, UMR 8203, Villejuif 94805, France. Electronic address: angelo.paci@gustaveroussy.fr.
authors.forename: Angelo
authors.initials: A
authors.lastname: Paci
mesh_terms: D014408:Biomarkers, Tumor / Q000097:blood* / Q000737:chemistry / Q000378:metabolism; D045744:Cell Line, Tumor; D002853:Chromatography, Liquid / Q000379:methods*; D005977:Glutarates / Q000097:blood* / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007521:Isocitrate Dehydrogenase / Q000378:metabolism*; D015470:Leukemia, Myeloid, Acute / Q000378:metabolism; D016014:Linear Models; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013237:Stereoisomerism; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D014408:Biomarkers, Tumor; D005977:Glutarates; C019417:alpha-hydroxyglutarate; D007521:Isocitrate Dehydrogenase
keywords: 2-Hydroxyglutaric acid; Biomarker; Endogenous compounds; Isocitrate dehydrogenase mutations; Liquid tandem mass spectrometry; Oncometabolite