LC-MS/MS method after derivation for the determination of

LC-MS/MS method after derivation for the determination of L- and D- isomers of 2-
hydroxyglutaric acid in biological fluids: application as biomarker of IDH mutations in
cancer.
Vianney POINSIGNON1, Lionel MERCIER1, Koïchi NAKABAYASHI2, Alexandre
LALLI1, Muriel DAVID2, Sophie BROUTIN1, Stéphane De BOTTON2, Angelo PACI1,3,4,5
(1) Gustave Roussy Cancer Campus Grand Paris, Service de Pharmacologie et d'Analyse
du Médicament (SIPAM), Villejuif, France-94805.
(2) INSERM U 985, Institut Gustave Roussy, Villejuif, France-94805.
(3) Université Paris-Sud, Laboratoire de Vectorologie et Thérapeutiques Anticancéreuses,
UMR 8203, Villejuif, France-94805;
(4) Centre National de la Recherche Scientifique (CNRS), Laboratoire de Vectorologie et
Thérapeutiques Anticancéreuses, UMR 8203, Villejuif, France-94805;
(5) Gustave Roussy Cancer Campus Grand Paris, Laboratoire de Vectorologie et
Thérapeutiques Anticancéreuses, UMR 8203, Villejuif, France-94805;
A recent update of the hallmarks of cancer includes metabolism with deregulating cellular energetics.
Moreover activating mutations of isocitrate deshydrogenase (IDH) leading to the production of 2-
hydroxyglutaric acid (2-HGA) have been described in hematologic malignancies and solid tumors.
Therefore, 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 human serum for clinical applications as diagnostic and predictive biomarker.
Deuterated (R,S)-2-hydroxyglutaric acid (disodium salt) is used as internal standard and added
to serum aliquots before a solid phase extraction on STRATATM-X-A (200 mg – 3 mL) 33µm
cartridges. A derivatization step with (+)-o,o’-diacetyl-L-tartaric anhydride permitted to
separate the two enantiomers without chiral stationary phase, on a C18 column (Agilent
ZorbaxSB®, 4.6 x 150 mm, 5 µM) combined to a XevoTQ mass spectrometer with an
electrospray ionization (ESI) source. This method allows standard curves to be linear over the
range 0.338-135.04 µmol/L with r² values > 0.995 and low matrix effects (<7.1%). It is an
accurate (bias <5.6% RSD) and precise (CV value for repeatability <6.2% and for
intermediate precision <5.6%) method which was validated according ICH Q2(R1) and
current EMA guidelines and is suitable for the analysis of patient samples.