title: UPLC-MS/MS method for quantification of the azathioprine metabolites 6-mercaptoguanosine and 6-methylmercaptopurine riboside in peripheral blood mononuclear cells.
abstract: In the treatment of inflammatory bowel diseases, the use of azathioprine is increasing over the time. It has been demonstrated that the effectiveness of this therapy is modulated by the metabolism of azathioprine, which is mainly exerted by both thiopurine methyl-transferase and inosine triphosphatase enzymes. Several studies reported chromatographic methods to determine the amount of its metabolites in erythrocytes, but there are not reported methods to dose them in peripheral blood mononuclear cells (PBMCs). The development of a method capable to quantify azathioprine nucleoside metabolites in this compartment could give better information on drug penetration and metabolism in the active site. In this work, we validated a new chromatographic method suitable for the monitoring of the two major biologically active ribonucleos(t)ide metabolites of azathioprine in PBMCs: 6-thioguanosine and 6-methyl-mercaptopurine riboside. After PBMCs extraction from blood through separation on density gradient, samples underwent a de-phosphorylation procedure with acid phosphatase (only one aliquot for each sample) and were then treated with a protein precipitation protocol in acetonitrile, followed by UPLC-tandem-mass spectrometry analysis. The calibration curve for each metabolite in PBMC fitted a least squares model (weighed 1/X) from 0.048 to 25ng (r(2)=0.998). Both accuracy and precision parameters fitted FDA guidelines. We tested this method by monitoring the concentrations of each metabolite in PBMC from eight inflammatory bowel diseases affected patients, receiving azathioprine maintenance therapy with optimal results.
authors: Unit of Infectious Diseases, University of Turin, Department of Medical Sciences, Amedeo di Savoia Hospital, Turin, Italy. Electronic address: amedeodenicolo@libero.it.
authors: Amedeo
authors: A
authors: De Nicolò
authors: Unit of Infectious Diseases, University of Turin, Department of Medical Sciences, Amedeo di Savoia Hospital, Turin, Italy.
authors: Danilo
authors: D
authors: Agnesod
authors: Unit of Infectious Diseases, University of Turin, Department of Medical Sciences, Amedeo di Savoia Hospital, Turin, Italy.
authors: Marco
authors: M
authors: Simiele
authors: Dipartimento di Scienza e Tecnologia del Farmaco, University of Turin, Turin, Italy.
authors: Danila
authors: D
authors: Riganò
authors: Unit of Gastroenterology, University of Turin, Department of Medical Sciences, San Giovanni Battista Hospital, Turin, Italy.
authors: Alessandro
authors: A
authors: Adriani
authors: Dipartimento di Scienza e Tecnologia del Farmaco, University of Turin, Turin, Italy.
authors: Roberto
authors: R
authors: Canaparo
authors: Unit of Gastroenterology, University of Turin, Department of Medical Sciences, San Giovanni Battista Hospital, Turin, Italy.
authors: Marco
authors: M
authors: Astegiano
authors: Unit of Gastroenterology, University of Turin, Department of Medical Sciences, San Giovanni Battista Hospital, Turin, Italy.
authors: Mario
authors: M
authors: Rizzetto
authors: Unit of Infectious Diseases, University of Turin, Department of Medical Sciences, Amedeo di Savoia Hospital, Turin, Italy.
authors: Giovanni
authors: G
authors: Di Perri
authors: Unit of Infectious Diseases, University of Turin, Department of Medical Sciences, Amedeo di Savoia Hospital, Turin, Italy.
authors: Antonio
authors: A
authors: D'Avolio
mesh_terms: D001379:Azathioprine / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D006151:Guanosine / Q000031:analogs & derivatives* / Q000737:chemistry; D006801:Humans; D015212:Inflammatory Bowel Diseases / Q000175:diagnosis; D007963:Leukocytes, Mononuclear / Q000737:chemistry*; D008778:Methylthioinosine / Q000737:chemistry*; D053719:Tandem Mass Spectrometry / Q000379:methods; D013872:Thionucleosides / Q000737:chemistry*
chemical_list: D013872:Thionucleosides; D006151:Guanosine; D008778:Methylthioinosine; C005783:6-thioguanosine; D001379:Azathioprine
keywords: Intracellular determination; Nucleosides; Tandem-mass detector; UPLC