Comprehensive Quantitative and Qualitative Liquid Chromatography–Radioisotope–Mass Spectrometry Analysis for Safety Testing of Tolbutamide Metabolites Without Standard Samples

Tozuka, Zenzaburo; Aoyama, Shigeru; Nozawa, Kohei; Akita, Shoji; Oh-hara, Toshinari; Adachi, Yasuhisa; Ninomiya, Shinichi

doi:10.1002/jps.22646

Cited by 10 publications

(5 citation statements)

References 16 publications

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“…As depicted in Scheme 2, tolbutamide (TBM) is metabolized in vivo via benzylic oxidation also, with the hydroxymethyl product reported as a major metabolite. 20,21 The first oxidation of TBM occurred at 1.3 V, as measured by cyclic voltammetry (CV). This falls within the potential range at which benzylic oxidation products of tolyl substrates have been isolated.…”

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confidence: 99%

“…The reaction outcome was shown to depend on the oxidation potential of each substrate: oxidation products were isolated in good yields when a substrate’s oxidation occurred below 2.3 V versus the standard calomel electrode (SCE) reference. As depicted in Scheme , tolbutamide ( TBM ) is metabolized in vivo via benzylic oxidation also, with the hydroxymethyl product reported as a major metabolite. , The first oxidation of TBM occurred at 1.3 V, as measured by cyclic voltammetry (CV). This falls within the potential range at which benzylic oxidation products of tolyl substrates have been isolated.…”

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Preparative Microfluidic Electrosynthesis of Drug Metabolites

Stalder

Roth

2013

ACS Med. Chem. Lett.

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In vivo, a drug molecule undergoes its first chemical transformation within the liver via CYP450-catalyzed oxidation. The chemical outcome of the first pass hepatic oxidation is key information to any drug development process. Electrochemistry can be used to simulate CYP450 oxidation, yet it is often confined to the analytical scale, hampering product isolation and full characterization. In an effort to replicate hepatic oxidations, while retaining high throughput at the preparative scale, microfluidic technology and electrochemistry are combined in this study by using a microfluidic electrochemical cell. Several commercial drugs were subjected to continuous-flow electrolysis. They were chosen for their various chemical reactivity: their metabolites in vivo are generated via aromatic hydroxylation, alkyl oxidation, glutathione conjugation, or sulfoxidation. It is demonstrated that such metabolites can be synthesized by flow electrolysis at the 10 to 100 mg scale, and the purified products are fully characterized. KEYWORDS: Drug metabolites, electrochemistry, microfluidic synthesis, continuous-flow oxidation T he first step toward elimination of xenobiotic compounds in vivo occurs predominantly through first pass hepatic oxidation. In the liver, the oxidation takes place at the iron center of the porphyrin that constitutes the catalytic site of the CYP450 family of enzymes. 1,2 The oxidized metabolites (Phase I) may undergo subsequent transformations (Phase II) including, among others, conjugation with glutathione (GSH). This is of particular importance in the design of new drug candidates since their toxicity levels are regulated by such metabolic processes. 3,4 Significant research effort has been devoted to develop synthetic methodologies to simulate the metabolism of drugs. These methodologies fall into four distinct categories: microsomal incubation, porphyrin-catalyzed chemical oxidation, Fenton-type reactions, and electrochemical oxidations. 5,6 Electrosynthetic transformations offer the advantage of atom economy since the oxidation takes place at the surface of an electrode, which provides the electron from a current source. 7 These transformations are thus heterogeneous reactions by definition. To achieve high conversion, the substrate solution must be conductive, and the ratio of electrode surface-tosolution volume ought to be as high as possible. While electrosynthesis has been extensively performed in batch mode, 8,9 continuous-flow technology provides increased electrode surface ratios by design. 9 Additionally, at the microfluidic scale the distance between the working electrode and the counter electrode is shortened. This can help overcome conversion limitations linked to solution resistivity, and even enable electrolyte-free reactions. 10 In the past few years, the study of drug metabolites has benefited from the emergence of in-line electrochemical/mass spectrometry (EC-MS) systems used to generate phase I and phase II metabolites. 13,14 Unfortunately, full structure elucidation is limi...

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Preparative Microfluidic Electrosynthesis of Drug Metabolites

Stalder

Roth

2013

ACS Med. Chem. Lett.

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“…Asterisks indicate unknown components, which were also found in the blank analysis (incubation time = 0). B -MS/MS spectrum of HTB is accompanied by proposed structural formulae [27]. Calculated monoisotopic mass is indicated in blue-colored font.…”

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confidence: 99%

Combination of on‐line CE assay with MS detection for the study of drug metabolism by cytochromes P450

et al. 2015

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A new CE-MS method with enzymatic reaction inside the capillary was developed for the study of drug metabolism by cytochromes P450. This automated method, based on the transverse diffusion of laminar flow profiles methodology, is comprised of the injection of substrates and enzyme, their mixing, incubation, and separation of the reaction products, all performed by CE, and their detection, identification, and quantification by MS. The developed and validated method was finally used to conduct a kinetic study of cytochrome P450 isoform 2C9 or human liver microsomes with diclofenac in order to demonstrate its practical functionality. All the estimated kinetic values--apparent Michaelis constants and apparent maximum reaction velocities were in agreement with literature data obtained using other techniques. In addition, the consumption of reactants was in the tens of nL per analysis. The method's usability was further demonstrated on tolbutamide, the other probe substrate of cytochrome P450 isoform 2C9. As a result, the method is conceptually applicable for the screening of any other cytochrome P450 isoform and its substrates and inhibitors after adapting the incubation and separation conditions.

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“…Low amounts of metabolites and parent drug in samples are measured by sufficiently sensitive LC-MS/MS, where the analysis of higher amounts allows detection on RAD and due to response factor enables calculating of metabolite concentration. The best way to perform analyses is to combine RAD with MS after liquid chromatography with splitting flow in order to obtain peaks of the metabolites and parent at the same retention times on both detectors [51]. RAD is convenient for such analyses because of the large dynamic range but its use is limited by the availability of radiolabeled standards.…”

Section: Quantification Using Response Factormentioning

confidence: 99%