Autoxidation Kinetics of Tetrahydrobiopterin-Giving Quinonoid Dihydrobiopterin the Consideration It Deserves.

In humans, tetrahydrobiopterin (H4Bip) is the cofactor of several essential hydroxylation reactions which dysfunction cause very serious diseases at any age. Hence, the determination of pterins in biological media is of outmost importance in the diagnosis and monitoring of H4Bip deficiency. More than half a century after the discovery of the physiological role of H4Bip and the recent advent of gene therapy for dopamine and serotonin disorders linked to H4Bip deficiency, the quantification of quinonoid dihydrobiopterin (qH2Bip), the transient intermediate of H4Bip, has not been considered yet. This is mainly due to its short half-life, which goes from 0.9 to 5 min according to previous studies. Based on our recent disclosure of the specific MS/MS transition of qH2Bip, here, we developed an efficient HPLC-MS/MS method to achieve the separation of qH2Bip from H4Bip and other oxidation products in less than 3.5 min. The application of this method to the investigation of H4Bip autoxidation kinetics clearly shows that qH2Bip's half-life is much longer than previously reported, and mostly longer than that of H4Bip, irrespective of the considered experimental conditions. These findings definitely confirm that an accurate method of H4Bip analysis should include the quantification of qH2Bip.

Quantification of Monoamine Neurotransmitter Metabolites and Cofactors in Cerebrospinal Fluid: State-of-the-Art.

Inborn errors of monoamine neurotransmitter metabolism are rare diseases characterized by nonspecific neurological symptoms. These symptoms appear in early childhood and correspond to movement disorders, epilepsy, sleep disorders and/or mental disability. Cerebrospinal fluid biomarkers have been identified and validated to allow specific diagnosis of these diseases. Biomarkers of inborn errors of monoamine neurotransmitter metabolites are divided in two groups: monoamine neurotransmitter metabolites and pterins. Biomarkers quantification in cerebrospinal fluid is based on high-performance liquid chromatography separation coupled to electrochemical detection, fluorescence detection, or mass spectrometry. The following article reviews the advances in the proposed routine methods for the measurement of these analytes in cerebrospinal fluid. The purpose of this review is to compare the various proposed methods in terms of sample preparation, chromatographic conditions and detection modes. Despite the broad range of proposed methods, quantification of inborn errors of monoamine neurotransmitter biomarkers remains a great challenge, given the complexity of biological fluids and the low amounts of analytes that are present in cerebrospinal fluid.

Multianalytical Approach for Deciphering the Specific MS/MS Transition and Overcoming the Challenge of the Separation of a Transient Intermediate, Quinonoid Dihydrobiopterin.

Despite recent technological developments in analytical chemistry, separation and direct characterization of transient intermediates remain an analytical challenge. Among these, separation and direct characterization of quinonoid dihydrobiopterin (qH2Bip), a transient intermediate of tetrahydrobiopterin (H4Bip)-dependent hydroxylation reactions, essential in living organisms, with important and varied human pathophysiological impacts, are a clear illustration. H4Bip regeneration may be impaired by competitive nonenzymatic autoxidation reactions, such as isomerization of qH2Bip into a more stable 7,8-H2Bip (H2Bip) isomer, and subsequent nonenzymatic oxidation reactions. The quinonoid qH2Bip intermediate thus plays a key role in H4Bip-dependent hydroxylation reactions. However, only a few experimental results have indirectly confirmed this finding while revealing the difficulty of isolating qH2Bip from H4Bip-containing solutions. As a result, no current H4Bip assay method allows this isomer to be quantified even by liquid chromatography-tandem mass spectrometry (MS/MS). Here, we report isolation, structural characterization, and abundance of qH2Bip formed upon H4Bip autoxidation using three methods integrated into MS/MS. First, we characterized the structure of the two observed H2B isomers using IR photodissociation spectroscopy in conjunction with quantum chemical calculations. Then, we used differential ion mobility spectrometry to fully separate all oxidized forms of H4Bip including qH2Bip. These data are consistent and show that qH2Bip can also be unambiguously identified thanks to its specific MS/MS transition. This finding paves the way for the quantification of qH2Bip with MS/MS methods. Most importantly, the half-life value of this intermediate is nearly equivalent to that of H4Bip (tens of minutes), suggesting that an accurate method of H4Bip analysis should include the quantification of qH2Bip.

Quantification of monoamine biomarkers in cerebrospinal fluid: Comparison of a UHPLC-MS/MS method with a UHPLC coupled to fluorescence detection method.

Inborn errors of monoamine neurotransmitter metabolism are rare genetic diseases classified as catecholamine and serotonin metabolism disorders or neurotransmitter transportopathies. To diagnose these orphan diseases, monoamine metabolites have been identified and validated as cerebrospinal fluid (CSF) biomarkers: 5-hydroxy-tryptophane, 5-hydroxy-indol-acetic acid, 3-ortho-methyl-DOPA, homovanillic acid, and 3-methoxy-4-hydroxyphenylglycol. The present work presents a UHPLC-MS/MS method developed for the quantification of these metabolites in CSF and compares it with a previously described UHPLC with fluorescence detection (UHPLC-FD) method. MS/MS detection was performed in positive electrospray ionization and multiple reaction monitoring mode. The UHPLC-MS/MS and UHPLC-FD methods were validated in terms of accuracy, linearity, precision and matrix effect. The lower limits of quantification (LLOQ) ranged between 0.5 and 10 nm and between 1 and 5 nm for the UHPLC-MS/MS method and the UHPLC-FD one, respectively. We verified the applicability of both methods by analyzing 30 CSF samples. The measured concentrations were comparable with the reference values described in the literature. The two methods allowed pathological samples to be distinguished from healthy ones for clinical diagnosis. UHPLC-MS/MS and UHPLC-FD methods exhibited very close LLOQs. As the UHPLC-MS/MS method is more selective, it allows faster analysis with a run time of 6 min per run vs. 10 min for the UHPLC-FD method.

A rapid and sensitive method for the quantification of dopamine and serotonin metabolites in cerebrospinal fluid based on UHPLC with fluorescence detection.

Inborn errors of dopamine and serotonin metabolism are diseases caused by deficiencies in enzymes belonging to metabolic pathways. The specific diagnosis of these inborn illnesses is based on the identification and quantification of biomarkers in cerebrospinal fluid (CSF), especially: 5-hydroxy-tryptophane (5-HTP), 5-hydroxy-indol-acetic acid (5-HIAA), 3-ortho-methyl-DOPA (3-OMD), homovanillic acid (HVA) and 3-methoxy-4-hydroxyphenylglycol (MHPG). In the present work, we propose a novel ultrahigh performance liquid chromatography (UHPLC) method coupled to fluorescence detection (FD) to quantify simultaneously the five dopamine and serotonin metabolites. This method efficiently separates the five molecules in less than 10 min. A complete validation of the proposed method was performed in terms of accuracy, linearity, precision, and lower limit of quantification (LLOQ). Depending on the compound, the obtained LLOQs are between 1 nM and 5 nM, thus allowing to measure concentrations as low as in CSF samples. We also verified the method applicability by analyzing 10 CSF samples in triplicates. The obtained results showed satisfactory repeatability and an ability of this method to clearly distinguish healthy samples from pathologic samples, hence, demonstrating, the method suitability for diagnosing inborn errors of dopamine and serotonin metabolism. Therefore, the proposed UHPLC-FD method appears as a reliable alternative to the current gold standard for the quantification of these biomarkers, which is based on UHPLC coupled to electrochemical detection (ECD).