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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.

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).

A series of reversed phases bonded with several functional groups was investigated for separation of anthraquinone derivatives, following the previous work, dedicated to the selectivity of octadecyl silica bonded phases. Considering wide diversity of substitutions in hydrophobic anthraquinone skeleton, interactions like dipole-dipole, π-π or H-bond acceptor/donor, as well as inclusion complexes formation can be employed to improve separation. In this study, several phases with grafts like cyano, nitro, aromatic, PEG, diol, calixarene and cyclodextrin were used with water-acetonitrile gradient for separation of thirty anthraquinoids' standards. The evaluation of performances was measured using the symmetry parameter and the number of critical pairs of peaks formed. The results point out the aromatic and calixarene bonded silica as the most interesting in terms of symmetry and critical pairs number. Finally we tested the performance of Caltrex Resorcinaren, Pursuit XRs DP and Luna Phenyl-Hexyl on real samples of anthraquinone natural dye extracted from a red thread taken from a 15th C. tapestry. We observed and compared the retention behaviour of some new anthraquinoids additional to our standards set and showing behaviour particular to substituted anthraquinone carboxylic acids.

A comparison of chromatograms obtained in a series of separation conditions for a given complex mixture may be done with a series of chromatographic descriptors. In this study, we used two descriptors: the number of critical pairs and symmetry of peaks, further rescaled and converted to the corresponding critical pairs' coefficient (CPc) and symmetry coefficient (Sc). Considering the difficulty of appreciating global separation quality using CPc and Sc criteria separately, as their respective values are usually uncorrelated, a double-criteria cross-evaluation system was required. For that purpose we tested the commonly used multi-criteria decision-making method – Derringer's desirability function (D) – as well as the recently introduced sum of ranking differences (SRD). To facilitate the graphical comparison of both approaches, the desirability function (D) was used in the inverse form (Dinv). The advantages and drawbacks of both evaluation methods, especially the respective under- or over-evaluation of outliers, caused us to introduce a new ranking approach, separation system suitability (3S). The obtained suitability rankings for the three tested approaches (Dinv, SRD and 3S) are different; nevertheless, 3S appears to be the most balanced and the easiest to interpret as well. The approach developed for selection of suitable systems was applied to the problem of separation of complex mixtures through the analysis of a series of standards of anthraquinone derivatives. To judge the pertinence of this evaluation, a sample containing a number of natural anthraquinones extracted from the bark of Indian mulberry (Morinda citrifolia) was analysed. In conclusion, the proposed methodology for the cross-evaluation of the series of chromatograms using single specific descriptors (CPc and Sc) through a global composite descriptor (3S) significantly simplifies the decision that separation systems are the most suitable for the separation of complex target mixtures of compounds.