According to previous methods several
According to previous methods, several mobile phase compositions and gradient programmes were assayed to get the best resolved peaks for HA and MHA , , . Considering the slightly structural differences of the analytes, a mobile phase consisting in two eluents of different polarity and pH permitted to establish the necessary gradient in order to properly separate HA and MHA. This was achieved by a gradual and linear slight increase of eluent B, the less polar eluent of the mobile phase, during the first minutes of the chromatographic run. Just after the separation of both analytes, the proportion of acetonitrile was markedly incremented aiming to completely elute less polar urinary compounds, which consequently incremented the chromatographic run up to the final 11min. Moreover, sodium octanesulphonate as ion-pairing reagent was added to the mobile phase in order to improve chromatographic separation of these order us and polar compounds. Fig. 1 shows the chromatograms of the standard solutions and of urine samples. The proposed method accomplished an acceptable separation between HA and MHA with a chromatographic resolution (R) of 1.5. HA and MHA were identified on the basis of the retention time by comparison with the standard.
The present UHPLC method reduces considerably the time required for urinary determination of HA and MHA in comparison with previously published HPLC-FL methods, resulting in turn in decreased reagent costs and reduced environmental impact , , . The reliability of this UHPLC method for routine analysis of urine samples was assessed in terms of linearity, sensitivity, precision and recovery.
Conclusions The proposed UHPLC method allows the satisfactory determination of urinary HA and MHA in less than 11min. The use of MCX SPE cartridges was effective for the selective purification and concentration of HA and MHA in human urine. Overall sample treatment procedure achieved a final concentration of the analytes of fifty-fold in relation to initial urine content. Online post-column derivatization of the analytes with OPA permitted the sensitive detection of the analytes while minimizing sample manipulation prior to UHPLC injection. Unequivocal identification of HA and MHA OPA derivatives in standard and in urine samples has been accomplished through UHPLC-ITD-FTMS. To our knowledge, this is the first UHPLC-FL method with OPA post-column derivatization used to determine HA and MHA in human urine; thus becoming a potential new approach for the routine diagnosis of histamine intolerant individuals. Further studies involving more volunteers are needed to validate MHA as a biomarker for the diagnosis of HIT.
Funding This work was supported by Direcció General de Recerca of the Generalitat de Catalunya (SGR 2014-1438) and the Interministerial Commission for Science and Technology (CICYT) of the Ministerio de Educación, Cultura y Deporte (AGL 2012-39995). Oriol Comas-Basté is a recipient of a doctoral fellowship from the University of Barcelona (APIF 2015).
Early data suggesting a role for histamine in memory consolidation: inhibitory avoidance learning Histamine is synthetized in neurons whose cell bodies are in the tuberomamillary nucleus of the hypothalamus (Panula, Yang, & Costa, 1984). These cells project to multiple brain areas involving different histamine receptor types, which suggests that histaminergic neurons are probably organized into functionally distinct circuits, impinging on different brain regions and displaying selective control mechanisms (Munari, Provensi, Passani, & Blandina, 2013). This could imply separate and independent functions of subsets of histamine neurons according to their respective origin and terminal projections (Blandina, Munari, Provensi, & Passani, 2012). Histaminergic fibers from the tuberomamillary nucleus projects to the septum/diagonal band nucleus (Wouterlood, Gaykema, Steinbusch, Watanabe, & Wada, 1988), and to the dorsal hippocampus and the basolateral amygdala, regions critically involved in memory consolidation and retrieval (Izquierdo et al., 2016, Myskiw et al., 2016).