The analytical approach for the simultaneous determination o
The analytical approach for the simultaneous determination of HA and its metabolites is complex. There are commercial kits that allow the determination of HA through ELISA and of its metabolites through radioimmunoassay (RIA) techniques. However, these immunological-based techniques do not allow simultaneous determination of HA and its metabolites, while, in the case of RIA, they also involve complications related to the use of radioactive material . Alternatively, chromatographic methods, mainly based on high performance liquid chromatography (HPLC) coupled with various detection systems, appear to be the most appropriate for the simultaneous separation and quantification of these compounds . Few HPLC methods are available in the literature for the simultaneous determination of HA and MHA, mainly focused on the quantification of both compounds in laboratory animal plasma and other biological specimens, such as brain or intestinal tissues , , , , , . Although UV detection has been widely used, the high sensitivity and specificity necessary to detect these compounds in samples such as blood makes fluorescence (FL) or mass spectrometry (MS) detection systems more suitable for this purpose. More recently, ultra high performance liquid chromatography (UHPLC) has been proposed for the simultaneous determination of HA and MHA in mice hair and cerebrospinal fluid , , . A fast chromatographic procedure (UHPLC) coupled with FL detection could be an advantageous approach for the routine determination of these analytes in human urine.
In order to have a new approach for the diagnosis of histamine intolerance, the aim of this work was to develop and validate a rapid and reliable method to quantifity HA and MHA in urine. An UHPLC procedure coupled with an on-line o-phthaldehyde (OPA) post-column derivatization and FL detection has been validated in terms of linearity, sensitivity, precision and recovery. Structural analysis of HA and MHA OPA derivatives using UHPLC-ITD-FTMS was carried out to unequivocally identify these compounds in urine samples.
Material and methods
Results and discussion To properly determine total content of HA and MHA in urine, sample preparation consisted in the ursolic acid hydrolysis (0.1M HCl) combined with heat treatment. As reported in previous works, higher levels of these compounds were obtained in hydrolysed urine than in non-hydrolysed samples . In order to set final sample hydrolysis parameters, several time and temperature conditions were assayed. Total conversion of conjugated analytes to free HA and MHA was achieved after submitting the sample at 90°C for 30min. Purification and concentration procedure through MCX SPE cartridges was implemented attending the low concentration of HA and MHA in urine and the need to minimize potential interferences of this matrix. When loading urine sample onto the SPE cartridge, acid pH values achieved by previous acidic hydrolysis facilitated the strong interaction of positively charged amine groups of HA and MHA with the sulfonic anion of MCX sorbent. A proper cleanup step washing with 0.1M HCl allowed the reduction of matrix interferences. Later elution with a basic 5% NH4OH in methanol (v/v) solution ensured cleavage of the electrostatic interactions between ammonium ion of HA and MHA and sulfonic anion of the sorbent. The use of MCX SPE cartridges allowed five-fold pre-concentration of analytes and greater sample concentration was achieved by eluate evaporation to dryness with a centrifugal vacuum concentrator. Overall analytical procedure achieved a final concentration of the analytes of fifty-fold in relation to initial urine content. Due to the low natural fluorescence of the analytes, the use of a derivatizing reagent in order to detect HA and MHA and increase the sensitivity of the method was required. The presence of amino groups in the structures of HA and MHA makes both compounds suitable for the derivatization with a large number of fluorogenic reagents, being OPA, fluorescamina and DBD-F (4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole) the most commonly used , , . In this method, an online post-column OPA derivatization procedure was used, which ensured a high reproducibility by minimizing sample manipulation prior to the injection. Moreover, OPA reacts rapidly with amines in the presence of a reducing agent, improving detection sensitivity, reducing the polarity of original amino compounds and increasing method selectivity . The current method provides a significant improvement in comparison with some previous methods that mostly used pre-column derivatization techniques, which could face problems related to the low stability of OPA-amine derivatives , .