Design, synthesis and biological evaluation of new aryl thiosemicarbazone as antichagasic candidates

The present work reports on the synthesis, biological assaying and docking studies of a series of 12 aryl thiosemicarbazones, which were planned to act over two main enzymes, cruzain and trypanothione reductase. These enzymes are used as targets of trypanocidal activity in Chagas disease control with a minimal mutagenic profile. Three p-nitroaromatic thiosemicarbazones showed high activity against Trypanosoma cruzi in in vitro assays (IC50 < 57 μM), and no mutagenic profile was observed in micronucleous tests. Although the in vitro inhibition test showed that 10-μM doses of eight compounds inhibited cruzain activity, no correlation was found between cruzain inhibition and trypanocidal activity. © 2013 Elsevier Masson SAS. All rights reserved.

In vitro and in vivo biological behavior of CDHA/OCP/β-TCP scaffold

The biological behavior of a new bioactive material composed of calcium-deficient hydroxyapatite, octacalcium phosphate, and beta-tricalcium phosphate was investigated by in vitro indirect and direct cytotoxicity, cell adhesion and proliferation tests, and by in vivo subcutaneous and bone implantation in rats. The results of the in vitro studies showed that the material is biocompatible and no cytotoxic. Slightly poorer initial cell adhesion and lower cell proliferation than in control was observed, which were attributed to the reactivity and roughness of the material surface, In vivo results showed that the material is biodegradable and bioactive in bone tissue, but only biocompatible and partially biodegradable in soft tissue.

Use of solvents and environmental friendly materials for applications in Green Chemistry

The present Thesis studies three alternative solvent groups as sustainable replacement of traditional organic solvents. Some aspects of fluorinated solvents, supercritical fluids and ionic liquids, have been analysed with a critical approach and their effective “greenness” has been evaluated from the points of view of the synthesis, the properties and the applications. In particular, the attention has been put on the environmental and human health issues, evaluating the eco-toxicity, the toxicity and the persistence, to underline that applicability and sustainability are subjects with equal importance. The “green” features of fluorous solvents and supercritical fluids are almost well-established; in particular supercritical carbon dioxide (scCO2) is probably the “greenest” solvent among the alternative solvent systems developed in the last years, enabling to combine numerous advantages both from the point of view of industrial/technological applications and eco-compatibility. In the Thesis the analysis of these two classes of alternative solvents has been mainly focused on their applicability, rather than the evaluation of their environmental impact. Specifically they have been evaluated as alternative media for non-aqueous biocatalysis. For this purpose, the hydrophobic ion pairing (HIP), which allows solubilising enzymes in apolar solvents by an ion pairing between the protein and a surfactant, has been investigated as effective enzymatic derivatisation technique to improve the catalytic activity under homogeneous conditions in non conventional media. The results showed that the complex enzyme-surfactant was much more active both in fluorous solvents and in supercritical carbon dioxide than the native form of the enzyme. Ionic liquids, especially imidazolium salts, have been proposed some years ago as “fully green” alternative solvents; however this epithet does not take into account several “brown” aspects such as their synthesis from petro-chemical starting materials, their considerable eco-toxicity, toxicity and resistance to biodegradation, and the difficulty of clearly outline applications in which ionic liquids are really more advantageous than traditional solvents. For all of these reasons in this Thesis a critical analysis of ionic liquids has been focused on three main topics: i) alternative synthesis by introducing structural moieties which could reduce the toxicity of the most known liquid salts, and by using starting materials from renewable resources; ii) on the evaluation of their environmental impact through eco-toxicological tests (Daphnia magna and Vibrio fischeri acute toxicity tests, and algal growth inhibition), toxicity tests (MTT test, AChE inhibition and LDH release tests) and fate and rate of aerobic biodegradation in soil and water; iii) and on the demonstration of their effectiveness as reaction media in organo-catalysis and as extractive solvents in the recovery of vegetable oil from terrestrial and aquatic biomass. The results about eco-toxicity tests with Daphnia magna, Vibrio fischeri and algae, and toxicity assay using cultured cell lines, clearly indicate that the difference in toxicity between alkyl and oxygenated cations relies in differences of polarity, according to the general trend of decreasing toxicity by decreasing the lipophilicity. Independently by the biological approach in fact, all the results are in agreement, showing a lower toxicity for compounds with oxygenated lateral chains than for those having purely alkyl lateral chains. These findings indicate that an appropriate choice of cation and anion structures is important not only to design the IL with improved and suitable chemico-physical properties but also to obtain safer and eco-friendly ILs. Moreover there is a clear indication that the composition of the abiotic environment has to be taken into account when the toxicity of ILs in various biological test systems is analysed, because, for example, the data reported in the Thesis indicate a significant influence of salinity variations on algal toxicity. Aerobic biodegradation of four imidazolium ionic liquids, two alkylated and two oxygenated, in soil was evaluated for the first time. Alkyl ionic liquids were shown to be biodegradable over the 6 months test period, and in contrast no significant mineralisation was observed with oxygenated derivatives. A different result was observed in the aerobic biodegradation of alkylated and oxygenated pyridinium ionic liquids in water because all the ionic liquids were almost completely degraded after 10 days, independently by the number of oxygen in the lateral chain of the cation. The synthesis of new ionic liquids by using renewable feedstock as starting materials, has been developed through the synthesis of furan-based ion pairs from furfural. The new ammonium salts were synthesised in very good yields, good purity of the products and wide versatility, combining low melting points with high decomposition temperatures and reduced viscosities. Regarding the possible applications as surfactants and biocides, furan-based salts could be a valuable alternative to benzyltributylammonium salts and benzalkonium chloride that are produced from non-renewable resources. A new procedure for the allylation of ketones and aldehydes with tetraallyltin in ionic liquids was developed. The reaction afforded high yields both in sulfonate-containing ILs and in ILs without sulfonate upon addition of a small amount of sulfonic acid. The checked reaction resulted in peculiar chemoselectivity favouring aliphatic substrates towards aromatic ketones and good stereoselectivity in the allylation of levoglucosenone. Finally ILs-based systems could be easily and successfully recycled, making the described procedure environmentally benign. The potential role of switchable polarity solvents as a green technology for the extraction of vegetable oil from terrestrial and aquatic biomass has been investigated. The extraction efficiency of terrestrial biomass rich in triacylglycerols, as soy bean flakes and sunflower seeds, was comparable to those of traditional organic solvents, being the yield of vegetable oils recovery very similar. Switchable polarity solvents as been also exploited for the first time in the extraction of hydrocarbons from the microalga Botryococcus braunii, demonstrating the efficiency of the process for the extraction of both dried microalgal biomass and directly of the aqueous growth medium. The switchable polarity solvents exhibited better extraction efficiency than conventional solvents, both with dried and liquid samples. This is an important issue considering that the harvest and the dewatering of algal biomass have a large impact on overall costs and energy balance.

Identification of stock units of Mullus barbatus (Linnaeus, 1758) in the Italian seas: development of genomic 2b-RAD markers and biological variation analysis.

This thesis is settled within the STOCKMAPPING project, which represents one of the studies that were developed in the framework of RITMARE Flagship project. The main goals of STOCKMAPPING were the creation of a genomic mapping for stocks of demersal target species and the assembling of a database of population genomic, in order to identify stocks and stocks boundaries. The thesis focuses on three main objectives representing the core for the initial assessment of the methodologies and structure that would be applied to the entire STOCKMAPPING project: individuation of an analytical design to identify and locate stocks and stocks boundaries of Mullus barbatus, application of a multidisciplinary approach to validate biological methods and an initial assessment and improvement for the genotyping by sequencing technique utilized (2b-RAD). The first step is the individuation of an analytical design that has to take in to account the biological characteristics of red mullet and being representative for STOCKMAPPING commitments. In this framework a reduction and selection steps was needed due to budget reduction. Sampling areas were ranked according the individuation of four priorities. To guarantee a multidisciplinary approach the biological data associated to the collected samples were used to investigate differences between sampling areas and GSAs. Genomic techniques were applied to red mullet for the first time so an initial assessment of molecular protocols for DNA extraction and 2b-RAD processing were needed. At the end 192 good quality DNAs have been extracted and eight samples have been processed with 2b-RAD. Utilizing the software Stacks for sequences analyses a great number of SNPs markers among the eight samples have been identified. Several tests have been performed changing the main parameter of the Stacks pipeline in order to identify the most explicative and functional sets of parameters.

Screening of Indian Plants for Biological Activity : Part I

Two hundred and eighty-five botanically identifide plant materials have been extracted with 50 percent ethanol and the extracts put through a wide biological screen of 61 tests. These include tests for antibacterial, antifungal, anthelminthic, antiprotozoal, anticancer, antifertillity, hypoglycaemic and a wide range of pharmacological activities.

Screening of Indian Plants for Biological Activity: Part II

Three hundred botanically identified plant materials have been extracted with 50% ethanol and the extracts put through a wide biological screen. These include tests for antibacterial, anticancer, antifertility, antifungal, anthelminthic, antiprotozoal, antiviral and pharmacological activities. Biological activities have been confirmed in fractions of fifty-six of these extracts.

Design, synthesis and pharmacological characterization of analogs of 2-aminoethyl diphenylborinate (2-APB), a known store-operated calcium channel blocker, for inhibition of TRPV6-mediated calcium transport

2-Aminoethyl diphenylborinate (2-APB) is a known modulator of the IP3 receptor, the calcium ATPase SERCA, the calcium release-activated calcium channel Orai and TRP channels. More recently, it was shown that 2-APB is an efficient inhibitor of the epithelial calcium channel TRPV6 which is overexpressed in prostate cancer. We have conducted a structure-activity relationship study of 2-APB congeners to understand their inhibitory mode of action on TRPV6. Whereas modifying the aminoethyl moiety did not significantly change TRPV6 inhibition, substitution of the phenyl rings of 2-APB did. Our data show that the diaryl borinate moiety is required for biological activity and that the substitution pattern of the aryl rings can influence TRPV6 versus SOCE inhibition. We have also discovered that 2-APB is hydrolyzed and transesterified within minutes in solution.

Zebrafish como organismo-modelo para análises de efeitos comportamentais e toxicológicas da cetamina empregando cromatografia em fase gasosa e estatística multivariada

A cetamina é uma droga amplamente utilizada e o seu uso inadequado tem sido associado à graves consequências para a saúde humana. Embora as propriedades farmacológicas deste agente em doses terapêuticas sejam bem conhecidas, existem poucos estudos sobre os efeitos secundários induzidos por doses não-terapêuticas, incluindo os efeitos nos estados de ansiedade e agressividade. Neste contexto, os modelos animais são uma etapa importante na investigação e elucidação do mecanismo de ação a nível comportamental. O zebrafish (Danio rerio) é um novo organismo-modelo, interessante e promissor, uma vez que apresenta alta similaridade fisiológica, genética e neuroquímica com seres humanos, respostas comportamentais bem definidas e rápida absorção de compostos de interesse em meio aquoso além de apresentar uma série de vantagens em relação aos modelos mamíferos tais como manutenção de baixo custo, prática e executável em espaços reduzidos. Nesse sentido, faz-se necessário a execução de ensaios comportamentais em conjunto com análises estatísticas robustas e rápidas tais como ANOVA e Métodos Multivariados; e também o desenvolvimento de métodos analíticos sensíveis, precisos e rápidos para determinação de compostos de interesse em matrizes biológicas provenientes do animal. Os objetivos do presente trabalho foram a investigação dos efeitos da cetamina sobre a ansiedade e a agressividade em zebrafish adulto empregando Testes de Claro-Escuro e Testes do Espelho e métodos estatísticos univariados (ANOVA) e multivariados (PCA, HCA e SIMCA) assim como o desenvolvimento de método analítico para determinação da cetamina em matriz biológica proveniente do animal, empregando Extração Líquido-Líquido e Cromatografia em Fase Gasosa acoplada ao Detector de Nitrogênio-Fósforo (GC-NPD). Os resultados comportamentais indicaram que a cetamina produziu um efeito significativo dose-dependente em zebrafish adulto na latência à área clara, no número de cruzamentos entre as áreas e no tempo de exploração da área clara. Os resultados das análises SIMCA e PCA mostraram uma maior similaridade entre o grupo controle e os grupos de tratamento expostos às doses mais baixas (5 e 20 mg L-1) e entre os grupos expostos às doses de 40 e 60 mg L-1. Na análise por PCA, dois componentes principais responderam por 88,74% de toda a informação do sistema, sendo que 62,59% da informação cumulativa do sistema foi descrito pela primeira componente principal. As classificações HCA e SIMCA seguiram uma evolução lógica na distribuição das amostras por classes. As doses mais altas de cetamina induziram uma distribuição mais homogênea das amostras enquanto as doses mais baixas e o controle resultaram em distribuições mais dispersas. No Teste do Espelho, a cetamina não induziu efeitos significativos no comportamento dos animais. Estes resultados sugerem que a cetamina é modulador de comportamentos ansiosos, sem efeitos indutores de agressividade. Os resultados da validação do método cromatográfico indicaram uma extração com valores de recuperação entre 33,65% e 70,89%. A curva de calibração foi linear com valor de R2 superior a 0,99. O limite de detecção (LOD) foi de 1 ng e o limite de quantificação (LOQ) foi de 5 ng. A exatidão do método cromatográfico manteve-se entre - 24,83% e - 1,258%, a precisão intra-ensaio entre 2,67 e 14,5% e a precisão inter-ensaio entre 1,93 e 13,9%.

Interactions of different arsenic species with thols: Chemical and biological implications

Arsenic has been classified as a group I carcinogen. It has been ranked number one in the CERCLA priority list of hazardous substances due to its frequency, toxicity and potential for human exposure. Paradoxically, arsenic has been employed as a successful chemotherapeutic agent for acute promyelocytic leukemia and has found some success in multiple myeloma. Since arsenic toxicity and efficacy is species dependent, a speciation method, based on the complementary use of reverse phase and cation exchange chromatography, was developed. Inductively coupled plasma mass spectrometer (ICP-MS), as an element specific detector, and electrospray ionization mass spectrometer (ESI-MS), as a molecule specific detector, were employed. Low detection limits in the µg. L−1 range on the ICP-MS and mg. L−1 range on the ESI-MS were obtained. The developed methods were validated against each other through the use of a Deming plot. With the developed speciation method, the effects of both pH on the stability of As species and reduced glutathione (GSH) concentration on the formation and stability of arsenic glutathione complexes were studied. To identify arsenicals in multiple myeloma (MM) cell lines post arsenic trioxide (ATO) and darinaparsin (DAR) incubation, an extraction method based on the use of ultrasonic probe was developed. Extraction tools and solvents were evaluated and the effect of GSH concentration on the quantitation of arsenic glutathione (As-GSH) complexes in MM cell extracts was studied. The developed method was employed for the identification of metabolites in DAR incubated cell lines where the effect of extraction pH, DAR incubation concentration and incubation time on the relative distribution of the As metabolites was assessed. A new arsenic species, dimethyarsinothioyl glutathione (DMMTA V-GS), a pentavalent thiolated arsenical, was identified in the cell extracts through the use of liquid chromatography tandem mass spectrometry. The formation of the new metabolite in the extracts was dependent on the decomposition of s-dimethylarsino glutathione (DMA(GS)). These results have major implications in both the medical and toxicological fields of As because they involve the metabolism of a chemotherapeutic agent and the role sulfur compounds play in this mechanism.

Evaluation of screening tests for predicting older driver performance and safety assessed by an on-road test

A number of tests and test batteries are available for the prediction of older driver safety, but many of these have not been validated against standardized driving outcome measures. The aim of this study was to evaluate a series of previously described screening tests in terms of their ability to predict the potential for safe and unsafe driving. Participants included 79 community-dwelling older drivers (M=72.16 years, SD=5.46; range 65-88 years; 57 males and 22 females) who completed a previously validated multi-disciplinary driving assessment, a hazard perception test, a hazard change detection test and a battery of vision and cognitive tests. Participants also completed a standardized on-road driving assessment. The multi-disciplinary test battery had the highest predictive ability with a sensitivity of 80% and a specificity of 73%, followed by the hazard perception test which demonstrated a sensitivity of 75% and a specificity of 61%. These findings suggest that a relatively simple and practical battery of tests from a range of domains has the capacity to predict safe and unsafe driving in older adults.

Sensorimotor and postural control factors associated with driving safety in a community-dwelling older driver population

Background. To establish whether sensorimotor function and balance are associated with on-road driving performance in older adults. Methods. The performance of 270 community-living adults aged 70–88 years recruited via the electoral roll was measured on a battery of peripheral sensation, strength, flexibility, reaction time, and balance tests and on a standardized measure of on-road driving performance. Results. Forty-seven participants (17.4%) were classified as unsafe based on their driving assessment. Unsafe driving was associated with reduced peripheral sensation, lower limb weakness, reduced neck range of motion, slow reaction time, and poor balance in univariate analyses. Multivariate logistic regression analysis identified poor vibration sensitivity, reduced quadriceps strength, and increased sway on a foam surface with eyes closed as significant and independent risk factors for unsafe driving. These variables classified participants into safe and unsafe drivers with a sensitivity of 74% and specificity of 70%. Conclusions. A number of sensorimotor and balance measures were associated with driver safety and the multivariate model comprising measures of sensation, strength, and balance was highly predictive of unsafe driving in this sample. These findings highlight important determinants of driver safety and may assist in developing efficacious driver safety strategies for older drivers.

Tailored interventions based on exhaled nitric oxide versus clinical symptoms for asthma in children and adults

Background The measurement of severity and control of asthma in both children and adults can be based on subjective or objective measures. It has been advocated that fractional exhaled nitric oxide (FeNO) can be used to monitor airway inflammation as it correlates with some markers of asthma. Interventions for asthma therapies have been traditionally based on symptoms and/or spirometry. Objectives To evaluate the efficacy of tailoring asthma interventions based on exhaled nitric oxide in comparison to clinical symptoms (with or without spirometry/peak flow) for asthma related outcomes in children and adults. Search methods We searched the Cochrane Airways Group Specialised Register of Trials, the Cochrane Central Register of Controlled Trials (CENTRAL), MEDLINE, EMBASE and reference lists of articles. The last search was completed in February 2009. Selection criteria All randomised controlled comparisons of adjustment of asthma therapy based on exhaled nitric oxide compared to traditional methods (primarily clinical symptoms and spirometry/peak flow). Data collection and analysis Results of searches were reviewed against pre-determined criteria for inclusion. Relevant studies were independently selected in duplicate. Two authors independently assessed trial quality and extracted data. Authors were contacted for further information with response from one. Main results Two studies have been added for this update, which now includes six (2 adults and 4 children/adolescent) studies; these studies differed in a variety of ways including definition of asthma exacerbations, FeNO cut off levels, the way in which FeNO was used to adjust therapy and duration of study. Of 1053 participants randomised, 1010 completed the trials. In the meta-analysis, there was no significant difference between groups for the primary outcome of asthma exacerbations or for other outcomes (clinical symptoms, FeNO level and spirometry). In post-hoc analysis, a significant reduction in mean final daily dose inhaled corticosteroid per adult was found in the group where treatment was based on FeNO in comparison to clinical symptoms, (mean difference -450 mcg; 95% CI -677 to -223 mcg budesonide equivalent/day). However, the total amount of inhaled corticosteroid used in one of the adult studies was 11% greater in the FeNO arm. In contrast, in the paediatric studies, there was a significant increase in inhaled corticosteroid dose in the FeNO strategy arm (mean difference of 140 mcg; 95% CI 29 to 251, mcg budesonide equivalent/day). Authors' conclusions Tailoring the dose of inhaled corticosteroids based on exhaled nitric oxide in comparison to clinical symptoms was carried out in different ways in the six studies and found only modest benefit at best and potentially higher doses of inhaled corticosteroids in children. The role of utilising exhaled nitric oxide to tailor the dose of inhaled corticosteroids cannot be routinely recommended for clinical practice at this stage and remains uncertain.

Primary and secondary neural networks of auditory prepulse inhibition: a functional magnetic resonance imaging study of sensorimotor gating of the human acoustic startle response

Feedforward inhibition deficits have been consistently demonstrated in a range of neuropsychiatric conditions using prepulse inhibition (PPI) of the acoustic startle eye-blink reflex when assessing sensorimotor gating. While PPI can be recorded in acutely decerebrated rats, behavioural, pharmacological and psychophysiological studies suggest the involvement of a complex neural network extending from brainstem nuclei to higher order cortical areas. The current functional magnetic resonance imaging study investigated the neural network underlying PPI and its association with electromyographically (EMG) recorded PPI of the acoustic startle eye-blink reflex in 16 healthy volunteers. A sparse imaging design was employed to model signal changes in blood oxygenation level-dependent (BOLD) responses to acoustic startle probes that were preceded by a prepulse at 120 ms or 480 ms stimulus onset asynchrony or without prepulse. Sensorimotor gating was EMG confirmed for the 120-ms prepulse condition, while startle responses in the 480-ms prepulse condition did not differ from startle alone. Multiple regression analysis of BOLD contrasts identified activation in pons, thalamus, caudate nuclei, left angular gyrus and bilaterally in anterior cingulate, associated with EMGrecorded sensorimotor gating. Planned contrasts confirmed increased pons activation for startle alone vs 120-ms prepulse condition, while increased anterior superior frontal gyrus activation was confirmed for the reverse contrast. Our findings are consistent with a primary pontine circuitry of sensorimotor gating that interconnects with inferior parietal, superior temporal, frontal and prefrontal cortices via thalamus and striatum. PPI processes in the prefrontal, frontal and superior temporal cortex were functionally distinct from sensorimotor gating.

Reproducible and label-free biosensor for the selective extraction and rapid detection of proteins in biological fluids

Erythropoietin (EPO), a glycoprotein hormone of ∼34 kDa, is an important hematopoietic growth factor, mainly produced in the kidney and controls the number of red blood cells circulating in the blood stream. Sensitive and rapid recombinant human EPO (rHuEPO) detection tools that improve on the current laborious EPO detection techniques are in high demand for both clinical and sports industry. A sensitive aptamer-functionalized biosensor (aptasensor) has been developed by controlled growth of gold nanostructures (AuNS) over a gold substrate (pAu/AuNS). The aptasensor selectively binds to rHuEPO and, therefore, was used to extract and detect the drug from horse plasma by surface enhanced Raman spectroscopy (SERS). Due to the nanogap separation between the nanostructures, the high population and distribution of hot spots on the pAu/AuNS substrate surface, strong signal enhancement was acquired. By using wide area illumination (WAI) setting for the Raman detection, a low RSD of 4.92% over 150 SERS measurements was achieved. The significant reproducibility of the new biosensor addresses the serious problem of SERS signal inconsistency that hampers the use of the technique in the field. The WAI setting is compatible with handheld Raman devices. Therefore, the new aptasensor can be used for the selective extraction of rHuEPO from biological fluids and subsequently screened with handheld Raman spectrometer for SERS based in-field protein detection.

Thin-Layer Chromatography with Ultraviolet and Mass Spectrometric Detection: From Preparative-Layer to Miniaturized Ultra-Thin-Layer Technique

The present challenge in drug discovery is to synthesize new compounds efficiently in minimal time. The trend is towards carefully designed and well-characterized compound libraries because fast and effective synthesis methods easily produce thousands of new compounds. The need for rapid and reliable analysis methods is increased at the same time. Quality assessment, including the identification and purity tests, is highly important since false (negative or positive) results, for instance in tests of biological activity or determination of early-ADME parameters in vitro (the pharmacokinetic study of drug absorption, distribution, metabolism, and excretion), must be avoided. This thesis summarizes the principles of classical planar chromatographic separation combined with ultraviolet (UV) and mass spectrometric (MS) detection, and introduces powerful, rapid, easy, low-cost, and alternative tools and techniques for qualitative and quantitative analysis of small drug or drug-like molecules. High performance thin-layer chromatography (HPTLC) was introduced and evaluated for fast semi-quantitative assessment of the purity of synthesis target compounds. HPTLC methods were compared with the liquid chromatography (LC) methods. Electrospray ionization mass spectrometry (ESI MS) and atmospheric pressure matrix-assisted laser desorption/ionization MS (AP MALDI MS) were used to identify and confirm the product zones on the plate. AP MALDI MS was rapid, and easy to carry out directly on the plate without scraping. The PLC method was used to isolate target compounds from crude synthesized products and purify them for bioactivity and preliminary ADME tests. Ultra-thin-layer chromatography (UTLC) with AP MALDI MS and desorption electrospray ionization mass spectrometry (DESI MS) was introduced and studied for the first time. Because of the thinner adsorbent layer, the monolithic UTLC plate provided 10 100 times better sensitivity in MALDI analysis than did HPTLC plates. The limits of detection (LODs) down to low picomole range were demonstrated for UTLC AP MALDI and UTLC DESI MS. In a comparison of AP and vacuum MALDI MS detection for UTLC plates, desorption from the irregular surface of the plates with the combination of an external AP MALDI ion source and an ion trap instrument provided clearly less variation in mass accuracy than the vacuum MALDI time-of-flight (TOF) instrument. The performance of the two-dimensional (2D) UTLC separation with AP MALDI MS method was studied for the first time. The influence of the urine matrix on the separation and the repeatability was evaluated with benzodiazepines as model substances in human urine. The applicability of 2D UTLC AP MALDI MS was demonstrated in the detection of metabolites in an authentic urine sample.