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Antimicrobial resistance is a silent pandemic considered a public health concern worldwide. Strategic therapies are needed to replace antibacterials that are now ineffective. One approach entails the use of well-known antibacterials along with adjuvants that possess non-antibiotic properties but can extend the lifespan and enhance the effectiveness of the treatment, while also improving the suppression of resistance. In this regard, a group of uniform materials based on organic salts (GUMBOS) presents an alternative to this problem allowing the combination of antibacterials with adjuvants. Fluoroquinolones are a family of antibacterials used to treat respiratory and urinary tract infections with broad-spectrum activity. Ciprofloxacin and moxifloxacin-based GUMBOS were synthesized via anion exchange reactions with lithium and sodium salts. Structural characterization, thermal stability and octanol/water partition ratios were evaluated. The antibacterial profiles of most GUMBOS were comparable to their cationic counterparts when tested against Gram-positive S. aureus and Gram-negative E. coli, except for deoxycholate anion, which demonstrated the least effective antibacterial activity. Additionally, some GUMBOS were less cytotoxic to L929 fibroblast cells and non-hemolytic to red blood cells. Therefore, these agents exhibit promise as an alternative approach to combining drugs for treating infections caused by resistant bacteria.
Assuntos
Fluoroquinolonas , Sais , Fluoroquinolonas/farmacologia , Sais/farmacologia , Sais/química , Escherichia coli , Staphylococcus aureus , Antibacterianos/química , Ânions , Testes de Sensibilidade MicrobianaRESUMO
The aim of this study is the development of an automated method for myeloperoxidase activity evaluation and its application in testing the inhibitory action of different plant extracts on the activity of the enzyme. This enzyme has its concentration increased in inflammatory and infectious processes, so it is a possible target to limit these processes. Therefore, an automatic sequential in-jection analysis (SIA) system was optimized and demonstrated that it is possible to obtain results with satisfactory accuracy and precision. With the developed method, plant extracts were studied, as promising candidates for MPO inhibition. In the group of selected plant extracts, IC50 values from 0.029 ± 0.002 mg/mL to 35.4 ± 3.5 mg/mL were obtained. Arbutus unedo L. proved to be the most inhibitory extract for MPO based on its phenolic compound content. The coupling of an automatic SIA method to MPO inhibition assays is a good alternative to other conventional methods, due to its simplicity and speed. This work also supports the pharmacological use of these species that inhibit MPO, and exhibit activity that may be related to the treatment of infection and inflammation.
Assuntos
Ericaceae , Extratos Vegetais , Antioxidantes/farmacologia , Peroxidase , Fenóis/análise , Extratos Vegetais/análise , Extratos Vegetais/farmacologiaRESUMO
A miniaturized microsequential injection/lab-on-valve (µSIA-LOV) system was developed and shown to be a useful alternative to perform inhibitory studies on acetylcholinesterase. These studies are essential for the evaluation of the potential therapeutic effect of drugs commonly used in the treatment of Alzheimer's disease. Donepezil, galantamine, and rivastigmine were tested, in addition to compounds based on the xanthone scaffold. Four of these xanthone derivatives were identified as having EC50 values between 676 and 4466 µmol/l, showing a potential inhibitory effect higher than the clinical agent rivastigmine. The developed automatic system added advantages of reduction of reagents and sample consumption (around 55 µl per analysis), lower cost per analysis, and the generation of less waste (around 1.2 ml per analysis). The µSIA-LOV system is also a robust, rapid, reliable, and simple system to use. Docking studies suggested a possible mode of interaction with the target acetylcholinesterase protein.
Assuntos
Inibidores da Colinesterase/farmacologia , Simulação de Acoplamento Molecular , Xantonas/farmacologia , Animais , Inibidores da Colinesterase/química , Donepezila/farmacologia , Electrophorus , Análise de Injeção de Fluxo/métodos , Galantamina/farmacologia , Humanos , Reprodutibilidade dos Testes , Rivastigmina/farmacologia , Relação Estrutura-Atividade , Xantonas/químicaRESUMO
In this work, two solid-phases based on imidazolium-based ionic liquids were obtained and characterized for solid-phase extraction of fluoroquinolones. The process of immobilization was performed replacing a toxic reagent by UV-irradiation to get a harmless process. The obtained solid-phases were characterized by nuclear magnetic resonance spectroscopy and elemental analysis. Each solid-phase was packed in a cartridge and was used in solid-phase extraction processes for norfloxacin and ciprofloxacin, after the optimization of some parameters such as the elution solvent, the eluent volume and, the sample volume to be used during the loading step. The developed solid-phases with immobilized ionic liquids were successfully implemented for the studied compounds and indicate high probabilities to be useful in solid-phase extractions of other fluoroquinolones.
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An automatic assay was developed that is intended to be a generic tool for evaluation of a horseradish peroxidase activity in different ionic liquids (ILs). Ionic liquids with different characteristics were used and their effects on the enzymatic reaction, were compared with those obtained with conventional organic solvents. In addition, ILs were tested as solvents for the enzyme substrate (bisphenol A (BPA)). ILs were shown to be a good alternative to conventional organic solvents from either the effect on enzymatic activity or the solubilization of bisphenol. Since bisphenol A is an endocrine disruptor frequently used in plastic industries, it was also applied the developed enzymatic methodology for quantification of this compound in real beverage samples. To increase the sensitivity (already increased by the use of an IL) and the selectivity of the methodology, a sample pre-treatment using a molecular recognition solid phase extraction was applied. Finally, the methodology presented detection and quantification limits of 7.73 × 10-4 and 1.29 × 10-3 mmol L-1 and a linear range up to 1.00 mmol L-1, allowing accurate and reliable quantifications of bisphenol in beer and cola drink samples. This work confirmed the potential of a sequential injection analysis (SIA) system as a simple, versatile, robust, and rapid analytical tool for automating enzymatic assays in ILs medium and, at the same time, showed it to be a relevant automatic alternative for routine determinations of bisphenol A in food samples.
Assuntos
Compostos Benzidrílicos/análise , Biocatálise , Análise de Alimentos/métodos , Líquidos Iônicos/química , Fenóis/análise , Disruptores Endócrinos , Extração em Fase Sólida , SolventesRESUMO
Antipsychotic (AP) drugs are becoming accumulated in terrestrial and aqueous resources due to their actual consumption. Thus, the search of methods for assessing the contamination load of these drugs is mandatory. The COD is a key parameter used for monitoring water quality upon the assessment of the effect of polluting agents on the oxygen level. Thus, the present work aims to assess the chemical oxygen demand (COD) levels of several typical and atypical antipsychotic drugs in order to obtain structure-activity relationships. It was implemented the titrimetric method with potassium dichromate as oxidant and a digestion step of 2h, followed by the measurement of remained unreduced dichromate by titration. After that, an automated sequential injection analysis (SIA) method was, also, used aiming to overcome some drawbacks of the titrimetric method. The results obtained showed a relationship between the chemical structures of antipsychotic drugs and their COD values, where the presence of aromatic rings and oxidable groups give higher COD values. It was obtained a good compliance between the results of the reference batch procedure and the SIA system, and the APs were clustered in two groups, with the values ratio between the methodologies, of 2 or 4, in the case of lower or higher COD values, respectively. The SIA methodology is capable of operating as a screening method, in any stage of a synthetic process, being also more environmentally friendly, and cost-effective. Besides, the studies presented open promising perspectives for the improvement of the effectiveness of pharmaceutical removal from the waste effluents, by assessing COD values.
Assuntos
Antipsicóticos/análise , Análise da Demanda Biológica de Oxigênio/métodos , Águas Residuárias/análise , Poluentes Químicos da Água/análise , Antipsicóticos/química , Oxirredução , Oxigênio/análise , Relação Estrutura-Atividade , Titulometria , Eliminação de Resíduos Líquidos , Poluentes Químicos da Água/química , Qualidade da ÁguaRESUMO
A novel automated fluorimetric technique was developed for the assessment of the chemical oxygen demand (COD) of ionic liquids (ILs) and combined with a photodegradation step to promote IL degradation. The method was implemented on a sequential injection analysis (SIA) system and is based on the reduction of cerium(IV) in the presence of irradiated ILs. Compounds incorporating the chloride anion were found to exhibit higher COD values and 1-butyl-3-methylimidazolium chloride ([bmim]+ [Cl]- ), 1-butyl-1-methylpyrrolidinium chloride ([bmpyr]+ [Cl]- ), and1-hexyl-3-methylimidazolium chloride ([hmim]+ [Cl]- ) also exhibited considerable photodegradability, whereas the cholinium cation and methanesulfonate and tetrafluoroborate anions showed resistance to photolysis. The developed methodology proved to be a simple, affordable, and robust method, showing good repeatability under the tested conditions (rsd <3.5 %, n=10). Therefore, it is expected that the developed approach can be used as a screening method for the preliminary evaluation of compounds' potential impact in the aquatic field. Additionally, the photolysis step presents an attractive option to promote degradation of ILs prior to their release into wastewater.
Assuntos
Automação , Fluorometria , Líquidos Iônicos/química , Oxigênio/química , Processos FotoquímicosRESUMO
Immobilization of quantum dots (QDs) onto solid supports could improve their applicability in the development of sensing platforms and solid-phase reactors by allowing the implementation of reusable surfaces and the execution of repetitive procedures. As the reactivity of QDs relies mostly on their surface chemistry, immobilization could also limit the disruption of solution stability that could prevent stable measurements. Herein, distinct strategies to immobilize QDs onto porous aminated supports, such as physical adsorption and the establishment of chemical linking, were evaluated. This work explores the influence of QD capping and size, concentration, pH, and contact time between the support and the QDs. Maximum QD retention was obtained for physical adsorption assays. Freundlich and Langmuir isotherms were used to analyze the equilibrium data. Gibbs free energy, enthalpy, and entropy were calculated and the stability of immobilized QDs was confirmed.
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Chronic wounds (CW) present a significant healthcare challenge due to their prolonged healing time and associated complications. To effectively treat these wounds and prevent further deterioration, monitoring their healing progress is crucial. Traditional wound assessment methods relying on visual inspection and subjective evaluation are prone to inter-observer variability. Biomarkers play a critical role in objectively evaluating wound status and predicting healing outcomes, providing quantitative measures of wound healing progress, inflammation, infection, and tissue regeneration. Recent attention has been devoted to identifying and validating CW biomarkers. Various studies have investigated potential biomarkers, including growth factors, cytokines, proteases, and extracellular matrix components, shedding light on the complex molecular and cellular processes within CW. This knowledge enables a more targeted and personalized approach to wound management. Accurate and sensitive techniques are necessary for detecting CW biomarkers. Thus, this review compares and discusses the use of electrochemical and optical sensors for biomarker determination. The advantages and disadvantages of these sensors are highlighted. Differences in detection capabilities and characteristics such as non-invasiveness, portability, high sensitivity, specificity, simplicity, cost-effectiveness, compatibility with point-of-care applications, and real-time monitoring of wound biomarkers will be pointed out and compared. In summary, this work provides an overview of CW, explores the emerging field of CW biomarkers, and discusses methods for detecting these biomarkers, with a specific focus on optical and electrochemical sensors. The potential of further research and development in this field for advancing wound care and improving patient outcomes will also be noted.
Assuntos
Técnicas Biossensoriais , Humanos , Biomarcadores/análise , Cicatrização , Atenção à SaúdeRESUMO
Two automatic methodologies were developed to perform the loading and release assays of drugs from porous silicon (PSi) nanoparticles. 5-Fluorouracil (5-FU) was selected as a model drug, and different functionalized PSi nanoparticles were used. While for drug loading studies the reproducible characteristics of flow systems were explored regarding mixture and volumes taken, in the drug release flow methodology versatility in accommodating different devices around the valve were tested. Fluorescent properties of 5-FU were used with detection limit of 9â¯×â¯10-4 mgâ¯L-1 and a linear response up to 5â¯mgâ¯L-1 The drug loading and release procedures were optimized in sequential injection analysis (SIA) systems obtaining a huge economy regarding the time spent (4â¯min towards 2â¯h). Batch and SIA methods were tested and compared for the different behaviours of the PSi nanoparticles towards both methodologies and no noteworthy differences were obtained with Student's t-test for loading with a calculated t value of 2.04 showing the absence of statistical differences. All the results present a RSD less than 10%. So, the developed automatic methodologies are a viable alternative to load drugs and to study the release profiles from PSi nanoparticles.
Assuntos
Antineoplásicos/química , Portadores de Fármacos/química , Fluoruracila/química , Nanopartículas/química , Silício/química , Tecnologia Farmacêutica/métodos , Liberação Controlada de Fármacos , Porosidade , Propriedades de SuperfícieRESUMO
Acylase I (ACY I) plays a role in the detoxication and bioactivation of xenobiotics as well in other physiological functions. In this context, an automated ACY I assay for the evaluation of ionic liquids' (ILs) toxicity was developed. The assay was implemented in a sequential injection analysis (SIA) system and was applied to eight commercially available ILs. The SIA methodology was based on the deacetylation of N-acetyl-l-methionine with production of l-methionine, which was determined using fluorescamine. ACY I inhibition in the presence of ILs was monitored by the decrease of fluorescence intensity. The obtained results confirmed the influence of ILs' structural elements on its toxicity and revealed that pyridinium and phosphonium cations, longer alkyl side chains and tetrafluoroborate anion displayed higher toxic effect on enzyme activity. The developed methodology proved to be robust and exhibited good repeatability (RSD < 1.3%, n = 10), leading also to a reduction of reagents consumption and effluents production. Thus, it is expected that the proposed assay can be used as a novel tool for ILs' toxicity screening.
Assuntos
Amidoidrolases/metabolismo , Bioensaio/métodos , Líquidos Iônicos/toxicidade , Ânions/química , Cátions/química , Líquidos Iônicos/química , Metionina/análogos & derivados , Metionina/metabolismoRESUMO
This work carries out for the first time the comparison between the physical and chemical immobilization of choline oxidase onto aminated silica-based porous supports. The influence on the immobilization efficiency of concentration, pH, temperature and contact time between the support and choline oxidase, was evaluated. The immobilization efficiency was estimated taking into consideration the choline oxidase activity, which was assessed by using cadmium telluride (CdTe) quantum dots (QDs), obtained by hydrothermal synthesis, as photoluminescent probes. Hydrogen peroxide produced by enzyme activity was capable of quenching CdTe QDs photoluminescence. The magnitude of the PL quenching process was directly related with the enzyme activity. By comparing the chemical process with the physical adsorption, it was observed that the latter provided the highest choline oxidase immobilization. The equilibrium data were analyzed using Langmuir and Freundlich isotherms and kinetic data were fitted to the pseudo-first-order and pseudo-second-order models. Thermodynamic parameters, such as Gibbs free energy and entropy were also calculated. These results will certainly contribute to the development of new sensing schemes for choline, taking into account the growing demand for its quantification in biological samples.
Assuntos
Oxirredutases do Álcool/química , Oxirredutases do Álcool/metabolismo , Adsorção , Técnicas Biossensoriais , Compostos de Cádmio/química , Colina/análise , Estabilidade Enzimática , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Cinética , Porosidade , Pontos Quânticos/química , Pontos Quânticos/metabolismo , Dióxido de Silício , Telúrio/química , TermodinâmicaRESUMO
The aim of this work was to develop a simple, automatic system for the evaluation of cationic surfactants by combining sequential injection analysis and the sensitized effect of cationic surfactants on the reaction between metal ions and chelating dyes. This reaction is based on the increase in absorbance of the complex formed among molybdenum, bromopyrogallol red and increasing concentrations of cationic surfactants. Under optimum conditions, two calibration plots were obtained for a concentration range between 2.50 x 10(-7) mol dm(-3) (detection limit) and 5.00 x 10(-4) mol dm(-3) of cetylpyridinium chloride, used as standard. Solubilization of water insoluble complexes formed for concentrations of cationic surfactants greater than 1.00 x 10(-4) mol dm(-3) were successfully achieved with Triton X-405. RSD values lower than 5.0% were obtained in all cases. The quality of the results obtained for 18 water samples were evaluated by comparison with conventional methods, with no statistically significant differences for a 95% confidence level.
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This paper presents a new way to the synthesis of uniform and size-controlled silver nanoparticles by means of microreaction technology. It complies with the philosophy of green chemistry by developing a process that prevents pollution at source-by automation of the manipulations using microtubes manifolds and with the use of benign reagents and photochemistry to enhance the reaction of synthesis of Ag nanoparticles. Effect of hydrodynamic parameters (reagent volumes and volume flow rate) and concentrations (reducer and stabilizer), temperature, pH and UV irradiation time on morphology and size of nanoparticles was studied. The silver nanoparticles has been characterized by transmission electronic microscopy (TEM), electron diffraction X-ray spectroscopy (EDS), UV-vis spectra analysis, dynamic light scattering (DLS) and zeta potential measurements. Particles are mostly spherical in shape and have average sizes between 7 and 20 nm. The particle size can be controlled by changing not only the flow rate and UV light time exposition but also the reducer/AgNO3 concentration ratio. This is a sustainable and cost-saving methodology that guarantees not only reproducible synthesis, but also the changing of NPs characteristics at time by simple control of the fluid transport.
Assuntos
Química Verde/instrumentação , Nanopartículas Metálicas/química , Técnicas Analíticas Microfluídicas/instrumentação , Nanotecnologia/instrumentação , Prata/química , Desenho de Equipamento , Análise de Injeção de Fluxo/instrumentação , Nanopartículas Metálicas/ultraestrutura , Tamanho da PartículaRESUMO
Nanoparticles (NPs) exhibit a number of distinctive and entrancing properties that explain their ever increasing application in analytical chemistry, mainly as chemosensors, signaling tags, catalysts, analytical signal enhancers, reactive species generators, analyte recognition and scavenging/separation entities. The prospect of associating NPs with automated flow-based analytical is undoubtedly a challenging perspective as it would permit confined, cost-effective and reliable analysis, within a shorter timeframe, while exploiting the features of NPs. This article aims at examining state-of-the-art on continuous flow analysis and microfluidic approaches involving NPs such as noble metals (gold and silver), magnetic materials, carbon, silica or quantum dots. Emphasis is devoted to NP format, main practical achievements and fields of application. In this context, the functionalization of NPs with distinct chemical species and ligands is debated in what concerns the motivations and strengths of developed approaches. The utilization of NPs to improve detector's performance in electrochemical application is out of the scope of this review. The works discussed in this review were published in the period of time comprised between the years 2000 and 2013.
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Nanopartículas/química , Automação , Carbono/química , Nanopartículas de Magnetita/química , Nanopartículas Metálicas/química , Microfluídica/instrumentação , Microfluídica/métodos , Porosidade , Pontos Quânticos/química , Dióxido de Silício/químicaRESUMO
In the present work an automatic methodology, based on sequential injection analysis, (SIA) for the kinetic evaluation of α-chymotrypsin (α-CT) activity in cetyltrimethylammonium bromide (CTAB)/ionic liquid (IL) mixed micelles, was developed. The α-CT-catalysed hydrolysis of N-succinyl-l-phenylalanine-p-nitroanilide (SPpNA) was monitored by following the change in absorbance at 410nm brought about by the formation of p-nitroaniline (pNA). The influence of parameters such as reagents concentration, flow rate, as well as reaction coil length, on the sensitivity and performance of the SIA system were studied and the optimum reaction conditions were subsequently selected. The effect of CTAB/IL micelles on the catalytic constant Kcat and apparent Michaelis-Menten constant (KM) for SPpNA hydrolysis was then studied. The kinetic assays evidence that CTAB/ILs mixed micellar systems can induce α-CT superactivity. In order to perform a critical evaluation of the obtained results, CMC and average micellar size of CTAB/hmim[Cl], CTAB/bmim[Cl], CTAB/bmpyr[Cl] and CTAB/bmpy[Cl] mixed micelles were evaluated by fluorescence and dynamic light scattering, respectively. The SIA methodology showed to be an interesting tool for evaluation of α-CT activity in mixed micelles as it proved to be robust and exhibited good repeatability in all the assay conditions leading also to a reduction of the consumption of solvents as well as of effluent production.
Assuntos
Compostos de Cetrimônio/farmacologia , Quimotripsina/metabolismo , Análise de Injeção de Fluxo/métodos , Líquidos Iônicos/farmacologia , Micelas , Animais , Catálise , Bovinos , Cetrimônio , Ensaios Enzimáticos , CinéticaRESUMO
A novel strategy for immobilization of CdTe quantum dots (QDs) onto amino functionalized solid supports was developed. QDs capped with compounds holding an amino group were covalently bonded to the substrate under mild reaction conditions, exhibiting great stability and strong luminescence.
Assuntos
Cisteína/química , Glutationa/química , Pontos Quânticos , Aminas/química , Compostos de Cádmio/química , Glutationa/metabolismo , Microscopia de Fluorescência , Espectroscopia de Infravermelho com Transformada de Fourier , Propriedades de Superfície , Telúrio/químicaRESUMO
According to the current demands of environmentally friendly analytical chemistry and with a view to achieving lower reagent consumption with improved analytical performance, an automatic methodology composed of a photoreactor and fluorimetric detection (λ(exc)=287 nm, λ(em)=378 nm) was developed. To this end, a sequential injection analysis (SIA) system was developed for indomethacin determination using ultra-violet (UV) light which promotes an increase in the fluorescence of indomethacin. This increase in sensitivity makes it possible to apply this methodology to a dissolution test and to determine indomethacin in pharmaceutical formulations. The calibration graph for indomethacin was linear between 4.10 × 10(-6) and 9.00 × 10(-5) mol L(-1)and the detection limit was 1.23 × 10(-6) mol L(-1). The method was proven to be reproducible with a R.S.D.<5% and sampling rate of approximately 20 per hour. The potential effect of several compounds commonly used as excipients on analytical signals was studied and no interfering effect was observed. Statistical evaluation at the 95% confidence level showed good agreement between the results obtained for the pharmaceutical samples with both the SIA system and comparison batch procedures.
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According to the current demands of Green Analytical Chemistry and regarding the need for lower reagent consumption with improved analytical performance, an automatic methodology with a flow-through optosensor incorporating solid-phase spectrophotometric detection was developed. The sensor used in this work was based on the redox state of thionine whose oxidized form is blue and reduced form is colorless with monitoring carried out at 621 nm. This redox indicator was immobilized on gel beads and subsequently packed into a flow-through cell. It was then assembled into a sequential injection system and was shown to be an excellent alternative to monitor enzymatic redox reactions automatically as the redox catalysis is performed by glucose dehydrogenase. This enzyme is a representative dehydrogenase enzyme and uses NAD(+) as cofactor, promoting the oxidation of glucose to glucono-lactone and reduction of NAD(+) to NADH. The produced NADH promotes color depletion on the surface of the sensor. The calibration graph for glucose was linear between 5.74 x 10(-4) and 2.00 x 10(-3) mol L(-1) and detection limit was 1.72 x 10(-4) mol L(-1). Glucose concentration in different samples including sera, salines, perfusion solutions, powder for preparing oral solutions and solutions for hemodialysis was determined. The method proved to be reproducible with a RSD < 5% for glucose determinations.
Assuntos
Técnicas Biossensoriais/métodos , Análise de Injeção de Fluxo/métodos , Glucose/análise , Fenotiazinas/química , Biocatálise , Glucose 1-Desidrogenase/metabolismo , NAD/química , Oxirredução , EspectrofotometriaRESUMO
The estimation of the time since death known as postmortem interval (PMI) is a main issue in the field of forensic science and legal medicine. In this work it is proposed a sequential injection system for the determination of hypoxanthine and potassium in the same sample of vitreous humor since the concentrations of both parameters change with PMI and the vitreous humor has been regarded as the ideal extracellular fluid for these kinds of determinations. By measuring both parameters the accuracy of estimation of PMI can be increased, and the effects of factors which influence the values in postmortem chemistry minimized. Hypoxanthine determination is based on its oxidation to uric acid (290 nm), catalyzed by immobilized xanthine oxidase, and the quantification of potassium levels in vitreous humor was performed using a tubular potassium ion-selective electrode. With a unique analytical cycle both analytes were evaluated being potassium levels determined during the degradation of hypoxanthine in the enzymatic reactor. Working concentration ranges between 6.04-40.00 micromol L(-1) and 7.00 x 10(-5) to 1.00 x 10(-1)mmol L(-1) were obtained, for hypoxanthine and potassium, respectively. The method proved to be reproducible with R.S.D. <5% for hypoxanthine and <3% for potassium. Sampling rate was approximately 30 per hour for the sequential determination of both parameters being 15 and 60 determinations per hour if hypoxanthine or potassium, where evaluated independently. Statistical evaluation at the 95% confidence level showed good agreement between the results obtained, for the vitreous humor samples, with both the SIA system and the comparison batch procedures. Moreover the methodology has low environmental impact in agreement with the demands of green analytical chemistry as only 2.7 mL of chemical waste is produced during both determinations.