Determinação do período de estabilidade de soluções volumétricas rotineiramente utilizadas em laboratórios de análises químicas / Determination of the stability period of volumetric solutions routinely used in chemical analysis laboratories / Determinación del período de estabilidad de las soluciones volumétricas utilizadas habitualmente en los laboratorios de análisis químicos

As soluções volumétricas são rotineiramente utilizadas nos laboratórios, principalmente nos processos de síntese de produtos e nas análises quantitativas de matéria-prima e/ou produto acabado, entretanto poucos são os estudos que abordam a estabilidade destas soluções. Considerando que a qualidade das soluções volumétricas pode afetar os procedimentos de análises químicas e consequentemente induzir a erros, e ainda que, a Farmacopeia Brasileira (2010) não cita tempo máximo de utilização dessas soluções padronizadas, a avaliação da estabilidade das mesmas é importante. Sendo assim, o objetivo do trabalho foi avaliar a estabilidadede 10 soluções volumétricas, empregadas rotineiramente em laboratórios de análises químicas, com o intuito de estabelecer o período que essas soluções permanecem estáveis, isto é, sem sofrer alteração na concentração. As metodologias de preparo e padronização das soluções volumétricas seguiram os métodos descritos na Farmacopeia Brasileira (2010), sendo as mesmas padronizadas no momento do preparo e a cada 20 dias, por um período de 180 dias. As soluções contendo ácidos e bases, bem como as soluções de iodato de potássio e nitrato de prata, permaneceram constantes durante o período de análises. As soluções de EDTA, iodo, nitrito de sódio, permanganato de potássio e tiossulfato de sódio apresentaram estabilidade inferior a 180 dias, tornando necessária a realização de padronização periódica. As soluções volumétricas utilizadas nos laboratórios apresentam diferentes estabilidades, o que ressalta a importância da determinação do período que as mesmas se mantêmcom as concentrações estáveis, evitando possíveis alterações de resultados nas análises químicas.

Volumetric solutions are routinely used in laboratories, mainly in product synthesis processes and in quantitative analyzes of raw materials and/or finished products, however there are few studies that address the stability of these solutions. Considering that the quality of volumetric solutions can affect chemical analysis procedures and consequently induce errors, and even though the Brazilian Pharmacopoeia (2010) does not mention the maximum time for using these standardized solutions, the evaluation of their stability is important. Therefore, the aim of this work was to evaluate the stability of 10 volumetric solutions, routinely used in chemical analysis laboratories, in order to establish the period that these solutions remain stable without changing their concentrations. The methodologies for preparing and standardizing the volumetric solutions followed the methods described in the Brazilian Pharmacopoeia (2010), being standardized at the time of preparation and every 20 days, for a period of 180 days. Solutions containing acids and bases, as well as potassium iodate and silver nitrate solutions, were stable during the analysis period. The solutions of EDTA, iodine, sodium nitrite, potassium permanganate and sodium thiosulfate showed stability less than 180 days, making it necessary to carry out periodic standardization of these solutions. The volumetric solutions used in the laboratories have different stabilities, which highlights the importance of determining the period in which they remain stable, avoiding possible changes in results in chemical analyzes.

Las soluciones volumétricas se utilizan de forma rutinaria en los laboratorios, principalmente en los procesos de síntesis de productos y en el análisis cuantitativo de materias primas y/o productos acabados. Sin embargo, existen pocos estudios que aborden la estabilidad de estas soluciones. Considerando que la calidad de las soluciones volumétricas puede afectar los procedimientos de análisis químico y consecuentemente inducir a errores, y también que, la Farmacopea Brasileña (2010) no menciona el tiempo máximo de uso de estas soluciones estandarizadas, la evaluación de su estabilidad es importante. Así, el objetivo del trabajo fue evaluar la estabilidad de 10 soluciones volumétricas, utilizadas rutinariamente en los laboratorios de análisis químico, con el fin de establecer el período en que estas soluciones permanecen estables, es decir, sin sufrir alteraciones en la concentración. Las metodologías de preparación y estandarización de las soluciones volumétricas siguieron los métodos descritos en la Farmacopea Brasileña (2010), siendo las mismas estandarizadas en el momento de la preparación y cada 20 días, por un período de 180 días. Las soluciones que contienen ácidos y bases, así como las soluciones de yodato de potasio y nitrato de plata, permanecieron constantes durante el periodo de análisis. Las soluciones de EDTA, yodo, nitrito de sodio, permanganato de potasio y tiosulfato de sodio fueron estables durante menos de 180 días, por lo que fue necesario realizar estandarizaciones periódicas. Las soluciones volumétricas utilizadas en los laboratorios presentan diferentes estabilidades, lo que pone de manifiesto la importancia de determinar el periodo que permanecen con concentraciones estables, evitando posibles cambios en los resultados en los análisis químicos.

Determination of Total Sulfur, Sulfate, Sulfite, Thiosulfate, and Sulfolipids in Plants.

In response to oxidative stress the biosynthesis of the ROS scavenger, glutathione is induced. This requires the induction of the sulfate reduction pathway for an adequate supply of cysteine, the precursor for glutathione. Cysteine also acts as the sulfur donor for the sulfuration of the molybdenum cofactor, crucial for the last step of ABA biosynthesis. Sulfate and sulfite are, respectively, the precursor and intermediate for cysteine biosynthesis and there is evidence for stress-induced sulfate uptake and further downstream, enhanced sulfite generation by 5'-phosphosulfate (APS) reductase (APR, EC 1.8.99.2) activity. Sulfite reductase (SiR, E.C.1.8.7.1) protects the chloroplast against toxic levels of sulfite by reducing it to sulfide. In case of sulfite accumulation as a result of air pollution or stress-induced premature senescence, such as in extended darkness, sulfite can be oxidized to sulfate by sulfite oxidase. Additionally sulfite can be catalyzed to thiosulfate by sulfurtransferases or to UDP-sulfoquinovose by SQD1, being the first step toward sulfolipid biosynthesis.Determination of total sulfur in plants can be accomplished using many techniques such as ICP-AES, high-frequency induction furnace, high performance ion chromatography, sulfur combustion analysis, and colorimetric titration. Here we describe a total sulfur detection method in plants by elemental analyzer (EA). The used EA method is simple, sensitive, and accurate, and can be applied for the determination of total S content in plants.Sulfate anions in the soil are the main source of sulfur, required for normal growth and development, of plants. Plants take up sulfate ions from the soil, which are then reduced and incorporated into organic matter. Plant sulfate content can be determined by ion chromatography with carbonate eluents.Sulfite is an intermediate in the reductive assimilation of sulfate to the essential amino acids cysteine and methionine, and is cytotoxic above a certain threshold if not rapidly metabolized and can wreak havoc at the cellular and whole plant levels. Plant sulfite content affects carbon and nitrogen homeostasis Therefore, methods capable of determining sulfite levels in plants are of major importance. Here we present two robust laboratory protocols which can be used for sulfite detection in plants.Thiosulfate is an essential sulfur intermediate less toxic than sulfite which is accumulating in plants in response to sulfite accumulation. The complexity of thiosulfate detection is linked to its chemical properties. Here we present a rapid, sensitive, and accurate colorimetric method based on the enzymatic conversion of thiosulfate to thiocyanate.The plant sulfolipid sulfoquinovosyldiacylglycerol (SQDG) accounts for a large fraction of organic sulfur in the biosphere. Aside from sulfur amino acids, SQDG represents a considerable sink for sulfate in plants and is the only sulfur-containing anionic glycerolipid that is found in the photosynthetic membranes of plastids. We present the separation of sulfolipids from other fatty acids in two simple ways: by one- and two-dimensional thin-layer chromatography.

Testing the degradation effects of three reagents on various antineoplastic compounds.

OBJECTIVE: Studies for decontamination of antineoplastic compounds have been conducted for decades. Nevertheless, recent studies indicate the contamination of work place in hospitals, and the exposure of workers. In this study, to develop an effective cleaning method for contaminated environments, the degradation efficacies of antineoplastic compounds by reagents were evaluated. METHODS: The degradation efficacies of various combinations of three reagents (sodium hypochlorite, sodium thiosulfate, and sodium hydroxide) were evaluated with four antineoplastic compounds (cyclophosphamide, epirubicin, cisplatin, and carboplatin). The residues of antineoplastic compounds were measured with high-performance liquid chromatography. RESULTS: Of the three reagents, sodium hypochlorite was the most effective to all antineoplastic compounds used in this study. Although sodium hypochlorite degraded 86.6% of cyclophosphamide, it degraded other three antineoplastic compounds completely. The combination with sodium hypochlorite and sodium thiosulfate degraded only 3.3% of cyclophosphamide, since sodium thiosulfate inhibited the degradation ability of sodium hypochlorite. Similarly, the combinations used in all three reagents failed to degrade cyclophosphamide. CONCLUSION: Although three of four antineoplastic compounds were degraded successfully, any combinations of three reagents could not degrade cyclophosphamide completely. However, the addition of sodium thiosulfate which inhibits the corrosion of metal by sodium hypochlorite is essential for daily cleaning. Therefore, the evaluation of reaction time before the addition of sodium thiosulfate may be required. We will continue to investigate the reagents for degradation.

Toxicological analysis of 17 autopsy cases of hydrogen sulfide poisoning resulting from the inhalation of intentionally generated hydrogen sulfide gas.

Although many cases of fatal hydrogen sulfide poisoning have been reported, in most of these cases, it resulted from the accidental inhalation of hydrogen sulfide gas. In recent years, we experienced 17 autopsy cases of fatal hydrogen sulfide poisoning due to the inhalation of intentionally generated hydrogen sulfide gas. In this study, the concentrations of sulfide and thiosulfate in blood, urine, cerebrospinal fluid and pleural effusion were examined using GC/MS. The sulfide concentrations were blood: 0.11-31.84, urine: 0.01-1.28, cerebrospinal fluid: 0.02-1.59 and pleural effusion: 2.00-8.59 (µg/ml), while the thiosulfate concentrations were blood: 0-0.648, urine: 0-2.669, cerebrospinal fluid: 0.004-0.314 and pleural effusion: 0.019-0.140 (µmol/ml). In previous reports, the blood concentration of thiosulfate was said to be higher than that of sulfide in hydrogen sulfide poisoning cases, although the latter was higher than the former in 8 of the 14 cases examined in this study. These results are believed to be strongly influenced by the atmospheric concentration of hydrogen sulfide the victims were exposed to and the time interval between exposure and death.

Surface analysis under ambient conditions using plasma-assisted desorption/ionization mass spectrometry.

A novel plasma-assisted desorption/ionization (PADI) method that can be coupled with atmospheric pressure sampling mass spectrometry to yield mass spectral information under ambient conditions of pressure and humidity from a range of surfaces without the requirement for sample preparation or additives is reported. PADI is carried out by generating a nonthermal plasma which interacts directly with the surface of the analyte. Desorption and ionization then occur at the surface, and ions are sampled by the mass spectrometer. The PADI technique is demonstrated and compared with desorption electrospray ionization (DESI) for the detection of active ingredients in a range of over-the-counter and prescription pharmaceutical formulations, including nonsterodial anti-inflammatory drugs (mefenamic acid, Ibugel, and ibuprofen), analgesics (paracetamol, Anadin Extra), and Beecham's "all in one" cold and flu remedy. PADI has also been successfully applied to the analysis of nicotine in tobacco and thiosulfates in garlic. PADI experiments have been performed using a prototype source interfaced with a Waters Platform LCZ single-quadrupole mass spectrometer with limited modifications and a Hiden Analytical HPR-60 molecular beam mass spectrometer (MBMS). The ability of PADI to rapidly detect active ingredients in pharmaceuticals without the need for prior sample preparation, solvents, or exposed high voltages demonstrates the potential of the technique for high-throughput screening in a pharmaceutical or forensic environment.

Selective detection of thiosulfate-containing peptides using tandem mass spectrometry.

Incubation of proteins or peptides containing disulfide bonds (S-S) with sodium sulfite (Na(2)SO(3)) cleaves S-S bonds producing approximately equimolar amounts of free thiols (-SH) and thiosulfates (-S-SO(3)H), a process known as sulfitolysis. Proteins and peptides containing thiosulfates were separated by reverse-phase high-performance liquid chromatography (RP-HPLC) and characterized by mass spectrometry (MS) and peptide mapping. The mass of the thiosulfate-containing peptide formed from oxidized insulin B chain was 3478.02 Da, 80 Da greater than the reduced peptide and corresponding precisely to addition of sulfur trioxide (SO(3)). Disulfide bond cleavage was also observed using RP-HPLC and MS after incubation of the intramolecular homodimer of mouse S100A8 (mass 20614 Da). The mass of HPLC-separated A8-SH was 10308 Da, and 10388 Da for A8-S-SO(3)H. Loss of SO(3) from multiply charged precursor ions was generally observed at elevated declustering potentials in the source region or within q(2) at relatively low collision energies (approximately 20 V). The characteristic loss of SO(3) at low collision energies preceded peptide backbone fragmentations at higher collision energies. Accurate mass measurement and charge-state discrimination, using a hybrid quadrupole time-of-flight mass spectrometer, allowed specific detection of thiosulfate-containing peptides. An information-dependent acquisition method, where the switch criterion was loss of m/z 79.9568, specifically identified 11 thiosulfate-containing peptides using nano-LC/MS from a tryptic digest of bovine serum albumin (BSA).

Reduced glutathione accelerates the oxidative damage produced by sodium n-propylthiosulfate, one of the causative agents of onion-induced hemolytic anemia in dogs.

The oxidative effects of sodium n-propylthiosulfate, one of the causative agents of onion-induced hemolytic anemia in dogs, were investigated in vitro using three types of canine erythrocytes, which are differentiated by the concentration of reduced glutathione and the composition of intracellular cations. After incubation with sodium n-propylthiosulfate, the methemoglobin concentration and Heinz body count in all three types of erythrocytes increased and a decrease in the erythrocyte reduced glutathione concentration was then observed. The erythrocytes containing high concentrations of potassium and reduced glutathione (approximately five times the normal values) were more susceptible to oxidative damage by sodium n-propylthiosulfate than were the normal canine erythrocytes. The susceptibility of the erythrocytes containing high potassium and normal reduced glutathione concentrations was intermediate between those of erythrocytes containing high concentrations of potassium and reduced glutathione and normal canine erythrocytes. In addition, the depletion of erythrocyte reduced glutathione by 1-chloro-2, 4-dinitrobenzene resulted in a marked decrease in the oxidative injury induced by sodium n-propylthiosulfate in erythrocytes containing high concentrations of potassium and reduced glutathione. The generation of superoxide in erythrocytes containing high concentrations of potassium and reduced glutathione was 4.1 times higher than that in normal canine erythrocytes when the cells were incubated with sodium n-propylthiosulfate. These observations indicate that erythrocyte reduced glutathione, which is known as an antioxidant, accelerates the oxidative damage produced by sodium n-propylthiosulfate.

The glutathione dependence of inorganic sulfate formation from L- or D-cysteine in isolated rat hepatocytes.

The GSH dependence of the metabolic pathways involved in the conversion of cysteine to sulfate in intact cells has been investigated. It was found that hepatocyte-catalysed sulfate formation from added L-cysteine did not occur if hepatocyte GSH was depleted beforehand, but was restored when GSH levels recovered. Furthermore, sulfate formation did not recover in GSH-depleted hepatocytes if GSH synthesis was prevented with buthionine sulfoximine. Thiosulfate formation was, however, markedly enhanced in GSH-depleted hepatocytes. These results suggest that thiosulfate is an intermediate in the formation of inorganic sulfate from L-cysteine and that GSH was required for the conversion of thiosulfate to inorganic sulfate. Much less sulfate was formed if the cysteine was replaced with cysteinesulfinate. Furthermore, sulfate formation from L-cysteine was markedly inhibited by the addition of the transaminase inhibitor DL-cycloserine or the gamma-cystathionase inhibitor DL-propargylglycine. The major routes of sulfate formation from L-cysteine therefore seems to involve pathways that do not involve L-cysteinesulfinate. Similar amounts of sulfate were formed from D-cysteine as L-cysteine. Thiosulfate instead of sulfate was also formed in GSH-depleted hepatocytes. However, sulfate formation from D-cysteine differed from L-cysteine in that it was inhibited by the D-aminoacid oxidase inhibitor sodium benzoate and was not affected by transaminase or gamma-cystathionase inhibitors. These results suggest that thiosulfate is an intermediate in sulfate formation from D-cysteine and involves the oxidation of D-cysteine by D-amino acid oxidase to form beta-mercaptopyruvate.

A new enzymatic method for the determination of inulin.

A new enzymatic method for the determination of inulin in plasma and urine, using inulase (EC 3.2.1.7), fructokinase (EC 2.7.1.4), phosphoglucoisomerase (EC 5.3.1.9) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49) is described. The assay is based on the hydrolysis of inulin or Inutest (INutest which is the injectable form of inulin), by inulase and the determination of fructose released. The assay was linear up to 2 g/L of Inutest. The within-batch and between batch coefficients of variation were 2.3% and 2.2%, respectively. Recovery of added Inutest from plasma and urine was 98-102%. There was no interference from glucose (27.7 mmol/L), fructose (1.7 mmol/L) or mannose (1.7 mmol/L). When inulin clearance (using this method) and thiosulphate clearance were compared in 37 patients the inulin clearance was 9.3 mL/min (12%) lower than the thiosulphate clearance. We conclude that this enzymatic method is a simple and specific method.

Low-molecular-weight thiols in streptomycetes and their potential role as antioxidants.

The intracellular low-molecular-weight thiols present in five gram-positive Streptomyces species and one Flavobacterium species were analyzed by high-performance liquid chromatography after fluorescence labeling with monobromobimane. Bacteria were chosen to include penicillin and cephalosporin beta-lactam producers and nonproducers. No significant amount of glutathione was found in any of the streptomycetes. Major intracellular thiols in all strains examined were cysteine, coenzyme A, sulfide, thiosulfate, and an unknown thiol designated U17. Those streptomycetes that make beta-lactam antibiotics also produce significant amounts of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (ACV), a key intermediate in their biosynthesis. In Streptomyces clavuligerus, a potent producer of beta-lactams, the level of ACV was low during the early phase of growth and increased rapidly toward the end of exponential growth, paralleling that of antibiotic production. These and other observations indicate that ACV does not function as a protective thiol in streptomycetes. U17 may have this role since it was the major thiol in all streptomycetes and appeared to occur at levels about 10-fold higher than those of the other thiols measured, including ACV. Purification and amino acid analysis of U17 indicated that it contains cysteine and an unusual amine that is not one of the common amino acids. This thiol is identical to an unknown thiol found previously in Micrococcus roseus and Streptomyces griseus. A high level of ergothioneine was found in Streptomyces lactamdurans, and several unidentified thiols were detected in this and other streptomycetes.

Reaction of S-(2-amino-2-carboxyethylsulfonyl)-L-cysteine with thiosulfate: synthesis of L-alanine sulfodisulfane and application to the determination of thiosulfate.

A new reaction of S-(2-amino-2-carboxyethylsulfonyl)-L-cysteine (ACESC) with thiosulfate is described. The reaction proceeded quantitatively in formic or acetic acid solutions, yielding equimolar amounts of L-alanine sulfodisulfane (2-amino-2-carboxyethyl sulfodisulfane) and L-alanine 3-sulfinic acid. L-Alanine sulfodisulfane was obtained as pure monosodium salt; the yield was 92% of the theoretical. A new method is described for the determination of thiosulfate. The method is based on the quantitative reaction between ACESC and thiosulfate, and L-alanine sulfodisulfane, one of the reaction products, was determined using acid ninhydrin reagent 2 of M. K. Gaitonde (1967, Biochem. J. 104, 627-633). The recovery was over 95%. When samples contained sulfite in addition to thiosulfate, S-sulfo-L-cysteine (T. Ubuka et al., 1982, Anal. Biochem. 126, 273-277) was produced in addition to L-alanine sulfodisulfane by the treatment with ACESC. Both products were separated by a small Dowex 1 column and determined with the acid ninhydrin reagent 2. The recoveries were over 95%. The new method was applied to the thiosulfate sulfurtransferase reaction, in which thiosulfate, a substrate, and sulfite, a product, were determined separately.

High-performance liquid chromatographic measurement of exogenous thiosulfate in urine and plasma.

A simple technique using reverse-phase ion-pair liquid chromatography for measurement of exogenous thiosulfate is described. Accurate measurement of thiosulfate in plasma and urine was permitted by precolumn derivatization with monobromobimane, a substance that readily yields fluorescent compounds upon reaction with a variety of biologically important nucleophiles including glutathione, cysteine, and sulfite. Using an injection volume of 50 microliters, as little as 0.16 nmol of thiosulfate was reliably measured. The interassay precision of the method was reflected by a coefficient of variation of 7.7% while the coefficient of variation for interassay analysis was 2.6%. Recovery of thiosulfate from plasma was 96.9 +/- 3.2% and greater than 98% from urine. The simplicity, sensitivity, and precision of the method make it ideal for the study of thiosulfate and other important nucleophiles in body fluids.