Simultaneous quantification of peroxidase and ascorbic acid in biosamples with an automatic system based on a Fe(iii)/methylthymol blue-carbon dot simulative enzyme.

Peroxidase (POD) and ascorbic acid (AsA) usually coexist in organisms to synergistically protect them from reactive oxygen damage, and their contents undergo dynamic changes under different physiological conditions. What's more, the response of POD-catalytic activity in spectrophotometry has to be corrected using the content of concomitant AsA because we found that there is an extinction reaction between AsA and chromogenic products obtained from POD catalysis. With these implications, by skilfully using the chromogenic and the extinction phenomena in the guaiacol/POD/H2O2 reaction, an automatic analysis system for simultaneous quantification of POD (73-440 U L-1) and AsA (4-60 mg L-1) was successfully established based on flow injection analysis (FIA). Furthermore, under acidic conditions (0.5 mol L-1 of HCl), hydrothermal synthesis (250 °C for 1 h) was used for synthesizing new carbon dots (sPOD-CDs) of methylthymol blue (0.08 g L-1)/FeCl3 (0.8 g L-1), which is a simulative enzyme for POD, and it was first used for catalyzing the guaiacol/H2O2 reaction within the FIA system to replace natural HRP in the extinction reaction. This sPOD-CD solution has no background absorption and its concentration shows excellent correlation with simulative POD-activity. Finally, after optimization, this FIA system was utilized to testify that the reducibility of AsA is due to ascorbate ions and to determine POD and AsA in some plant samples. The standard addition recovery experiment showed that there was no interference from the matrix in real samples (recoveries: 95%-105%), and the obtained POD and AsA results were also consistent with the reference experiments (relative deviation ≤ 2.80%, t-test ≥ 0.07). The proposed FIA system is characterized by high sample-throughput (40 samples per h), better repeatability (relative standard deviation ≤ 1.4%), etc.

Multifactorial study and kinetics of signal development in ferrous-methylthymol blue-gelatin gel dosimeters.

PURPOSE: To develop and characterize a ferrous-methylthymol blue-gelatin gel dosimeter with low optical background and appropriate additives for reduced rate of auto-oxidation and diffusion. METHODS: A mixed-level multifactorial design of experiments was used to test the effects of the concentrations of sulfuric acid, 5-nitro-1,10-phenanthroline (Nn), and glyoxal (Gx) on the background absorbance, dose sensitivity, and auto-oxidation of the tested gel dosimeter. The dosimetric properties of the proposed ferrous-methylthymol blue-gelatin dosimeter, doped with Nn and Gx, were compared with the undoped formulation and with ferrous-xylenol orange-gelatin gel dosimeters. Irradiations were performed in both small-scale cuvette samples and large 400-mL bulk samples. In addition to that, a new kinetic model for the signal development postirradiation was derived based on chemical principles and used for comparison of the different formulations. RESULTS: The new formulation showed a reduced auto-oxidation rate, while maintaining low background absorbance relative to the common ferrous-xylenol orange-gelatin gel dosimeter. Compared with undoped ferrous-xylenol orange or ferrous-methylthymol blue gels, the dose sensitivity of the new formulation is approximately 2 to 3 times lower, but remains clinically adequate. A previously unreported dose rate dependence of the dose sensitivity was observed, and a new kinetic model for the signal development postirradiation was used to investigate this effect. Similar dose rate dependences in gels containing either methylthymol blue or xylenol orange, with or without doping with Nn and Gx, were observed, suggesting that the low ferrous ammonium sulfate concentrations used in studied formulations were responsible for this effect. CONCLUSIONS: A multifactorial design of experiments and a new kinetic model for the signal development postirradiation were successfully employed to optimize the composition and characterize the properties of a new ferrous-methylthymol blue-gelatin gel dosimeter doped with 5-nitro-1,10-phenanthroline and glyoxal. Concrete recommendations were provided for precise dosimetry using the new formulation.

Application of a new version of GA-RBF neural network for simultaneous spectrophotometric determination of Zn(II), Fe(II), Co(II) and Cu(II) in real samples: An exploratory study of their complexation abilities toward MTB.

The current study for the first time is devoted to the application of whole space genetic algorithm-radial basis function network (wsGA-RBFN) method to determine the content micro minerals of Zn(2+), Fe(2+), Co(2+) and Cu(2+) based on their complexes formation with methylthymol blue (MTB) spectrophotometrically in various pharmaceutical products and vegetable samples. Advantage of wsGA-RBFN compared to GA-RBFN is that centers can be located in any point of the samples spaces. Initially, the parameters controlling behavior of the system were investigated and optimum conditions were selected. Then, an exploratory analysis of complex systems was carried out by chemometrics approaches such as SVD, EFA, MCR-ALS and RAFA. The optimal parameters and conditions for constructing the proposed model of wsGA-RBFN were obtained from processing the data set of synthetic samples. Finally, wsGA-RBFN was successfully applied to the simultaneous determination of Zn(2+), Fe(2+), Co(2+) and Cu(2+) in tomato, white cabbage, red cabbage and lettuce and pharmaceutical products included iron, zinc, multi complete and B12 ampoule.

Simultaneous voltammetric determination of ascorbic acid, dopamine and uric acid using polybromothymol blue film-modified glassy carbon electrode.

A sensitive and selective electrochemical method for simultaneous determination of ascorbic acid (AA), dopamine (DA), and uric acid (UA) using an electropolymerized bromothymol blue (BTB)-modified glassy carbon electrode (GCE) was developed. The electrochemically synthesized film was investigated using electrochemical impedance spectroscopy and voltammetric methods. The electrochemical behavior of the polymer-modified electrode depends on film thickness, i.e., the electropolymyerization time. The poly-BTB-modified GCE shows excellent electrocatalytic activity toward the oxidation of AA, DA, and UA in phosphate buffer solution (pH 5.0). The voltametric peak separations of AA/DA, DA/UA, and AA/UA on this modified electrode are 118 mV, 298 mV, and 455 mV, respectively. Therefore the voltammetric responses of these three compounds can be resolved well on the polymer-modified electrode, and simultaneous determination of these three compounds can be achieved. In addition, this modified electrode can be successfully applied to determine AA and DA in injection and UA in urine samples without interference.

Direct colorimetric detection of copper(II) ions in sampling using diffusive gradients in thin-films.

A novel binding phase was developed for use in diffusive gradients in thin-film (DGT) sampling for Cu(II) by employing methylthymol blue as a chelating and chromogenic agent. Methylthymol blue was adsorbed onto beads of Dowex 1 x 8 resin (200-400 mesh) and the resin beads were then immobilised onto an adhesive disc. Analysis of exposed binding discs by either UV-vis spectrophotometry or computer imaging densitometry provided robust quantification of adsorbed Cu(II) in the 0.2-1 microg cm(-2) range, allowing detection at microg L(-1) concentrations in the test solution (ca. 17 microg L(-1) for a 24 h deployment), and in good agreement with established DGT theory. The method was shown to be a potential replacement for binding phases based on Chelex 100 where a colorimetric response to a specific metal is desired.

Experimental correlation between the pKa value of sulfonphthaleins with the nature of the substituents groups.

This work presents the results obtained from a spectrophotometry study performed on some indicators of the sulfonphtaleins like phenol red (PR), thymol blue (TB), bromothymol blue (BTB), xylenol orange (XO) and methylthymol blue (MTB). During the first stage the acidity constants of some of the indicators were determined using the data from spectrophotometry, potentiometry and with the use of the software SQUAD. These were as follows: for the equilibrium 2H+BTB<-->H(2)BTB, log beta(2)=15.069+/-0.046 and for H+BTB<-->HBTB, log beta(1)=8.311+/-0.044. For the XO and the MTB five values were calculated for each, namely, for MTB: log beta(5)=42.035, log beta(4)=38.567+/-0.058, log beta(3)=32.257+/-0.057, log beta(2)=23.785+/-0.057, and log beta(1)=12.974+/-0.045 while for XO: log beta(5)=40.120+/-0.102, log beta(4)=35.158+/-0.062, log beta(3)=29.102+/-0.053, log beta(2)=21.237+/-0.044, and log beta(1)=11.682+/-0.044. During the second stage, a study was conducted on the effect of the substituents present in the indicators to determine the effect of different functional groups on the pK(a) value corresponding to the last indicator's dissociation.

Determination of fluoride and oxalate using the indicator reaction of Zr(IV) with methylthymol blue adsorbed on silica gel.

Solid-phase spectrophotometric and visual test-methods of fluoride and oxalate determination are proposed. The methods are based on the competitive reactions of ZrOCl2 with methylthymol blue immobilized on silica gel and fluoride or oxalate in solution. Absorbance of the solid-phase reagent at 590 nm decreases with the growth of fluoride and oxalate contents in solution. The developed methods demonstrate high selectivity. The interference of Bi(III) and SO4(2-), PO4(3-) is eliminated by the addition of 0.01 mol L(-1) solution of ascorbic acid and 0.01 mol L(-1) of BaCl2, respectively. To eliminate the fluoride interference with oxalate determination 1x10(-3) mol L(-1) solution of Ca(NO3)2 at pH 1.5 was added. The anions of the organic acids were destructed prior to F- determination by ultrasonic exposition (44 kHz, intensity of < or = 10 W cm(-2) for 3 min). The proposed methods were applied to the analysis of mineral water, toothpaste and biological fluids.

[Determination of trace manganese in Mongolian medicine by methylthymol blue-Mn(II)-H2O2 catalytic system].

A new catalytic kinetic spectrophotometry method for the determination of trace manganese based on the oxidation reaction of H2O2 with methylthymol blue in Na2B4O7-NaOH medium has been developed. The range of the determination range was 0.20-40.00 microg x L(-1). The apparent activation energy and apparent rate constant were 73.24 kJ x mol(-1) and 5.62 x 10(-4) x s(-1), respectively, and the detection limit was 6.4 x 10(-10) g x mL(-1). The method has been used to determine trace manganese in Mongolian medicine with satisfactory result.

Direct and selective flow-injection method for the simultaneous spectrophotometric determination of calcium and magnesium in red and white wines using online dilution based on "Zone Sampling".

The present work reports a selective and simple flow injection method for the direct and simultaneous determination of calcium and magnesium ions in red, rose, and white wines. Both ions react with methylthymol blue (MTB) at a strongly basic medium to form colored complexes that are monitored spectrophotometrically (lambda(max) = 610 nm). The simultaneous determination is achieved by online masking of magnesium by 8-hydroxyquinoline (8-HQ). Incorporating an online dilution mode based on the "zone sampling" technique in the FI system, the determination of both analytes was achieved without any pretreatment of the samples, in the range 0-350 mg L(-1) and 0-200 mg L(-1) for Ca(II) and Mg(II), respectively. The 3 sigma detection limits were quite satisfactory (2.1 and 1.8 mg L(-1) for Ca(II) and Mg(II) respectively), and the precision was 1.2% (at a mixture of 100.0 mg L(-1) Ca(II) + 100.0 mg L(-1) Mg(II), n = 12). A detailed study of interferences proved that the proposed method is highly selective. The application of the method to the direct analysis of red, rose, and white wines yielded excellent results compared with those obtained by using FAAS as a reference method (e(r) < 2.8%).

Flow injection manifold for the direct spectrophotometric determination of bismuth in pharmaceutical products using Methylthymol Blue as a chromogenic reagent.

A new simple and rapid flow injection method is reported for the direct determination of bismuth in pharmaceutical products. Methylthymol Blue (MTB) was used as a chromogenic reagent and the absorbance of the colored Bi(III)-MTB complex produced was monitored at 548 nm. The various chemical and physical variables were optimized and a study of interfering ions was also carried out. Linear calibration graphs were obtained from 0 to 100 mg l-1 Bi(m) (120 injections per hour). The precision was very good (sr = 1.3%) and the limit of detection was cL = 0.150 mg l-1. The average accuracy was also very good (er = 0.75%) and was evaluated by comparison of the results obtained with those claimed by the manufacturers. The method was found to be adequately selective, considering the ions that the samples contain.

[Spectrophotometric study on the interaction of protein with acid dye].

Spectrophotometric characters of protein interaction with chromazurol S(CAS), bromocresol green (BCG), bromopyrogallol red (BPR) and methylthymol blue (MTB) have been studied in acid and alkaline medium respectively. Protein can combine the above mentioned dye to reduce absorptivity of dye solution of CAS and BCG at pH 3.8 and pH 4.0, to add absorptivity of dye solution of BPR and MTB at pH 3.8 and pH 10.8 for the bathochromic shift of wave length of maximal absorption of them to add 10 nm and 20 nm respectively. The linear relationship holds between the reduced or added absorptivity of dye and the optimum concentration range of protein. The interaction mechanism of protein and dye has been discussed tentatively.

Interference of magnetic resonance imaging contrast agents with the serum calcium measurement technique using colorimetric reagents.

A possible interaction between either linear (Gd-DTPA-BMA and Gd-DTPA) or macrocyclic (Gd-DOTA) gadolinium complexes used as magnetic resonance imaging (MRI) contrast agents and colorimetric technique reagents for the measurement of serum calcium was evaluated on human serum pools, and its mechanism was investigated by means of UV spectrometry and electro-spray ionization mass spectrometry (ESI-MS). The highest concentration tested was 2.5 mM (corresponding to a putative strictly intravascular distribution of the compound) and the lowest dose was 0.2 mM (i.e. about two elimination half lives). Serum calcium was dosed in duplicate by conventional colorimetric techniques involving o-cresol-phthalein complexone (OCP) or methylthymol blue (MTB) as reagents. No interference was detected when mixing Gd DOTA with serum, whatever the concentration. Gd DTPA (2.5 mM) did not interfere with the colorimetric technique either. Conversely, the Gd DTPA-BMA solution induced a concentration-related variation in apparent calcium levels. In the UV experiments, solutions of 2.5 mM MRI contrast media were mixed with OCP or MTB and UV absorption spectra were recorded between 400 and 800 nm. For Gd-DOTA/OCP and Gd-DOTA/MTB, no significant variations in the absorbance were detected. However, in the presence of Gd DTPA BMA, the absorbance of OCP and MTB showed substantial and immediate variations over time. The ESI-MS studies showed a complete displacement of Gd3+ ion in the case of Gd-DTPA BMA. In the presence of OCP, we observed the disappearance of Gd-DTPA BMA and the formation of the free ligand DTPA BMA and a new complex Gd OCP with an original stoichiometry of 2/2. Such a phenomenon did not occur in the case of Gd DOTA and Gd DTPA. The decomplexation of Gd-DTPA BMA in the presence of OCP can probably be explained by the weaker thermodynamic stability of Gd-DTPA BMA compared to that of Gd-DOTA and Gd DTPA.

[Determination of the content of serum calcium with methylthymol blue as chromogenic reagent].

It is reported in this paper that calcium can be determined with MTB as chromogenic reagent by isoabsorption dual-wavelength elimination. The measuring wavelength and referential wavelength were 613.7 and 590.0nm, respectively. No masking reagent was necessary in the analysis. The average recovery and RSD of calcium are 98.98% and 0.81%, respectively. Compared with titration, this method is rapid, simple and precise.

Comparison of the determination of magnesium by methylthymol blue spectrophotometry and atomic absorption spectrophotometry.

Plasma samples (n = 155) of 30 patients on an intensive ward were analysed for magnesium simultaneously by atomic absorption spectrophotometry (AAS) and methylthymol blue spectrophotometry. Methylthymol blue spectrophotometry was performed at the bedside, using two different multianalysers, Easy ST 1 and Easy ST 2, Merck, D-Darmstadt. Precision was 12.2% (Easy ST 1) and 17.1% (Easy ST 2), and the average value was 0.89 mmol/l, which was above the expected range (0.72-0.88 mmol/l). Accuracy was 16.25% (Easy ST 1) and 8.75% (Easy ST 2). Analyser 2 was more accurate (8.75% versus 6.25%) but less precise (17.1% versus 12.2%) than analyser 1. Precision of AAS was between the expected values of 0.69 and 0.84 mmol/l. Easy ST and AAS gave significantly different values (p < 0.0001) for 155 measurements. Comparison of AAS and methylthymol blue spectrophotometery showed that methylthymol blue spectrophotometry produced higher values than AAS (mean difference 0.186 mmol/l). Furthermore, analyses of 40 samples of a standardized plasma concentration with methylthymol blue spectrophotometry showed a very low precision (15.3%). Easy ST cannot be assigned for urinary measurements of magnesium. Experimentally measured samples gave unaccountable results.

Five methods for determining urinary calcium compared.

We compared frequently used methods for calcium in urine with respect to linearity, analytical recovery, within- and between-batch imprecision, bias, and practicability. We assayed serum, lyophilized urine, native urine, and an aqueous reference solution of calcium carbonate. We found that atomic absorption spectrometry and the Corning 940 Analyzer have the widest ranges of linearity; the methylthymol blue method has the poorest analytical recovery. All methods--the aforementioned three plus the Du Pont aca and Technicon RA-1000 methods--had acceptable precision, although random errors were found with the methylthymol blue method, and, except for one type of commercial lyophilized urine assayed by the Technicon method, there were no matrix problems or difficulties with bias. We cannot recommend the methylthymol blue method, but evidently urinary calcium assays can be adequately done with many currently available methods. Intralaboratory attention to methodology should give improved performance in assessment programs.

Methods for the estimation of serum magnesium in clinical laboratories.

A variety of methods have been reported for the quantitative analysis of magnesium in serum and other biological fluids. The methods may be classified into the following groups: magnesium ammonium phosphate; 8-hydroxyquinoline; titan yellow; dihydroxyazobenzene using fluorimetry; Schwarzenbach ethylenediaminetetraacetic acid; atomic emission spectrophotometry; atomic absorption spectrophotometry; ion chromatography, and carboxanilide, calmagite and related dye methods. These methods are reviewed with specific emphasis on the limitations for their use in clinical laboratories.

Depression of serum calcium by increased plasma free fatty acids in the rat: a mechanism for hypocalcemia in acute pancreatitis.

Some patients with hypertriglyceridemia and acute pancreatitis have marked hypocalcemia and high levels of plasma free fatty acids (FFAs). This study tests the hypothesis that increased plasma FFAs can significantly reduce the calcium level in vivo, a phenomenon which is different from local formation of calcium soaps due to lipolysis of adipose tissue lipids. Free fatty acid elevation was induced in rats by the administration of heparin and by the infusion of triglycerides. The results show that, compared with controls, induction of elevated FFA (from 1.57 +/- 0.08 mEq/L to 5.64 +/- 0.35, mean +/- SEM) causes the concentration of calcium to fall rapidly (from 9.04 +/- 0.06 mg/dl to 8.42 +/- 0.10, p less than 0.001). There is a significant (p less than 0.001) positive correlation between spontaneous baseline concentration of FFA and the responsiveness of calcium concentration to FFA challenge. At near-normal levels of FFA there is a significant (p less than 0.001) correlation between the magnitude of increased FFA concentration and decreased calcium concentration. Additional studies in vivo and in vitro show that elevated plasma triglycerides per se did not interfere with measurement of calcium concentration; however, FFA-albumin complexes bind calcium and lower its measured value. These findings suggest that (a) changes in the concentration of FFA occurring spontaneously may affect measured serum calcium concentration; (b) the observed depression of serum calcium concentration may be due in part to intravascular sequestration of calcium by FFA, but increased flux of circulating calcium-FFA complexes into extravascular and intracellular sites may also be important; (c) the markedly increased FFA concentration in some patients with acute pancreatitis may contribute significantly to hypocalcemia and calcium flux in these patients. As parathyroid hormone secretion, function, or integrity may be impaired in pancreatitis, the depressant effect of FFA could be even greater in that disease than in this model.

New rapid kit for determining calcium in serum and urine evaluated.

We describe the performance of a colorimetric reagent kit (Diagnostic Systems Laboratories, Inc., Webster, TX 77598) for measuring calcium directly in urine, serum, and ultrafiltered serum, and compare the results with those obtained with atomic absorption spectrophotometry. The CV for within-run precision was 2.8 and 2.1% for urine and whole serum, respectively (n = 10 each). Between-run precision for urine, whole serum, and ultrafiltered serum was 1.9, 1.6, and 2.2%, respectively (n = 8 to 10). Analytical recovery of added calcium from three different urine and serum specimens, to which three different concentrations of calcium had been added, was 101.9 (SD 0.3%) for urine and 100.9 (SD 0.2%) for serum. Assay of 30 urine specimens, 15 ultrafiltered serums, and 20 whole serums by both the kit and atomic absorption spectrophotometry demonstrated correlation coefficients of 0.993, 0.828, and 0.751, respectively. Mg2+, hemolysis, or lipemia does not interfere. Compared with atomic absorption spectrophotometry, the calcium kit procedure is rapid and simple.

[Evaluation of the methylthymol blue and orthocresolphthalein direct methods of determination of serum calcium]. / Valutazione dei metodi diretti al blu di metiltimolo e all'ortocresolftaleina per la determinazione del calcio nel siero.

Two methods for the direct colorimetric determination of serum calcium (BMT and OCFT) have been evaluated and compared with atomic absorption spectrophotometry (AAS). Both colorimetric methods resulted simple to use, but temperature-sensitive: values for the ratio absorbance at 10 degrees C/absorbance at 45 degrees C were 1.6 (BMT) and 1.8 (OCFT). When readings were taken at 37 degrees C good precision was achieved with both methods (between-the-series coefficients of variation were 3.5 divided by 2.2% for OFCT and 1.9 divided by 3.4% for BMT). No interference from bilirubin (up to 180 mumol/l) was observed with both methods; slight positive interference from haemoglobin (1 g/l) was observed only with OCFT method. Results from the two colorimetric methods closely agreed in the range 1.0 divided by 4.5 mmol/l: statistical analysis of comparison data gave BMT = 0.0963 + 0.9521 OCFT, r = 0.9820. From the results of direct comparison with AAS (103 serum specimens, concentration spanning from 1.0 to 4.5 mmol/l) statistical analysis gave: BMT (y)/AAS(x): y = 0.0721 + 1.0226x (r = 0.9678); OCFT(y)AAS(x): y = -0.0208 + 1.0720x (r = 0.9836): it is concluded that both methods show a slight positive bias with respect to AAS.