Interference study on precipitating reagents used in a sensitive color reaction for high-density lipoprotein cholesterol determination

A spectrophotometric study was carried out which describes the effect of the inclusion of potassium ferrocyanide into an enzyme reagent system as a protective device against the competitive interaction of bilirubin in the indicator reaction. A previously described system for depicting how bilirubin interacts competitively in a modified 4-aminoantipyrene-phenolic peroxidase-peroxide coupled reaction was used as a template to judge the effectiveness of the protective action. The results obtained indicate that the protection is not total, but that the close approximation may make this a worthwhile modification in procedure to consider for the determination of total, free, and/or high density lipoprotein cholesterol. At the same time it might be inferred that the interference of bilirubin can be diminished chemically for other oxidase-peroxidase coupled systems. A search for even better solutions to this interference problem may be possible. For us, this lead has pointed the way in other similar directions.In addition, this study reports on a preliminary investigation as to the site of coupling of phenolic compounds with 4-aminoantipyrene.     

Interaction of hemoglobin in elevated serum total bilirubin determinations

A spectrophotometric study was carried out in an effort to elucidate how hemoglobin in serum interferes in the diazo coupling reaction used for the determination of bilirubin. Because it is difficult to see hemoglobin in neonatal specimens in which bilirubin is elevated without obtaining spectra of the mixtures, this can be a real problem when handling these specimens while using this particular reaction. From the spectrophotometric evidence described here, one can infer that hemoglobin can act in two ways. In one way, the formation of ferriprotoporphyin generates an oxidizing agent which can convert bilirubin to an oxidized product precluding it from taking part in a diazo coupling reaction. In the other way, the same ferriprotoporphyrin seems capable of oxidizing the azobilirubin complex after it has reached equilibrium again causing some lowering of the results obtained. It may be reasonable to assume that the quicker the analytical event can occur, the more accurate the result might be. If the matrix of reaction can be changed, and the reaction of diazo coupling also speeded up as a feature of the change, then it may be possible to avoid the interference from hemoglobin almost entirely. High concentrations of hemoglobin were tested here in an attempt to clearly show how the interference might take place.     

Cholesterol study: Reaction plateau and kinetic determinations

A study has been described of a comparison between reaction plateaus and kinetic modes for measuring cholesterol. A similar investigation of bilirubin reactions under the same procedural conditions was also carried out because this compound is a major interference which is frequently encountered in abnormal serums. The findings indicate that bilirubin is a more sensitive reactant than cholesterol by LB reaction but that its effect as an interference can be lessened by a kinetic approach providing that the effect of H₂O on reaction velocity and molar absorptivity can be minimized. An iron reaction even though more sensitive proved less workable in the kinetic mode owing to reaction velocity and the presence of air bubbles. However, its reaction plateau characteristics were superior to the LB reaction because of a favorable ratio of colors generated for the two reacting constituents. Based on this study, it is predictable that a kinetic approach is quite workable for the LB reaction and could be made more workable for the iron reaction if, in the latter case, conditions perhaps such as temperature and reaction media, could be altered to slow the rate of color formation.     

Spectrophotometric study of bilirubin and hemoglobin interactions in several hydrogen peroxide generating procedures

The interference effects of bilirubin and hemoglobin have been described for the peroxidase-hydrogen peroxide oxidation of a hydrogen donor and the catalase-hydrogen peroxide oxidation of methanol to formaldehyde. A competition between bilirubin and the intended hydrogen donor is shown for the substitute analyte, hydrogen peroxide, with a resultant diminution of color due to the loss of intended reaction. No inhibition of peroxidase action appears to take place; its action when complexed with hydrogen peroxide is directed toward the competing hydrogen donor, bilirubin. The final color measured appeared to be partially compensatory, that is the sum of intended color plus the color of residual bilirubin. The subtraction of a serum blank representing a static system will result in a lowered value and a larger error. Hemoglobin, with its strong Soret band can, if its concentration is excessive, cause a major interference in reactions such as the Hantzsch reaction which result in overlapping bands at the reaction wavelength. Samples which are both hemolyzed and jaundiced would present as formidable blanking problems. Further studies on bilirubin and its glucuronide and their individual effect on the peroxidase-peroxide reaction are presently in progress.     

Bilirubin and hemoglobin interferences in direct colorimetric cholesterol reactions using enzyme reagents

The interferences of bilirubin and hemoglobin were tested in two cholesterol procedures in which enzymes were used as chemical reagents. Both procedures used similar approaches with cholesterol esterase to free cholesterol from its esters and cholesterol oxidase to generate hydrogen peroxide from the total free cholesterol resulting. From that common start, one procedure then used catalase to generate formaldehyde from methanol and the peroxide produced from cholesterol, and the formaldehyde was then reacted with acetylacetone to produce a yellow chromogen, while the other procedure used peroxidase to catalyze a reaction directly between peroxide and 4-aminoantipyrine plus phenol to generate a pink chromogen. Bilirubin and hemoglobin were shown to produce some interference by reacting competitively with peroxide in both systems and by contributing residual absorbance at the wavelengths of measurement of each of the chromogens. Since bilirubin showed a spectral change, static blanking with sample blanks caused overcorrections. However, the elimination of a sample blank for either procedure could result in a favorable compensating error because the residual color of bilirubin could substitute in part at least for the lost reactivity of the peroxide used up in reaction with the bilirubin of the sample.     

A sensitive reaction for dilute cholesterol determinations

A sensitive reaction for the peroxidase-coupled sequence of the determination of a dilute total cholesterol mixture of free and esterified forms is described. Substitution of a chlorinated auxochrome of phenol made water soluble by sulfonation through a synthetic procedure previously described created a severalfold enhancement factor which magnified considerably the sensitivity of one equilibrium reaction over the other. This enabled the determination of dilute cholesterol solutions to be carried out with high absorbance signals across the range of 0.0–10.0 mg/liter (0–0.0259 mmol/liter) of cholesterol, a range which includes and exceeds the normal values one would encounter in dilute solutions such as cerebrospinal fluid. Important potential interference factors including increased protein concentrations and calcium and magnesium were considered and studied. The formidable interacting compound, bilirubin, which is competitive with the 4-aminoantipyrene and sodium 2-hydroxy-3, 5-dichlorobenzenesulfonate mixture in the peroxidase sequence was also studied, its interference characteristics were determined and an alternative methodology suggested for obviating this perturbing effect. It is believed that the simple substitution of the auxochrome derivative is a useful contribution not only here but in studies presently ongoing involving needed sensitivity for cholesterol fractions which are considerably lower than the serum total cholesterol concentrations most commonly determined.     

Interesting interferences in a direct serum creatinine reaction

A spectrophotometric study on a direct picric acid reaction for creatinine in severely jaundiced serums is described. A problem appears to be caused by the oxidation of bilirubin which minimizes rising absorbance when using continuous measurement. Simple examples of interferences with the kinetic mode are shown along with the hitherto unreported interference of the drug, Cephalothin, which also undergoes a picric acid reaction. A procedure in which a delta absorbance is obtained after decolorization of the Jaffé complex by acidification is shown as one available means for obviating the bilirubin effect. However, the theory that Jaffé-reactive interferences do not decolorize with the same acid treatment is not totally applicable when the drug, Cephalothin, is present.     

Study of a fluorometric-enzymatic method for bilirubin based on chemically modified bilirubin-oxidase and multivariate calibration

The chemical derivatization of bilirubin oxidase (BOx) with a fluorescein derivative (FS) yields a chemically modified enzyme (BOx-FS), with excitation and emission maxima at 487 and 520 nm, respectively. During the oxygen oxidation reaction of bilirubins, in the presence of the modified enzyme, the change in the fluorescence of the modified enzyme depends on the concentration and type of bilirubin. This effect can be used for analytical purposes. Firstly, a theoretical-experimental study of the analytical system was carried out. The mechanism responsible for the fluorescence variation was clarified, a mathematical model developed and the variables affecting the fluorescence changes optimized. The concentration ranges in which the model can be applied (up to 12 mg bilirubin l(-1)), and the precision of the measurement (about 4%) were established for the three bilirubins. The application of the methodology to the simultaneous determination of direct and total bilirubins were studied by applying multivariate calibration methods to the whole kinetic profiles. A reduced calibration matrix (derived from a 5(3) base matrix) is proposed for calibration and different numerical methods were tested: Principal Components Regression (PCR), Partial Least Squares Regression (PLS) and Artificial Neural Networks (ANN). The simultaneous determination of direct and total bilirubin (average validation errors of about 9 and 10%, respectively) can be carried out from a single run. Furthermore, a semi-quantitative speciation of the three bilirubins (free, conjugated and albumin-bonded bilirubin) may be simultaneously obtained.     

Bilirubin degradation by uncoupled cytochrome P450. Comparison with a chemical oxidation system and characterization of the products by high-performance liquid chromatography/electrospray ionization mass spectrometry

Bilirubin is a protective antioxidant; however, when its conjugation and excretion are impaired, as in neonatal and hereditary jaundice, bilirubin accumulates and may cause severe neurotoxicity. Degradation of bilirubin takes place (a) on interaction with oxidative free radicals and (b) when cytochrome P450 (CYP) enzymes are uncoupled by polyhalogenated substrate analogues. The products of pathways (a) and (b) above have now been characterized by high-performance liquid chromatography/electrospray ionization mass spectrometry (HPLC/ESI-MS) and the mechanisms of fragmentation in part clarified. Oxidation of bilirubin by uncoupled CYP1A5 and by a Fe-EDTA/H2O2 system produced both biliverdin and an identical profile of dipyrrolic fragments, as detected by positive ESI-MS. A similar profile of oxidation products was found from mesobilirubin, all showing the expected increase in mass, thus providing direct evidence for fragmentation at the central methene bridge of the tetrapyrroles. Two degradation products, also detected by negative ESI-MS, were characterized as dipyrroles retaining the central bridge carbon, with one or two oxygen atom(s) bound (probably as the aldehyde and hydroperoxide derivatives). Ions compatible with propentdyopents and bilifuscins were also detected, but here the assignment was less certain. It is concluded that the first step in the oxidation of bilirubin may be hydrogen abstraction at the central methene bridge. This is followed either by loss of another hydrogen to give biliverdin, or by oxygen binding and fragmentation. Fe-EDTA/H2O2 and uncoupled CYP(Fe=O) may both initiate the reaction, the latter in an attempt to reduce the ferryl oxygen to water. These studies shed light on the CYP uncoupling mechanism and are of potential significance for the therapy of severe jaundice.     

Quantum dynamic calculation for gas-phase nucleophilic substitution (SN2) reactions

According to experimental data, gas-phase S_N2 reactions have low efficiency characterized by the ratio k(T)/k_c, where k_c is the collision rate constant. The energy profile of the reaction pathway is a double-well curve, and the top of the central barrier may be located either above or below the energy level of the reagents. Nonempirical calculations of the potential surfaces for S_N2 reactions have shown that their dynamics may be described in the collinear collision approximation. The probability of a reactive collision is determined by the interference of direct processes and processes of decay of quasibound states arising upon formation of the pre-reaction complex. The reflection probability in the direct process is determined by the degree of excitation of the reactive system in channels that are closed at the central barrier point, which depends on the curvature of the reaction pathway and the depth of the potential well for the pre-reaction complex. Depending on these factors, the direct process may correspond either to total transmission or to total reflection (low reaction efficiency). In the first case, interference of direct processes with decay processes decreases the reaction probability; in the second case, such interference increases the reaction probability. Calculations of the thermal reaction rate constants k(T) have shown good agreement with experimental data.Translated from Teoreticheskaya i Éksperimental'naya Khimiya, Vol. 22, No. 4, pp. 435–443, July–August, 1986.The author acknowledges M. V. Bazilevskii, A. M. Berezhkovskii, V. A. Tikhomirov, and G. É. Chudinov for useful discussions.     

氢醌对胆红素氧化过程的促进作用及其分析意义

研究了氢醌与胆红素的作用过程，发现氢醌在碱性条件下可促使胆红素氧化，且胆红素被氧化成胆绿素，此反应过程被证实为自由基反应，考察了介质条件，抗氧化剂等反应过程的影响及其它酚类物质对胆红素氧化的作用情况。     

Novel synthesis of 5-iodo-1,2,3-triazoles using an aqueous iodination system under air

A novel aqueous iodination system was developed for the synthesis of 5-iodo-1,2,3-triazoles under air. This reaction system has high efficiency and excellent chemo-selectivity with wide functional group tolerance. In addition, this method can be utilized for the modification of biomolecules such as riboses and nucleosides, and for the double labeling of biomolecules when coupled with other reaction types such as the Sonogashira reaction.     

Spectrophotometric study of influences on the direct ferric perchlorate method for the determination of serum cholesterol

A spectrophotometric study of several influences on a direct colorimetric determination of serum cholesterol has been described. Interferences of in vitro and in vivo types were considered, and it was found that certain compounds such as bromide, uracils, and bilirubin could exert both positive and negative interfering influences on the reaction of ferric perchlorate with cholesterol in an ethyl acetate-ethanol-sulfuric acid medium. Some interferences such as bilirubin are noncompeting side reactions which are absolute in their interference capabilities, while others such as the uracils and bromide have an entirely different influence. In the latter circumstance, the fine structures of spectra are altered by a nonadditive phenomenon of the reactant and hyperchromic (bromide), and hypochromic (uracils) effects take place which appear to result in relative rather than absolute errors.     

Chromatographic Determination of Total Selenium in Biofortified Allium sp. following Piazselenol Formation and Micro-Solid-Phase Extraction

Herein, a method based on selective piazselenol formation is applied for total selenium determination in biofortified Allium species. Piazselenol is formed by reacting Se(IV) with an aromatic diamine, namely 4-nitro-1,2-phenylenediamine, in acidic medium. Samples were digested in a nitric acid/hydrogen peroxide open system, followed by selenate reduction in hydrochloric acid. Reaction conditions were optimized in terms of pH, temperature, reaction time, and other auxiliary reagents for interference removal, namely, EDTA and hydroxylamine. For the extraction of the selectively formed 4-nitro-piazselenol, micro-solid-phase extraction (μSPE) was applied, and the analysis and detection of the corresponding complex was performed by HPLC coupled with DAD. An external standard calibration curve was developed (R² = 0.9994) with good sensitivity, and was used to calculate the total selenium content from several Allium plants material, with good intermediate precision (RSD% < 16%). The accuracy of the method was evaluated using both, a comparison with an accepted reference method from our previously published data, as well as three certified reference material with recoveries between 84–126%. The limit of detection was determined to be 0.35 μg/g (in solids) and 1.1 μg/L (in solution), while the limit of quantification was 1.07 μg/g and 3.4 μg/L (in solution). Using the proposed method, selenium content can be quickly and accurately determined in several types of samples. In addition, this study present experimental conditions for overcoming the interferences that might be encountered in selenium determination using piazselenol.     

Electrochemical Hydride Generation:A Novel Sample Introduction Method for the Non-dispersive Atomic Fluorescence Spectrometry

Production of volatile covalent hydrides of environmentally important elements (As、Se、Bi、Ge、Sn、Sb and Pb) by reaction with sodium (potassium) tetrahydroborate (NaBH₄) in acidic media for determination by atomic absorption has been widely used in routine analysis. However, this technique has several disadvantages. NaBH₄ is a potential source of contamination, its aqueous solution is unstable. As an alternative to chemical hydride generation, hydride generation by electrochemical reduction (EcHG) has been reported in recent years by many authors^[1-3]. Compared with NaBH₄ hydride generation system, the EcHG with subsequent detection by atomic absorption spectrometry offers the high sensitivity, low detection limits, and the absence of interference from the transition metals. However, the hydrogen produced by EcHG reduced greatly, this led to the interference caused by hydride forming elements was serious due to the lack of free hydrogen (H·) radical in the quartz tube during the atomization. EcHG coupled with atomic fluorescence spectrometry system uses Ar-H2 mini-flame as an atomizer, in which the gaseous phase interference can be expected eliminated. In addition, non-dispersive atomic fluorescence spectrometry (NDAFS) system with a quite single structure, is a very sensitive and feasible method for the measurement of elements that form volatile hydrides. By combining with electrochemical hydride generation as a sample introduction technique, it is possible to develop a potential interface for chromatography or capillary electrophoresis in speciation analysis.     

Development of a novel analytical method for determination of chondroitin sulfate using an in-capillary enzyme reaction

A novel analytical method for determination of total amount of chondroitin sulfate (CS) based on its conversion to desulfated chondro-disaccharide via an enzyme-catalyzed reaction, was developed. Using the in-capillary enzyme reaction, the method was also applied to the successful construction of an on-line analytical system. Within this system, electrophoretic migration was used to mix zones containing the enzyme mixture (chondroitinase ABC, chondro-4-sulfatase, chondro-6-sulfatase and 2-o-sulfatase) and the substrate (CS). The reaction was then allowed to proceed in the presence of a weak electric field and, finally, the product (desulfated chondro-disaccharide) of enzyme reaction migrated to the detector under the influence of an applied electric field. A polyvinyl alcohol-coated capillary was used to reduce protein adsorption. Desulfated chondro-disaccharide was successfully migrated toward the anode in 10 mM Tris-acetate buffer (pH 7.0) under reversed polarity and detected at 232 nm. The established method was validated and demonstrated to be applicable in the determination of total amount of CS in a commercial ophthalmic solution. No interference from the formulation excipients was observed. Good linearity was obtained, with correlation coefficients above 0.999. Recoveries and precisions ranged from 100.0 to 100.5%, and from 0.2 to 0.6% of the relative standard deviation, respectively. Good agreement was obtained between the established method and traditional photometric method based on carbazole reaction. In this study, application of the method to disaccharide compositional analysis was also performed.     

In vitro determination of diamine oxidase activity in food matrices by an enzymatic assay coupled to UHPLC-FL

Intestinal diamine oxidase (DAO) acts as a protective barrier against exogenous histamine. A deficit of DAO activity can lead to the appearance of histamine intolerance, a clinical condition that may be treated by a low-histamine diet and oral DAO supplementation to enhance intestinal histamine degradation. As sources of DAO, porcine kidneys and certain legume seedlings are suitable components for the formulation of a DAO supplement. The aim of this work was to develop a rapid and reliable methodology for the in vitro determination of DAO activity in food matrices based on an enzymatic assay coupled to UHPLC-FL. The proposed method showed a satisfactory linearity and sensitivity and provided a relative standard deviation lower than 3%, guaranteeing method precision, and a mean recovery greater than 99% both for lyophilized pea sprouts and porcine kidney protein extracts. A high specificity is a key attribute of this method due to the use of histamine as the reaction substrate and the direct quantification of its degradation. Moreover, the lack of interference of catalase and hydrogen peroxide is another advantage in comparison with previously published methods. Lyophilized pea sprouts showed the greatest histamine-degrading activity (0.40 ± 0.01 mU/mg), followed by porcine kidney protein extracts (0.23 ± 0.01 mU/mg) and commercial DAO supplements (0.09 ± 0.06 mU/mg). This technique could be used as a tool to validate the DAO activity of food matrices of potential interest for the treatment of histamine intolerance.

     

Evaluation of on-line desalter-inductively coupled plasma-mass spectrometry system for determination of Cr(III), Cr(VI), and total chromium concentrations in natural water and urine samples

We have developed a simple and convenient method for the determination of Cr(III), Cr(VI), and the total chromium concentrations in natural water and urine samples that use a flow injection on-line desalter-inductively coupled plasma-mass spectrometry system. When using aqueous ammonium chloride (pH 8) as the stripping solution, the severe interference from sodium in the matrix can be eliminated prior to inductively coupled plasma-mass spectrometry measurement, and the Cr(VI) level can be determined directly. To determine the total concentration of Cr in natural water and urine samples, we used H₂O₂ or HNO₃ to decompose the organic matter and convert all chromium species into the Cr(VI) oxidation state. To overcome the spectral interference caused by the matrix chloride ion in the resulting solutions, we employed cool plasma to successfully suppress chloride-based molecular ion interference during the inductively coupled plasma-mass spectrometry measurement. By significantly eliminating interference from the cationic and anionic components in the matrices prior to the inductively coupled plasma-mass spectrometry measurement, we found that the detection limit reached 0.18 μg L^− 1 (based on 3 sigma). We validated this method through the analysis of the total chromium content in two reference materials (NIST 1643c and 2670E) and through measuring the recovery in spiked samples.     

Kinetics of the formation of biliverdin during the photochemical oxidation of bilirubin monitored by column liquid chromatography

In the photochemical oxidation of bilirubin, biliverdin is formed as the primary product and is further degraded. This photooxidation is especially significant in the presence of riboflavin. Column liquid chromatography was used to monitor the kinetics of this reaction. The biliverdin concentration amounts to a maximum of ca. 38% of the total loss of bilirubin in experiments in vitro. It is probable that this mechanism is also operative during phototherapy. The formation of a product of the photooxidation of biliverdin that has not yet been identified has been observed; the product behaves as a dimer. A method for the determination of biliverdin in the blood of newborn infants has been developed. It has been found that the biliverdin content increases during hyperbilirubinaemia.     

Sensitive o-toluidine reagent for glucose assay at moderately elevated temperatures

A reagent is developed for increasing the sensitivity of the direct o-toluidine procedure for glucose so that the reaction may be carried out at temperatures as low as 37 °C.The sensitivity is achieved by eliminating all water from the system, except for a minute percentage introduced with the samples and by using a high boric acid concentration. Under these conditions protein does not precipitate and lipids remain dissolved. At 55 °C, interference from bilirubin at a concentration of 20 mg/100 ml is 3.5%.Blood may be collected with sodium fluoride since it does not interfere in the procedure. The rate of color development is slower for aqueous standards than it is for serum at the lower temperatures. This may be corrected by preparing the standards in a 6% albumin solution.Results obtained by applying the reagent with the continuous flow system of analysis (Technicon), and with the discrete sample analyzers, Beckman DSA, Lars Jungberg Autolab (Sweden), Robot Chemist, and RaBA (Japan) are reported.The reagent is useful as a spray reagent for sugar identification on thin-layer chromatography plates. Heating at different temperatures permits the differentiation between certain sugars with similar R_f values.