Acidic Phosphate Buffer Research Articles

Liquid chromatographic method for simultaneous determination of mycophenolic acid and its phenol- and acylglucuronide metabolites in plasma

A simple, sensitive and reproducible HPLC method is presented for the simultaneous determination of mycophenolic acid (MPA) and its metabolites phenolic MPA-glucuronide (MPAG) and acyl glucuronide (AcMPAG) in human plasma. Sample purification requires protein precipitation with 0.1 M phosphoric acid/acetonitrile in the presence of Epilan D as an internal standard (IS). Separation was performed by reversed-phase HPLC, using a Zorbax SB-C18 column, 32% acetonitrile and a 40 mM phosphoric acid buffer at pH 3.0 as mobile phase; column temperature was 50 °C, flow rate 1.4 ml/min, and measurement by UV detection was at 215 nm (run time 12 min). The method requires only 50 μl plasma. Detection limits were 0.1 μg/ml for MPA and AcMPAG, and 2.0 μg/ml for MPAG, respectively. Mean absolute recovery of all three analytes was >95%. This analytical method for the determination of MPA and its metabolites is a reliable and convenient procedure that meets the criteria for application in routine clinical drug monitoring and pharmacokinetic studies.

Direct tris(2,2'-bipyridyl)ruthenium (II) electrochemiluminescence detection of polyamines separated by capillary electrophoresis.

Capillary electrophoresis (CE) with tris(2,2'-bipyridyl) ruthenium (II) (Ru(bpy)3(2+)) electrochemiluminescence (ECL) detection technique was developed for the analysis of four polyamines (putrescine (Put), cadaverine (Cad), spermidine (Spd), and spermine (Spm)) analysis. The four polyamines contain different amine groups, which have different ECL activity. There are several parameters which influence the resolution and ECL peak intensities, including the buffer pH and concentrations, separation voltage, sample injection, electrode materials, and Ru(bpy)3(2+) concentrations. Polyamines are separated by capillary zone electrophoresis in an uncoated fused-silica capillary (50 cmx25 micro m (ID) filled with acidic phosphate buffer (200 mmol/L phosphate, pH 2.0) - 1mol/L phosphoric acid (9:1 v/v) and a separation voltage of 5 kV (25 micro A), with end-column Ru(bpy)3(2+) ECL detection. A 5 mmol/L Ru(bpy)3(2+) solution plus 200 mmol/L phosphate buffer (pH 11.0) is added into the reagent reservoir. The calibration curve is linear over a concentration range of two or three orders of magnitude for the polyamines. The analysis time is less than 25 min. Detection limits for Put and Cad are 1.9x10(-7) mol/L and 7.6x10(-9) mol/L for Spd and Spm, respectively. Intraday and interday relative standard deviations of ECL peak intensities are less than 8%. The main advantages of this CE-ECL detection technique for polyamines analysis presented herein are the omission of chemical derivatization of the analytes and the high selectivity.

Model studies on the stability of folic acid and 5-methyltetrahydrofolic acid degradation during thermal treatment in combination with high hydrostatic pressure.

Stability of folic acid and 5-methyltetrahydrofolic acid in phosphate buffer (0.2 M; pH 7) toward thermal (above 65 degrees C) and combined high pressure (up to 800 MPa)/thermal (20 up to 65 degrees C) treatments was studied on a kinetic basis. Residual folate concentration after thermal and high pressure/thermal treatments was measured using reverse phase liquid chromatography. The degradation of both folates followed first-order reaction kinetics. At ambient pressure, the estimated Arrhenius activation energy (E(a)) values of folic acid and 5-methyltetrahydrofolic acid thermal degradation were 51.66 and 79.98 kJ mol(-1), respectively. It was noticed that the stability of folic acid toward thermal and combined high pressure thermal treatments was much higher than 5-methyltetrahydrofolic acid. High-pressure treatments at room temperature or higher (up to 60 degrees C) had no or little effect on folic acid. In the whole P/T area studied, the rate constant of 5-methyltetrahydrofolic acid degradation was enhanced by increasing pressure, and a remarkable synergistic effect of pressure and temperature on 5-methyltetrahydrofolic acid degradation occurred at temperatures above 40 degrees C. A model to describe the combined pressure and temperature effect on the 5-methyltetrahydrofolic acid degradation rate constant is presented.

Assignment of Soret MLCT band of reduced form of copper binuclear cluster in cytochrome c oxidase film

Low concentration of dithionite results in the reduction of Cu-Cu binuclear and heme a active sites of the cytochrome c oxidase thin solid film immersed in the acidic phosphate buffer, but Fe-Cu binuclear center keeps in the oxidation state. It manifests as a negative peak at 426 nm and a positive one at ∼408 nm in the difference spectra induced by dithionite. The former implies decrease of the oxidized form of heme a center, that is, Fe a 3+ →Fe a 2+ · And the latter results from the contribution of metal-ligand charge transfer (MLCT) transition in the reduced binuclear Cu-Cu cluster, rather than from that of heme a center. This stronger Soret MLCT band must be helpful to overcoming the difficulty in distinguishing the weaker copper sign from the stronger one of iron when studying copper-iron protein.

Kinetics and Mechanisms of the Reduction of Chromium(VI) by 2-Mercaptoethanesulfonic Acid in Aqueous Solution: Difference in the Mechanistic Process of Reduction with Noncarboxylate Thiols

The kinetics of the reaction of chromium(VI) with 2-mercaptoethanesulfonic acid (MESA) has been studied spectrophotometrically at wavelength, 435 nm, and ionic strength, I, of 0.50 mol dm-3 (NaClO4) over the ranges: 16.0 < θ < 29.5 °C, 2.6 < pH < 7.1 (citric acid-phosphate buffer), [Cr(VI)]T = 2.0 χ 10-4 mol dm-3, 1.0 χ 10-2 < [MESA]T < 4.0 χ 10-2 mol dm-3. The reaction occurs giving clear indication of the formation of an intermediate, which is assumed to be a typical Cr(VI)-thioester. The decay of the thioester then occurs via a series of electron transfer reactions to give Cr(III). The formation and decay rates of this intermediate have been investigated kinetically and a detailed mechanism for the overall reaction proposed. Experimental data were fitted to rate equations derived from this mechanism. The specific rate constants and corresponding activation parameters were also deduced. The results show that MESA without carboxylate groups, react differently when compared to other thiols having such groups present. This confirms the participating role of carboxylate groups in the formation of the Cr(VI)-thioester. The catalytic effect of added Zn2+ was investigated and its overall role in the reaction of Cr(VI) with MESA was explained.

Analysis of chitin oligosaccharides by capillary electrophoresis with laser-induced fluorescence.

A method, using capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection for analyzing chitin oligosaccharides is described. Chitin oligosaccharides were derivatized with 9-aminopyrene-1,4,6-trisulfonate (APTS) via reductive amination at 37 degrees C for 16 h (optimized conditions). The APTS-chitin oligosaccharides were analyzed using either an acidic citric acid-phosphate buffer or an alkaline borate buffer. The effects of buffer types, buffer pH values, and buffer concentrations on the separation were examined. The analytes were successfully separated by using a pH 4.6 citric acid-phosphate within 19 min. The APTS-derivatized chitin monosaccharide (D-glucosamine) migrated first. The analytes were also completely separated by using a pH 9.0 borate buffer within 24 min. Moreover, the specificity of enzyme digestion on chitin polysaccharides using the optimized APTS labeling procedure and the CE-LIF method was demonstrated.

Screening approach for chiral separation of pharmaceuticals: Part II. Reversed-phase liquid chromatography

A screening strategy for the rapid separation of drug enantiomers by reversed-phase liquid chromatography was developed using three cellulose/amylose stationary phases. The key point to achieve enantioselectivity is the control of the compound ionisation. Only two mobile phases, i.e. an acidic phosphate buffer (pH 2.0) containing a chaotropic salt (KPF 6) and a borate buffer (pH 9.0) mixed with acetonitrile, are used in the proposed strategy. This strategy was successfully applied to a set of 37 diverse chiral pharmaceuticals. Satisfactory enantioselectivity was achieved for 89% of them.

Kinetic Study on the Removal of Organic Pollutants by an Electrochemical Oxidation Process

This paper deals with the kinetics of electrochemical oxidation of 2,6-dichlorophenol and p-hydroxybenzoic acid in phosphate buffer at pH = 7. On the basis of experimental results, a mathematical model was formulated which effectively describes the trend in time of the concentration of the organic compounds involved in the electrochemical oxidation process. The proposed kinetic model is based on a reaction mechanism which takes into account both the direct oxidation of the organic substrate at the electrode surface and the indirect oxidation, in the liquid phase, by means of electrogenerated oxidizing agents. The model well interprets the complex effect on the removal efficiency of such operating parameters as the current density, the linear velocity of the electrolyte, and the ratio between the anode surface and solution volume.

Cracking of cherry tomatoes in solution

Tomato fruit cracking occurs both during ripening and after harvest. Cracked fruits cannot be marketed and the cracks form sites for fungal penetration and infection. An assay based on immersion of the fruit in water was developed to study factors involved in fruit cracking. Adding calcium to the water reduced cracking whereas chelating agents increased cracking. Mineral analysis of the fruit following calcium treatment demonstrated an increase in bound calcium, while CDTA reduced the amount of soluble calcium. Decrease in fruit weight associated with water loss during storage was correlated with a decrease in the cracking potential of the fruit. Conversely, ripening during storage resulted in an increase in the cracking potential. Immersion of the fruit in acidic phosphate or citrate buffers promoted cracking whereas neutral or basic buffers prevented cracking. The cracking potential of cherry tomatoes was high after morning harvest, and it declined at noon and was low after evening harvest. It is anticipated that this study will assist to evaluate positive or negative practices which may influence cracking of cherry tomatoes after harvest.

Simple method for determination of paraquat in plasma and serum of human patients by high-performance liquid chromatography

A simple and fast HPLC system is presented for quantifying paraquat in human plasma and serum using 1,1′-diethyl-4,4′-bipyridyldiylium (diethyl paraquat) as an internal standard. An octadecyl–silica column is used with an eluent of 10% acetonitrile (v/v) containing sodium 1-octanesulphonic acid (3.0 m M) and a diethylamine–orthophosphoric acid buffer (pH 3). Unlike with other techniques, sample treatment requires only the precipitation of protein contents by 6% perchloric acid (v/v) in methanol. The method has a limit of detection of 0.1 μg/ml and is linear up to 10 μg/ml. The serum of four patients and the plasma of one patient with paraquat intoxication’s were analysed and positive identification and quantification was readily achieved. One of those patients survived, partially given the rapid disclosure of his levels of paraquat. Therefore, this method is suitable for quantification of paraquat in toxicological samples. It may be used as a prognostic tool in critical case detoxification and to quickly identify potentially salvageable patients for enrolment in new hemofiltration studies.

A switch in the electron transfer from heme a to binuclear centre of cytochrome c oxidase

New experimental evidence that a switch controls the reduction of the heme a3-CuB binuclear centre has been observed in the N2-dried thin film of purified cytochrome oxidase. When immersing the enzyme film into the acid phosphate buffer with extremely low concentration of dithionite, a spectrum was given to show a reduction of heme a with no electrons resting on CuA. By increasing dithionite, electrons could be accumulated gradually on CuA, but the binuclear centre still remains in the oxidized state. When the accumulation of electrons on CuA and/or heme a exceeded a threshold, a turnover of reduction of the binuclear centre and oxidation of heme a occurred abruptly. This switch-like action is pH-dependent.

Determination of trace lead, cadmium and mercury by on-line column enrichment followed by RP-HPLC as metal-tetra-(4-bromophenyl)-porphyrin chelates

This paper reports the utilization of tetra-(4-bromophenyl)-porphyrin (T 4BPP) as a chelating reagent using Waters Xterra™ RP 18 column for the on-line column enrichment and the separation of trace lead, cadmium and mercury ions by reversed-phase high-performance liquid chromatography (RP-HPLC) with photodiode array detector. When the Hg–T 4BPP, Pb–T 4BPP and Cd–T 4BPP chelates were injected into the injector and sent to the enrichment column with 0.05 mol l −1 of pH 10.0 pyrrolidine–phosphoric acid buffer solution (containing 10% of tetrahydrofuran (THF)) as mobile phase. The chelates were retained on the top of the enrichment column. After the enrichment is finished, by switching the valve of six-ports switching valve, the retained metal–T 4BPP chelates will be eluted by mobile phase in reverse direction and will travel towards analytical column. With THF (containing 0.05 mol l −1, pH 10.0 pyrrolidine–phosphoric acid buffer salt) and 0.05 mol l −1, pH 10.0 pyrrolidine–phosphoric acid buffer solution (containinging 10% THF) gradient elution as mobile phase, the chelates separation on the analytical column was satisfactory. The linearity ranges are 0.01–120 μg l −1 for each metal ion. The detection limits (S/N=3) of lead, cadmium and mercury are 1.0, 0.5 and 1.0 ng l −1, respectively. This method can be applied to the determination (μg l −1) level of lead, cadmium and mercury in drinking water with satisfactory results.

Profiling and screening analysis of 27 aromatic amino acids by capillary electrophoresis in dual modes.

An efficient capillary electrophoretic (CE) profiling and screening system based on dual modes of capillary zone electrophoresis (CZE) and micellar electrokinetic capillary chromatography (MEKC) was developed for the simultaneous determination of 23 nonprotein amino acids (NPAAs) and 4 protein amino acids with aromatic moiety. It involves separation by an uncoated fused-silica capillary under phosphoric acid buffer in CZE mode and by another uncoated fused-silica capillary under neutral sodium dihydrogen phosphate buffer containing sodium dodecyl sulfate in MEKC mode. Migration orders of the amino acids studied on the two separation modes under each optimum condition were very different. The repeatability of migration times measured by the CZE and MEKC was found to be better than 4.8 and 3.4%, respectively, thereby enabling to cross-check the identification of each amino acid. The method linearity and limit of detection of the CZE for each amino acid were found to be adequate for the assay of aromatic amino acids. When the present CE profiling and screening analysis in dual modes was applied to plant seeds, NPAAs such as mimosine from Mimosa pudica Linné, and 2-phenylglycine from Lindera erythrocarpa Makino were positively detected along with tryptophan, phenylalanine and tyrosine.

Fundamental studies on selectivity in 3-phase liquid-phase microextraction (LPME) of basic drugs

Recently, we introduced an inexpensive and disposable hollow fibre based device for liquid-phase microextraction (LPME) where ionic analytes typically are extracted from 1–4 mL aqueous samples (such as plasma and urine) through an organic solvent immobilized in the pores of a polypropylene hollow fibre and into a 10–25 μL volume of acceptor phase present inside the hollow fibre. Because of the substantial volume ratio of the sample relative to the acceptor phase, ionic analytes may be preconcentrated considerably during LPME (typical preconcentration factors of 50 to 150). In addition, during LPME from biological samples such as plasma and urine, the majority of matrix components are not extracted into the acceptor phase resulting in excellent sample clean-up. For the extraction of basic drugs, dilute solutions of HCl or acidic phosphate buffers have been utilized as acceptor phase without further optimization. In the present work therefore, systematic studies have been performed to explore new acidic acceptor phase possibilities in combination with dihexyl ether as a standard organic solvent in the pores of the hollow fibre. In general, 10 mM solutions of HCl, H2SO4, and phosphate pH 3.3 were found to provide the highest extraction recoveries. pH was found to play a major role in adjusting the selectivity of LPME, whereas minor selectivity alterations were observed on changing the type of acid in the acceptor phase. For weakly basic drugs, an increased level of HCl in the acceptor phase may provide higher recovery, and for certain drugs, addition of ethanol to the acceptor phase may improve the extractability.

Comparison of conventional microparticulate and a monolithic reversed-phase column for high-efficiency fast liquid chromatography of basic compounds

The effect of mobile phase flow on column efficiency for a neutral compound together with weak and strong bases was compared for conventional microparticulate (3/3.5 μm and 5 μm) silica RP columns and a monolithic silica RP. For benzene, the minimum plate height (Hmin) at optimum flow-rate (uopt) for weak bases was similar for the 5 μm and the monolith phases. However, the monolith generated much flatter Van Deemter curves, such that at high flow-rate (5 ml min−1) the plate height was nearly 3.5 times lower on the monolith. For weak bases analysed in unbuffered mobile phases, and stronger bases with acid phosphate buffer, increased tailing was obtained on the monolith compared with the conventional phases. Nevertheless, Van Deemter plots on the monolith still showed some advantages over particulate phases, even when asymmetry factor was included in the calculation of the plate height. However, at pH 7 considerable tailing of strong bases was found using the monolith; it is not clear whether this results from unique features of the monolith structure, or whether it is due merely to usual problems of silica activity. Van Deemter plots for conventional phases may be improved considerably by operating the column at elevated temperatures. At pH 3, these improvements are influenced to a considerable extent by increases in Dm, as shown by measurements of Dm using the Taylor–Aris procedure. However, at pH 7.0, improvements are much too substantial to be explainable wholly on this basis.

Mechanism of reduction of chromium(VI) ion by 2-mercaptosuccinic acid in aqueous solution

The kinetics of the reduction of the chromium(VI) ion by 2-mercaptosuccinic acid (thiomalic acid) were studied by rapid scanning stopped flow spectrophotometry. The conditions used were [Cr(VI)] T=0.20 mM, [MSA] T=5–90 mM, 3.0≤pH≤5.6 in citric acid–phosphate buffer, or 3.3≤pH≤5.4 in 0.40 M acetic acid–acetate buffer, 20.0≤ T≤35.0 °C at I=0.50 M (NaClO 4). Spectrophotometric titration at 350 nm indicates the stoichiometry of the reaction to be 1:3. The kinetics of both formation and decay of the intermediate chromium(VI) thioester were followed at λ max=425 nm and rate expressions, specific rate constants and corresponding activation parameters were derived from the proposed mechanism. The acetic acid–acetate buffer was found to catalyze the formation but not the decay rate of the intermediate. The citric acid–phosphate buffer and dissolved oxygen did not have any significant effect on the reaction rates. The justification of the mechanism was discussed in terms of standard biological conditions.

Use of capillary electrophoresis to monitor wheat maturation

The maturation of wheat varieties with different harvest times has been examined by high-performance capillary electrophoresis. The unique proteins of the albumin, gliadin and glutenin fractions of Hungarian winter wheat cultivars Bankuti 1201 (early harvest time), Martonvasari 23 (medium harvest time), and Martonvasari 15 (semi-late harvest time) were analysed. An acidic phosphate buffer containing a polymeric additive and organic modifiers was used in capillary zone electrophoresis mode. Formation of albumin followed the same time scale, and the patterns were quite similar, for all three cultivars. For gliadins and glutenins the time scale and patterns were different and some correlation was observed between harvest time and gliadin formation.

Optical Method for the in-situ Measurement of the pH-value During High Pressure Treatment of Foods

This contribution presents a novel optical method to determine the pH-value during the high pressure treatment of foods. Up to date an appropriate online measurement device does not exist. The present study makes use of a multivariate approach, using the optical properties of pH indicators. An indicator mix of 16 different pH-sensitive substances is applied. The calibration of the system is performed taking five different buffer substances (TRIS, CAPS, citric, phosphoric and acetic acid buffers), applying thermodynamical considerations, to calculate the pH-value at high pressure. The corresponding spectra of the indicator mix is measured and fed to a multivariate data model together with calculated pH values. Different chemometric models have been constructed to estimate the pH value based upon the spectrum of the indicator mix. The calibration device covers a pH range from 2 to 8. After calibration it was possible to determine the pH value at pressures up to 450 MPa with an accuracy of 0.02 pH units in 9 different test media.

Chelating Activity of Advanced Glycation End-product Inhibitors

The advanced glycation end-product (AGE) hypothesis proposes that accelerated chemical modification of proteins by glucose during hyperglycemia contributes to the pathogenesis of diabetic complications. The two most commonly measured AGEs, N(epsilon)-(carboxymethyl)lysine and pentosidine, are glycoxidation products, formed from glucose by sequential glycation and autoxidation reactions. Although several compounds have been developed as AGE inhibitors and are being tested in animal models of diabetes and in clinical trials, the mechanism of action of these inhibitors is poorly understood. In general, they are thought to function as nucleophilic traps for reactive carbonyl intermediates in the formation of AGEs; however alternative mechanisms of actions, such as chelation, have not been rigorously examined. To distinguish between the carbonyl trapping and antioxidant activity of AGE inhibitors, we have measured the chelating activity of the inhibitors by determining the concentration required for 50% inhibition of the rate of copper-catalyzed autoxidation of ascorbic acid in phosphate buffer. All AGE inhibitors studied were chelators of copper, as measured by inhibition of metal-catalyzed autoxidation of ascorbate. Apparent binding constants for copper ranged from approximately 2 mm for aminoguanidine and pyridoxamine, to 10-100 microm for carnosine, phenazinediamine, OPB-9195 and tenilsetam. The AGE-breakers, phenacylthiazolium and phenacyldimethylthiazolium bromide, and their hydrolysis products, were among the most potent inhibitors of ascorbate oxidation. We conclude that, at millimolar concentrations of AGE inhibitors used in many in vitro studies, inhibition of AGE formation results primarily from the chelating or antioxidant activity of the AGE inhibitors, rather than their carbonyl trapping activity. Further, at therapeutic concentrations, the chelating activity of AGE inhibitors and AGE-breakers may contribute to their inhibition of AGE formation and protection against development of diabetic complications.

Synthesis of pH-Degradable Nonionic Surfactants and Their Applications in Microemulsions

An oil-soluble pH-degradable nonionic surfactant with poly(ethylene glycol) monomethyl ether as the hydrophile and a cyclic ketal as the hydrophobe was synthesized for use in microemulsion-based protein extraction. The surfactant solubilized water in isooctane. Dynamic light-scattering measurements showed formation of fairly monodisperse water-in-oil microemulsions of radii 4−6 nm, with very strong intermicellar attractive interactions. The ternary phase diagram for the system surfactant/water/isooctane at 23 °C consists of one-, two-, and three-phase regions as well as gel-like phases. The well-known “fish” pattern occurred for the phase diagram of temperature vs surfactant concentration at a fixed ratio of water-to-oil (1/1 g/g). The surfactant remained stable at neutral pH for several days but degraded rapidly when a mildly acidic phosphate buffer (pH = 5) was encapsulated in the water-in-oil microemulsion solution. Degradation occurred more rapidly when the microemulsion solution was brought in contact with an equal volume of pH 5 buffer solution in the presence of agitation. The encapsulation of protein (lysozyme) and its subsequent release upon contact with pH 5 buffer were observed, with 70% recovery of lysozyme mass in 0.5 h and 90% recovery in 1.0 h. The specific activity of the recovered lysozyme was within 90.4 ± 4.0% of the value for untreated lysozyme.