Improved detection of multi-phosphorylated peptides in the presence of phosphoric acid in liquid chromatography/mass spectrometry

In contrast to lower phosphorylation states (e.g. the tryptic monophosphopeptide FQpSEEQQQTEDELQDK from bovine beta-casein), the specific detection of multi-phosphorylated peptides (e.g. the tetraphosphopeptide RELEELNVPGEIVEpSLpSpSpSEESITR from tryptic digestion of bovine beta-casein) has often been problematic for liquid chromatographic mass spectrometric (LC/MS) analysis owing to their high affinity for adsorption to exposed surfaces. We observed an enhancement in the overall detection of phosphopeptides on addition of phosphoric acid (0.1-1.0%) to the sample solution; a 10-fold increase in sensitivity was determined for the detection of two tryptic phosphopeptides and also a significant improvement in the detection of the tetraphosphopeptide. Using capillary LC with ion trap tandem MS for detection and identification, the achievable detection limits were 50 fmol and 50 pmol for the monophosphopeptide and the tetraphosphopeptide, respectively. Phosphoric acid is believed to act as a blocking agent to available silanol groups on both the silica capillary surface and the C(18)-bonded stationary phase silica surface.     

Effect of surfactants on the stability of thin liquid film flow on a rotating disk

The effect of surfactants on surface instabilities of thin liquid film flow on a rotating disk was studied at different flow rates Q (0.5相似文献   

Evaluation of the adsorption affinity of proteins to calcium hydroxyapatites by desorption and pre-adsorption methods

The adsorption affinity of bovine serum albumin (BSA) and lysozyme (LSZ) to calcium hydroxyapatite (CaHAP) was evaluated by desorption and two step adsorption methods. These experiments were carried out at 15°C in a 1×10⁻⁴ mol dm⁻³ KCl solution of pH 6.0. BSA molecules were scarcely desorbed, exhibiting an irreversible adsorption of BSA, though LSZ slightly desorbed. This result supports our previous findings that LSZ adsorbs weakly onto phosphate ions exposed on ac or bc faces of CaHAP while BSA adsorbs strongly onto positively charged sites on ac or bc faces of CaHAP. The amount of adsorbed LSZ was markedly increased by the pre-adsorption of BSA, where LSZ was adsorbed onto BSA-covered CaHAP. On the other hand, the amount of adsorbed BSA was not changed by the pre-adsorption of LSZ. In both pre-adsorption systems it was confirmed by an HPLC method that no protein molecule pre-adsorbed was desorbed after the post-adsorption procedure. Therefore, it was interpreted that the enhancement of adsorption of positively charged LSZ is induced by an electrostatic attractive force through pre-adsorption of negatively charged BSA molecules with a high coverage. However, since the coverage of LSZ onto CaHAP is considerably low, no stimulation of BSA adsorption occurred on the LSZ-covered surface. The formation of double protein adsorbed layers consisting of pre- and post-adsorbed proteins was proposed.     

Therapeutic monitoring of imipramine and desipramine by micellar liquid chromatography with direct injection and electrochemical detection

A micellar liquid chromatographic (MLC) procedure was developed for the clinical monitoring of imipramine and its active metabolite, desipramine. The determination of these highly hydrophobic substances was carried out after direct injection of the serum samples using a mobile phase composed of 0.15 m SDS--6% (v/v) pentanol buffered at pH 7, pumped at 1.5 mL/min into a C(18) column (250 x 4.6 mm), and electrochemical detection at 650 mV. Using this MLC method, calibration was linear (r > 0.995) and the limits of detection (ng/mL) were 0.34 and 0.24 for imipramine and desipramine, respectively. Repeatabilities and intermediate precision were tested at three different concentrations in the calibration range and a CV (%) below 2.2 was obtained. In this MLC procedure, the serum is determined without treatment, thus allowing repeated serial injections without changes in retention factors, and reducing the time and consumables required to carry out the pretreatment process. The assay method can be applied to the routine determination of serum imipramine and its metabolite in therapeutic drug monitoring.     

Determination of norfloxacin in pharmaceuticals,human serum,and urine using a luminol—dissolved oxygen chemiluminescence system

A sensitive chemiluminescence method, based on the enhancive effect of norfloxacin on the reaction between luminol and dissolved oxygen in a flow injection system, was proposed for the determination of norfloxacin. The increment of the chemiluminiscence intensity was proportional to the concentration of norfloxacin, giving a calibration graph linear over the concentration from 0.4 ng mL⁻¹ to 400.0 ng mL⁻¹ (r ² = 0.9988) with the detection limit of 0.1 ng mL⁻¹ (3 × σ _noise). At the flow rate of 2.0 mL min⁻¹, a complete determination of norfloxacin, including sampling and washing, could be accomplished in 30 s with the relative standard deviation lower than 3.0 %. The proposed method was applied successfully to determine norfloxacin in pharmaceuticals, human urine, and serum. Possible mechanism of the reaction was also discussed.     

Thermal denaturation of bovine serum albumin and its oligomers and derivativespH dependence

In a previous paper, we report a preliminary DSC study on bovine (BSA) and human (HSA) serum albumins. However, at accurate HPLC analysis the commercial proteins show three peaks: Fraction V-I, probably globulins (as declared by the producers), Fraction V-II (about 15–18% of the product) and Fraction V-III that represents pure BSA or HSA. A hypothesis is that the Fraction II is a covalent dimer, or trimer or a mixture of both, generated during the scalf-life of the commercial product. Denaturation enthalpies of the purified Fraction V-III and Fraction V-II of BSA, have been determined calorimetrically, at changing thepH, and the results of both compared with those obtained on the untreated protein. Few calorimetric experiments have been also carried on a BSA monomer derivative with sulphidril group protected. Computer program have been developed for the deconvolution of exo- and endothermic effects and for the analysis of thermal denaturation profiles.     

Determination of levodopa methyl ester and its metabolites in rat serum by CZE with amperometric detection

A reliable and reproducible method, capillary zone electrophoresis with amperometric detection (CZE–AD), has been developed for separation and quantification of levodopa methyl ester (LDME) and its biotransformation products levodopa (L-DOPA) and dopamine (DA) in rat serum. A carbon-disk electrode was used as working electrode. The optimum conditions for CZE detection were 50 mmol L^–1 phosphate solution at pH 7.0 as running buffer, 17 kV as separation voltage, 1.0 V (vs Ag/AgCl, 3.0 mol L^–1) as detection potential, and sample injection for 8 s at 17 kV. The linear ranges were from 2.4×10^–2 to 2.2 g mL^–1 for LDME, 2.9×10^–1 to 49.5 g mL^–1 for L-DOPA, and 1.4×10^–2 to 1.5 g mL^–1 for DA with correlation coefficients of 0.9997, 0.9994, and 0.9999, respectively. The detection limits for LDME, L-DOPA, and DA were 14.6, 98.0, and 9.7 ng mL^–1, respectively. Recoveries were 80.3% for LDME, 93.5% for L-DOPA, and 86.5% for DA. This method was applied to serum samples after intravenous injection of LDME and L-DOPA to rats.     

Simplified Solid-Phase Extraction Procedure and Liquid Chromatographic Determination of Celecoxib in Rat Serum

A simplified solid phase extraction method, eliminating a preliminary protein precipitation has been developed for the determination of celecoxib in rat plasma. The technique included a solid phase extraction of the serum samples on a [poly (divinylbenzene-co-N-vinylpyrrolidone)] sorbent. After conditioning, the cartridge was loaded with 0.5 mL of acidified serum containing internal standard. Elution was made with 1 mL of a mixture of acetonitrile and methanol (1/1, v/v). After evaporation of the eluate to dryness and reconstitution with methanol, the samples were analyzed on an octadecyl bonded phase with several mobile phases containing acetonitrile and a phosphate buffer. Detection was carried out using a Photodiode Array Detector. Full validation of the proposed method was provided (linearity range: 0.01–2 mg. L^–1, average extraction efficiency: 92.4%; average intra-day variability: 4.6% with an accuracy of 94.8%; average interday variability: 5% with an accuracy of 95.3%, limit of detection: 0.005 mg. L^–1, limit of quantification: 0.002 mg. L^–1). The proposed method was successfully utilised to quantify celecoxib in rat plasma for a pharmacokinetic study.Revised: 26 January and 23 April 2004     

A fluorescence spectroscopic study of the interaction between epristeride and bovin serum albumine and its analytical application

A new spectrofluorimetric method to determine epristeride (EP) has been developed, which based on the EP has a strong ability to quench the intrinsic fluorescence of bovine serum albumin (BSA). There was the relationship between the fluorescence quenching intensity of BSA (ΔF = F_BSA − F_BSA-EP) and the concentration EP. The quenching mechanism was investigated with the quenching type, the association constants, the number of binding sites and basic thermodynamic parameters. The method had been successfully applied to the analysis of EP in real samples and the obtained results were in good agreement with the results of official method-HPLC.     

Interaction between peroxide ion and phenol group which are coordinated to the same iron(III) ion

We have prepared several new iron(III) complexes with ligands which contain a phenol group; these are tetradentate [(X-phpy)H, X and H(phpy) represent the substituents on the phenol ring and N,N-bis(2-pyridylmethyl)-N-(2-hydroxybenzyl)amine, respectively] and pentadentate ligands [(R-enph-X)H; R=ethyl(Et) or methyl(Me) derivative and H(Me-enph) denotes N,N-bis(2-pyridylmethyl)-N″-methyl-N″-(2″-hydroxyl-benzylamine)ethylenediamine] and have determined the crystal structures of Fe(phpy)Cl₂, Fe(5-NO₂-phpy)Cl₂, and Fe(Me-enph)ClPF₆, which are of a mononuclear six-coordinate iron(III) complex with coordination of one or two chloride ion(s). These compounds are highly colored (dark violet) due to the coordination of phenol group to an iron(III) atom. When hydrogen peroxide was added to the solution of the iron(III) complex, a color change occurs with bleaching of the violet color, indicating that oxidative degradation of the phenol moiety occurred in the ligand system. The bleaching of the violet color was also observed by the addition of t-butylhydroperoxide. The rate of the disappearance of the violet color is highly dependent on the substituent on the phenol ring; introduction of an electron-withdrawing group in the phenol ring decreases the rate of bleaching, suggesting that disappearance of the violet band should be due to a chemical reaction between the phenol group and a peroxide adduct of the iron(III) species with an η¹-coordination mode and that in this reaction the peroxide adduct acts as an electrophile towards phenol ring. The intramolecular interaction between the phenol moiety and an iron(III)-peroxide adduct may induce activation of the peroxide ion, and this was supported by several facts that the solution containing an iron(III) complex and hydrogen peroxide exhibits high activities for degradation of nucleosides and albumin.