On-line stacking capillary electrophoresis for analysis of methotrexate and its eight metabolites in whole blood

Therapeutic drug monitoring of methotrexate (MTX) and its metabolites is significant for the evaluation of treatment response of acute lymphoblastic leukemia and the prevention of adverse effects. Those eight metabolites including 7-hydroxymethotrexate, MTX-polyglutamate derivatives (MTX-(Glu)(n), n=2-7), and 2,4-diamino-N(10)-methylpteroic acid, especially the MTX-(Glu)(n), only exist in blood cells and also present antifolate effects. Therefore, whole blood analysis has importance in clinical therapy. This study combined solid-phase extraction and on-line stacking capillary electrophoresis to examine the levels of MTX and its eight metabolites in whole blood. A higher conductivity buffer (HCB) was used to accumulate large amount of samples into a narrow zone, which resulted in effective stacking and sharp peaks. A fused-silica capillary was filled with phosphate buffer (80 mM, pH 2.5) containing 15% v/v tetrahydrofuran and 100 mM sodium dodecyl sulfate. Then HCB (100 mM phosphate, pH 2.5; 1 psi, 60 s) was injected for accumulating large volume of analytes (1 psi, 200 s). Owing to the pH difference between sample zone and HCB, dynamic pH junction was generated for focusing. Meanwhile, sweeping made further stacking by using sodium dodecyl sulfate in phosphate buffer. The limits of detection (S/N=3) were 0.1 microM for MTX, 0.2 microM for 7-hydroxymethotrexate and 2,4-diamino-N(10)-methylpteroic acid, 0.3 microM for MTX-(Glu)(n=2--5), and 0.5 microM for MTX-(Glu)(n=6--7). During validation, this method showed good linearity (r> / =0.9914) and reproducibility (relative standard deviation and relative error, both less than 13%). The applications were performed to monitor blood MTX and its metabolites in acute lymphoblastic leukemia patients. The blood concentrations could provide some information related to therapeutic response and adverse effects.     

Analysis of erythromycin in biological fluids by high-performance liquid chromatography with electrochemical detection

A simple and sensitive high-performance liquid chromatographic assay was developed for the quantitative determination of major erythromycin components and their potential metabolites or degradation products in plasma and urine. An ether extract of alkalized plasma sample was chromatographed on a reversed-phase column and the components in the column effluent were monitored by an electrochemical detector. The recovery of the drug from extraction was virtually 100%. The detection limits for erythromycin A in plasma were 5-10 ng/ml and 30 ng/ml using 1 and 0.2 ml of sample, respectively. For urine samples, a simple one-step deproteinization with two volumes of acetonitrile was satisfactory for analysis. The method has been evaluated in plasma and urine from dogs receiving oral or intravenous erythromycin A. The standard curves for potential metabolites or degradation products were not constructed due to the lack of sufficient samples.     

Studies of the triplet state of the proton-transfer tautomer in salicylaldehydes

The spectroscopy and dynamics of the low-lying triplet state of the proton-transfer tautomer in salicylaldehydes have been studied via internal heavy-atom effects coupled with a sensitive near-IR detecting system. For 3,5-diiodosalicylaldehyde a weak proton-transfer keto-tautomer phosphorescence was resolved with a maximum at 710 nm (τ_p1.8 μs, Φ_obs5.23×10⁻⁴) in a 77 K methylcyclohexane glass. The results, in combination with the time-resolved thermal lensing experiment, further deduced the triplet-state population yield and radiative decay rate to be 0.20 (298 K) and 3.12×10² s⁻¹, respectively. Consequently, the energetics and dynamics of the triplet states during a proton transfer cycle are discussed in detail.     

Modeling particle deposition from fully developed turbulent flow

An unsteady state transfer of immersed particles within the interval between the arrival of eddies is solved by use of the Laplace transform schemes. The mean particle flux and the mean particle transport mechanisms are automatically considered on the average sublayer growth period by formulating the mean distributions as a stochastic process with the aid of exponentially distributed density function. The proposed relationship for the particle deposition velocity of average time domain obtained by this analysis is expressed as the form of analytical equation, with the inclusion of the effects of Brownian diffusion, turbulent eddy diffusivity, turbophoresis, and thermophoresis. The solution of this equation is in reasonable agreement with the measured deposition velocities for three distinct categories. This mathematical framework offers a simple computation tool of practical use to aerosol engineers and can further extend by including appropriate forces in the analytical formulation through the equilibrium among acceleration terms.     

Genotyping of intron 22 inversion of factor VIII gene for diagnosis of hemophilia A by inverse-shifting polymerase chain reaction and capillary electrophoresis

This is the first capillary electrophoresis (CE) analysis for diagnosis of hemophilia A (HA). The intron 22 inversion of factor VIII gene (F8) causes 40–50 % of severe bleeding disorder of HA in all human populations. Consequently, identification of the disease-causing mutations is becoming increasingly important for accurate genetic counseling and prenatal diagnosis. In this study, the key steps of inverse-shifting polymerase chain reaction (IS-PCR) and of short-end injection capillary electrophoresis were used for more specific and rapid genotyping of intron 22 inversion of F8. In IS-PCR, three specific primers were used to amplify 512-bp amplicon for wild type and 584-bp amplicon for patients with intron 22 inversion. The capillary gel electrophoresis (CGE) system was performed using 1× Tris–borate–EDTA (TBE) buffer containing 0.3 % (w/v) polyethylene oxide (PEO). The PCR amplicons were electrokinetically injected at 10 kV for 10 s at a temperature of 25 °C. The optimal short-end injection CGE was applied to detect the F8 gene of HA patients and carriers within 5 min. Intron 22 inversion was indeed found on some HA patients (13/35, 37.1 %). All genotyping results showed good agreement with DNA sequencing method and long-distance polymerase chain reaction (LD-PCR). The IS-PCR combined with short-end injection CGE method was feasible and efficient for intron 22 inversion screening of F8 in the HA populations.     

Activation mechanisms of butyrylcholinesterase by 2,4,6-trinitrotoluene, 3,3-dimethylbutyl-N-n-butylcarbamate, and 2-trimethylsilyl-ethyl-N-n-butylcarbamate

The goal of this work was to propose a possible mechanism for the butyrylcholinesterase activation by 2,4,6-trinitrotoluene (TNT), 3,3-dimethylbutyl-N-n-butylcarbamate (1), and 2-trimethylsilyl-ethyl-N-n-butylcarbamate (2). Kinetically, TNT, and compounds 1 and 2 were characterized as the nonessential activators of butyrylcholinesterase. TNT, and compounds 1 and 2 were hydrophobic compounds and were proposed to bind to the hydrophobic activator binding site, which was located outside the active site gorge of the enzyme. The conformational change from a normal active site gorge to a more accessible active site gorge of the enzyme was proposed after binding of TNT, and compounds 1 and 2 to the activator binding site of the enzyme. Therefore, TNT, and compounds 1 and 2 may act as the excess of butyrylcholine in the substrate activator for the butyrylcholinesterase catalyzed reactions.     

Mononitrosyl tris(thiolate) iron complex [Fe(NO)(SPh)3]- and dinitrosyl iron complex [(EtS)2Fe(NO)2]-: formation pathway of dinitrosyl iron complexes (DNICs) from nitrosylation of biomimetic rubredoxin [Fe(SR)4]2-/1- (R = Ph, Et)

Nitrosylation of the biomimetic reduced- and oxidized-form rubredoxin [Fe(SR)4]2-/1- (R = Ph, Et) in a 1:1 stoichiometry led to the formation of the extremely air- and light-sensitive mononitrosyl tris(thiolate) iron complexes (MNICs) [Fe(NO)(SR)3]- along with byproducts [SR]- or (RS)2. Transformation of [Fe(NO)(SR)3]- into dinitrosyl iron complexes (DNICs) [(RS)2Fe(NO)2]- and Roussin's red ester [Fe2(mu-SR)2(NO)4] occurs rapidly under addition of 1 equiv of NO(g) and [NO]+, respectively. Obviously, the mononitrosyl tris(thiolate) complex [Fe(NO)(SR)3]- acts as an intermediate when the biomimetic oxidized- and reduced-form rubredoxin [Fe(SR)4]2-/1- exposed to NO(g) were modified to form dinitrosyl iron complexes [(RS)2Fe(NO)2]-. Presumably, NO binding to the electron-deficient [Fe(III)(SR)4]- and [Fe(III)(NO)(SR)3]- complexes triggers reductive elimination of dialkyl/diphenyl disulfide, while binding of NO radical to the reduced-form [Fe(II)(SR)4]2- induces the thiolate-ligand elimination. Protonation of [Fe(NO)(SEt)3]- yielding [Fe(NO)(SPh)3]- by adding 3 equiv of thiophenol and transformation of [Fe(NO)(SPh)3]- to [Fe(NO)(SEt)3]- in the presence of 3 equiv of [SEt]-, respectively, demonstrated that complexes [Fe(NO)(SPh)3]- and [Fe(NO)(SEt)3]- are chemically interconvertible. Mononitrosyl tris(thiolate) iron complex [Fe(NO)(SPh)3]- and dinitrosyl iron complex [(EtS)2Fe(NO)2]- were isolated and characterized by X-ray diffraction. The mean NO bond distances of 1.181(7) A (or 1.191(7) A) in complex [(EtS)2Fe(NO)2]- are nearly at the upper end of the 1.178(3)-1.160(6) A for the anionic {Fe(NO)2}9 DNICs, while the mean FeN(O) distances of 1.674(6) A (or 1.679(6) A) exactly fall in the range of 1.695(3)-1.661(4) A for the anionic {Fe(NO)2}9 DNICs.