Comparative parametric method 5 (PM5) study of trans-stilbene

In this work we report a comparative Austin method 1 (AM1), parametric method 3 (PM3), and parametric method 5 (PM5) studies for trans-stilbene in its ground, excited (singlet and triplet), and ionic (positive and negative polarons and bipolarons) states. We evaluated the accuracy of the recently developed PM5 method. PM5 and AM1 predict a non-planar ground and singlet states for trans-stilbene, while PM3 predicts planar ones, which is in agreement with the available experimental data. In general the PM3 and PM5 bond lengths are superior to AM1 while AM1 bond angles are superior to PM3 and PM5 when compared with available experimental data. The PM5 underestimates the cis–trans isomerization energy and and it is not a quite reliable method for the calculation of relative IP values. The presumed PM5 superior performance against AM1 and PM3 was not observed for the stilbene structures.     

Alcohol biomarker analysis: simultaneous determination of 5-hydroxytryptophol glucuronide and 5-hydroxyindoleacetic acid by direct injection of urine using ultra-performance liquid chromatography-tandem mass spectrometry

A direct ultra-performance liquid chromatography-tandem mass spectrometry method (UPLC-MS/MS) for simultaneous measurement of urinary 5-hydroxytryptophol glucuronide (GTOL) and 5-hydroxyindoleacetic acid (5-HIAA) was developed. The GTOL/5-HIAA ratio is used as an alcohol biomarker with clinical and forensic applications. The method involved dilution of the urine sample with deuterated analogues (internal standards), reversed-phase chromatography with gradient elution, electrospray ionisation and monitoring of two product ions per analyte in selected reaction monitoring mode. The measuring ranges were 6.7-10 000 nmol/l for GTOL and 0.07-100 micromol/l for 5-HIAA. The intra- and inter-assay imprecision, expressed as the coefficient of variation, was below 7%. Influence from ion suppression was noted for both compounds but was compensated for by the use of co-eluting internal standards. The accuracy in analytical recovery of added substance to urine samples was 96 and 98%, respectively, for GTOL and 5-HIAA. Method comparison with GC-MS for GTOL in 25 authentic patient samples confirmed the accuracy of the method with a median ratio between methods (GC-MS to UPLC-MS/MS) of 1.14 (r(2) = 0.975). The difference is explained by the fact that the GC-MS method also measures unconjugated 5-hydroxytryptophol naturally present in urine. The comparison with data for 5-HIAA obtained by an HPLC method demonstrated a median ratio of 1.05 between the methods. The UPLC-MS/MS method was capable of measuring endogenous GTOL and 5-HIAA levels in urine, which agreed with the literature data. In conclusion, a fully validated and robust direct method for the routine measurement of urinary GTOL and 5-HIAA was developed.     

Simultaneous measurement of serotonin and 5-hydroxyindoleacetic acid in rat brain using a liquid chromatographic method with electrochemical detection.

A simple and sensitive method for the simultaneous measurement of 5-hydroxytryptamine and its main metabolite 5-hydroxyindoleacetic acid in rat brain is described. Brain tissue samples were only deproteinated and, without further extraction, were injected directly onto a high-performance liquid chromatography column and detected electrochemically. The detection limit for 5-hydroxytryptamine and 5-hydroxyindoleacetic acid was 16 and 8 g per injection, respectively. Within 8 min, the total separation of 5-hydroxytryptamine and 5-hydroxyindoleacetic acid is achieved. With this method over 100 analyses can be performed in a single working day. Brain samples from young and old, male and female Brown Norway rats were analysed for indoles by this method. The ratio 5-hydroxytryptamine 5-hydroxyindoleacetic acid, an index for serotonin turnover, showed significant differences between age groups and genders in the cortex, and a significant difference between genders in the hypothalamus.     

Improved method for the determination of 5,6-dihydroxytryptamine and 5,7-dihydroxytryptamine in tissue using high-performance liquid chromatography with electrochemical detection.

A liquid chromatographic method with electrochemical detection is described for the determination of the 5-hydroxytryptamine (5-HT) neurotoxins 5,6-dihydroxytryptamine (5,6-DHT) and 5,7-dihydroxytryptamine (5,7-DHT) in rat brain tissue. This method has also been used for the determination of 5-hydroxyindoleacetic acid, homovanillic acid and 5-HT in other tissue samples. The method is based on extraction of the indoles from brain samples with perchloric acid followed by reversed-phase liquid chromatography with electrochemical detection. The detection limit is 1 ng per 100 mg of tissue. This paper describes a quick and reliable method of assaying the 5-HT neurotoxins 5,6-DHT and 5,7-DHT in brain tissue, which is improved compared to currently available assays.     

Development of a competitive ELISA for the quantification of F5 conjugate in HER2-targeted STEALTH immunoliposome doxorubicin in plasma samples

HER2 (human epidermal growth factor receptor 2, erbB2, or neu) is overexpressed by a large number of tumor types and has been identified as an important target for cancer therapy. F5 is a single-chain human antibody fragment that recognizes HER2 receptor and is covalently conjugated to PEGylated lipid to form F5 conjugate (F5CG) in the product HER2 targeted STEALTH immunoliposome doxorubicin. Here we described the method development of a competitive enzyme-linked immunosorbent assay (ELISA) for the determination of total concentration of F5 conjugate in plasma samples. The method involved the biotinylation of F5CG, detergent treatment of plasma sample to solubilize F5CG into monomeric form, and competitive ELISA for solubilized F5CG competitively binding to anti-F5CG antibody with biotinylated F5CG for the determination of total F5CG in plasma. The detection range of this method was from 0.2 ng/mL to 125 ng/mL for F5CG in plasma. The lower limit of quantification (LLOQ) was 0.2 ng/mL. This method was established and used for the measurement of F5CG concentration to provide information about F5CG circulation after the administration of immunoliposome in preclinical studies.      

常温凝胶渗透色谱法分析C5加氢石油树脂的相对分子量及分布

通过凝胶渗透色谱（GPC）,采用普适校准法对C5加氢石油树脂的相对分子质量及分布进行测定.方法选取适合有效的小分子物质作为标准物质,测得样品的数均分子量约Mn=500,重均分子量约Mw=900。结果表明,方法不仅数据准确、可靠、重现性好,而且精密度高,绘出的图谱具有直观性,这对研究石油树脂的机械性能、流动性和成型加工性等有着极其重要的意义,为C5加氢石油树脂生产工艺提供了行之有效的分析方法。     

Liquid chromatographic analysis of glucosamine in commercial dietary supplements using indirect fluorescence detection

A method of using indirect fluorescence detection is evaluated for the analysis of glucosamine in commercial dietary supplements following chromatographic separation. In this method, the eluting analyte, glucosamine, was detected by monitoring an increase in the fluorescence signal for L-tryptophan (L-Trp) or DL-5-methoxytryptophan (5-MTP) after glucosamine complexed with a copper(II) ion and released either L-Trp or 5-MTP from a copper(II) complex, which is introduced postcolumn. The fluorescence of L-Trp and 5-MTP are quenched when complexed with the copper(II) ion. The results obtained using indirect fluorescence detection are compared with the results obtained for precolumn derivatization with phenylisothiocyanate. Statistical analysis is performed to compare the results obtained for the two postcolumn interaction components, Cu(L-Trp)2 and Cu(5-MTP)2, as well as the results obtained using the indirect fluorescence detection method and a precolumn derivatization method. The indirect fluorescence detection method provided an alternative to precolumn derivatization for determining the concentration of glucosamine in commercial dietary supplements. The stability of the glucosamine-o-phthalaldehyde-3-mercaptopropionic acid derivative is also evaluated to investigate the applicability of the popular precolumn derivatization reagent, o-phthalaldehyde-3-mercaptopropionic acid, for this analysis.     

Reactions of methyl 3-hydroxythiophene-2-carboxylate. Part 4. Synthesis of methyl 5-azolyl-3-hydroxythiophene-2-carboxylates

The indirect introduction of an azolyl group in position 5 of compound 1 by an easy two-step procedure taking place at room temperature is described. A similar procedure yields the 4-chloro derivatives of these 5-azolyl compounds. The same method is applied for the introduction of a 5-azido group and from the 5-azido compounds, 5-v-triazolyl derivatives are obtained by a known method.     

Nucleoside 5''-phosphordiamidates

A one-flask method is reported for the preparation of the 5'-phosphordiamidate of adenosine (3a), cytidine (3b), guanosine (3c), uridine (3d) and thymidine (3e), and also for that of adenosine 5'-(N,N'-dimethyl) phosphordiamidate (4a), adenosine 5'-(N,N,N',N'-tetramethyl) phosphordiamidate (5a) and adenosine 5'-phosphor-dimorpholidate (6a). The method is based on the aminolysis of nucleoside 5'-phosphordichloridates performed in situ by NH₄OH, or aqueous amine solutions, respectively.     

Simultaneous assay of organic fluorine compounds in biological fluids by fluorine-19 nuclear magnetic resonance spectrometry over a large spectral width: Application to Fluoropyrimidines

¹⁹F-n.m.r. spectrometry is used as a direct method to assay simultaneously in body fluids (urine, plasma) all the fluorinated metabolites of a new antineoplastic drug fluoropyrimidine, 5′-deoxy-5-fluorouridine. The resonances of these metabolites are spread over a large spectral width (about 100 ppm). The calibration graphs for 5′-deoxy-5-fluorouridine and its major metabolite, α-fluoro-β-alanine, are linear over the range 10^?5–10^?1 M; the accuracy is 3–5% for urine samples and 5–7% for plasma samples. The method is applied to the determination of all the metabolites in urine from a patient.     

The solvent cage effect: is there a spin barrier to recombination of transition metal radicals?

This investigation explored whether there is a spin barrier to recombination of first- and second-row transition metal-centered radicals in a radical cage pair. To answer this question, the recombination efficiencies of photochemically generated radical cage pairs (denoted as FcP) were measured in the presence and absence of an external heavy atom probe. Two methods were employed for measuring the cage effect. The first method was femtosecond pump-probe transient absorption spectroscopy, which directly measured FcP from reaction kinetics, and the second method (referred to herein as the "steady-state" method) obtained FcP from quantum yields for the radical trapping reaction with CCl4 as a function of solvent viscosity. Both methods generated radical cage pairs by photolysis (lambda = 515 nm for the pump probe method and lambda = 546 nm for the steady-state method) of Cp'2Mo2(CO)6 (Cp' = eta(5)-C5H4CH3). In addition, radical cage pairs generated from Cp'2Fe2(CO)4 and Cp*2TiCl2 (Cp* = eta(5)-C5(CH3)5) were studied by the steady-state method. The pump-probe method used p-dichlorobenzene as the heavy atom perturber, whereas the steady-state method used iodobenzene. For both methods and for all the radical caged pairs investigated, there were no observable heavy atom effects, from which it is concluded there is no spin barrier to recombination.     

Ion-pairing high-performance liquid chromatographic method for the determination of 5-aminosalicylic acid and related impurities in bulk chemical

An ion-pairing high-performance liquid chromatographic method has been developed for the determination of 5-aminosalicylic acid (5-ASA) bulk chemical in the presence of thirteen potential synthetic process impurities. In addition, the method is suitable for the determination of the in process intermediate, 5-nitrosalicylic acid. A selective method was achieved on a Hypersil-BDS reversed-phase column using 1-heptanesulfonic acid sodium salt as the ion-pairing reagent in a 0.08 M sodium phosphate buffer (pH 2) containing 0.005 M 1-heptanesulfonic acid sodium salt and 0.07 M sodium chloride-methanol-tetrahydrofuran (85:11:4, v/v/v) isocratic mobile phase. The method was validated using a multi-day, intra-laboratory protocol. The validation addressed linearity, accuracy, precision, sensitivity, and ruggedness of the method. The validated method characterizes the purity of 5-ASA bulk chemical.     

Rapid determination of 5-hydroxytryptamine in whole blood by liquid chromatography with fluorimetric detection.

A rapid and simple reversed-phase (using muBondapak C18 as the stationary phase) liquid chromatographic method with fluorimetric detection is described for the quantitation of 5-hydroxytryptamine in whole blood. The rapidity and simplicity of the method are explained by the absence of a pretreatment. 5-Fluoro-dl-tryptophan was used as internal standard. The mobile phase was 0.01 M phosphate buffer (pH 4.5) with 0.0025 M 1-heptanesulfonic acid and 20% methanol. The detection wavelength were 302 nm for excitation and 340 nm for emission. Analysis time was 10 min with retention times for 5-hydroxytryptamine of 9 min and for 5-fluoro-dl-tryptophan of 7 min. This method is proposed for biological exploration of psychiatric disorders involving 5-hydroxytryptamine and would be useful for tryptophan.     

锆-氟-7-碘-8-羟基喹啉-5-磺酸-EDTA-表面活性剂体系的荧光光度研究

在pH5.5～7.0的HAc-NaAc缓冲溶液中,锆与氟、7-碘-8-羟基喹啉-5-磺酸(H_2QSI)、EDTA及十六烷基三甲基溴化铵(CTMAB)形成五元荧光络合物,其组成为Zr(Ⅳ):F:H_2QSI:EDTA:CTMAB=1:2:1:1:4.络合物的最大激发波长(λ_(ex))为365nm,最大发射波长(λ_(em))为 500 nm.由此建立了锆的选择性好、灵敏度高的荧光测定新方法.方法的检测限为 0.8μg/L.测锆的线性范围为 0.0016～1.04 mg/L.应用于合金中锆的测定,结果满意.     

Simultaneous determination of 5'-monophosphate nucleotides in infant formulas by HPLC-MS

A method was developed for simultaneous determination of 5'-monophosphate nucleotides, adenosine 5'-monophosphate, cytidine 5'-monophosphate, guanosine 5'-monophosphate, inosine 5'-monophosphate, and uridine 5'-monophosphate in infant formulas by high-performance liquid chromatography-mass spectrometry equipped with electrospray ionization source. The complete chromatographic separation of five nucleotides was achieved through a Symmetry C(18) column, after a binary gradient elution with water containing 0.1% formic acid and acetonitrile as mobile phase. The multi-reaction monitoring mode was applied for tandem mass spectrometry analysis. The established method was further validated by determining the linearity (R(2) > 0.999), recovery (92.0-105.0%), and precision (relative standard deviation ≤6.97%). To verify the applicability of the method, thirty commercially available infant formulas were randomly purchased from the supermarkets in Hangzhou, China, and then analyzed. The results showed that the developed method is validated, sensitive, and reliable for quantitation of nucleotides in infant formulas.     

Preliminary study of the use of dansyl chloride to determine cyclic-3',5'-AMP in tissues.

A highly sensitive method for the determination of cyclic-3',5'-AMP has been developed which involves the reaction of the substance with dansyl chloride and the subsequent separation of the dansyl-cyclic-3',5'-AMP derivative by thin-layer chromatography. Experiments with standard solutions of 3H-cyclic-3',5'-AMP have shown that there is a direct relationship between the amount of dansyl-3H-cyclic-3',5'-AMP recovered and that dansylated. The procedure is exceedingly sensitive, allowing milligram quantities of material to be analysed for its endogeneous cyclic-3',5'-AMP content. With the use of 14C-adenine as substrate, this method permits the separation of 14C-cyclic-3',5'-AMP formed from the substrate and other 14C-containing compounds, thus allowing the turn-over of cyclic-3'-5'-AMP to be studied. The usefullness of the method is demonstrated by analysing the turn-over and endogenous content of cyclic-3'-5'-AMP in rat nervous tissue.     

An Assay for Pyrroline 5-Carboxylate Based on its Interaction with Cysteine

Abstract

Pyrroline 5-carboxylate, a potent effector of redox dependent pathways, is a nutritionally responsive plasma constituent and is present in tissue culture medium conditioned by a variety of cells. Studies of P5C production and release both in vivo and in vitro have become increasingly important. To date, the assay used is a specific, sensitive radioenzymatic method. Unfortunately, the necessary materials (purified P5C reductase and NADP[3H], labeled in the transferable position) are not readily available. We now describe a method using commercially available reactants. This new method, based on the interaction of P5C with cysteine, is sensitive and specific; the presence of a wide variety of compounds only minimally affected the assay. [S³⁵] Cysteine at a defined specific activity is reacted with P5C and the P5C-CYS adduct can be separated from the reactants by cation exchange column chromatography. To optimize the assay conditions, we characterized the time course, pH dependence and cysteine concentrations for adduct formation. Studies showed that the sulfhydryl group of cysteine is critical in the formation of the P5C-CYS adduct. Comparison of this new method to the previously published radioenzymatic method showed close correlation over a wide range of P5C concentrations in conditioned media.     

Reversed-phase high-performance liquid chromatographic method for the quantification of 5-hydroxymethylfurfural as the major degradation product of glucose in infusion fluids

Analysis of 5-hydroxymethylfurfural (5-HMF) is an important indicator of degradation in glucose infusion fluids. Current pharmacopoeial methods for analysis are insensitive and non-specific. A method is described here, incorporating an internal standard, to give a sensitive, accurate and precise method, suitable for use in the quality control of glucose infusion fluids, and with possible applications for the quantitative determination of 5-HMF in food and drink, bacterial cultures and hydrothermolysed wood.     

Complexometric titration of Ti(IV) with 2-(5-chloro-2-pyridylazo)-5-dimethylaminophenol as indicator

A method for determination of titanium by a reverse titration with EDTA in the presence of hydrogen peroxide, with 2-(5-chloro-2-pyridylazo)-5-dimethylaminophenol as indicator, is presented; use of the method for analysis of titanium paints is also described.     

Sensitive and rapid simultaneous quantitation of leucovorin and its major active metabolite 5-methyl-tetrahydrofolate in human plasma using a liquid chromatography coupled with triple quadruple mass spectrometry

Bioanalysis of an endogenous compound such as leucovorin is never an easy task on a liquid chromatography tandem mass spectrometer (LC–MSMS). Unless it is necessary, regulatory guidance discourages working with surrogate matrices for calibration curve standard preparation. Herein, a selective and sensitive liquid chromatography–tandem mass spectrometry method for simultaneous determination of leucovorin and 5-methyl tetrahydrofolic acid in human plasma was developed and validated. Stable labeled internal standards, i.e. leucovorin D₄ and 5- methyl tetrahydrofolic acid ¹³C₅, were used as internal standards to track and compensate the parent compounds during processing and extraction from plasma. The method involves a rapid solid-phase extraction from plasma followed by reverse-phase gradient chromatography and mass spectrometry detection with a total run time of 5 min. The method was developed and validated from 5 to 2,202 ng/ml for leucovorin and from 5 to 1,300 ng/ml for 5-methyl tetrahydrofolic acid. The mean recoveries for leucovorin and 5-methyl tetrahydrofolic acid were 100.4 and 100.9% respectively. The validated method enabled the simultaneous analysis of leucovorin and 5-methyl tetrahydrofolic acid in samples from clinical pharmacokinetic studies of leucovorin. The peak concentrations of leucovorin and 5-methyl tetrahydrofolic acid were 651–883 and 518–635 ng/ml, respectively, in fasted and fed conditions. The terminal half-life values for leucovorin and 5-methyl tetrahydrofolic acid were 9.3–10.5 and 9.2–17.6 h, respectively.