Simultaneous amperometric determination of lignin peroxidase and manganese peroxidase activities in compost bioremediation using artificial neural networks

High-performance liquid chromatography (HPLC) and fluorescence derivatization were applied for a nanogram-level N-nitrosodimethylamine (NDMA) analysis of water samples. For the analysis of N-nitrosodimethylamine, samples were first denitrosated by a mixed solution of hydrobromic acid and acetic acid to produce dimethylamine, which was derivatized with dansyl chloride for HPLC fluorescence detection. Fluorescence detection was optimized with excitation and emission wavelengths of 340 and 530 nm, respectively. pH adjustment after denitrosation was necessary to maximize fluorescence intensity with pHs in the range of 9-12. A dansyl chloride concentration of 500 mg l⁻¹ was found to be optimal for measuring a fluorescence signal. An instrumental detection limit of 0.1 ng of NDMA was possible with fluorescence derivatization. The NDMA in water samples was extracted by continuous solid-phase extraction using Ambersorb 572. Although the determination of NDMA was variable at lower concentrations (less than 200 ng l⁻¹), it was observed that the NDMA detection limit with this method could be lowered to a concentration of 10 ng l⁻¹. Another benefit of this method can be found in its selectivity for NDMA. Unlike gas chromatographic (GC) methods, this method generates a distinct peak for NDMA without interference even in the complex matrix of wastewater effluents. The HPLC with fluorescence derivatization method may be applicable for determining NDMA in water and wastewater samples for various research purposes and for screening environmental samples.     

Amino Acid Profiling of Protein Hydrolysates Using Liquid Chromatography and Fluorescence Detection

Abstract

A liquid chromatography procedure is described for separating the amino acids in protein hydrolysates. The proteins are hydrolyzed with hydrochloric acid and an aliquot of the hydrolysate is derivatized with dansyl chloride reagent. The derivatization procedure takes only 2 minutes using a reaction temperature of 100°C. The dansylated amino acids are chromatographed using a reversed-phase C₈ column and a multi-step, nonlinear gradient elution solvent program which is readily achieved using a microprocessor-controlled liquid chromatograph. Chromatography is complete in approximately 40 min. The procedure is useful for characterizing proteins and may also be used to analyse intact dansylated polypeptides. Chromatograms showing the amino acid profile of chymotrypsin, albumin and histone are given.     

Quantitation of P-Aminohippuric Acid and N-Acetyl-P-Aminohippuric Acid from Blood by HPLC

Abstract

P-aminohippuric acid, N-acetyl-p-aminohippuric acid, and p-aminobenzoic acid can be separated by a reverse-phase, isocratic high-pressure liquid chromatographic procedure in less than 4 minutes. The eluent is 10 mM sodium phosphate buffer, pH 3.5, containing 30% methanol. Detection is by absorbance at 270 nm; quantitation is accomplished by peak height. Linear response was obtained from 3 to 1600 nmoles of each compound in aqueous standards and in blood deproteinized with perchloric acid.     

Determination of Volatile N-Nitrosamines in Food by High-Performance Liquid Chromatography with Fluorescence Detection

A procedure is proposed for the determination of the mass fraction of volatile N-nitrosamines in food by high-performance liquid chromatography with fluorescence detection. A high sensitivity and selectivity of determination is attained by the precolumn operation of obtaining derivatives secondary amines, which were obtained by the denitrosation of initial nitrosamines with 5-dimethylamino-1-naphthalenesulfonyl chloride (dansyl chloride). A mixture of concentrated hydrobromic acid and glacial acetic acid was proposed as the reducing agent for the denitrosation reaction.     

Reductive Isopropylation of Amino Groups in Lysine Containing Peptides

Abstract

Model peptides, Gly-Gly-Lys-Arg, Arg-Lys-Asp-Val-Tyr, and Pro-Gly-Lys-Ala-Arg were reductively alkylated with [²H₆]acetone and sodium borohydride to assess the effects on peptide behavior. Lysine residues were converted to ?-N-isopropyllysine which eluted between phenylalanine and histidine on amino acid analysis. Amino terminal groups were also modified to an extent which depended on the particular peptide (glycine 100%, arginine 30%, and proline 10%-20%). High voltage paper electrophoresis of native and isopropylated peptides showed similar properties except for minor decreases in the mobility of the modified peptides due mainly to increased molecular weight. Isopropyllysine was not an effective substrate for trypsin, and α-N-isopropyl-amino acids did not form dansyl chloride derivatives. These findings should aid in the location, by peptide mapping techniques, of specific modified residues in reductively isopropylated proteins.     

High-Pressure Liquid Chromatography Separation of Potential Impurities of Altretamine

Abstract

Two isocratic liquid chromatographic separation methods and UV detection were developed to allow for sensitive and specific analysis of potential impurities in Altretamine using a reversed phase C₁₈ column and mixtures of water-acetonitrile as mobile phase. Linear calibration curves for each of the possible contaminants of Altretamine, in the range of 0.25–125 μg/ml, were also obtained. The detection limit for all of the compounds (except cyanuric acid) was less than 0.25 μg/ml. Several Altretamine lots were examined and their impurities identified. Hydrolysis of cyanuric chloride to cyanuric acid was studied and shown to follow 1^st order kinetics. Evidence for the formation of chlorohydroxytriazine intermediates during the hydrolysis of cyanuric chloride to cyanuric acid is given.     

Chiral capillary electrophoresis with cationic pyrrolidinium‐β‐cyclodextrin derivatives as chiral selectors

New single‐isomer, cationic β‐cyclodextrins, including mono‐6‐deoxy‐6‐pyrrolidine‐β‐cyclodextrin chloride (pyCDCl), mono‐6‐deoxy‐6‐(N‐methyl‐pyrrolidine)‐β‐cyclodextrin chloride (N‐CH₃‐pyCDCl), mono‐6‐deoxy‐6‐(N‐(2‐hydroxyethyl)‐pyrrolidine)‐β‐cyclodextrin chloride (N‐EtOH‐pyCDCl), mono‐6‐deoxy‐6‐(2‐hydroxymethyl‐pyrrolidine)‐β‐cyclodextrin chloride (2‐MeOH‐pyCDCl) were synthesized and used as chiral selectors in capillary electrophoresis for the enantioseparation of carboxylic and hydroxycarboxylic acids and dansyl amino acids. The unsubstituted pyCDCl exhibited the greatest resolving ability. Most analytes were resolved over a wide range of pH from 6.0 to 9.0 with this chiral selector. In general, increasing pH led to a decrease in resolution. The effective mobilities of all the analytes were found to decrease with increasing CD concentration. The optimal concentration for most carboxylic acids and dansyl amino acid was in the range 5–7.5 mM and >15 mM for hydroxycarboxylic acids. ¹H NMR experiments provided direct evidence of inclusion in the CD cavity.     

Simultaneous High Performance Liquid Chromatographic Determination of Theophylline and Ethylenediamine in Aminophylline Dosage Forms as Their Dansyl Derivatives

Abstract

A simple HPLC method has been developed for the assay of the components of aminophylline (theophylline: ethylenediamine 2:1) in solid and liquid dosage forms. Following the extraction of tablets into or the dilution of a liquid dosage form with water, the aqueous extract was reacted with dansyl chloride in an alkaline medium. The mixture of the dansyl derivatives of theophylline and ethylediamine was analyzed on an reversed phase μBondapak Cia column, using a methanol-water-acetic acid-triethylamine (60–65: 33–38: 1. 5:0. 5) mobile phase delivered at the rate of 1.5 mL/min, and a detection wavelength of 254 nm. In addition to exhibiting excellent accuracy and precision, the method yielded detector responses that were linearly related to concentrations of theophylline and ethylenediamine in aminophylline of up to 7 mg of theophylline, and of up to 10 mg     

Temperature and Solute Molecular Size Effects on the Retention and Enantioselectivity of a Series of D, L Dansyl Amino Acids on a Vancomycin-Based Chiral Stationary Phase

Summary The influence of column temperature (0–28 °C) and solute molecular size on the retention and enantioselectivity of a series of D, L dansyl amino acids with a non-polar side chain (valine, leucine, phenylalanine and tryptophan) were investigated using a vancomycin-based chiral stationary phase (CSP). The enthalpic and entropic terms for the solute-CSP association were determined from the linear vant Hoff plots. Two solute groups were distinguished in relation to these thermodynamic quantities: the solute group I (dansyl valine, dansyl leucine, dansyl phenylalanine) for which large negative values of enthalpic terms were obtained and the solute group II (dansyl tryptophan) for which H value was much less negative. The enthalpy-entropy compensation study revealed that the interaction mechanism was identical for the group I solute enantiomers but changed for D, L dansyl tryptophan. This was further exemplified as the group I compound enantiomers were resolved over the temperature range while the enantiomers of dansyl tryptophan were not separated in the operating conditions. Relationships between both the solute retention factors and apparent enantioselectivity, and the accessible surface area of the amino acid side chain indicated that when the solute molecular size increased (i) the retention was enhanced by the hydrophobic effect and (ii) the chiral discrimination decreased dependent, at least in part, on a steric hindrance phenomenon at the vancomycin aglycone pocket.     

Chemically enhanced liquid chromatography/tandem mass spectrometry determination of glutamic acid in the diffusion medium of retinal cells

A rapid, reproducible and highly sensitive method, based on liquid chromatography mass spectrometry, was developed for the determination of the excitatory amino acid glutamic acid released in the diffusion medium of control, ischemic and mutant cells from retinas. Signal intensity of glutamic acid was enhanced by dansyl chloride derivatization giving rise to a detection limit in the order of pmol/mL. Further, in HPLC-ESI-MS detection an MS-friendly dansyl group to glutamic acid enhanced both ionization efficiency in the ESI source and collision-activated dissociation in the collision cell. The sample processing procedure included liquid-liquid extraction, derivatization with dansyl chloride and a final cation-exchange extraction to generate clean extracts for LC/MS/MS analysis. This approach has been validated as sensitive, linear (20-300 ng/mL), accurate and precise for the differential quantification of glutamic acid in the diffusion medium of retina cells. This is the first report of using chemical derivatization to enhance MS/MS detection of the glutamic acid released in the diffusion medium of wild-type and mutant retina cells, under ischemic conditions.     

丹酰氯柱前衍生仲胺的高效液相色谱-电化学检测方法的研究

立了一种灵敏检测仲胺的新方法。首次以丹酰氯作为仲胺的电化学检测衍生试剂,对分离检测条件、仲胺的衍生条件及电化学定量检测条件作了详细的研究。以甲醇－２０ｍｍｏｌ／Ｌ的邻苯二甲酸氢钾缓冲液（７０∶３０,ｐＨ３．３）为淋洗液,采用安培检测器在Ｅ＝＋１．１Ｖ电压条件下,在ＰＥＲＫＩＮ－ＥＬＭＥＲ／ＨＳ－３Ｃ１８（８３ｍｍ×４．６ｍｍｉ．ｄ．,３μｍ）柱上实现了二甲胺、二乙胺和哌啶的良好反相分离和灵敏检测,线性范围超过两个数量级。二甲胺、二乙胺和哌啶的检出限分别为０．０２４,０．６０,０．０９０ｐｇ。     

Simple and rapid quantitative determination of lysinoalanine and protein hydrolysate amino acids by high-performance liquid chromatography after derivatization with dansyl chloride

Summary A rapid and simple method for the determination of both lysinoalanine (LAL) and protein hydrolysate amino acids after derivatization with dansyl chloride (5-dimethylaminoaphtalene-1sulfonyl chloride) and separation with RP-HPLC (UV detection) is presented. LAL is analysed in less than 15 minutes and complete separation of 22 amino acids is achieved in less than 30 minutes using single linear gradients of solvents (phosphate buffer and acetonitrile). Quantitative results obtained by HPLC compare well with results of the ion-exchange chromatography (amino acid analyser). The importance of the duration of the derivatization reaction and of the excess of reagent is discussed. As examples, the results of the determination of LAL in two samples of base treated α-casein and 22 samples of soy protein and the results of the analysis of amino acids in two balanced diet mixtures are presented. Presented at the 15th International Symposium on Chromatography, Nürnberg, October 1984     

Determination of Uric Acid in Human Serum: Reversed-Phase Liquid Chromatography with Electrochemical Detection

Abstract

A method for the simultaneous determination of uric acid in human serum by reversed-phase high-performance liquid chromatography with electrochemical detection has been developed. Human serum (0.5 ml) was mixed with 0.5 ml of 0.2 N perchloric acid solution and the mixture was centrifuged at 3,000 g for 20 min. An aliquot (10 μl) of the supernatant (deproteinized human serum) was injected into the chromatographic system employed in this study. The assay limit for quantitation was about 10 pg for uric acid. Complete separation of uric acid was achieved in about 8 min under the present chromatographic conditions.     

Screening for amino acid disorders by thin-layer chromatography of the dansyl amino acids

Summary A procedure is described for the screening of disorders of amino acid metabolism or tranpsort. The amino acids and other reactive constituents present in a small volume of deproteinized plasma or urine are derivatized with dansyl chloride. Desalting or concentrating of urine is not required. The fluorescent derivatives are separated by two-dimensional thin-layer chromatography and visualized by ultraviolet radiation. The time of development is less than 1 hr. The derivatives of thirty-seven amino acids and related constituents found in physiological fluids have been mapped in the system. A standard, reflective of normal plasma, is used to aid in identification of those derivatives generally observed on plasma and urine chromatograms and in detection of aminoacidemias. Aminoacidurias are detected by observing derivatives that in relation to others are present in greater than normal amounts.

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Analytischer Nachweis von Störungen des Aminosäurestoffwechsels durch Dünnschicht-Chromatographie der Dansylverbindungen

Zusammenfassung Dansylderivate von Aminosäuren und anderen reaktionsfähigen Stoffen in kleinen Volumina enteiweißten Plasmas oder Harns wurden hergestellt. Entsalzung oder Einengung des Harns ist nicht notwendig. Die fluoreszierenden Derivate werden durch zweidimensionale Dünnschichtchromatographie getrennt und im UV nachgewiesen. Die dazu erforderliche Zeit ist kürzer als 1 Stunde. Die Derivate von 37 Aminosäuren und verwandten Verbindungen in physiologischen Flüssigkeiten wurden erfaßt. In üblicherweise zur Beobachtung gelangenden Plasma- bzw. Harnchromatogrammen feststellbare Aminosäuren dienen zum Vergleich mit abnormalen Ergebnissen.

     

Catalytic NBP Spot Test for the Detection of Trichothecene Mycotoxin T-2

Abstract

Thorium(IV)salts were found to act as catalysts for the alkylation of 4-(p-nitrobenzyl)pyridine by T-2 toxin. Using thorium(IV)chloride as the catalyst, a detection procedure for T-2 toxin was developed that required a heating step consisting of only 2 minutes at 90°C. The limit of detection of T-2 toxin with this procedure was found to be 1 microgram.     

Gas Chromatographic–Mass Spectrometric Method for Quantitation of Trimethylsilyl Derivatives of Nerve Agent Degradation Products in Human Plasma, Using Strong Anion–Exchange Solid-Phase Extraction

For unequivocal proof of the use of nerve agents such as sarin, soman, cyclohexylsarin, VX, and Russian VX, a simple and accurate method, gas chromatography–mass spectrometry (GC–MS) after trimethylsilyl derivatization, was explored for simultaneous determination of the corresponding alkyl methylphosphonic acids (AMPAs) and of methylphosphonic acid (MPA) in human plasma. GC–MS analysis was performed after solid-phase extraction, with a strong anion-exchange cartridge, from plasma samples previously deproteinized with mercuric acetate, and then derivatization with bis(trimethylsilyl)trifluoroacetamide containing 5% trimethylchlorosilane. All five AMPA derivatives and the MPA derivative were separated to baseline within 11 minutes without interference. Linear calibration plots were obtained over concentrations ranging from 50 ng mL⁻¹ to 5 µg mL⁻¹. The relative standard deviation of recoveries ranged from 1.9 to 9.7% and detection limits were 22 ng mL⁻¹ or below.Revised: 3 and 23 May 2005     

A Simplified Neutron Activation Analysis Method for Copper and Manganese

Abstract

An activation analysis procedure has been developed to determine manganese and copper serum values in one milliliter of serum or less rapidly and with good precision. The sample is irradiated in a neutron flux of 10¹²cm^?2 sec^?1 for two hours followed by nitric acid digestion. The activity plus carrier is absorbed from saturated lithium chloride solution on Dowexl-8 contained in a syringe. After washing the resin with more lithium chloride, the syringe is counted in a five inch well crystal multichannel spectrometer. The carrier is washed off the syringe by dilute acid and analyzed. The sample concentration is determined by comparison to irradiated standards correcting the unknown for decay and chemical yield.     

Rapid Method for the Micro-Determination of Hydrazine by Amperometric Titration With Potassium Iodate

Abstract

An amperometric method, with potassium iodate as the titrant, for the rapid and precise determination of micro amounts of hydrazine salts is described. Hydrazine dihydrochloride, hydrazine sulfate and hydrazine hydrate could be quantitatively analyzed at the concentration range of 4 × 10^?7 -4×10^?3 M in the presence of 5 M hydrochloric acid. Hydrazine salts, 2×10^?4 -4×10^?3 M, were titrated in 5 minutes with a relative error and a relative standard deviation of 0.1%. It was also found that hydrazine dihydrochloride can be precisely determined, without any interference, even in the presence of hydroxylamine which is ten times as much as the former.

The suitable applied potential between the rotating platinum indicator microelectrode and the silver plate-silver chloride reference electrode was + 0.7V.     

Development and validation of UPLC‐MS/MS method for simultaneous determination of gestodene and ethinyl estradiol in rat plasma

A selective and sensitive ultra‐performance liquid chromatography method with tandem mass spectrometric detection for simultaneous determination of gestodene (GES) and ethinyl estradiol (EE) in rat plasma was developed and validated. GES, EE and the internal standard, norgestrel, were extracted with ethyl acetate, derivatized (EE only) with dansyl chloride and then back‐extracted into diethyl ether‐hexane (2:1, v/v). The separation was performed on an ACQUITY UPLC? BEH C₁₈ column with gradient elution using mobile phase consisting of acetonitrile and water (both containing 0.1% formic acid). The detection was carried out by means of electrospray ionization (ESI) mass spectrometry in positive ion mode with multiple‐reaction monitoring. Calibration curves of GES and EE were linear (r² ≥ 0.99) over the concentration ranges 1.59–159 and 0.196–78.4 ng/mL, respectively. The intra‐ and inter‐day precisions were not more than 6.9 and 12.9% for GES and 10.6 and 9.0% for EE, and the accuracies were ?2.5–8.0% for GES, and ?7.2–0.19% for EE, respectively. The method herein described was superior to previous methods and was applicable to the pharmacokinetic study of GES and EE in rats. Copyright © 2009 John Wiley & Sons, Ltd.     

Synthesis and characterization of new thiadiazole derivatives bearing a pyrazole moiety

A series of thidiazole derivatives (4, 7) from pyrazole-3-carboxylic acid chloride (2) and pyrazole-3,4-dicarboxylic acid chloride derivatives (6) were synthesized and characterized. The structures of the new compounds were confirmed byelemental analysis, NMR (¹H and ¹³C) and IR spectra. The molecular and crystal structure of 4-benzoyl-N-[5-(methylthio)-1,3,4-thiadiazol-2-yl]-1,5-di-phenyl-1H-pyrazole-3-carboxamide(4d)was determined by single crystal X-ray diffraction method.