High Performance Liquid Chromatographic Determination of Neomycin in Milk Using a Hisep Column

Abstract

A high performance liquid chromatographic (HPLC) method for the determination of neomycin in milk is described. Milk is passed directly through an amberlite CG-50 ion exchange resin column, and the neomycin which is retained on the column is derivatized with ortho-phthalaldehyde (OPA) reagent. The derivatized neomycin is eluted from the column with potassium borate buffer/methanol and analyzed by HPLC. A HISEP HPLC column with fluororoetric detection was used. Recoveries ranged from 94 to 102% in samples fortified between 0.1 to 5ppra levels. The detection limit is 50ppb.     

Preparation and Characterization of 61,6n-DI-O-(α-D-Galactopyranosyl)Cyclomaltooctaoses

ABSTRACT

Four positional isomers of 6¹,6ⁿ-di-O-(D-galactopyranosyl)cyclomaltooctaoses (cG₈s, γ-cyclodextrins) (n = 2?5) were chemically synthesized using the trichloroacetimidate method. The desired compounds having two α-(1→6)-linkages were isolated from a mixture of configurational isomers by HPLC, and their structures were confirmed by ¹³C NMR spectroscopy and FAB-high resolution mass spectra (HRMS). The elution behavior of their four positional isomers on an ODS column by HPLC is discussed.     

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.     

RP-HPLC assay for 1,2-5,6-dianhydro-3,4-disuccinyl-galactitol and its metabolites in blood plasma and liver

Summary A method involving pre-column derivatization and HPLC assay is described for measuring submicrogram quantities of 1,2-5,6-dianhydro-3,4-disuccinyl-galactitol (1,2-5,6-dianhydro-3,4-bis(carboxypropionyl)-galactitol), an effective cytostatic drug and its metabolites in blood plasma and liver homogenate. The substance and its metabolites were derivatized with sodium pentamethylene-dithiocarbamate to form different bis(dithiocarbamoyl) esters, which can be detected by UV absorbance at 254 and 280 nm. The directly derivatized products were then extracted into CHCl₃, and after sample preparation resolved by RP-HPLC on SAS-Hypersil column.     

Cellular Fatty Acid Composition of Vibrio parahaemolyticus by Reversed-Phase High-Performance Liquid Chromatography

Abstract

High-performance liquid chromatography was used to separate and identify cellular fatty acids isolated from Vibrio parahaemolyticus, a gram-negative estuarine microorganism associated with seafood-borne enteritis in man. Fatty acids were isolated from statically grown bacterial cultures, saponified, and derivatized with an ultraviolet tag. Aliquots of derivatized fatty acids were injected onto a reversed-phase column with water:acetonitrile gradient as the mobile phase and ultraviolet detection at 254 nm. The predominant fatty acids found for the V. parahaemolyticus strains studied were C12, C14, C16:1, C16, C18:1, and C18. In addition, previously unreported fatty acids C13, C17, C19, and C21 were identified. Comparison of HPLC with GLC fatty acid separations showed good agreement with the exception that HPLC was able to resolve previously unidentified fatty acid constituents.     

Spectroscopic Characterization of the Geminal Isomer of Lewisite

Abstract

Lewisite is generally a mixture of several components with the trans isomer of lewisite being the predominant compound. A geminal isomer has not been previously reported as one of the components of the mixture. In the lewisite samples we examined, the geminal isomer, dichloro(l-chlorovinyl)arsine, comprised 2.7 per cent of the total material compared to 95.2 and less than 1 per cent, respectively, for the trans and cis isomers. The remaining fraction was not identified. The geminal isomer of lewisite has been characterized along with the trans and cis isomers using several spectroscopic techniques. Proton NMR of the geminal isomer produced a coupling constant consistent with vinylic protons in a geminal configuration. Mass spectrometry and infrared spectroscopy characterizations were based on an ethanedithiol derivative of the lewisite isomers with gas chromatography used to first separate the derivatized isomers. The electron ionization massspectra of the trans and cis derivatives were very similar, but significant differences were observed in the mass spectrum of the geminal form. Infrared absorption spectra were obtained for the trans and geminal derivatives with significant differences observed between the two, but the method was not sensitive enough to detect the cis isomer.

     

Mass spectrometric analysis of four regioisomers of F2-isoprostanes formed by free radical oxidation of arachidonic acid

F₂-isoprostanes are complex metabolites of arachidonic acid generated via nonenzymatic free radical oxidation and are isomeric to prostaglandin F_2α, enzymatically produced by prostaglandin H₂ synthase. In theory, four distinct regioisomeric families are possible. These regioisomeric families have a common 1,3-diol cyclopentane structural feature, but differ by the comparative length of two attached alkyl chains and the position of a third hydroxyl group. Eight synthetic PGF_2α isomers were found separable by capillary gas chromatography (GC) and reversed-phase high-performance liquid chromatography (HPLC). Electrospray ionization tandem mass spectrometry was used to detect the elution of these isomers from the HPLC column by monitoring the characteristic loss of 44 u (C₂H₄O) from the 1,3-diol cyclopentane ring. Catalytic reduction, derivatization, and electron ionization mass spectrometric techniques were used to obtain definitive information as to the location of the side chain hydroxyl position in these isomers through abundant α-cleavage ions. Free radical oxidation of arachidonic acid was used to generate a complex mixture of F₂-isoprostanes, which were separated by HPLC and capillary GC. Members of each of the four specific regioisomeric isoprostane families could be identified in this mixture from the predicted α-cleavage ions. Although many epimers within a single family type could be separated, the four regioisomeric families were substantially superimposed in their HPLC and GC elution. The Type I and Type IV regioisomers were the major F₂-isoprostane products, but the complexity of the isomers required more than a simple GC-mass spectrometry assay to precisely identify a particular stereoisomer within a regioisomeric family (e. g., 8-epi-PGF_2α). Type I F₂-isoprostanes are unique noncyclooxygenase products and may be more specific targets to measure lipid peroxidation in vivo.     

HPLC Separation of Tautomeric Compounds of 4-Aminoisoxazolyl-1,2-naphthoquinones. II

Abstract

HPLC separations and quantitative analysis are described for a mixture of 4-aminoisoxazolyl-1,2-naph-thoquinone isomers. This assay is simple, rapid and stability indicating because the precursors and isomerization products can be monitored simultaneously. The results obtained are in agreement with those obtained by UV spectroscopy.     

Derivatizing Reagents Based on Ferrocene for HPLC-Ecd Determination of Peptides and Proteins

Abstract

Several ferrocenic compounds for derivatization of peptides and proteins were synthesized and then tested by reaction with bovine serum albumin (BSA). The reactivity of the reagents and the electroactivity of the derivatized BSA were estimated by the height of the ECD-signal after an HPLC analysis run. The most suitable reagent was 3-ferrocenylpropionic anhydride which reacts with BSA within 15 minutes at room temperature. The anhydride presented itself as a stable compound which can be synthesized in high yields. Up to pH 9–10 it is only slowly hydrolyzed, and its derivatization products are highly electroactive. Another reagent is especially suited to derivatize those proteins whose isoelectric points are higher than 10: Ferrocenylmethyl-succinimidyl-glycine-hydrochloride. This compound develops its derivatization activity only with pH values higher than 9, but it is rather difficult to prepare.     

Derivatization of Ketosteroids for High-Performance Liquid Chromatography with Electrochemical Detection

Abstract

A highly sensitive and selective derivatization of ketosteroids for use in high-performance liquid chromatography with electrochemical detection (HPLC/EC) is described. When the detector response for three phenylhydrazone derivatives was compared with one another using dehydroepiandrosterone, the p-nitrophenylhydrazone showed the highest sensitivity with a detection limit of 200 pg. Dehydroepiandrosterone and other principal 17-ketosteroids in human blood were quantitatively derivatized into p-nitrophenylhydrazones and efficiently separated by HPLC/EC on a μBondapak C_{1 8} column using 0.5%. NH₄H₂PO₄/methanol (2:7, v/v).     

Separtion of Menthol Isomers by Normal Phase High Performance Liquid Chromatography (1)

Abstract

An HPLC method has been developed for the separtion of the four isomers of methol using isocaratic and normal phase ethyl acetate/isooctane systems. This method has used to detect and measure these isomers in peppermint techniques. It is more rapid than GC which in addition requires unstable columns for similar analysis. Beacause solvent and column are normal phase and isocratic, the method and sample preparation are very simple.     

An Investigation of the Metabolism of Amitriptyline Using High Performance Liquid Chromatography

Abstract

A systematic study of the metabolism of the antidepressant amitriptyline was conducted using an inbred strain of rats. Variables affecting rat liver metabolism in vitro that were examined include age of the rat, the substrate concentration, and pH. A liquid-liquid extraction with hexane-butanol and back extraction into phosphoric acid was developed to provide efficient extraction for the wide range of polarity exhibited by amitriptyline and seven metabolites (amitriptyline-N-oxide, cis and trans isomers of 10-hydroxy-amitriptyline, cis and trans isomers of 10-hydroxynortriptyline, nortriptyline, and desmethylnortriptyline). HPLC was performed with 5 μm C-8 reversed phase column and a methanol/sodium phosphate buffer/amine modifier mobile phase.     

Enantiomeric separation of D,L-tryptophan and D,L-kynurenine by HPLC using pre-column fluorescence derivatization with R(-)-DBD-PyNCS

The enantiomeric separation of d ,l ‐tryptophan (Trp) and d ,l ‐kynurenine (KYN) was investigated by high‐performance liquid chromatography using pre‐column fluorescence derivatization with a chiral fluorescent labeling reagent, R(−)‐4‐(3‐isothiocyanatopyrrolidin‐1‐yl)‐7‐ (N,N‐dimethylaminosulfonyl)‐2,1,3‐benzoxadiazole [R(−)‐DBD‐PyNCS]. Using an octadecylsilica column, namely, an Inertsil ODS‐3 column (250 × 2.0 mm; i.d., 3 µm), four fluorescence peaks of D‐ and l ‐Trp as well as d ‐ and l ‐KYN derivatized with R(−)‐DBD‐PyNCS were clearly observed, and their chemical structures were confirmed by HPLC–time‐of‐flight–mass spectrometry. Simultaneous separation was achieved under the mobile phase condition of 1.5% acetic acid in H₂O–CH₃CN (60:40), and the separation factors of d ,l ‐Trp and d ,l ‐KYN derivatized with R(−)‐DBD‐PyNCS were 1.22 and 1.19, respectively. Fluorescence detection was carried out by setting the emission wavelength at 565 nm, and the excitation wavelength at 440 nm, and the detection limits were approximately 0.3–0.5 pmol (signal‐to‐noise ratio of 3). Copyright © 2010 John Wiley & Sons, Ltd.     

New high-performance liquid chromatographic method for sensitive determination of pheomelanin in biological materials by precolumn fluorescence derivatization with naphthalene-2,3-dicarboxaldehyde

Pheomelanin is an important type of melanin distributed in the skin, eye and hair in the mammal, which is of great social, clinical and cosmetic significance. In this study, a new HPLC method with fluorescence detection is described originally for the sensitive determination of pheomelanin in biological materials. The pheomelanin polymer is decomposed into two specific degradation products, 3-amino-4-hydroxyphenylalanine (3-AHP) and 4-amino-3- hydroxyphenylalanine (4-AHP) with hydriodic acid. Then the two AHP isomers are derivatized using a fluorescent probe naphthalene-2,3-dicarboxaldehyde in the presence of cyanide. The resulting highly stable 2-substituted 1-cyanobenz[f] isoindole derivatives were separated on a 5NH(2)-MS aminopropyl packed HPLC column with binary isocratic elution profile and detected fluorimetrically. The assay shows high sensitivity of 0.11nM (2.2fmol per injection, the lowest reported) at signal-to-noise ratio of 3 for each AHPs, good accuracy and precision (RSDs<3.1%), and linearity (range of 0.02-10microM, r>0.995). The results obtained by using fluorescence detection have been compared with other detection systems (electrochemical and UV). The sensitivity can increase from 100 to respect electrochemical detection and 30000 times respect UV detection. The method has been used for the quantitative determination of pheomelanin in various biological samples, including cell cultures from five types of melanoma cell lines of human and rat origin, hair samples of various colors, melanoma tissue and the urines from human melanoma patients and healthy subjects. This original application of HPLC-fluorescence detection represents a powerful tool for investigating pheomelanin synthesis in vitro and in vivo under physiological and pathophysiological conditions.     

New method for high-performance liquid chromatographic separation and fluorescence detection of ginsenosides

A novel pre-column derivatization method for the quantitative determination of ginsenosides by HPLC with fluorescence detection was established. The double bond at the C₂₄–C₂₅ position of ginsenoside was converted into an aldehyde group by means of ozonolysis. Then the aldehyde group reacts with FMOC-hydrazine forming the ginsenoside FMOC-hydrazone. The derivatized products were separated by RP-HPLC with gradient elution. The detection limits of ginsenosides Rg₁ and Rb₁ were 2.0 ng (about 2.5 pmol) and 1.0 ng (about 0.9 pmol), respectively. This method can be used for all ginsenosides having the C₂₄–C₂₅ double bond.     

应用C_(30)色谱柱高效液相色谱法同时分析枇杷叶中三萜酸类同分异构体

建立了一种快速、简便测定中药枇杷叶中4种三萜酸成分的液相色谱分析方法。枇杷叶经甲醇提取并定容后,采用C30色谱柱进行分离,以乙腈-水(95∶5,v/v)为流动相进行洗脱,流速为1.0 mL/min,于210 nm波长下检测。对不同产地枇杷叶中的山楂酸、科罗索酸、齐墩果酸和熊果酸4种同分异构体进行了测定和比较。结果表明,该方法分离度好(分离度(R)≥2.2),精密度高(RSD≤1.1%),线性关系良好(r≥0.999 2),重现性良好(RSD≤4.4%),加标回收率范围为95.4%~101.7%(RSD≤4.8%),满足定量要求。该方法简便、快速,结果可靠,可作为枇杷叶质量评价的方法。     

Photoisomerization kinetics of trifloxystrobin

The photoisomerization kinetics of trifloxystrobin (TFS) in acetone under artificial sunlight is reported. HPLC analysis showed the TFS, a strobilurine fungicide of EE conformation, was converted into an equilibrium mixture of four isomers after illumination for 7 h. The isomers were identified as EZ, EE, ZZ, and ZE and were separated in the crystalline form by preparative HPLC and characterized by use of a variety of spectroscopic techniques. The quantum yield and reaction constants for the isomerization reactions were determined. The detailed spectral features of the individual isomers measured by UV, IR, Raman, NMR and mass spectroscopy are presented and compared. The spectra of the isomers were found to be very characteristic, with good analytical significance.     

Study of the inhibitory effect of ferulic acid on short-chain fatty acids in milk samples by dispersive liquid–liquid micro-extraction in combination with fluorescence derivatization

A sensitive high-performance liquid chromatography (HPLC) method was developed to study the influence of ferulic acid on the formation of volatile fatty acids and lactic acid in milk and soybean milk samples. Volatile fatty acids were extracted by liquid–liquid micro-extraction using chloroform and acetonitrile as the extraction and disperser solvents, respectively. The analytes were derivatized with 2-(5-benzoacridine)ethyl-p-toluenesulfonate that showed excellent fluorescence property and made the sensitive HPLC analysis of short-chain fatty acids become possible. The optimized HPLC sensitivity was in the range of 1.1–1.9?µg?L^?1. Ferulic acid was added in milk and soybean milk samples to study its preservative effect. The results indicated that ferulic acid with concentration of 0.2% (m/v) could effectively reduce the formation of short-chain fatty acids.     

Analysis of Taurine in Blood Plasma of Epileptic Patients Using an Improved Isocratic HPLC Method for Amino Acids

Abstract

An efficient, reversed-phase HPLC method is described in which 21 derivatized amino acids were isocratically separated in less than 90 minutes. An internal standard was used to improve precision and accuracy. The method, which separated taurine from α-, β-, and γ-aminobutyric acids, was used to quantify taurine levels in 83 blood plasma samples. A statistical comparison was made between taurine levels found in the plasma of epileptic and non-epileptic children.     

Thermal degradation of phytate produces all four possible inositol pentakisphosphates as determined by ion chromatography and 1H and 31P NMR spectroscopy

Abstract

Decomposition of phytate has recently been shown to occur under mild conditions in the solid state, giving rise to a complex mixture of lower inositol phosphates. In this study, the reaction products of this thermal, abiotic degradation of phytate were separated using ion chromatography and the most highly phosphorylated products subsequently identified using 1D and 2D NMR spectroscopy. Two late eluting fractions were each shown to be a mixture of two specific inositol pentakisphosphate isomers. The peak with shorter retention time contained Ins(1,2,3,4,6)P₅ and DL-Ins(1,2,3,4,5)P₅, while the later eluting fraction contained Ins(1,3,4,5,6)P₅ and DL-Ins(1,2,4,5,6)P₅. The formation of all four possible inositol pentakisphosphate isomers by thermal degradation of phytate contrasts with the selective production of unique inositol pentakisphosphate isomers during enzymatic phytate degradation and therefore suggests a method for differentiating abiotic and biotic processes in environmental samples, including soils and decomposing plant biomass.