Mass Spectrometry and Liquid Chromatography - Mass Spectrometry of β-Amino ß-Nitrostilbenes

Abstract

(Z)-α-amino ß-nitrostilbenes are obtained by the reaction of either ammonia or primary or secondary amines on (Z)-α, ß-dinitrostilbene (DNS). The fragmentometric study of these compounds by electron impact and chemical ionization was carried out. The conditions in which it may be possible to determine the purity of these compounds were obtained by HPLC on reversed phase.

However the resolution factor between DNS and benzyl- or phenyl-aminonitroenamine is not sufficient for UV detection; only liquid chromatography-mass spectra coupling with negative ionization enables the detection of the possible presence of DNS in these nitro enamino derivatives.     

Electrochemical Detection of Retinoids Using Normal Phase HPLC

Abstract

Non-aqueous electrochemical (EC) detection of 13-cis-retinoic acid, all-trans retinoic, acitretin and vitamin A palmitate in non-aqueous solvents are reported.

Non-aqueous (EC) detection allows for normal-phase chromato-graphy of these compounds prior to detection. The normal phase system used a mobile phase of HEX/THF/AcOH for the separation of all four compounds. The stationary phase was either silica or PVA-sil. The lipophilic salts, t-butylammoniumtetrafluoroborate or t-butyl-ammoniumhexafluorophosphate necessary for EC detection were added post-column.

The limit of detection (LOD) for EC detection of these compounds is approximately 1 ng on column compared with an LOD by UV absorption of 2 ng on column.

The linear detection for these compounds with the EC detector was about two orders of magnitude.     

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.     

A New Reagent for Determination of Isocyanates in Working Atmospheres by HPLC Using UV or Fluorescence Detection

Abstract

A new liquid chromatographic method with increased sensitivity has been developed for the determination of isocyanates common in industrial environments. The isocyanates are converted to stable urea derivatives by reaction with 9-(N-methylaminomethyl)-anthracene. These derivatives were analyzed using high performance liquid chromatography on a bonded octadecylsilyl phase using isocratic elution with acetonitrile/water and detected either by a UV or a fluorescence detector. The method was applied to toluene 2, 4- and 2, 6-diisocyanate (2, 4- and 2, 6-TDI), hexamethylene diisocyanate (HDI) and 4, 4-diphenylmethane diisocyanate (MDI).

The influence of various salts on the retention of the reagent amine was studied, as well as the separation of the urea derivatives on different C₁₈-phases. The detection limit is about 1 · 10^?4 mg/m³ for the isocyanates investigated, using either UV or fluorescence detection. This means that the new method is ten to twenty times more sensitive than the previously described reversed phase LC method, which utilized N-4-nitrobenzyl-N-n-propylamine as reagent.     

HPLC Determination of Methylphenidate in Human Plasma

Abstract

A simple, rapid and sensitive method for measuring methylphenidate in human plasma by HPLC has been developed. After the addition of the internal standard, ethylphenidate, the two compounds are extracted under basic conditions. The residue obtained is resuspended in acetonitrile and analysed on an ODS reversed phase column with detection by UV absorbance at 192 nm. The limit of sensitivity is 5 ng/ml and the procedure is linear over the 5–50 ng/ml concentration range.     

Simultaneous Determination of Diazepam and its metabolites in Plasma by High-Performance Liquid Chromatography

Abstract

A reversed phase high-performance liquid chromatographic method (HPLC) for the simultaneous determination of diazepam and its three active metabolites, nordazepam, oxazepam and temazepam, in plasma was proposed. The compounds were isolated by solid-phase extraction. The chromatographic mobile phase was metanol-water (55:45, v/v) at a flow rate of 1 mL/min. UV detection was performed concurrently at 240 and 254 nm.     

HPLC Analysis of Adriamycine as N-2,4-Dinitrophenyladriamycine

Abstract

Derivatization of adriamycine (I) by reaction with 2,4-dinitrofluorobenzene, followed by HPLC analysis on reversed phase (RP-18 μ-Bondapak) or on normal phase (μ-porasil) and detection at 482 nm, provides a fast method (R_f=4.0 min) for determination of adriamycine [as N-2,4-dinitrophenyladriamycine (III)] in the 1–10 ppm range. Reaction with 2,4-dinitrofluoro[^?14C]benzene, followed by HPLC separation and detection on a γ counter, extends the detection limit to 0.04 ppm adriamycine.     

Liquid Chromatographic Determination of 4-Amino-N-(2,6-dimethylphenyl)-benzamide in Rat Serum and Urine

Abstract

The compound 4-amino-N-(2,6-dimethylphenyl)-benzamide has shown potential as a new anticonvulsant. A method for the liquid chromatographic determination of serum and urine concentrations of the compound and its N-acetylated metabolite was developed for pharmacokinetic studies. Quantitation was achieved via UV detection at 275 nm following isocratic reversed phase (C₁₈) separation using a ternary solvent system of water:acetonitrile:acetic acid (60:39:1) at a flow rate of 1.5 mL/min. The compounds were isolated from a 50 μL sample of serum using solid phase extraction with prior protein precipitation. The compounds and internal standard were eluted from the extraction column with acetonitrile. Isolation from urine was achieved similarly with the exclusion of protein precipitation. The assay procedure is useful for the determination of concentrations of parent compound from 0.68 to 204.6 μg/mL.     

Off-Line and On-Line Preconcentration Techniques for the Determination of Phenylureas in Freshwaters

Abstract

Phenylurea herbicides are analysed by reversed-phase liquid chromatography using UV detection at 244 nm after a concentration step in order to determine ppb or sub-ppb levels in drinking and river waters. With an average UV detection limit of 5 ng, a 500 ml sample volume is necessary to reach the 10 ppt level for spiked LC grade water samples and enables easy determination of concentrations below the ppb level for river water samples. Off-line and on-line methods are compared for the concentration step. Off-line concentration consists in a liquid sorption on n-octadecyl silica (C18) and elution by a suitable organic solvent. Polar phenylureas have low retention volumes on C18 silica and consequently the length of the concentration column has to be 10 cm to concentrate them at the ppb level from 100 ml of water and longer for lower levels of detection. Nevertheless, we show that increasing the size of the concentration column does not improve the limits of detection because of the numerous interferences also concentrated when percolating high volumes of water. On-line technology can be used only with short precolumns and requires a sorbent with a great retention for phenylureas. The copolymer-based PRP-1 is found to be an excellent sorbent and it is then possible to apply on-line precolumn technology with preconcentration through two precolumns (10 × 21 mm ID) in series, the first one being packed with C18 silica and the other with the PRP-1 polymer. Interfering compounds are then trapped onto the first precolumn acting as a filter and common phenylurea-breakthrough volumes on the PRP-1 precolumn are higher than 500 ml. Knowing the amounts preconcentrated on both precolumns and using UV and electrochemical detection help the identification of phenylureas in river water.     

Differential Measurement of Cortisol and Cortisone in Human Saliva by HPLC with Uv Detection

Abstract

Both cortisol and its dehydro metabolite cortisone are present in normal human saliva. A method for differential Measurement of both compounds in 1 ml samples of saliva by HPLC/UV is described. the method uses an extraction column having a cyclodextrin bonded phase to retain the compounds of interest while allowing elution of interfering compounds. A steroid-bearing fraction is eluted from the cyclodextrin column, dried, reconstituted in a weak mobile phase, and injected on a reversed phase HPLC/UV system provided with an injector-mounted reversed phase extraction column. Samples containing corticosteroid concentrations as low as 0.5 ng/ml can be effectively analyzed by this method.     

Simultaneous Determination of Diazepam,Oxazepam and Temazepam in Spiked Urine By Hplc

ABSTRACT|Oxazepam and temazepam are two minor metabolites of diazepam. These three benzodiazepines may be found in presence of each other in biological fluids.

Therefore, in this study an HPLC method was developed to separate and analyze them. Benzodiazepines have ability to form inclusion complexes with p-cyclodextrin (β-CyD). According to the degree of binding constant with β-CyD, these compounds can be separated by β-CyD bound to silica (cyclobond column) as the stationary phase using HPLC.

The development and validation of the HPLC procedure for the separation and determination of these compounds in mixtures were studied. The mobile-phase system consisted of phosphate buffer (pH 7): methanol [75:25], with flow rate 0.8 ml min^?1 and UV detection at 240 nm was used.

The calibration graphs were rectilinear from 0.1-2.5 μg/ml and coefficients of variation were <2% for the three compounds in bulk forms.

The method was used to analyse these bezodiazepines in spiked urine containing all three compounds in combination. Recoveries were 97-99.8%

The limit of detection and limit of quantitation were 0.05 μg/ml and 0.1 μg/ml, respectively.

The described method is selective, rapid, simple, reproducible and accurate.     

Analysis of Phosmet and Azinphos-Methyl in Apples by High-Performance Liquid Chromatography

Abstract

A high-performance liquid chromatography (HPLC) method has been developed to analyze two organophosphate insecticides (phosmet and azinphosmethyl) in apples. The procedure includes a novel extraction whereby whole apples are sonicated for 2 min in 100 ml of MeOH to remove the pesticides. Reversed-phase HPLC separation was accomplished with an Ultremex C18 column and acetonitrile:methanol:water as the eluent. Detection was at 224 nm for phosmet and 300 nm for azinphos-methyl. For both pesticides the limit of detection was 0.5 ppb and the linearity was from 1 to 405 ng injected. Average recoveries were 80% for phosmet and 86% for azinphos-methyl. Thirteen apple varieties comprising 240 apples were analyzed from supermarkets and roadside stands for phosmet (amount found ranged from none detected to 1233 ppb) and azinphos-methyl (amount found ranged from none detected to 388 ppb). Confirmation of phosmet and azinphos-methyl was made by UV spectral scans.     

Electrochemical Detection in Liquid Chromatography: Application to Organometallic Speciation

Abstract

The development of a new technique for the measurement of organometallic species is presented. It combines the resolution of high performance liquid chromatography with sensitive electrochemical detection used in a reductive mode. Past difficulties with this detection system have been overcome including the choice of a suitable working electrode and purification of the solvent.

The redox behavior of the organometals in the chromatographic solvent was studied by cyclic voltammetry in order to optimize the detector cell potential.

A separation of organomercurials and a multielement organometal mixture demonstrate the applicability of the system. Linear calibration curves can be obtained over a wide concentration range the detection limit for trimethyllead cation is about 0.1 ng.     

Determination of phenyltin compounds by reversed-phase liquid chromatography

An analytical procedure for the determination of phenyltin compounds in environmental sample waters was studied. Chromatography of mono-, di- tri-phenyltin (MPT, DPT and TPT) was performed on a reversed-phase C₁₈ column with the mobile phase comprising methanol/10⁻² M H₃PO₄ (80:20 v/v) at pH 3 and UV detection at 214 nm. To enhance the sensitivity of the detection system, the post-column reaction between morin or 3-hydroxyflavone and phenyltin compounds was formed before fluorescence detection. Several parameters affecting the fluorescence intensity were studied systematically, including the optimum condition for the post-column reagent that was also compatible with the eluent. The parameters concerned in this study were the pH, the percentage of Triton X-100, the ratio of fluorigenic reagent to phenyltin compounds and the amount of methanol in the eluent. Detection limits before the preconcentration process were in the region of 1.5 ppb for TPT and 150–250 ppb for MPT and DPT, respectively. Utilizing solid-phase extraction on a C₁₈ cartridge for sample clean-up as well as preconcentration successfully reduced the detection limit of TPT to the level of ng dm⁻³ and can be applied to seawater analysis. Recovery in the range 95.0–98.0% was obtained by developing the optimum elution profile in the preconcentration step. © 1998 John Wiley & Sons, Ltd.     

A Sensitive High Performance Liquid Chromatography Assay for Trospectomycin. An Aminocyclitol Antibiótic,in Human Plasma and Serum

Abstract

This report describes a sensitive, selective and robust assay for the quantification of trospectomycin, an aminocyclitol antibiotic, in human plasma and serum.

This is the first published High Performance Liquid Chramatography (HELC) bioanalytical method for a member of this class of compound.

The method involves the selective solid phase extraction of 6′-n-propyl spectinomycin and the internal standard 6′-n-butyl spectinonycin from 0.5 ml of biofluid, efficient reversed phase high pressure liquid chromatography with post column oxidation, reaction with o-phthaldialdehyde and fluorescence detection. The limit of quantification from 0.5 ml of biofluid is 10 ng/ml.     

The Analysis of Sulphonamide Drug Residues in Pork Muscle using Automated Dialysis

ABSTRACT

The aim of this study was to assess the suitability of automated dialysis, using a commercial system, for the analysis of sulphonamides in porcine tissue. The system involves automated dialysis, followed by trace enrichment of the dialysate prior to HPLC determination. The procedure was applied to the analysis of nine sulphonamide drug residues using reversed phase HPLC as the method of determination. Muscle samples were blended in saline, centrifuged and the supernatant was filtered before dialysis for an optimised time of 11 min. The resulting dialysate was concentrated on a reversed phase trace enrichment cartridge prior to HPLC analysis with UV detection at 280 nm. The developed method was evaluated by carrying out intra- and inter-assays on fortified porcine muscle. Mean recoveries, evaluated from the inter-assay study, were 80% or higher for the nine sulphonamides studied and the limit of determination for the method was 40 ng g^?1.     

Assay of Arginine-Esterase Activities by Reversed Phase High Performance Liquid Chromatography

Abstract

Application of a high performance liquid chromato-graphic technique to assay of arginine-esterase activities is presented. Enzyme reaction was carried out with benzoyl-L-arginine ethylester as a substrate and analysis was performed on a reversed phase chromato-graphic system using a μ Bondapak C₁₈ or a Radial-PAK A column and buffered aqueous methanol as the mobile phase. The enzyme activities were determined by the peak height of cleaved product (benzoyl-L-arginine). The minimum detection limit for benzoyl-L-arginine was 0.02 nM on each column. The generality of this method was demonstrated by its application to determination of plasmin activity, and so it might be suitable for both kinetic studies and routine assays of plasmin-like es-terases.     

High-Performance Liquid Chromatography of Domoic Acid,a Marine Neurotoxin,with Application to Shellfish and Plankton

Abstract

A recent outbreak of poisoning resulting from the consumption of cultured blue mussels (Mytilus edulis L.) from a localized area in Eastern Canada has been attributed to the presence of domoic acid (1), a relatively rare neurotoxic amino acid, previously found only in some algae of the family Rhodomelaceae. Studies on aqueous extracts of shellfish tissue indicated that the toxin and several of its isomers could be separated (and isolated in sufficient amounts for subsequent structural identification) by reversed-phase high-performance liquid chromatography (HPLC) with ultraviolet (UV) diode array detection (DAD). Aqueous acetonitrile containing 0.1% v/v trifluoroacetic acid was used as mobile phase. As the retention time and characteristic UV absorption spectrum of 1 (λ_max = 242 nm) permit unequivocal identification, the HPLC-DAD procedure was refined with a microbore column to provide a rapid (5 min), sensitive (0.3 ng detection limit) and reproducible assay method for the determination of 1 in shellfish tissue. Extraction was accomplished by boiling homogenized shellfish tissue for 5 min with distilled water. Extracts were taken through an octadecylsilica solid phase extraction clean-up prior to HPLC. This method has been applied to a variety of shellfish and phytoplankton samples.

BRIEF

Reversed-phase HPLC with ultraviolet diode array detection was used to analyze shellfish tissue and phytoplankton extracts for domoic acid. A rapid (5 min) and sensitive (0.3 ng detection limit) assay is presented.     

β-Naphthol as a Chromophore and Fluorophore Forming Reagent in the HPLC Determination of Alkyl Halides

Abstract

A suitable chromophore- and fluorophore- forming reagent for the detection of alkyl halide water contaminants is β-naphtol. The ether derivatives are separated from each other and excess β-naphthol reagent by reverse, phase HPLC and are detected by ultraviolet absorption or fluorescence. A method for the determination of alkyl halides is presented. The method is applicable to concentration levels in the ppm range for UV detection and in the ppb concentration range using fluorescence detection.     

Spectrofluorimetric Determination of Nanogram Amounts of Cadmium with Benzyl-2-Pyridylketone 2-Pyridylhydrazone

Abstract

A rapid, simple and sensitive spectrofluorimetric determination of cadmium based on the formation of a fluorescent chelate with benzyl-2-pyridylketone 2-pyridylhydrazone is described. In darkness, the fluorescence development is instantaneous and remains stable for 1 h. The detection limit is 11 ppb. The influence of reaction variables and the effect of foreign ions are discussed.