Fluorogenic Labelling of Carbonylcompounds with 7-Hydrazine-4-nitrobenzo-2-oxa-1,3-diazole (NBD-H)

Abstract

A method for the prechromatographic fluorescence derivatization of carbonyl compounds with 7-hydrazino-4-nitrobenzo-2-oxa-1,3-diazole (NBD-H) is presented. The separation and quantitation of the hydrazones is carried out by TLC and HPLC on silica gel and RP-ma-terials. Detection limits obtained for benzaldehyde by TLC with fluorodensitometric evaluation are 5 ng/spot and by HPLC with fluorescence detection 200 pg.     

Quantitative Analysis of a Chloroquinolin-Ethenyl Phenyl Derivative Using Thin Layers Impregnated With Di-P-Toluyl Tartaricacid

Abstract

Thin Layer Chromatography (TLC) is an established method for the evaluation of final product drug and intermediate impurity profiles. Quantitative TLC has gained credibility within the Pharmaceutical industry as a result of the latest developments in an availability of scanning technology. In the present paper we wish to report a quantitative TLC method for the determination of some potential impurities which may exist in a final bulk drug MK0679. In order to improve the selectivity of the chromatographic method, di-p-toluyl tartaric acid was impregnated on the stationary phase. Utilizing the modified layer, complete separation of the known impurity was obtained. The calibration curves for all components studied were linear and the detection limits obtained were less than 5ng.     

A New Detector System for Continuous-Flow TLC

Abstract

A new detector system for continuous-flow TLC is described that provides possibilities both for optical and for electrochemical measurements with low detection limits.     

LCEC Detector with Graphite-Teflon Electode

Abstract

A compression molded graphite-Teflon electrode is evaluated as a detector for the electrochemical detection of phenolic compounds, separated by reverse-phase high-performance liquid chromatography. Detection limits for these phenolic compounds range from 0.5–1 ng. Repetitive injections yield peak heights with RSDs less than 1%. Passivation of electrode surface by phenols is discussed. In addition, construction of a flow cell to be used in conjunction with graphite-Teflon electrode is also described.     

Comparison of Electrochemical and Spectro-Photometric Detection in Hrp-Based Elisa for Tobacco Mosaic Virus

ABSTRACT

An electrochemical method was compared with a spectrophotometric method using a horseradish peroxidase (HRP)-based indirect enzyme-linked immunosorbent assay (ELISA) with direct antigen coating (DAC) technique for the determination of tobacco mosaic virus (TMV). In substrates for a spectrophotometric HRP-based ELISA method, was selected as the substrate for the electrochemical method as well as the spectrophotometric method. 2, 3-Diaminophenazine is the product of the oxidation of OPD with H₂O₂ catalyzed by HRP in the selected conditions. It is electroactive and has a sensitive voltammetric response at -0.93 V (vs. Ag/AgCl) in alkaline solution. The detection limit of free HRP, by second-order derivative linear-sweep voltammetry, is 1.1 mU/L. This method can be further used to detect TMV antigen. The peak current is linear with TMV concentration in the range of 0.25-5.0 ng/mL. The detection limit for TMV is 0.25 ng/mL and the highest dilution ratio detected for the infected leaf sap is 1:102400. Compared with the classical OPD ELISA spectrophotometric method, the detection limits of the electrochemical method for the clarified TMV and the infected leaf sap detection are about ten and four times lower, respectively.     

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.     

Determination of Aldehydes and Ketones in Fuel Ethanol by High-Performance Liquid Chromatography with Electrochemical Detection

A new methodology was developed for analysis of aldehydes and ketones in fuel ethanol by high-performance liquid chromatography (HPLC) coupled to electrochemical detection. The electrochemical oxidation of 5-hydroxymethylfurfural, 2-furfuraldehyde, butyraldehyde, acetone and methyl ethyl ketone derivatized with 2,4-dinitrophenylhydrazine (DNPH) at glassy carbon electrode present a well defined wave at +0.94 V; +0.99 V; +1.29 V; +1.15 V and +1.18 V, respectively which are the basis for its determination on electrochemical detector. The carbonyl compounds derivatized were separated by a reverse-phase column under isocratic conditions with a mobile phase containing a binary mixture of methanol / LiClO_4(aq) at a concentration of 1.0 × 10⁻³ mol L⁻¹ (80:20 v/v) and a flow-rate of 1.1mL min⁻¹ . The optimum potential for the electrochemical detection of aldehydes-DNPH and ketones-DNPH was +1.0 V vs. Ag/AgCl. The analytical curve of aldehydes-DNPH and ketones-DNPH presented linearity over the range 5.0 to 400.0 ng mL⁻¹, with detection limits of 1.7 to 2.0 ng mL⁻¹ and quantification limits from 5.0 to 6.2 ng mL⁻¹, using injection volume of 20 μL. The proposed methodology was simple, low time-consuming (15 min/analysis) and presented analytical recovery higher than 95%.     

Analytical approach to determine biogenic amines in urine using microextraction in packed syringe and liquid chromatography coupled to electrochemical detection

The goal of this work was to develop and validate an analytical method for the detection and quantification of the biogenic amines serotonin (5‐HT), dopamine (DA) and norepinephrine (NE), using microextraction in packed syringe (MEPS) and liquid chromatography coupled to electrochemical detection (HPLC‐ED) in urine. The method was validated according to internationally accepted guidelines from the Food and Drug Administration. Linearity was established between 50 and 1000 ng/mL for 5‐HT and between 5 and 1000 ng/mL for DA and NE, with determination coefficients (R²) >0.99 for all compounds. The limits of quantification and detection were respectively 50 and 20 ng/mL for 5‐HT, and 5 and 2 ng/mL for DA and NE. Within‐ and between‐run precision ranged from 0.84 to 9.41%, while accuracy ranged from 0.79 to 12.76% for all compounds. The intermediate precision and accuracy were 1.50–8.36 and 0.54–13.51%, respectively. The method was found suitable for clinical routine analysis of the studied compounds, using a sample volume of 0.5 mL. This is the first study employing a commercially available MEPS column for the simultaneous detection and quantification of 5‐HT, DA and NE in urine by coulometric detection. Copyright © 2012 John Wiley & Sons, Ltd.     

An optimized method for the determination of volatile and semi-volatile aldehydes and ketones in ambient particulate matter

A method has been developed to measure aldehydes and ketones associated with atmospheric particles. Carbonyl compounds from particulate material collected on Teflon-coated glass-fiber filters were simultaneously extracted and derivatized with an appropriate 2,4-dinitrophenylhydrazine (2,4-DNPH) solution. The efficiency of this procedure utilizing various 2,4-DNPH concentrations and solvent compositions was studied for 13 carbonyl compounds of atmospheric importance. These include formaldehyde, acetaldehyde, acetone, dicarbonyls such as glyoxal and methylglyoxal, and biogenic carbonyls such as pinonaldehyde and nopinone. An extraction solution containing 3 × 10^?2 M 2,4-DNPH, in 60% acetonitrile/40% water, and pH 3 was most efficient in extracting and derivatizing these aldehydes and ketones (83-100% recovery). Improved sample enrichment and 2,4-DNPH purification methods were developed that afforded detection limits of 0.009-5.6 ng m^?3. The relative standard deviation for replicate analyses were 1.9-10.1%. Carbonyl compounds in ambient particulate samples were quantified during a recent field study. Median values for nine carbonyl species ranged from 0.01-33.9 ng m^?3 during the study.     

Determination of Organic Peroxides by High Performance Liquid Chroma-Tography with Electrochemical Detection

Abstract

The application of electrochemical detection to the high performance liquid chromatographic determination of organic peroxides has been studied. The use of a buffered mobile phase was found to be critical to the successful analysis of samples containing hydroperoxides. Using amperometric detection, mixtures of peroxide containing compounds were readily determined. The sensitivity of the amperometric detector was in the one nanogram range for both benzoyl peroxide and cumene hydroperoxide. Polar-ographic detection was found to be a highly reproducible method for the analysis of samples containing peroxides as components of mixtures in the range of 5-2000 ng. The peroxide containing compounds determined in this manner were t-butyl hydroperoxide, cumene hydroperoxide, and 13-hydroperoxy-9(Z)-11(E)-octadecadien-oic acid. The polarographic detection system was used to obtain observed half-wave potentials for the peroxides under different chromatographic conditions. These observations correlated closely with literature results on the polarography of these compounds.     

Comparing Electrochemical,Fluorescence, and Ultraviolet Detectors for HPLC Analysis of the Decapeptide,Nafarelin

Abstract

This report describes a reverse-phase HPLC technique to determine the concentration of nafarelin (a decapeptide luteinizing hormone-releasing hormone analog) in aqueous solutions for intranasal administration. Pursuant to the method development we evaluated three different detectors with respect to sensitivity, linearity, specificity and reliability. The three detector types investigated were: spectrophotometric (225 nm), electrochemical (at +1.2 v), and fluorescence (excitation = 282 nm, emission = 332 nm). All three detectors gave satisfactorily linear response, and gave equivalent results for nafarelin samples assayed in parallel. The lower detection limits for the three detectors were: ultraviolet = 1.5 ng, electrochemical = 2.0 ng, and fluorescence = 0.6 ng. Thus, the three detector types are nearly equally sensitive for nafarelin analysis. For routine determinations the ultraviolet detector is superior to the electrochemical and fluorescence detectors with respect to convenience of operation.     

Determination of Sulfophenyl Carboxylic Acids in Agricultural Groundwater Samples by Liquid Chromatography with Fluorescence Detection

Abstract

We have developed an analytical method to determine six sulfophenyl carboxylic acids (SPC) in agricultural groundwater samples. It involves a solid-phase extraction (SPE) procedure and subsequent separation and determination using liquid chromatography with fluorescence detection (LC–FD).The quantification limits ranged between 0.7 and 1.2 ng ml^?1. The proposed method was proved satisfactorily for the detection and determination of these compounds in groundwater samples during a study into the biodegradation of linear alkylbenzene sulfonates (LAS) in an agricultural soil sampled from the irrigated plain to the west of Granada (Spain).     

A sensitive,specific method for the determination of tetraalkyllead compounds in air by gas chromatography/atomic absorption spectrometry

The development of a sensitive and specific method of determining individually the five tetraalkyllead compounds normally present in ambient air is described. The method is based on collection of the analytes on a porous polymer using a prefilter to destroy ozone and prevent decomposition of the sample during collection and storage. Two-stage thermal desorption, separation by gas chromatography and detection by modified flame atomic absorption spectrometry gives detection limits (3σ) of ca. 0.25 ng Pb m^?3 for tetramethyllead and 0.37 ng Pb m^?3 for tetraethyllead in an air sample of 80 dm³ collected over a 3–24 h period. Environmental sampling by this method in paralle with a wet chemical (iodine monochloride) method was used to validate the method. During the comparative study, higher organic lead levels were consistently found by the iodine monochloride method; it is postulated that this indicates the presence of vapour-phase tri-(or di)-alkyllead in excess of 1 ng Pb m^?3 in rural air. The versatility of the method is demonstrated by the results of atmospheric sampling at two locations, one rural and one at the kerbside in a city centre.     

Gradient Elution of Biogenic Amines and Derivatives in Reversed Phase Ion-Pair Partition Chromatography with Electrochemical and Fluorometric Detection

Abstract

The compatibility of gradient elution and reversed phase ion-pair partition systems combined with electrochemical and fluorometric detection has been investigated. The phase system consisting of buffers with perchlorate counter-ions as mobile phases and tri-n-butylphosphate as stationary phase allows the use of pH and counter-ion gradients. It appeared that (i) use of gradients is time saving and favourable with respect to detection limits and (ii) dual electrode detection may offer a solution to the problem of gradient-induced baseline shifting in electrochemical Electrochemical detectors are rather sensitive towards changes in the mobile phase. Applying the differential coulometric detection principle the gradient induced baseline shift is reduced and consequently the quantification of a number of compounds is facilitated, although noise levels are slightly increased.

Future research will be devoted to noise investigations in order to minimize detection limits.     

Use of SPE—TLC for Quality Control of Rhodiola rosea Extracts

SPE and TLC have been used for qualitative and quantitative analysis of salidroside, rosavin, rosarin, and rosin in commercially available dry extracts from Rhodiola rosea roots. The best separation of all the compounds was achieved on silica gel TLC plates with ethyl acetate—methanol—water, 77 + 13 + 10 (v/v), as mobile phase. UV detection was performed at λ = 215 nm for salidroside and at λ= 245 nm for the rosavins (rosavin, rosarin, and rosin). Detection limits for salidroside and the rosavins were 90 ng and 60 ng per spot, respectively. Results from quantitative analysis confirmed the manufacturer’s declaration of the amounts of salidroside and the rosavins in the extracts.

     

A simple and sensitive high‐performance liquid chromatography–electrochemical detection assay for the quantitative determination of monoamines and respective metabolites in six discrete brain regions of mice

A rapid, sensitive, and reproducible assay is described for the quantitative determination of the monoamine neurotransmitters dopamine, norepinephrine and serotonin, their metabolites, and the internal standard 3,4‐dihydroxybenzlyamine hydro‐bromide in mouse brain homogenate using high‐performance liquid chromatography with electrochemical detection. The method was validated in the following brain areas: frontal cortex, striatum, nucleus accumbens, hippocampus, substantia nigra pars compacta and ventral tegmental area. Biogenic amines and relevant metabolites were extracted from discrete brain regions using a simple protein precipitation procedure, and the chromatography was achieved using a C₁₈ column. The method was accurate over the linear range of 0.300–30 ng/mL (r = 0.999) for dopamine and 0.300–15 ng/mL (r = 0.999) for norepinephrine, 3,4‐dihydroxybenzlyamine hydro‐bromide, homovanillic acid and 5‐hydroxyindolacetic acid, with detection limits of ~0.125 ng/mL (5 pg on column) for each of these analytes. Accuracy and linearity for serotonin were observed throughout the concentration range of 0.625–30 ng/mL (r = 0.998) with an analytical detection limit of ~0.300 ng/mL (12 pg on column). Relative recoveries for all analytes were approximately ≥90% and the analytical run time was <10 min. The described method utilized minimal sample preparation procedures and was optimized to provide the sensitivity limits required for simultaneous monoamine and metabolite analysis in small, discrete brain tissue samples.     

Simultaneous Determination of Monoamines,Their Precursor Amino Acids,and Related Metabolites in Mice Brain by High‐Performance Liquid Chromatography with a Coulometric Electrode Array System

Abstract

A method based on high performance liquid chromatography with a coulometric electrode array system (HPLC‐coulometric electrode array) using C₁₈ column has been developed for the simultaneous determination of norepinephrine (NE), epinephrine (E), l‐3,4‐dihydroxyphenylalanine (l‐DOPA), p‐tyrosine (p‐TYR), dopamine (DA), m‐tyrosine (m‐TYR), 5‐hydroxytryptamine (5‐HT), homovanillic acid (HVA), and 5‐hydroxyindoleacetic acid (5‐HIAA) in mice brain. The chromatography was performed using a C₁₈ column (250 mm×4.6 mm i.d. and 5 µm) with sodium acetate buffer (pH 5.0, 0.05 M) and methanol as the mobile phase. Elution of analytes was carried out at a flow rate of 1.0 ml/min. The nine compounds were monitored using an ESA electrochemical detector. Potentials of three electrodes in series were set at 200, 500, and 700 mV, respectively. Optimization of the pH of the mobile phase and the proportion of methanol were also considered. The minimal detection limits were 2–8 ng/ml. Linear (r=0.99) detector performances were observed within a range of 10～2000 ng/ml. Recoveries for the nine compounds in spiked samples were over 90% and the relative standard deviations (RSD) were less than 4.0%.     

Determination of Benzidine and its Congeners by Liquid Chromatography with Electrochemical Detection

High performance liquid chromatography coupled with electrochemical detection (HPLC-EC) method has been developed for the determination of benzidine and its related congeners in wastewater at the low ng/mL level. With a µ-Bondapak C₁₈ column, 53% acetonitrile (pH 4.7), and electrochemical detection at +1.0 V applied potential, the detection limits range from 3 ng/mL for benzidine to 10 ng/mL for 3,3′-dichlorobenzidine, assuming a 100 µL injection. The detection limits can be further lowered to the sub-ng/mL level by incorporating a short precolumn into the HPLC system for on-line sample enrichment. Recoveries are generally greater than 80% — except for benzidine and 3,3′-diaminobenzidine which give 50–70% recoveries. Industrial wastewater samples from a coke-plant and a plant producing 3,3′-dichlorobenzidine based dyes were successfully studied via this method.     

Validation of the Method of Selected Polychlorinated Biphenyls Determination in Human Milk Samples

A method for determination of trace concentrations of individual PCB congeners in human milk was validated. The analytical procedure included the following steps: acetone : hexane extraction, clean-up of extracts with concentrated sulfuric acid and solid phase extraction (SPE) on Florisil. The identification and quantification of analytes in purified extracts were carried out by high-resolution gas chromatography (HRGC) with electron capture detection (ECD) and/or with low-resolution mass spectrometry (LRMS). Recoveries of 14 PCB congeners from spiked cow milk samples, based on HRGC-ECD were between 87.3 and 93.6%. The precision of analyte determination was established as close to or less than 10%. The detection limits ranged between 0.14 and 0.26 ng/g fat and the quantification limits between 0.57 and 0.86 ng/g fat. The method was linear and characterized by good correlation coefficients (>0.99) for most of the compounds studied. The quality of the method under validation was verified by the analysis of Standard Reference Material (CRM-450) and interlaboratory exercise.     

High Performance Liquid Chromatography-Electrochemical Assay for Monitoring the Formation of 8-Oxo-7,8-dihydroadenine and its Related 2′-Deoxyribonucleoside

Abstract

A sensitive method involving the combined use of reversed-phase high-performance liquid chromatography and electrochemical detection has been developed for monitoring the formation of 8-oxo-7,8-dihydroadenine and 8-oxo-7,8-dihydro-2′-deoxyadenosine. The limit of detection for both compounds is close to 0.2 ng under optimised conditions of detection at an oxidation potential of 850 mV. This assay allows the quantitative measurement of the hydroxylated purine components in aqueous solutions of adenine and 2′-deoxyadenosine exposed to a dose of ionizing radiation as low as 2 Gray (Gy).