Catecholamine: „On-Line” probenaufarbeitung und nachsäulenderivatisierung

Summary We developed an automated, two-column HPLC-method that can be used routinely to quantify the catecholamines norepinephrine and epinephrine in body fluids. The method is based on a new, laboratory-prepared SEC-HPAC column material, on the application of a microprocessor-controlled column-switching technique, and on the use of an integrated reaction system for postcolumn derivatization and trihydroxyindol-fluorescence monitoring. It allows the direct injection (upto 500 μl) of an appropriate biological fluid and distinguishes on its integrated sample-processing mode, its practicability, its chemoselectivity and-specivity, its detection limit (2 pg), its within- and between-run precision and its speed of analysis.

     

A high-performance liquid chromatographic method for determination of flecainide in human plasma using fluorescence detection

Summary A high performance liquid chromatographic method for the determination of flecainide in serum has been developed. The analysis is performed on a microparticulate silica column. The eluate is monitored by fluorescence detection at an excitation wavelength of 300nm and an emission wavelength of 370nm. No sources of interference were identified and a coefficient of variation of less than 8% was observed on repeated flecainide determinations. The method has a good reproducibility, specificity and accuracy, and can be applied in therapeutic drug monitoring of flecainide in patients.     

Determination of polycyclic aromatic hydrocarbons in limpet samples

Summary An HPLC method with UV and programmed fluorescence detection has been optimized and used to evaluate the levels of contamination of PAHs in lightly contaminated biota samples. A specific stationary phase was used that offers high selectivity for PAH separation. The proposed method was applied to limpet samples from Fuerteventura (Canary Island) with good results.     

High performance liquid chromatography with post-column photolysis of pesticides for generation of fluorophores

Summary Several classes of pesticides have been found to respond to a high performance liquid chromatography post-column reaction detector that employs UV photolysis with an optional reaction with o-phthalicdicarboxaldehyde-2-mercaptoethanol (OPA-MERC) reagent followed by fluorescence detection. Photolysis of most of the N-methylcarbamates, carbamoyl oximes, carbamothioic acids, dithiocarbamates, and phenylureas tested produced primary amines which reacted with OPA-MERC to form the respective derivatives. In some cases, substituted aromatic pesticides such as phenylcarbamates, phenylamides, and phenylureas photolyzed to chemical species which had native fluorescence. This technique was successfully applied to a method for pesticide analyses in groundwater and vegetation. For aldicarb sulfone, a pesticide that did not absorb UV light, use of acetone as a photosensitizer enhanced detection.     

An improved analytical method for determination of heterocyclic amines in chicken legs

Summary Several extraction, separation and detection methods for heterocyclic amines (HAs) in chicken legs were evaluated by liquid chromatography. Results showed that the most appropriate extraction method includes the removal of macrosubstances by centrifugation and subsequent purification using a PRS (propylsulfonic acid silica gel) and a C₁₈ cartridge, and the recovery obtained ranged between 51 and 89 %. For HPLC separation, a binary solvent system consisting of acetonitrile and 0.05 M ammonium acetate solution (pH 3.6) with gradient elution with flow rate of 1.0 mL min⁻¹ and detection at 258 nm was used to resolve 16 HAs. With fluorescence nine HAs could be detected by employing a programmable wavelength, and the sensitivity was 100–400 times higher than that by UV detection. The detection limits for UV and fluorescence detection were 0.02≈0.5 ng and 0.05≈3 pg respectively, with a signal-to-noise ratio 3. The presence of HAs in fried chicken legs was also determined.     

High Performance Liquid Chromatographic Assay of Methoclopramide in Plasma Using a Silica Gel Column and An Aqueous Meobile Phase

Abstract

A high performance liquid chromatographic method for quantitating matoclopramide in plasma is presented. Proteins ara precipitated from the plasma sample with acetonitri la containing the internal standard, procainamida. The treated samples ara than analyzad using an Ultrasphere Si column, an aqueous solution at pH 7 of 65% CH³CN and 5.0 mM (NH₄)₂HPO₄ as a mobile phase, and a fluorescence detector. The retention times for drug and intarna1 standard ara 11.2 and 13.2 min, respectively. The caibration curve is Linear from 0.89 to 44.5 ng/ml. The detection limit is 0.89 ng/ml [signaL/hoisa = 31] for 0.2 ml plasma samples Pracision is measured by intraday and intarday coefficients o f variation, which are less than 10%. This method is currently being used for pharmacokinetic studies of methoclopramide.     

A Smartphone-assisted Paper-based Analytical Device for Fluorescence Assay of Hg2+

Rapid, efficiency and portable detection systems in low-resource settings with limited laboratory equipment and technical expertise are urgently needed. Herein, an integrated platform composed of a paper-based analytical device and a smartphone detection system for Hg²⁺ onsite testing was developed. Nitrogen-doped carbon dots(N-CDs) were synthesized by a simple hydrothermal method using citric acid as the carbon source and ethanediamine as the nitrogen source, which gave out bright blue fluorescence under the excitation at 350 nm UV light and the absolute fluorescence quantum yield was 17.1%. The fluorescence of the prepared N-CDs can be effectively quenched by Hg²⁺. In addition, an external attachment of smartphone for illumination and external light interference was designed to trace the fluorescence signals, and a software application of Android system with simple operation program was developed to perform snapshot and image processing. The smartphone-assisted detection system was combined with the N-CDs decorated paper chip to achieve the sensitive detection of Hg²⁺ in water samples. This integrated method for reliable sensing of Hg²⁺ shows a good linear detection range of 10-800 μmol/L(R²=0.9595) with detection limit of 1.07×10^-8 mol/L.     

Fluorescence enhancement for aflatoxins in HPLC by post-column split-flow iodine addition from a solid-phase iodine reservoir

Summary A method for post-column derivatization of the highly carcinogenic aflatoxins with iodine has been developed. It involves splitting of the mobile phase used for the reversed phase HPLC separation. One part flows through the injection valve and the C₁₈ analytical column to achieve the separation. The other part flows through a column packed with solid iodine. The iodine-containing solution is recombined with the flow coming from the analytical column. The derivatization reaction takes place in a knitted open tubular reactor maintained at 60 °C. Detection is done by fluorescence measurement. Due to the low solubility of iodine in the mobile phase, the iodine solid-phase column can be used for very long periods of time before refilling is necessary. The analytical system consists of only one pump and therefore gives the opportunity to carry out low-cost post-column reaction detection. The method yields reproducible results, a linear response over at least two orders of magnitude and detection limits of about 1 ppb, both for standard solutions and for peanut butter samples.     

Development of capillary electrophoresis methods for quantitative determination of taurine in vehicle system and biological media

CE methods have been developed for the determination of taurine in pharmaceutical formulation (microemulsion) and in biological media such as sweat. The CE system with end-column pulsed amperometric detection has been found to be an interesting method in comparison with UV and fluorescence detection for its simplicity and rapidity. A gold-disk electrode of 100 mm diameter was used as the working electrode. The effects of a field decoupler at the end of the capillary, separation voltage, injection and pressure times were investigated. A detection limit of 4 x 10(-5) mol/L was reached using integrated pulsed amperometric detection, a method successfully applied to taurine analysis of the biological samples such as sweat. For taurine analysis of oil-in-water microemulsion, fluorescence detector was the favored method, the detection limit of which was 4 x 10(-11) mol/L.     

Direct determination of the enantiomeric purity of oxyphenonium using chiral HPLC with post-column extraction detection

Summary A method is described for the determination of the enantiomeric purity (enantiomeric excess) of the anticholinergic drug oxyphenonium. The method for this quaternary ammonium compound is based on the direct HPLC analysis with a chiral stationary phase. Two kinds of 1-acid glycoprotein-bonded phases were used.For the detection a post-column extraction with fluorescence detection of the ion-pair counter ion dimethoxyantracene sulphonate was used.     

Sensitive and selective bioanalytical assay for the determination of dobutamine in rat plasma samples

Summary Dobutamine is one of the synthetic catecholamines acting directly onβ ₁-receptors. For the analysis of dobutamine in rat plasma samples, a selective and sensitive liquid chromatographic method is described. After a simple liquid-liquid extraction, separation of the analyte was performed using a reversed-phase ion-pair system with an octyl modified silica column. The solute was detected by fluorescence detection, applying an excitation wavelength of 285nm and an emission wavelength of 313nm. The (im)possibilities of the application of the normally used assays for the isolation, concentration and quantitation of catecholamines are discussed. By the addition of a minimum amount of modifier to the mobile phase, the selectivity of the system was increased significantly. With this method the detection limit is 9ng/ml in 0.2ml plasma samples. The application of the method is shown in rat plasma samples by measuring the concentration-time curves to establish plasma level-effect relationships for this drug.     

Micro fluorescent analysis system integrating GaN-light-emitting-diode on a silicon platform

A micro fluorescent analysis system is proposed using silicon micromachining. GaN blue light-emitting diode (LED) monolithically integrated on a silicon substrate is used as a light source for the fluorescent analysis system. The blue light suits the excitation of several dyes used commonly in fluorescent analysis. Silicon photodiode (Si-PD) that matches the visible and near infrared fluorescent wavelengths of dyes is integrated on a silicon substrate. Polydimethylsiloxane (PDMS) micro-channels are also stacked for flowing dye-sensitized liquid. Therefore, the proposed system is an integrated system that can be composed on a silicon platform, i.e. a bottom layer of Si-PD, a middle layer of GaN-LED on silicon substrate and a top layer of micro PDMS channel. An aperture is opened into the GaN-LED layer by deep reactive ion etching to create a ring-shaped GaN-LED and a through-hole for detection. The light from the ring-shaped GaN-LED in the middle layer excites the dye-sensitized liquid in the top micro-channel layer. The fluorescence emitted from dye is detected by the Si-PD on the bottom layer at an angle larger than 90 degrees from the direction of excitation. Therefore, the detection optics consist basically of a dark-field illumination optical system. In order to evaluate the performance of the integrated system, fluorescence of fluorescein isothiocyanate (FITC) solution flowing in the micro channel is measured. From the measurement, the noise, sensitivity and limit of detection in the fabricated system are evaluated for FITC dye to be 0.57 pA, 1.21 pA μM(-1) and 469 nM, respectively. From these results, a compact fluorescence analysis system is demonstrated.     

Simultaneous Quantification of Puerarin and Daidzein in Rat Plasma by High-Performance Liquid Chromatography with Post-Column Modification and Fluorescence Detection

A sensitive HPLC method based on post-column modification and fluorescence detection has been developed for determination of puerarin and daidzein in rat plasma. Chromatographic separation was performed on a C₈ column with a linear gradient prepared from 0.5% aqueous acetic acid and 0.5% acetic acid in acetonitrile, delivered at a flow rate of 0.8 mL min⁻¹. Naringin was used as the internal standard. It was necessary to use acetic acid in the mobile phase to achieve good separation, but this led to fluorescence signal suppression, because puerarin and daidzein have native fluorescence at pH 8.0–9.0. To enhance the sensitivity, post-column modification with alkaline buffer was adopted. After this modification, detection sensitivity for puerarin and daizein increased more than 500-fold and 600-fold, respectively, compared with direct fluorescence detection. Signal-to-noise ratios for detection for puerarin were more than 150 times better than for UV detection after use of the same method of sample preparation. This sensitive analytical method was successfully used to determine pharmacokinetic data for puerarin and daidzein in rat plasma after oral administration of a single dose of Puerariae radix extract containing puerarin (approx. 8.4 mg) and daizein (approx. 5.9 mg) to male SD rats.     

Improved HPLC determination of fluoxetine and norfluoxetine in human plasma

Summary An HPLC method with fluorescence detection has been developed for the determination of fluoxetine and its main metabolite norfluoxetine in human plasma. Pretreatment of the biological samples by liquid-liquid extraction was used to improve the sensitivity of a previously published SPE procedure. The method uses 200 μL plasma and recovery is good for both analytes. On a C₈ column with a mixture of perchlorate buffer and acetonitrile as mobile phase fluoxetine, norfluoxetine and the internal standard (paroxetine) were eluted in less than 9 min, without interference from the biological matrix. Response for both analytes was linearly dependent on concentration over the range 2.5–500 ng mL⁻¹, and repeatability (RSD%) was <4%. The limit of detection was 1 ng mL⁻¹ for both fluoxetines. Application to plasma samples from depressed patients treated with fluoxetine gave good results. There was no interference from other common CNS drugs. This method seems to be a useful tool for clinical monitoring, because it requires small plasma samples and is highly sensitive and highly selective.     

Enhanced sensitivity by laser-induced fluorescence for the determination of calcitriol and other vitamin D3 metabolites in plasma

Summary A sensitive method for the determination of hydroxymetabolites of vitamin D₃, parficularly calcitriol (1,25-(OH)₂-D₃), in human plasma is reported. The method is based on the use of laser-induced fluorescence detection as an alternative to conventional fluorimetry in an integrated cleanup/preconcentration, HPLC separation and post-column derivatization system. The derivatization step is based on a dehydration reaction which takes place in secosteroid structures at high temperature in a strong-acid medium. A LOD of 0.01 pg mL⁻¹ (SNR=3) was obtained for each analyte with a linear dynamic range over 4 order of magnitude with excellent regression coefficients (≥0.9922) in all cases. The precision was studied at two concentration levels and the RSDs values (for n=5) were acceptable (between 2.6 and 4.7%). The method was also checked by applying it to human plasma spiked with the target analytes and excellent recoveries were obtained. This is the first time that these species have been determined at the sub-pg mL⁻¹ level.     

Determination of adapalene (CD271/differin®) and retinol in plasma and tissue by on-line solid-phase extraction and HPLC analysis

Summary Adapalene, the active constituent of Differin^?, is a novel potent retinoid (vitamin A analogue) for the topical treatment of acne vulgaris. The clinical usefulness of retinoids is limited by a number of side effects, such as teratogenicity and skin irritation. A method has been developed for simultaneous determination of adapalene and retinol in plasma and tissue in in vivo and in vitro studies for the determination of the pharmacokinetic profile and the influence of adapalene on the endogenous retinol level. The new method was developed by coupling an autosampler to an automated solid-phase extraction unit on-line with a gradient HPLC system using UV and fluorescence detection. The low detection limit (0.25 ng mL⁻¹ for adapalene), the small sample weight (50 mg) and the high degree of automation make this method convenient for analysis of biological samples in animal and human studies. Presented at the 21^st ISC held in Stuttgart, Germany, 15^th–20^th September, 1996.     

Selective and sensitive analysis of 1-nitropyrene in diesel exhaust particulate extract by multidimensional HPLC

Summary A multicolumn (MC) HPLC method for the determination of 1-nitropyrene (1-NP) at trace-levels via on-line reduction to 1-aminopyrene and fluorescence detection is presented. On the first column, packed with a pyrenebutyric acid amide stationary phase, the nitro-derivatives of PAHs are strongly retained and separated from other matrix components. The nitro-PAHs-containing fractions are transferred onto a RP₁₈-column via stepwise gradient elution and finally separated according to their various lipophilicities and sizes. To increase the overall selectivity and sensitivity of the multidimensional method (MD-HPLC system) post-column, on-line reduction of the nitro-PAHs to the respective amino-PAHs via a short catalysis column is performed thus enhancing the sensitivity significantly (to low pg levels). The applicability of this method for the determination of trace amounts of 1-NP in real samples (diesel particulate extracts) is demonstrated.     

New detection modes for the determination of cephalosporins and their decomposition products

Summary The feasibility of using fluorescence and electrochemical detection after high performance liquid chromatography separation is investigated for a sensitive and selective determination of cephalosporins and their decomposition products. The sensitivity and detectability of fluorescence and electrochemical detection are compared to those of UV absorption detection. The methods have been validated for the determination of cephalosporins in biological fluids and for stability studiesin vitro.     

Determination of picomole amounts of carbonyls as luminarin hydrazones by high-performance liquid chromatography with fluorescence detection

Summary Fluorogenic reagents (luminarin 3, luminarin 11 and luminarin 12), having a quinolizinocoumarin moiety as fluorophore and a carboxylic acid hydrazide function as reacting group, have been developed. These reagents were found to be highly sensitive fluorescence derivatization reagents for aldehydes and ketones in high-performance liquid chromatography. The reagents readily react with carbonyl compounds in aqueous sulphuric acid solution (0.1 M) at room temperature to produce the corresponding hydrazone derivatives, which can be separated on both reversed or normal-phase column. The structures of the derivatives were studied, together with their properties in reversed and normalphase chromatographic systems. UV absorbance, corrected fluorescence spectral data and quantum yields of luminarin 3, luminarin 11 and luminarin 12 are presented. The detection limits (signal to noise ratio=3) for aldehydes and ketones were in the sub-pmol range. Luminarin 3 was also applied to the determination of hydroxymethylfurfural (HMF) in orange juices and concentrates. The method for HMF involves the solid-liquid extraction of the juice by using a C-18 cartridge prior to derivatization and normal-phase separation of the derivative with fluorimetric detection at 387 nmex., 444 nm em. The calibration curve was linear for amounts of HMF ranging from 0.1 to 10 nmol. Intrarun relative standard deviation was 12.8% for 0.1 nmol and 2.6% for 1 nmol. Recovery studies indicated an average of 98.7±1.9% for juice concentrate and 99.8±3.2% for pasteurized juice.     

Trace enrichment and HPLC analysis of polycyclic aromatic hydrocarbons in environmental samples,using solid phase extraction in connection with UV/VIS diode-array and fluorescence detection

Summary Both the separation of polycyclic aromatic hydrocarbons (PAH's) on tailor made HPLC phases, and their chromatographic enrichment and quantitative determination are of great interest. Because of the generally low concentrations of PAH's in the environment, and the low limiting values in German legislation for drinking water, methods for selective enrichment and sensitive detection are indispensable. On account of their relatively high amounts in soil, the determination of PAH's is valuable for the assessment of the potential danger to ground water by waste materials. These requirements can be fulfilled by used of solid phase extraction on enrichment columns, and fluorescence or UV/VIS diode array detection. For the detection of PAH's in the picogram range, the wavelengths for excitation and emission were time programmed over the chromatogram. With this feature, it is possible to detect all the individual compounds at the highest sensitivity, over the entire analysis.