Determination of pyruvate and lactate in primary liver cell culture medium during hypoxia by on-line microdialysis-liquid chromatography

A microdialysis sampling device was constructed for the measurement of pyruvate and lactate in primary liver cell culture medium during hypoxia. It was composed of a Petri dish, a dialysis membrane and two transmission tubes within a hypoxia chamber. The dialysis membrane was located in the Petri dish such that it was immersed in the culture medium. Dialysates were collected and introduced by an on-line injector to a liquid chromatographic system for analysis of pyruvate and lactate. The detection limit of this assay was 0.2-2.0 microM with acceptable intra- and inter-assay reproducibilities. In order to validate the assay, primary liver cells were incubated in the Petri dish within a hypoxia chamber in an incubator. The baseline concentrations of pyruvate and lactate in primary liver cell culture medium were 10.6+/-5.6 and 607+/-143 microM, respectively. These levels drastically changed during hypoxia and reperfusion. In conclusion, the present assay provides a sensitive, direct measurement of pyruvate and lactate in culture medium while minimizing pretreatment procedures for sample preparation.     

On-line microwave sample pretreatment for the determination of mercury in blood by flow injection cold vapor atomic absorption spectrometry

A method for on-line treatment of whole blood in a microwave oven and determination of mercury by flow injection cold vapor atomic absorption spectrometry was developed. After dilution of the whole blood and addition of oxidant, all further treatment and measurement were performed automatically, on-line. Recoveries of five mercury compounds were complete. Good agreement between measured and recommended values of mercury in whole blood reference materials was obtained. Measured mercury values also agreed with results from other accepted methods. Sample throughput was about 45 measurements/hr. Detection limit (3s) in diluted sample was 0.1 μg/l corresponding to 1μg/l Hg in whole blood. The RSD value at 0.5 μg/l Hg in the diluted sample was 6–7% (11 measurements and 0.5 ml sample volume). Mercury concentrations between 1 and 150 μg/l in whole blood can be measured using this method. For three replicate measurements, 0.5 ml of whole blood is required.     

Automated determination of verapamil and norverapamil in human plasma with on-line coupling of dialysis to high-performance liquid chromatography and fluorometric detection

A fully automated method for the simultaneous determination of verapamil and its main metabolite norverapamil in human plasma is described. This method is based on on-line sample preparation using dialysis followed by clean-up and enrichment of the dialysate on a precolumn and subsequent HPLC analysis with fluorometric detection. All sample handling operations were performed automatically by a sample processor equipped with a robotic arm (ASTED system). The plasma samples were dialysed on a cellulose acetate membrane (cut-off: 15 kD) and the dialysate was purified and enriched on a short pre-column filled with cyanopropyl silica. Before starting dialysis, this trace enrichment column (TEC) was first conditioned with the HPLC mobile phase and then with pH 3.0 acetate buffer. 370 μl of plasma sample spiked with the internal standard (gallopamil) were dialysed in the static-pulsed mode. The solution at the donor side was pH 3.0 acetate buffer containing Triton X-100 while the acceptor solution was made of the same acetate buffer. When dialysis was discontinued, the analytes were desorbed from the TEC by the HPLC mobile phase and transferred to the C₁₈ analytical column by means of a switching valve. This mobile phase consisted of a mixture of acetonitrile, pH 3.0 acetate buffer and 2-aminoheptane. The influence of different parameters of the dialysis process on the recovery of verapamil and norverapamil has been studied. The effect of the volume, the aspirating and dispensing flow-rates of the dialysis solution has been investigated. The recoveries of verapamil and norverapamil in plasma were close to 75% and the limits of quantification were 5 ng/ml for both analytes. The method was found to be linear in the concentration range from 5 to 500 ng/ml (r²: 0.9996 for both analytes). The intra-day and inter-day reproducibilities at a concentration of 100 ng/ml were 2.3% and 5.6% for verapamil and 1.7% and 5.1% for norverapamil, respectively.     

Flow-injection Chemiluminescence Determination of Catecholamines

Catecholaminesarenowwidelyusedinthetreatmentofbronchialasthma,hypertension,heartfailureassociatedwithorganicheartdiseases,andcardiacsurgery'.Thedeterminationofcatacholaminesinpharmaceuticalpreparationshasappearedespeciallyattractive.Chemiluminescence(CL)hasbeenexploitedinanumberofanalyticalapplicationsowingtoitsgreatsensitivity.SomeCLreactionshavenotfoundmanyapplicationsduetotheinstabilityofoxidationreagentssuchashypochlorite.WeproposedthatunstablereagentsrequiredforCLreactioncanbegenerated…     

Membrane separation in flow injection systems

A critical review of membranes for on-line separations in flow-through dialysers is given. Historical perspectives are briefly outlined. The fundamental aspects of dialysis as the selective separation of species through a semi-permeable membrane are discussed. Work done on theoretical principles by a number of authors is described and a simplified model is given. The basic components, locations thereof and the influence of various parameters on dialysis efficiency are highlighted. The value of dialysis as sampling preparation system for chromatography and reactor systems is outlined and the suitability of dialyser/flow injection systems in dilution, preconcentration and multicomponent analysis is evaluated. The review is concluded with a brief look at the use of dialysis in proteinligand binding studies and microdialysis.     

Automated,continuous, and dynamic speciation of urinary arsenic in the bladder of living organisms using microdialysis sampling coupled on-line with high performance liquid chromatography and hydride generation atomic absorption spectrometry

An on-line and fully automated method was developed for the continuous and dynamic in vivo monitoring of four arsenic species [arsenite (AsIII), arsenate (AsV), monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA)] in urine of living organisms. In this method a microdialysis sampling technique was employed to couple on-line with high performance liquid chromatography (HPLC) and hydride generation atomic absorption spectrometry (HGAAS). Dialysates perfused through implanted microdialysis probes were collected with a sample loop of an on-line injector for direct and automated injection into HPLC system hyphenated with HGAAS. The saline (0.9% NaCl) solution was perfused at the rate of 1 microl min(-1) through the microdialysis probe and the dialysate was loaded into 50 microl of sample loop. The separation conditions were optimally selected to be in phosphate buffer solution at a pH 5.2 with a flow rate of 1.2 ml min(-1). The effluent from the HPLC was first mixed on-line at the exit of the column with HCl (1 M) solution and then mixed with a NaBH4 (0.2% m/v) solution. Based on the optimal conditions obtained, linear ranges of 2.5-50 ng ml(-1) for AsIII and 6.75-100 ng ml(-1) for the other three arsenic species were obtained. Detection limits of 1.00, 2.18, 1.03 and 2.17 ng ml(-1) were obtained for AsIII, DMA, MMA and AsV, respectively. Typical precision values of 3.4% (AsIII), 5.4% (DMA), 3.6% (MMA) and 7.5% (AsV) were obtained, respectively, at a 25 ng ml(-1) level. Recoveries close to 100%, relative to an aqueous standard, were observed for each species. The average in vivo recoveries of AsIII, DMA, MMA and AsV in rat bladder urine were 56+/-5%, 60+/-9%, 49+/-3% and 55+/-7%, respectively. The use of an on-line microdialysis-HPLC-HGAAS system permitted the determination of four urinary arsenic species in the bladder of an anesthetized rat with a temporal resolution of 50 min sampling.     

Quantitative measurement of male steroid hormones using automated on-line solid phase extraction-liquid chromatography-tandem mass spectrometry and comparison with radioimmunoassay

A specific and sensitive method using high-performance liquid chromatography-tandem mass spectrometry (LC-MS-MS) equipped with automatic on-line solid-phase extraction device for the quantitative measurement of anabolic hormone residues, 4-androstene-3,17-dione, testosterone and dihydrotestosterone in cell culture medium was developed. Steroid content in cell culture medium was determined directly without an additional sample preparation step. Separation of analytes from polar endogenous compounds was carried out on an automatic column-switching device coupled with a C4-alkyl-diol silica restricted-access solid-phase extraction column. The lipophilic fraction containing anabolic hormone residues were back-flushed on to a conventional C-18 reversed-phase column for the final chromatography. The analyte was ionized in an ElectroSpray interface under positive ion mode before entering a quadrupole mass analyzer. The lowest points of calibration curves were 0.05 ng ml(-1) for 4-androstene-3,17-dione and testosterone, and 1 ng ml(-1) dihydrotestosterone, respectively. A comparison with results from radioimmunoassay (RIA) is also presented.     

Simultaneous determination of catecholamines by ion chromatography with direct conductivity detection

A simple method for simultaneous determination of three catecholamines using ion chromatography (IC) with direct conductivity detection (CD) based on the ionization of catecholamines in acidic medium without chemical suppression is developed in the present paper. The method could be used for the determination of these catecholamines in pharmaceutical preparations for the purpose of drug quality control. The recovery of catecholamines was more than 97% (n=3) and the relative standard deviation (R.S.D.) (n=11) was less than 2.1%. In a single chromatographic run, norepinephrine (NE), epinephrine (E) and dopamine (DA) can be determined in less than 10 min. The detection limits were found to be 0.001 μg/ml for NE, 0.01 μg/ml for E and DA respectively. Linear ranges were 0.01–50 μg/ml for NE (r²=0.9998), 0.1–50 μg/ml for E (r²=0.9995) and DA (r²=0.9999), respectively.     

On-line microdialysis–high-performance liquid chromatographic determination of aniline and 2-chloroaniline in polymer industrial wastewater

Determination of aniline and 2-chloroaniline in polymer industrial wastewater was examined using high-performance liquid chromatography with on-line microdialysis. After dilution, aniline and 2-chloroaniline in the sample were diffused through a cellular dialysis membrane into the perfusion stream under controlled conditions. Conditions for obtaining optimum dialysis efficiency such as flow-rate and polarity modifier in the perfusion stream, pH and added salt in the sample solution, as well as chromatographic conditions were investigated. The results indicate that the dialysis achieved at a sample matrix pH value of 9.5 with 0.1 M NaCl addition, and the perfusate at 10-μl/min flow-rate offered optimum dialysis efficiency. The aniline and 2-chloroaniline were well separated in an acceptable time on a reversed-phase C₁₈ column eluted with 40% aqueous methanol solution at pH 7.0 and 1.0 ml/min flow-rate. The proposed method provided a very simple procedure to determine aniline and 2-chloroaniline in wastewater. Application was illustrated by the analysis of aniline and 2-chloroaniline in wastewater released from a polymer factory.     

On-line microdialysis for enhanced resolution and sensitivity during electrospray mass spectrometry of non-covalent complexes and competitive binding studies

Many proteins and macromolecules easily form metal adduct ions which impairs their analysis by mass spectrometry. The present study describes how the formation of undesired adducts can be minimized by on-line microdialysis for non-covalent binding studies of macromolecules with low molecular mass ligands with electrospray ionization mass spectrometry (ESI-MS). The technique was indispensable for protein-ligand studies due to reduction of unwanted adduct ions, and thus gave excellent resolution and a sensitivity improvement of at least 5 times. The core of the analytical system was a modified microdialysis device, which was operated in countercurrent mode. A novel technique based on microdialysis for competitive binding studies is also presented. The non-covalent complex between a protein and a ligand was formed in the sample vial prior to analysis. The complex was injected into an on-line microdialysis system where a competitive ligand was administered in the dialysis buffer outside of the fiber. The second ligand competitively displaced the first ligand through transport via the wall of the dialysis fiber, and the intact complexes were detected by ESI-MS.     

Diffusion and calibration properties of microdialysis sampling membranes in biological media

The diffusion and calibration properties for three commercially available microdialysis membranes (polycarbonate-polyether (PC), polyacrylonitrile (PAN), and cuprophan (CUP)) were evaluated. The analytes studied had molecular weights between 94 (phenol) and 1355 (vitamin B12). For each analyte-membrane pair, an effective membrane diffusion coefficient was calculated. Effective membrane diffusion coefficients varied considerably between the microdialysis membranes. For Vitamin B12, CUP and PAN membranes gave relative recovery values of greater than 20% at 0.5 microl min(-1), while the PC membrane had a 1% recovery. When backpressure was applied. PC and PAN membranes exhibited more ultrafiltration than CUP membranes. Ultrafiltration did not affect analyte relative recovery through either PC or PAN membranes. Effective membrane diffusion coefficients were not significantly altered for some membrane-analyte combinations when exposed to 4% bovine serum albumin or 0.3% fibrinogen. These data suggest that reductions in relative recovery during long-term microdialysis sampling experiments may be due to other physiologically relevant proteins or to tissue reactions near the dialysis membrane.     

Collection of in vivo-like liver cell secretome with alternative sample enrichment method using a hollow fiber bioreactor culture system combined with tangential flow filtration for secretomics analysis

A hollow fiber bioreactor (HFB) culture system coupled with a tangential flow filtration (TFF) device was used for HepG2 cell secretome analysis. In order to reduce the loss of low-molecular-weight proteins, two new features, the hollow fiber with 0.1 μm pore size and a TFF device with a membrane of 1kDa molecular weight cutoff, were added to the system described previously. The HFB culture system and the conventional dish culture method for secretome collection were compared side by side. It was observed that only a small fraction of cells (<0.01%) were lysed in the HFB culture system, in contrast to the 2.73% in the conventional dish culture. A total of 111 proteins were identified in the collected conditioned medium (CM) by liquid chromatography-tandem mass spectrometry (LC-MS/MS) with this improved collection procedure. Many of these proteins reported to be biomarkers for liver-related diseases. About 16% of the identified proteins were smaller than 20kDa, demonstrating that the modified collection system had the ability to reduce the loss of low-molecular-weight proteins, in contrast to our previous collection system. The percentage increase of proteins classified as extracellular space or plasma membrane between the conventional dish culture and the HFB culture system was 40-60%. We believed that in vivo-like culture environments could support liver cells to improve protein secretion than conventional dish cultures. We suggest that the combination of the HFB culture system, TFF device, and LC-MS/MS analysis, would be an efficient procedure for the collection and characterization of in vivo-like cell secretome.     

Automated dilution in flow injection analysis with double on-line dialysis. A system for the determination of chloride in industrial effluents and plating bath solutions

Summary An automated on-line system for the determination of chloride in industrial effluents and plating bath solutions based on the concept of flow injection analysis is described. Samples with a very high chloride content up to 6 g/100 ml (60 g/l) are analysed by using an automated in-manifold double on-line dialysis technique. The sampling rate is 100 samples per hour. The method is suitable for the analysis of chloride with a relative standard deviation of better than 0.8%. The measurement is based on the red-coloured iron thiocyanate complex.     

In vivo microdialysis/liquid chromatography/tandem mass spectrometry for the on-line monitoring of melatonin in rat

Liquid chromatography/tandem mass spectrometry (LC/MS/MS) has been coupled to in vivo microdialysis for on-line monitoring of melatonin in a freely moving rat for a period of 15 hours. A microdialysis probe was surgically implanted into the jugular vein of the rat, and deionized water was used as the perfusion medium at a flow rate of 1.0 microL/min. Microdialysis samples were collected in an on-line injector with sample injection every 30 minutes. Melatonin was dosed by intraperitoneal (i.p.) injection and then monitored by microdialysis/LC/MS/MS. The whole experiment, including the microdialysis sampling and sample injection into the LC/MS system, was fully automated. Metabolites of melatonin were identified off-line by LC/MSn experiments. Two metabolites were identified as 6-hydroxymelatonin and cyclic 2-hydroxymelatonin, consistent with ones found previously in the literature.     

Flow injection determination of nitrite by fluorescence quenching

A simple, sensitive and selective fluorimetric method for the determination of nitrite ion in waters using a merging zones flow injection system is described. The fluorimetric determination is based on the measurement of the quenching effect produced by nitrite on proflavine (3,6-diaminoacridine) fluorescence (λ_ex/λ_em=290/519 nm).

The optimum experimental conditions were investigated by merging 0.5 ml of the sample and 0.5 ml of a solution of 5 mg l⁻¹ of proflavine (in 0.1 M HCl) in a flow injection system, on-line connected to a flow-cell placed in the conventional sample compartment of a spectrofluorimeter. The selected carrier solution and final flow rate were 0.1 M HCl and 0.5 ml min⁻¹, respectively. A reaction coil of 2 ml was used. As a result of the simplicity of this system, a sample throughput of about 50 samples h⁻¹ can be achieved with the proposed methodology.

The detection limit was 1.1 ng ml⁻¹ (3σ criterion) of nitrite. The repeatability for five sample injections containing 100 ng ml⁻¹ of nitrite was ±0.3% and the observed linear range extended up to 400 ng ml⁻¹. Also, the effect of interferences from various metals and anions commonly present in waters was also studied.

The method was successfully applied to the determination of low levels of nitrite in different water samples (river, fountain, tap and commercial drinking waters).     

Development of a push-pull microdialysis sampling technique for the quantitative determination of proteins

Summary An on-line push-pull sampling technique has been developed for continuous analysis of proteins of molecular weight from 5.7 to 67 kDa. The characteristics of the system include gradient elution with a total cycle time of 21 min, membrane stability, unattended automatic operation, and adjustment of the sampling mode and extraction fraction (the ratio of the concentration of analyte in the dialysate to that in the sample) by varying the effective dialysis length. The push and pull flow rates were adjusted in a manner which enabled three different modes of operation. When push-pull microdialysis was compared with conventional microdialysis sampling, significantly higher extraction fractions were obtained for all five model proteins studied. The technique has been applied to the quantification of proteins in cell samples. On-line fractionation enabled complementary MS identification of the proteins present.     

Simultaneous determination of paracetamol and chlorpheniramine in human plasma by liquid chromatography-tandem mass spectrometry

An analytical method for the determination of paracetamol and chlorpheniramine in human plasma has been developed, validated and applied to the analysis of samples from a phase I clinical trial. The analytical method consists in the extraction of paracetamol and chlorpheniramine with diethyl ether, followed by the determination of both drugs by an LC–MS–MS method, using 2-acetamidophenol as internal standard. The intra-assay and inter-assay precision and accuracy of this technique were good and the limit of quantitation was 0.5 μg/ml of plasma for paracetamol and 0.2 ng/ml for chlorpheniramine. The concentration working range was established between 0.5 μg/ml and 25 μg/ml for paracetamol and between 0.2 ng/ml and 50 ng/ml for chlorpheniramine. This method has been used for analyzing more than 1200 human plasma samples from a clinical study with 24 volunteers.     

Analysis of glutamate in striatal microdialysates using capillary electrophoresis and laser-induced fluorescence detection

High-performance liquid chromatography (HPLC) with electrochemical detection has been used routinely to analyse the neurochemical constituents of brain microdialysates. However, conventional HPLC analysis requires large injection volumes and hence lengthy dialysis sampling times. Capillary electrophoresis (CE) is a rapid high-resolution separation technique with the ability to routinely handle very small sample volumes. If CE is coupled to a high-sensitivity detection system, such as laser-induced fluorescence (LIF), it becomes a powerful and rapid separation technique for the analysis of small-volume microdialysis samples.

These preliminary studies report reduced separation times for the excitatory amino acid glutamate, prederivatised with naphthalene 2,3-dialdehyde, and demonstrate its detection within small-volume brain microdialysis samples. The limit of detection for this system was 10⁻⁸ M.

Characterisation of striatal microdialysis samples comprised infusions of Ca²⁺-free artificial cerebrospinal fluid (aCSF) and Tetrodotoxin (TTx) (10 mM) to demonstrate that the detected transmitter is of neuronal origin and released in a calcium-dependent manner.

Removal of calcium from aCSF resulted in a decrease in glutamate in dialysis samples. Glutamate release significantly decreased (p<0.05) to ca. 40% of preinfusion control levels after 60 min and this level was maintained throughout the sampling period. These data suggest that glutamate release is, to some degree, a calcium-dependent process. TTx infusion (10 μM) produced a significant (p<0.05) reduction in glutamate release to ca. 10% of preinfusion levels. It would therefore appear that glutamate release is dependent on neuronal activity. In summary, we have demonstrated the establishment of CE-LIF and microdialysis for the measurement of glutamate.     

Investigation of on-line coupling electrothermal atomic absorption spectrometry with flow injection sorption preconcentration using a knotted reactor for totally automatic determination of lead in water samples

A flow injection on-line sorption preconcentration electrothermal atomic absorption spectrometric system for fully automatic determination of lead in water was investigated. The discrete non-flow-through nature of ETAAS, the limited capacity of the graphite tube and the relatively large volume of the knotted reactor (KR) are obstacles to overcome for the on-line coupling of the KR sorption preconcentration system with ETAAS. A new FI manifold has been developed with the aim of reducing the eluate volume and minimizing dispersion. The lead diethyldithiocarbamate complex was adsorbed on the inner walls of a knotted reactor made of PTFE tubing (100 cm long, 0.5 mm i.d.). After that, an air flow was introduced to remove the residual solution from the KR and the eluate delivery tube, then the adsorbed analyte chelate was quantitatively eluted into a delivery tube with 50 μl of ethanol. An air flow was used to propel the eluent from the eluent loop through the reactor and to introduce all the ethanolic eluate onto the platform of the transversely heated graphite tube atomizer, which was preheated to 80°C. With the use of the new FI manifold, the consumption of eluent was greatly reduced and dispersion was minimized. The adsorption efficiency was 58%, and the enhancement factor was 142 in the concentration range 0.01–0.05 μg l⁻¹ Pb at a sample loading rate of 6.8 ml min⁻¹ with 60 s preconcentration time. For the range 0.1–2.0 μg l⁻¹ of Pb a loading rate of 3.0 ml min⁻¹ and 30 s preconcentration time were chosen, resulting in an adsorption efficiency of 42% and an enhancement factor of 21, respectively. A detection limit (3σ) of 2.2 ng l⁻¹ of lead was obtained using a sample loading rate of 6.8 ml min⁻¹ and 60 s preconcentration. The relative standard deviation of the entire procedure was 4.9% at the 0.01 μg l⁻¹ Pb level with a loading rate of 6.8 ml min⁻¹ and 60 s preconcentration, and 2.9% at the 0.5 μg l⁻¹ Pb level with a 3.0 ml min⁻¹ loading rate and 30 s preconcentration. Efficient washing of the matrix from the reactor was critical, requiring the use of the standard addition method for seawater samples. The analytical results obtained for seawater and river water standard reference materials were in good agreement with the certified values.     

A stopped-flow microdialysis sampling-flow injection system for automated multivessel high resolution drug dissolution testing

A simple and robust flow injection on-line microdialysis system for multivessel drug dissolution testing is described. Microdialysis probes were used for sampling from the dissolution media. A stopped-flow dialysis mode with a 50-s stopped-flow period followed by 10-s injection at a perfusion rate of 2.8 ml min(-1) for each probe was used to achieve high resolution of dissolution events using relatively simple equipment and operation. The precisions obtained for simultaneous monitoring of dissolution profiles for six tablets were all better than 0.9% (RSD n=80) and the overall sampling frequency of the system was 360 and 60 h(-1) for each test vessel. The dissolution profiles of isoniazid fast-release tablet from three sources were determined to demonstrate the performance of the system.