Enrichment Column Research Articles

Semi-micro high-performance liquid chromatographic analysis of tiropramide in human plasma using column-switching

A rapid and sensitive column-switching semi-micro high-performance liquid chromatography method was developed for the direct analysis of tiropramide in human plasma. The plasma sample (100 μl) was directly injected onto Capcell Pak MF Ph-1 precolumn where deproteinization and analyte fractionation occurred. Tiropramide was then eluted into an enrichment column (Capcell Pak UG C 18) using acetonitrile–potassium phosphate (pH 7.0, 50 mM) (12:88, v/v) and was analyzed on a semi-micro C 18 analytical column using acetonitrile–potassium phosphate (pH 7.0, 10 mM) (50:50, v/v). The method showed excellent sensitivity (limit of quantification 5 ng/ml), and good precision (C.V.⩽5.9%), and speed (total analysis time 20 min). The calibration curve was linear in the concentration range 5–200 ng/ml (coefficient of determination equal to 0.998). This method was successfully applied to the pharmacokinetic study of tiropramide in volunteers.

Optimisation of a dialytic set-up for liquid chromatography: automated separation and preconcentration of ciprofloxacin

Continuous-flow and static dialysis coupled on-line to liquid chromatography was evaluated and an automated method for determination of ciprofloxacin in biological samples developed. A trace enrichment column packed with C 18 material and coupled with a continuous dialysis and reversed-phase HPLC system with fluorescence detection enabled determination of ciprofloxacin in human blood serum at the 0.1-nmol/l level. The amount of analyte preconcentrated and loaded on the HPLC system was linearly proportional to the concentration in the dialysed sample over more than 4 orders of magnitude (up to 1·10 −6 M). Data for linearity, repeatability and detectability for each particular set-up are given. The trace enrichment step eliminates band broadening caused by solvents different from those of the eluent and affecting retention of ciprofloxacin on the analytical column (increase in k′) due to the on-column change of eluent composition. In analysis of human serum samples phthalates leached from plastic materials may interfere due to coelution with the analyte.

Temperature-promoted large-volume solute enrichment in column-switching miniaturized liquid chromatography: determination of an antioxidant.

A two-valve sub-ambient temperature-promoted reversed-phase packed-capillary liquid-chromatography column-switching system has been tailored for sensitive determination of hydrophobic compounds. Such compounds are not easily dissolved in solvent mixtures of non-eluting properties that traditionally are used for solute enrichment in reversed-phase liquid chromatography. Enrichment-column solute focusing of large sample volumes was promoted by use of sub-ambient temperatures only, allowing the use of sample solvents that were stronger or equal to the mobile phase solvent strength. Subsequent column switching and enrichment-column temperature increment provided efficient low-dispersion back-flushed enrichment-column solute desorption onto the analytical column, where the solute was subjected to temperature-programmed gradient action. The antioxidant, Irganox 1076 (octadecyl 3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionate) extracted from low density polyethylene with 100% acetonitrile served as a hydrophobic model compound. The mobile phase consisted of acetonitrile containing 10 mM triethylamine and formic acid, and the 0.25 mm id enrichment-column and analytical column in lengths of 27 and 250 mm, respectively, were packed with 3.5 microm Kromasil C18 particles. Sample volumes of up to 500 microL were successfully focused on the enrichment column at 5 degrees C using loading flow rates of up to 40 microL min(-1) prior to temperature programming to 90 degrees C. The concentration limit of detection of Irganox 1076 was 6 ng mL(-1) when using an injection volume of 500 microL. The within-assay precision was in the range 3.5-6.8% (n = 6) while the between-day precision was 7.5% (n = 3) relative standard deviation. The method was linear within the investigated mass range 3-100 ng (R2 = 0.9993).

Determination of trace lead, cadmium and mercury by on-line column enrichment followed by RP-HPLC as metal-tetra-(4-bromophenyl)-porphyrin chelates

This paper reports the utilization of tetra-(4-bromophenyl)-porphyrin (T 4BPP) as a chelating reagent using Waters Xterra™ RP 18 column for the on-line column enrichment and the separation of trace lead, cadmium and mercury ions by reversed-phase high-performance liquid chromatography (RP-HPLC) with photodiode array detector. When the Hg–T 4BPP, Pb–T 4BPP and Cd–T 4BPP chelates were injected into the injector and sent to the enrichment column with 0.05 mol l −1 of pH 10.0 pyrrolidine–phosphoric acid buffer solution (containing 10% of tetrahydrofuran (THF)) as mobile phase. The chelates were retained on the top of the enrichment column. After the enrichment is finished, by switching the valve of six-ports switching valve, the retained metal–T 4BPP chelates will be eluted by mobile phase in reverse direction and will travel towards analytical column. With THF (containing 0.05 mol l −1, pH 10.0 pyrrolidine–phosphoric acid buffer salt) and 0.05 mol l −1, pH 10.0 pyrrolidine–phosphoric acid buffer solution (containinging 10% THF) gradient elution as mobile phase, the chelates separation on the analytical column was satisfactory. The linearity ranges are 0.01–120 μg l −1 for each metal ion. The detection limits (S/N=3) of lead, cadmium and mercury are 1.0, 0.5 and 1.0 ng l −1, respectively. This method can be applied to the determination (μg l −1) level of lead, cadmium and mercury in drinking water with satisfactory results.

Analysis of tofisopam in human serum by column-switching semi-micro high-performance liquid chromatography and evaluation of tofisopam bioequivalency.

A rapid and sensitive column-switching semi-micro HPLC method is described for the direct analysis of tofisopam in human serum. The sample (100 microL) was directly injected onto the precolumn (Capcell Pak MF Ph-1), where unretained proteins were eluted to waste. Tofisopam was then eluted into an enrichment column using 13% acetonitrile in 50 mM phosphate buffer (pH 7.0) containing 5 mM sodium octanesulfonate and subsequently into the analytical column using 43% acetonitrile in 0.1% phosphoric acid containing 5 mM sodium octanesulfonate. The detection limit (2 ng/mL), good precision (CV < or = 4.2%) and speed (total analysis time 24 min) of the present method were sufficient for drug monitoring. This method was successfully applied to a bioequivalence test of two commercial tofisopam tablets.

Local and Systemic T Cell Responses to Purified Proteins Following Cow's Milk Feeding in Guinea Pigs

Abstract Cell-mediated immune mechanisms may contribute to hypersensitivity reactions following ingestion of cow's milk. We assessed in vitro lymphoproliferation to purified cow's milk proteins in sensitized lymphocytes from the spleen, mesenteric lymph nodes and peripheral blood of guinea pigs consuming cow's milk. Blood and lymph node cells were quite responsive but splenocytes responded poorly. Orally sensitized guinea pigs responded best to a-casein, β-casein and β-lactoglobulin and minimally to a-lactalbumin. Even two months after oral exposure, guinea pig mesenteric lymph node lymphocytes still proliferated when cultured with a-casein and β-casein. Refeeding cow's milk to guinea pigs after a milk-free interval following the initial exposure did not boost the proliferative response and may have induced suppression. The effect of nylon wool column enrichment of T cells prior to culture demonstrated the essential role of antigen-presenting cells in T cell activation by cow's milk proteins. Our results suggest the optimum length of time required for oral sensitization to a-casein, β-casein and β-lactoglobulin is 2 weeks. However, prolonged feeding for 4 weeks with cow's milk down-regulated the antigenic response and upregulated the proliferative response to a polyclonal T cell mitogen (ConA). These results suggest that feeding cow's milk can induce antigen specific T cell responses which are both local and systemic. The lymphocyte proliferation assay with highly purified antigens could be useful in elucidating the fundamental cellular mechanisms of cow's milk allergy.

Enrichment of heavy water in flat-plate thermal diffusion columns inclined for improved performance

The optimum angles of inclination for maximum separation, maximum production rate and minimum column length in a flat-plate thermal-diffusion column for enrichment of heavy water have been determined. Essential improvement in performance is obtainable if the column is tilted at the optimum angle, instead of being placed vertically, so that the convection strength can be properly reduced and controlled, resulting in suppressing the undesirable remixing effect while still preserving the desirable cascading effect. Accordingly, considerable saving in fixed and operating charges can be achieved, particularly for lower production rate operations, or for smaller degree of separation.

Direct cocktail analysis of drug discovery compounds in pooled plasma samples using liquid chromatography–tandem mass spectrometry

Direct plasma injection technology coupled with a LC–MS/MS assay provides fast and straightforward method development and greatly reduces the time for the tedious sample preparation procedures. In this work, a simple and sensitive bioanalytical method based on direct plasma injection using a single column high-performance liquid chromatography (HPLC) and tandem mass spectrometry (MS/MS) was developed for direct cocktail analysis of double-pooled mouse plasma samples for the quantitative determination of small molecules. The overall goal was to improve the throughput of the rapid pharmacokinetic (PK) screening process for early drug discovery candidates. Each pooled plasma sample was diluted with working solution containing internal standard and then directly injected into a polymer-coated mixed-function column for sample clean-up, enrichment and chromatographic separation. The apparent on-column recovery of six drug candidates in mouse plasma samples was greater than 90%. The single HPLC column was linked to either an atmospheric pressure chemical ionization (APCI) or electrospray ionization (ESI) source as a part of MS/MS system. The total run cycle time using single column direct injection methods can be achieved within 4 min per sample. The analytical results obtained by the described direct injection methods were comparable with those obtained by semi-automated protein precipitation methods within ±15%. The advantages and challenges of using direct single column LC–MS/MS methods with two ionization sources in combination of sample pooling technique are discussed.

Trace determination of priority pesticides in water by means of high-speed on-line solid-phase extraction–liquid chromatography–tandem mass spectrometry using turbulent-flow chromatography columns for enrichment and a short monolithic column for fast liquid chromatographic separation

An integrated on-line SPE–HPLC–MS/MS system has been developed for the rapid analysis of various trace level priority pesticides in surface and drinking water. Eleven pesticides were included in this study, with various phenylureas, triazines and organophosphorous species among them. Use of turbulent-flow chromatography columns (TFC, 50×1 mm, 30–50 μm particle size) as extraction cartridges enables fast on-line SPE at high sampling flow-rate (5 ml/min). Polymeric and carbon based TFC columns (Oasis HLB, Cyclone, Hypercarb) allow complete extraction with good recoveries from water volumes up to 50 ml. On-line coupling to HPLC is performed with re-mixing of the organic TFC eluate with water in front of the analytical column to ensure efficient band focussing. For fast HPLC analysis, a short monolithic column is applied in combination with highly selective API–MS/MS detection. Matrix effects on the APCI–MS/MS signal were found to be reduced by the system to an acceptable minimum. Limits of detection, determined for 10-ml samples of river water were in the range between 0.4 and 13 ng/l typically, except trifluralin (approximately 280 ng/l), which is less susceptible to ionization under atmospheric pressure conditions. At an enriched water volume of 10 ml, the whole SPE–HPLC–MS/MS procedure requires less than 14 min. The method was successfully applied to the analysis of drinking and surface water samples taken from several sampling sites around the city of Leipzig, Germany. Concentrations measured (maximum: 16 ng/l simazine in river water) were far below the concentration limits scheduled by law.

Simultaneous determination of gold and copper by reversed-phase high-performance liquid chromatography using online column enrichment as their syn-phenyl-2-pyridylketoximate chelates.

Gold and copper ions react with syn-phenyl-2-pyridylketoxime (PPKO) at 40 degrees C to form neutral chelates. Metal-PPKO chelates subsequently become preconcentrated on a minicolumn packed with a divinylbenzene-methacrylate copolymer. By switching the valve, these chelates are separated on the silica-based phenyl column and detected with a photometric detector. These processes occur automatically except for chelation. The results of the chelation, preconcentration, and separation conditions studies are presented. Calibration curves for Au and Cu ions are linear from nanograms per milliliter (parts per billion) to micrograms per milliliter (parts per million) in the original solution. The precision for 0.5-ppm standards of Au and Cu is 2.1 and 0.9 relative standard deviation (%), respectively. The accuracy of the present method is verified for Au and Cu based on the analysis of a standard alloy of the National Institute of Standards and Technology (NIST). The limits of detection for Au and Cu are 16.7 and 0.6 ppb, respectively. The effects of foreign ions on the determination of Au and Cu are discussed.

On-line coupling of sol–gel-generated immunoaffinity columns with high-performance liquid chromatography

The paper demonstrates the possibility to use sol–gel-generated immunoaffinity columns as selective sample preparation step in on-line combination with HPLC. In the past sol–gel-generated immunoaffinity columns have only been included in off-line sample preparation schemes. Compared with conventional RP-materials on-line coupling of sol–gel-generated silica matrices with a pore structure designed to retain antibodies poses additional problems caused by their lower pressure tolerance and by the necessity to match the mobile phases not only to take into account the chromatographic properties but also the conformational stability of the antibodies. These problems have been overcome by an on-line system which can be regarded as a prototype for similar systems which exploit the selectivity of sol–gel immunoaffinity columns. The system consists of a sol–gel-generated immunoaffinity column coupled to an RP enrichment column and an analytical column. The practicality of such systems is demonstrated using the example of anti-pyrene immunoaffinity columns applied for the determination of pyrene in aqueous solutions.

Flow-injection preconcentration and electrothermal atomic absorption spectrometry determination of manganese in seawater

A procedure for the determination of manganese in seawater by flow-injection preconcentration coupled to electrothermal atomic absorption spectrometry was developed. The analyte is complexed by 1-(1-hydroxy-2-naphthylazo)-6-nitro-2-naphthol-4-sulphonic acid (Eriochrome Black T), its complexes are retained onto an anion exchange resin, and eluted with hydrochloric acid. A software driven preconcentration apparatus was set up and a new housing for the enrichment column was designed and adopted. After studying the effect of acidity and ligand concentration, the accuracy of the procedure was assessed by analysis of certified seawater.

Development and validation of a column-switching high-performance liquid chromatographic method for the determination of sanfetrinem in rat and dog plasma by direct injection

A direct injection column-switching HPLC method was developed and validated for quantification of sanfetrinem in rat and dog plasma. Following dilution with buffer, samples were directly injected onto the system. The analyte was retained in an enrichment column while endogenous plasma components were eluted to waste. Sanfetrinem was then back-flushed to the analytical column for separation and quantification with an ultraviolet detector. Sample batch size was increased by adding a washing phase of the enrichment column and by alternating the injections between two enrichment columns. The method is very simple and sample preparation is minimal. The method has been fully validated and shown to be specific, accurate and reproducible.

On-line dialysis and quantitative high-performance liquid chromatography analysis of iodixanol in human, rat and monkey plasma

A fully automated HPLC method for analysing the non-ionic X-ray contrast agent iodixanol in plasma samples, using on-line dialysis for sample preparation, was developed. Optimal conditions were obtained with a static dialysis donor solution of 110 μl and 4 ml of recipient solution (dialysate) pulsed onto a trace enrichment column, giving maximum 55% dialysis efficiency in less than the chromatographic run time of 20 min. Hence, one sample could be dialysed during the analysis of the previous. The maximum load of iodixanol on the trace enrichment column was 3.75 mg. Validation showed that the method was selective for iodixanol, sensitive down to 84 pmol/ml and had a high precision over a linear range up to 320 nmol/ml. Although developed for iodixanol, the method can easily be modified and applied to other substances with similar properties, i.e., substances having low protein binding and high water solubility, but strong enough stationary phase affinity to be retained by an appropriate trace enrichment column.

Multiresidue Determination of Fluoroquinolones in Eggs

A multiresidue method was developed for the determination of fluoroquinolones in eggs. Extraction of eggs with ammoniacal acetonitrile was followed by liquid-liquid defatting, solvent evaporation, and redissolution in a small volume of buffer. The fluoroquinolones were further purified by on-line microdialysis, concentrated on a trace enrichment column, and separated by reversed-phase liquid chromatography with fluorescence detection. Norfloxacin (NOR), ciprofloxacin (CIP), and sarafloxacin (SAR) were extracted from fortified eggs over a range of 2-200 microg/kg, with recoveries of 65.7-78.9%, 65.6-77.1%, and 67.6-110%, respectively. Enrofloxacin (ENRO) was extracted over a range of 1-100 microg/kg, with recoveries of 71.5-86.7%, whereas desethylene ciprofloxacin (DCIP) and danofloxacin (DANO) were extracted over a range of 0.2-20 microg/kg, with recoveries of 68.7-90.7% and 76.0-93.8%, respectively. The limits of quantitation for the 6 fluoroquinolones were as follows: DCIP and DANO, 0.3 microg/kg; ENRO, 1 microg/kg; NOR and CIP, 2 microg/kg; and SAR, 3 microg/kg. Both SAR and ENRO incurred eggs were also successfully analyzed using this method.

Direct analysis of plasma samples for drug discovery compounds using mixed-function column liquid chromatography tandem mass spectrometry.

A sensitive, efficient, high throughput, direct injection bioanalytical method based on a single column and high-performance liquid chromatography (HPLC) with tandem mass spectrometry (MS/MS) was developed for pharmacokinetic analysis of early drug discovery compounds in plasma samples. After mixing with a working solution containing an internal standard each plasma sample was directly injected into a polymer-coated mixed-function column for sample cleanup, enrichment and chromatographic separation. The stationary phase incorporates hydrophilic polyoxyethylene groups and hydrophobic groups to the polymer-coated silica. This allows proteins and macromolecules to pass through the column due to restricted access to the surface of the packing while retaining the drug molecules on the bonded hydrophobic phase. The analytes retained in the column with a largely aqueous liquid mobile phase were then chemically separated by switching to a strong organic mobile phase. The column effluent was diverted from waste to the mass spectrometer for analyte detection. Within 200 plasma sample injections the response ratio (analyte vs. internal standard, %CV = 4.6) and the retention times for analyte and internal standard were found consistent and no column deterioration was observed. The recoveries of test compound in various plasma samples were greater than 90%. The total analysis time was </=5 min per sample.

Trace Determination of Metal Ions by On-Line Column Enrichment Followed by HPLC as Metal-2-(5-nitro-2-pyridylazo)-5-[N-propyl-N-(3-sulfopropyl)-amino]phenol Chelates

Trace Determination of Metal Ions by On-Line Column Enrichment Followed by HPLC as Metal-2-(5-nitro-2-pyridylazo)-5-[N-propyl-N-(3-sulfopropyl)-amino]phenol Chelates

Highly sensitive flow analysis determination of orthophosphate using solid phase enrichment of phosphomolybdenum blue without need for organic solvents in elution

In a flow analysis configuration, orthophosphate has been enriched as phosphomolybdenum blue on reversed phase polymers (styrene-divinylbenzene (DVB), methacrylate, dextran), recovered by an alkaline solution without added organic solvent, and measured photometrically at 700nm. It was found that phosphomolybdenum blue is bound to those polymers mainly by fast adsorption but also partly by slow diffusion (which causes some carryover); the relative extent of both processes depends on the chemical nature of the polymer. With the enrichment column placed near the detector to minimize transport dispersion and an enrichment time of 120s; a signal improvement factor of 30–50 was obtained using styrene-DVB.

Determination of albendazole and its main metabolites in ovine plasma by liquid chromatography with dialysis as an integrated sample preparation technique

Albendazole is a benzimidazole derivative with a broad-spectrum activity against human and animal helminth parasites. In order to determine the main pharmacokinetic parameters in sheep after oral and intravenous administration of a new formulation of albendazole (an aqueous solution), a fully automated method was developed for the determination of this drug and its main metabolites, albendazole sulfoxide (active metabolite) and sulfone in ovine plasma. This method involves dialysis as purification step, followed by enrichment of the dialysate on a precolumn and liquid chromatography (LC). All sample handling operations were executed automatically by means of an ASTED XL system. After conditioning of the trace enrichment column (TEC) packed with octadecyl silica with pH 6.0 phosphate buffer containing sodium azide, the plasma sample, in which a protein releasing reagent (1 M HCl) containing Triton X-100 was automatically added, was loaded in the donor channel and dialysed on a cellulose acetate membrane in the static-pulsed mode. The dialysis liquid consisted of pH 2.5 phosphate buffer. By rotation of a switching valve, the analytes were eluted from the TEC in the back-flush mode by the LC mobile phase and transferred to the analytical column, packed with octyl silica. The chromatographic separation was performed at 35°C and the analytes were monitored photometrically at 295 nm. Due to the differences in hydrophobic character between albendazole and its metabolites, a gradient elution was applied. The mobile phase consisted of a mixture of acetonitrile and pH 6.0 phosphate buffer. The proportion of organic modifier was increased from 10.0 to 50.1% in 12.30 min, then from 50.1 to 66.9% in 1.70 min. First, the gradient conditions and the temperature were optimised for the LC separation using the DryLab software. Then, the influence of some parameters of the dialysis process on analyte recovery was investigated. Finally, the method developed was validated. The mean recoveries for albendazole and its metabolites were about 70 and 65%, respectively. The limits of quantification for albendazole and its metabolites were 10 and 7.5 ng/ml, respectively.

Automated liquid chromatographic determination of atenolol in plasma using dialysis and trace enrichment on a cation-exchange precolumn for sample handling

A fully automated method involving dialysis combined with trace enrichment was developed for the liquid chromatographic (LC) determination of atenolol, a hydrophilic β-blocking agent, in human plasma. The plasma samples were dialysed on a cellulose acetate membrane and the dialysate was reconcentrated on a short trace enrichment column (TEC) packed with a strong cation-exchange material. All sample handling operations can be executed automatically by a sample processor (ASTED system). After TEC conditioning, the plasma sample, to which the internal standard (sotalol, another hydrophilic β-blocker) was automatically added, was introduced in the donor channel and dialysed in the static/pulsed mode. The dialysis liquid consisted of 4.3 m M phosphoric acid. When the dialysis process was discontinued, the analytes were eluted from the TEC in the back-flush mode by the LC mobile phase and transferred to the analytical column, packed with octyl silica. The LC mobile phase consisted of phosphate buffer, pH 7.0–methanol (81:19; v/v) with 1-octanesulfonate. Atenolol and the internal standard were monitored photometrically at 225 nm. The different parameters influencing the dialysis and trace enrichment processes were optimised with respect to analyte recovery. The influence of two different kinds of cation-exchange material on analyte recovery and peak efficiency was also studied. The method was then validated in the concentration range 25–1000 ng/ml. The mean recovery for atenolol was 65% and the limit of quantitation was 25 ng/ml.