Restricted-access Column Research Articles

Online enrichment ability of restricted-access column coupled with high performance liquid chromatography by column switching technique for benazepril hydrochloride

The online enrichment ability of the restricted-access media (RAM) column coupled with high performance liquid chromatography by column switching technique for benazepril hydrochloride in plasma was studied. The RAM-HPLC system consisted of an RAM column as enrichment column and a C18 column as analytical column coupled via the column switching technique. The effects of the injection volume on the peak area and the systematic pressure were studied. When the injection volume was less than 100 microL, the peak area increased with the increase of the injection volume. However, when the injection volume was more than 80 microL, the pressure of whole system increased obviously. In order to protect the whole system, 80 microL was chosen as the maximum injection volume. The peak areas of ordinary injection and the large volume injection showed a good linear relationship. The enrichment ability of RAM-HPLC system was satisfactory. The system was successfully used for the separation and detection of the trace benazepril hydrochloride in rat plasma after its administration. The sensitivity of HPLC can be improved by RAM pre-enrichment. It is a simple and economic measurement method.

Rapid high‐performance liquid chromatography method for determination of tryptophan in gastric juice

To develop a rapid, high-performance liquid chromatography (HPLC) method for the determination of tryptophan in gastric juice to help the differentiation between gastric cancer and benign gastric disease. HPLC was performed on a restricted access material Shiseido Capcell Pak MF SCX SG80 column. Phosphate buffered solution (90mmol/L, pH 3.5)-acetonitrile (ACN; 80/20, V/V) was chosen as the mobile phase. Separation was done at room temperature using a constant flow rate of 1.0mL/min. Fluorescence emission signal intensity at 330nm excited by 288nm ultra violet light was detected and measured. Thirty-eight gastric juice samples from patients with gastric cancer and 48 gastric juice samples of patients with benign gastric disease were tested. A linear relationship in the range of 0.20-100mg/L was obtained between the concentration of tryptophan and its fluorescence emission signal intensity at 330nm. The limit of detection was 0.05mg/L. The recovery rate was 77.4-90.6%. An HPLC method based on strong cation-exchange restricted access columns for determination of concentration of tryptophan in gastric juice was developed. The method has excellent precision and stability. It is compatible with the analysis of gastric juice and has the potential to be used for gastric cancer screening.

Sulphonic acid strong cation-exchange restricted access columns in sample cleanup for profiling of endogeneous peptides in multidimensional liquid chromatography: Structure and function of strong cation-exchange restricted access materials

In this work, the pore structural parameters and size exclusion properties of LiChrospher strong cation-exchange and reverse phase restricted access materials (RAM) are analysed. The molecular weight size exclusion limit for polystyrenes was found to be about 17.7 kDa, while for standard proteins, the molecular weight size exclusion limit was higher, at approximately 25 kDa. The average pore diameter on a volume basis calculated from the pore network model changes from 8.5 nm (native LiChrospher) to 8.6 nm (diol derivative) to 8.2 nm (sulphonic acid derivative) to 6.9 nm ( n-octadecyl derivative). Additional characterisations were performed on restricted access materials with nitrogen sorption at 77 K, water adsorption at 25 °C, intrusion–extrusion of water (in order to evaluate the hydrophobic properties of the pores of the hydrophobic RAM), and zeta potential measurements by microelectrophoresis. For peptide analysis out of the biofluids, the strong cation-exchange functionality seems to be particularly suitable mainly because of the high loadability of the strong cation-exchange restricted access material (SCX-RAM) and the fact that one can work under non-denaturing conditions to perform effective chromatographic separations. For bacitracin, the dynamic capacity of the SCX-RAM columns does not reach its maximum value in the analysed range. For lysozyme, the dynamic capacity reaches a value of 0.08 mg/ml of column volume before column is overloaded. Additionally, the proper column operating conditions that lead to the total effective working time of the RAM column to be equal to approximately 500 injections (depending on the type of sample), is comprehensively described. The SCX-RAM column was used in the same system analysing urine samples for the period of 1 month (∼150 injections) with run-to-run reproducibility below 5% RSD and below 10% RSD for the relative fractions.

Selective immuno-supported liquid membrane (ISLM) extraction, enrichment and analysis of 2,4,6-trichlorophenol

2,4,6-Trichlorophenol (2,4,6-TCP) was extracted from liquid samples (standards and spiked river water) using immuno-supported liquid membrane (ISLM) extraction followed by fluorescence flow immunoassay (FFIA) detection of the immuno-extracted analyte. ISLM–FFIA involves simultaneous on-line sample clean-up, enrichment and detection of analytes. The analyte is transported from a continuously flowing aqueous sample phase (donor) through a supported liquid membrane (SLM), containing an immobilised organic solvent, to a stagnant aqueous phase (acceptor), containing excess anti-analyte antibody, where it is trapped as analyte–antibody complex. The acceptor content is then dispensed and mixed on-line with a fluorescent analyte tracer (2,4,6-TCP/NH/(CH 2) 3/CO/EDF) and the resulting labelled fraction, separated from the mixture by restricted access column, is an indirect measure of the analyte amount extracted in the acceptor. The performance of ISLM–FFIA was compared with a similar FFIA (without the extraction step) with the following results: the ISLM–FFIA configuration leads to improved assay sensitivity (LOD 27.92 ± 2.44 and 224 ± 5.86 μg L −1 for ISLM–FFIA and FFIA, respectively) and improved selectivity and recovery for 2,4,6-TCP in presence of other chlorophenols (e.g. 2,4,5-trichlorophenol and 4-chlorophenol) compared to FFIA alone.

Facile monitoring and evaluation of bacteria in a fermentation process using perfusion chromatography and polarimetry detection

AbstractA 670 nm diode laser polarimeter was used as a detector for living cells in perfusion chromatography. It was found that all microorganisms examined, including vegetal organisms such as algae and fungi as well as several strains of different bacteria, gave a levorotatory signal, producing negative peaks in the chromatograms. Most naturally occurring chiral molecules, such as sugars and amino acids are dextrorotatory and give positive polarimetric peaks. A hydrophilic OH‐functionalized polystyrene divinylbenzene spherical porous particle packing was used as a sieving and size exclusion material and packed in 27×0.95 cm columns. The theory of the close‐packing of spheres is summarized to show that common bacteria should be able to perfuse in the interparticle voids of the 50 μm packing. It is shown that bacteria are eluted with the exclusion volume; a selective permeation of polymer with molecular weight ranging from 103 to 2×106 is obtained, and total permeation occurs for small molecules. The restricted access columns are used to follow the changes in cell concentration in bacteria cultures. The growth of three different bacteria was studied using calibrated polarimetric signals.

Mass spectrometry–based bioassay for the screening of soluble orphan receptors

The suitability of a novel mass spectrometric (MS) based flow-injection bioassay format to rapidly detect ligands for soluble orphan receptors in complex matrices was demonstrated, using digoxin and antidigoxigenin FAb as the model ligand and the model protein target, respectively. In addition, characteristic MS and MSn (n = 2 or 3) data, that is, molecular mass as well as mass fingerprints, were obtained during a single run and provided a basis for the identification of the active compounds that were found. The flow-injection setup used is characterized by the integration of two restricted access columns (RA column) and a pH dissociation step. Bioactive compounds are separated both from unbound ligands as well as the targeted proteins, e.g., as described in the text. This approach allows the isolated bioactive molecules to be introduced in the ion trap MS detector in a clean and well-defined matrix, which minimizes background noise during sample analysis while simultaneously improving bioassay robustness. In this article, we demonstrate the potential of the bioassay format to detect ligands for soluble orphan receptors. Natural extracts, both spiked and unspiked with the model compound digoxin, were analyzed, which demonstrated that only those compounds possessing affinity for the model protein target were detected by MS. Using an antidigoxigenin FAb concentration of 2 μM, digoxin could be detected down to 250 nM in spiked plant extracts. In contrast, however, molecules that did not bond to the protein target were efficiently trapped by the RA column and did not show up in the MS spectra, thus illustrating the principle and potential of the bioassay format.

Selective extraction of small proteins from biological samples using a novel restricted access column with cation exchange properties

The determination of proteins utilising a polymer-based restricted access suppor material with ion exchange properties (IERAM) is outlined. Solid phase extraction coupled on-line with a mincrobore reversed phase HPLC system for the quantitation of small marker proteins is demonstrated. The cation-exchange restricted access packings were characterised with respect to their adsorption and desorption kinetics. The IERAM material was also investigated by capacity, selectivity, and biocompatibility determinations when applied to the quantification of small molecular weight proteins such as cytochrome C, Lysozyme, Ribonuclease A, Myoglobin, Insulin, human serum albumin, and a Tryptic inhibitor.

Analysis of neuropeptide Y and its metabolites by high-performance liquid chromatography–electrospray ionization mass spectrometry and integrated sample clean-up with a novel restricted-access sulphonic acid cation exchanger

A novel restricted access cation exchanger with sulphonic acid groups at the internal surface was proven to be highly suitable in the sample clean up of peptides on-line coupled to HPLC–electrospray ionization (ESI)-MS. Neuropeptide Y (NPY) and several of its fragments in plasma were subjected to the sample clean-up procedure. The peptides were eluted by a step gradient from the restricted access column, applying 10 m M phosphate buffer pH 3.5 from 5 to 20% (v/v) of acetonitrile with 1 M NaCl and transferred to a Micra ODS II column (33×4.6 mm). The separation of the peptides and their fragments was performed by a linear gradient from 20 to 60% (v/v) acetonitrile in water with 0.1% formic acid and 0.01% trifluoroacetic acid in 4 min at a flow-rate of 0.75 ml/min. An integrated and completely automated system composed of sample clean up–HPLC–ESI-MS was used to analyze real life samples. The sample volumes ranged between 20 and 100 μl. Peaks due to the fragments NPY 1–36, 3–36 and 13–36 in porcine plasma were identified by ESI-MS. The limit of detection was in the 5 nmol/ml range. The total analysis required 21 min and allowed the direct injection of plasma.

Enantioselective analysis of ( R)- and ( S)-atenolol in urine samples by a high-performance liquid chromatography column-switching setup

An HPLC column-switching method for the enantioselective determination of ( R, S)-atenolol in human urine was developed and validated. Diluted urine samples were injected onto a LiChrospher ADS restricted access column and atenolol was separated from most of the matrix components using 0.01 M Tris buffer. The atenolol peak was sharpened by a step gradient of 30% acetonitrile and the atenolol-containing fraction was switched onto an enantioselective column. Separation of the atenolol enantiomers was carried out on a Chirobiotic T (Teicoplanin) column using acetonitrile–methanol–acetic acid–triethylamine (55:45:0.3:0.2, v/v/v/v) as eluent. Detection of the effluent was performed by fluorescence measurement. Several experiments were carried out to suppress the high blank reading, which was efficiently achieved using Tris buffer in the first dimension. For the enantioselective analysis of ( R)- and ( S)-atenolol in plasma under the same conditions the sample capacity of the ADS column is considerably lower.

Development of fluorescence based flow immunoassays utilising restricted access columns

The development of high-speed flow immunoassay techniques is described. The principles are based on heterogeneous flow immunoassay interactions. High sample throughput can be used for screening small analytes in a number of biological matrices originating from samples of water from environmentally polluted areas, or biological fluids such as urine and plasma. The immunochemical detection principle is based on chromatographic separation of the immunocomplex formed (AbAg or AbAg*) and the free antigen (Ag) by a restricted access (RA) column, utilising size-exclusion and reversed phase mechanisms. A fluorescein-labelled analyte (Ag*) was used in the competitive assay format with fluorescence detection. Sample throughput was 80 h−1 and detection limits 1.4 nM (300pgml−1) for atrazine and 2.3 nM (500 pg ml−1) for the sum of triazines. Analyses could be performed at a sample throughput of 400 6 h−1 shift.

High sample throughput flow immunoassay utilising restricted access columns for the separation of bound and free label

A flow immunodetection system with high sample throughput capacity is described for the screening of various analytes. The immunochemical detection principle is based on the chromatographic separation of the formed immunocomplex (AbAg or AbAg*) and the free antigen (Ag) by a restricted access (RA) column, utilising size-exclusion and reversed-phase mechanism. A fluorescein labelled analyte (Ag*) was used in the competitive assay format with fluorescence detection. The speed and simplicity of the assay were the greatest advantages, allowing measurement of the analyte to be carried out in less than 1 min. The biocompatibility and capacity of the restricted access material allowed multiple injections of up to 5000, without any breakthrough of the fluorescent tracer molecule and thus need for regeneration. The flow immunoassay was developed using the well-known atrazine herbicide and some transformation products as model compounds, due to their human toxicity and widespread use. The sample throughput was 80 samples per hour and the detection limits were 1.4 n M (300 pg/ml) for atrazine (Ab I) and 2.3 n M (500 pg/ml) for the sum of triazines (Ab II–III). Different sample matrices, PBS buffer, creek water, and urine were successfully applied in the flow system without the need for any sample handling step. For plasma samples an additional clean-up step using solid-phase extraction had to be included. The resulting detection limits for atrazine in plasma and water samples using this clean-up and trace enrichment procedure were found to be 2 ng/ml and 20 pg/ml, respectively. The analysis could be performed at a sample throughput rate of 400 per 6-h working shift.

Quality control in the determination of cortisol in plasma/serum by using, on every sample, two different three-step separation methods including ultrafiltration, restricted-access high-performance liquid chromatography and reversed-phase high-performance liquid chromatography, and contrasting results to immunoassays

Tests of HPLC columns with restricted access, polymer covered alumina, polymer, and different ODS phases showed that base-acid compatible ODS columns gave the best peak shapes of cortisol, internal standard, as well as of plasma/serum (P/S) matrix components. Further trials with cortisol in P/S showed that three separation steps were essential in order to obtain chromatographic data which were superior to immunoassay data. Also, sufficient confidence in results required determination of each sample with two newly developed separation methods: (a) pre-separation with a restricted access column, concentration of the desired cut with a 20 mm base-acid compatible ODS column, and analysis with a 250 mm column filled with the same ODS; (b) pre-separation with an ultrafilter followed by the last two steps in (a). For detection UV was preferred over fluorescence. This twin multistep chromatography showed that immunoassays were very treacherous in that they produced a spectrum of results ranging from good to untenable without any warning whatever about functionality. The measurement of official controls, with reference values derived via gas chromatography-isotope dilution mass spectrometry, also demonstrated the superiority of the double HPLC method.