Conventional High-performance Liquid Chromatography Methods Research Articles

Optical enzyme sensor for determining l-lysine content using l-lysine oxidase from the rockfish Sebastes schlegeli

L-lysine (L-Lys) in living bodies is critical for metabolism; therefore, determination of its levels in food is important. Most enzymatic methods for L-Lys analysis are performed using L-lysine oxidase (LyOx), but commercially manufactured LyOx is generally not highly selective for L-Lys among amino acids. We previously isolated LyOx as an antibacterial protein secreted from the skin of the rockfish Sebastes schlegeli. In the present study, we developed an optical enzyme sensor system for rapid and continuous determination of L-Lys using this LyOx. The system comprised an immobilized LyOx membrane, an optical oxygen probe, a flow system, and a personal computer. The amount of L-Lys was detected as a decrease in the oxygen concentration due to the LyOx reaction. The specificity of the sensor was examined against various amino acids. The sensor response was specific for L-Lys. Good reproducibility was obtained in 58 assays. The response of the sensor using commercially prepared LyOx was unstable compared with the response using LyOx isolated in our laboratory. Our sensor system could be used for 5 weeks without our having to change the enzyme membrane. The calibration curve for a standard L-Lys solution was linear from 0.1 to 3.0 mmol L(-1). One assay could be completed within 2 min. The sensor was applied to determine the L-Lys content in food samples such as bonito cooking water and scallop hepatopancreas. The values obtained using the sensor and conventional high-performance liquid chromatography methods were well correlated.

On-line desalting–mass spectrometry system for the structural determination of hydrophilic metabolites, using a column switching technique and a volatile ion-pairing reagent

A novel desalting method, using a column switching technique and a volatile ion-pairing reagent, pentadecafluorooctanoic acid, was developed. This system allows hydrophilic and cationic compounds in a nonvolatile buffer to be directly introduced into a mass spectrometer for structural elucidation. The desalting procedure consists of four steps: (1) the fractionation of a target compound from a separation column, (2) the removal of salts with pentadecafluorooctanoic acid on the trap column, (3) the desorption of the compound from the trap column, and (4) the re-equilibration of the trap column with a pentadecafluorooctanoic acid solution. In this procedure, we investigated the methods for optimizing the desalting and re-equilibration steps. Various amino acids, including branched chain amino acids, aromatic amino acids, basic amino acids and methionine, after separation with phosphate buffer on a cation-exchange column, were successively desalted by this method, and were observed as protonated ions by mass spectrometry. This desalting system could be useful for the structural elucidation of unknown hydrophilic compounds eluted by conventional high-performance liquid chromatography methods, such as ion-exchange chromatography, with mobile phases containing nonvolatile salts. As an example, we present the structural elucidation of unknown metabolites in bovine serum.

Microemulsion liquid chromatographic method for characterisation of fosinopril sodium and fosinoprilat separation with chemometrical support

The properties of the eluent are the essential factors governing the efficiency in the high-performance liquid chromatography (HPLC) method. A novel approach in retention modelling in the liquid chromatographic separation of fosinopril sodium and its degradation product, fosinoprilat, applying a microemulsion as the mobile phase, was used. The modifications of the mobile phase included the changes to the type of the lipophilic phase, the type and concentration of co-surfactant and surfactant, as well as the pH of the mobile phase. In this study, a full factorial 2(3) design, as the optimal method for screening of the experiment, was applied for selecting factors which had an influence on separation. Optimisation was done by a central composite design. An appropriate resolution with reasonable retention times was obtained with a microemulsion containing 0.9% w/w of cyclohexane, 2.2% w/w of sodium dodecyl sulphate (SDS), 8.0% w/w of n-butanol and 88.9% of aqueous 25 mM disodium phosphate, the pH of which was adjusted to 2.8 with 85% orthophosphoric acid. Separations were performed on an X-Terra 50-mmx4.6-mm, 3.5-mum particle size column at 30 degrees C. UV detection was performed at 220 nm and with a flow rate of 0.3 mL min(-1). The established method was validated and applied for analysis of appropriate tablets. The proposed chromatographic procedure for the separation of fosinopril sodium and its degradation product is less expensive compared with the conventional reversed-phase HPLC method, as well as being simple and rapid. The optimised and validated method can be used for separation, identification and simultaneous determination of fosinopril sodium and fosinoprilat in bulk drug and in pharmaceutical dose forms.

LC–tandem MS in proteome characterization

The coupling of high-performance liquid chromatography (HPLC) to mass spectrometry (MS) has been routinely used for a number of years for the analysis of a wide variety of different compounds. In typical proteomic analyses, where enzymatic digestion is used to generate proteolytic peptides, the limited amount of sample restricts the utility of conventional HPLC methods for MS detection. As reduced column diameters and nanolitre per minute flow rates have become increasingly standard, the application of HPLC to the analysis of low-volume, low-abundance samples has now become readily achievable. A number of novel chromatographic methods have increased the utility of such approaches for proteome-wide analyses. However, there remain in proteomic analyses some important challenges, which are being addressed by state-of-the-art methodologies. This article reviews a number of pertinent considerations and technological advances in proteomic analyses using HPLC–tandem MS (MS 2).

Method Development for Cortisol and Cortisone by Micellar Liquid Chromatography Using Sodium Dodecyl Sulphate: Application to Urine Samples of Rugby Players

The chromatographic behavior of cortisol and cortisone using a micellar medium of sodium dodecyl sulphate (SDS) as surfactant, a Hypersil C18 (150- x 3.2-mm i.d., 5 microm) column, a flow rate of 0.5 mL/min, and UV absorbance detection at 245 nm is described. The effect of several organic modifiers and the surfactant concentration on the separation is studied. A mobile phase of 18 mM SDS and 8.3% tetrahydrofuran allows for the separation of cortisol and cortisone up to baseline. These results are also achieved by applying a bivariant optimization method. The proposed method is sensitive, reproducible, and selective. In addition, it is less expensive than conventional high-performance liquid chromatography methods for cortisol and cortisone. The method is applied to the determination of cortisol and cortisone in urine samples of rugby players before and after stress for doping control purposes.

DETERMINATION OF DRUG GLUCURONIDATION AND UDP-GLUCURONOSYLTRANSFERASE SELECTIVITY USING A 96-WELL RADIOMETRIC ASSAY

A rapid and sensitive radiometric assay for UDP-glucuronosyltransferase (UGT) is described. UGT substrates are incubated in 96-well plates with microsomes in the presence of [14C]UDP-glucuronic acid, and 14C-labeled glucuronidation products are separated from the unreacted nucleotide sugar by solid-phase extraction using 96-well extraction plates. The assay was validated with 15 structurally diverse UGT substrates containing acidic, phenolic, and hydroxyl reacting groups. Glucuronidation velocities for these compounds were determined using human, rat, and dog liver microsomes, and reaction kinetics were studied with 1-naphthol and 4-methylumbelliferone. Results obtained with the new assay confirmed the previously reported rank order of glucuronidation velocity of several typical UGT substrates and the finding that the glucuronidation of most of these compounds is significantly faster in dog than in human liver microsomes. UGT specificity of five compounds was determined using recombinant human UGTs. The major UGT isoforms identified were UGT1A6, UGT1A7, and UGT1A9 for 4-methylumbelliferone; UGT1A6 and UGT1A8 for 1-naphthol; UGT2B7 for naloxone; UGT1A3 and UGT2B7 for ketoprofen; and UGT1A4 for trifluoperazine. Identical results were obtained with a conventional high-performance liquid chromatography method coupled to mass spectrometric detection. The new assay should prove valuable for rapidly benchmarking recombinant UGTs and microsomal preparations from different species and tissues, identifying high-turnover compounds during drug discovery, and reaction phenotyping studies.

Determination of alkylphenols and alkylphenol polyethoxylates by reversed-phase high-performance liquid chromatography and solid-phase extraction.

A simple, accurate and reproducible reversed-phase high-performance liquid chromatography (HPLC) method was developed for the separation and characterisation of alkylphenols (APs) and alkylphenol polyethoxylates (APEOs), using a C18 octadecyl silica (ODS) column. APs and each APEO oligomer were separated successfully within a reasonable time without gradient elution. An excellent resolution was obtained, even for mixtures of APs and low EO number APEOs, which are otherwise difficult to separate using conventional normal-phase HPLC methods. This method, combined with solid-phase extraction, was highly applicable for the simultaneous determination of alkylphenols and alkylphenol ethoxylates in real samples.

Gradient Polyacrylamide Gel Electrophoresis for Determination of Molecular Weights of Heparin Preparations and Low-Molecular-Weight Heparin Derivatives

The Mr values of pharmaceutical heparins and low-molecular-weight (LMW) heparin derivatives were examined as part of a collaborative study to develop methods for their characterization. Standard methods of Mr determination rely on gel permeation high-performance liquid chromatography (HPLC). We report the use of gradient polyacrylamide gel electrophoresis (PAGE) to determine the Mr values of pharmaceutical heparins and LMW heparin derivatives. This approach offers certain advantages over the HPLC method. Gradient PAGE analysis was performed in parallel, on multiple samples, with the same standard curve. HPLC was performed serially. Gradient PAGE gave higher resolution than HPLC, and thus, a mixture of easily obtained standards was used in place of individual standards for the construction of a standard curve. Heparin and various LMW heparin samples were analyzed by both gradient PAGE and conventional gel permeation HPLC methods. The number-average Mr, weight-average Mr, and polydispersity were examined by both techniques and found to be similar. This study demonstrates that gradient PAGE analysis is a sensitive method for the determination of the Mr values of heparin and LMW heparin.

Simple Prediction of Stability Constants for Inclusion Complexes of β-Cyclodextrin with various Drug Molecules Using β-Cyclodextrin Bonded Phases(Cyclobond I Column)

AbstractThe stability constants(Kc) for inclusion complexes of cyclodextrin with sulfonamides, sulfonylureas and p-aminobenzoic acid esters were investigated by high performance liquid chromatography (HPLC) using the cyclodextrin bonded phase. The retention time(Rt) of these drugs on a Cyclobond® I column showed a good relationship to K, values obtained by the solubility method and/or conventional HPLC method. The advantages of this method were that the K, values could be rapidly predicted by a simple procedure using a minimum quantity of the drugs.

Simple Prediction of Stability Constants for Inclusion Complexes of β-Cyclodextrin with various Drug Molecules Using β-Cyclodextrin Bonded Phases(Cyclobond® I Column)

The stability constants(Kc) for inclusion complexes of cyclodextrin with sulfonamides, sulfonylureas and p-aminobenzoic acid esters were investigated by high performance liquid chromatography (HPLC) using the cyclodextrin bonded phase. The retention time(Rt) of these drugs on a Cyclobond® I column showed a good relationship to K, values obtained by the solubility method and/or conventional HPLC method. The advantages of this method were that the K, values could be rapidly predicted by a simple procedure using a minimum quantity of the drugs.

Post column derivatization methodology in high performance liquid chromatography (HPLC)

Post column derivatization procedures represent a powerful analytical tool. The methodology is suitable for trace and ultra trace analysis and offers enhancement of both detectability and specificity compared to conventional HPLC methods.