Column-switching HPLC System Research Articles

Determination of Free [formula omitted]-Proline and [formula omitted]-Leucine in the Brains of Mutant Mice Lacking [formula omitted]-Amino Acid Oxidase Activity

A new procedure to accurately measure a trace amount of d-proline in biological samples has been developed. This d-amino acid was derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole and was determined by a column-switching HPLC system, a combination of a micro-ODS column and a chiral column. The detection limit for d-proline spiked in a mouse cerebrum sample is 1 fmol (injection amount, S/N = 3). Within-day precision and day-to-day precision obtained for spiked d-proline (10 fmol) are 2.14 and 5.35% (RSD), respectively. Using the new method, the amount of free d-proline in eight brain regions and sera of mutant ddY/DAO− mice, lacking d-amino acid oxidase activity, and control ddY/DAO+ mice was determined. The amount of free d-leucine was also investigated. The amount and distribution of d-proline in the brains of ddY/DAO+ mice and ddY/DAO− mice are almost the same, and relatively high amounts of d-proline have been observed in the pituitary gland and in the pineal gland. On the other hand, the amount of d-leucine is different between the two strains. In the brains of ddY/DAO+ mice, a relatively high amount of d-leucine has been observed in the pineal gland compared with other regions. In the brains of ddY/DAO− mice, d-leucine amounts are approximately 10 times higher than those obtained in ddY/DAO+ mice and regional difference has not been observed, while the amounts of l-proline and l-leucine are not significantly different between the two strains. In the serum, the amounts of both free d-proline and d-leucine are significantly higher in the ddY/DAO− mice than those obtained in ddY/DAO+ mice.

Methylcellulose-immobilized reversed-phase precolumn for direct analysis of drugs in plasma by HPLC.

We evaluated a new restricted access media (RAM) precolumn for direct analysis of drugs in plasma using a column switching HPLC system. The new RAM material was prepared by the modification of the external surface of porous silica with hydrophilic methylcellulose (MC), followed by modification of the internal surface with octadecylsilane (ODS). The external surface of the MC-immobilized ODS silica material (MC-ODS) suppressed the adsorption of proteins, while the internal surface of MC-ODS retained various types of drugs, such as ketoprofen, propranolol, caffeine and atenolol in plasma samples. In addition, MC-ODS allowed direct analysis of drugs in a 1000-microL plasma sample to monitor trace amounts of analytes contained. Reduced efficiency and clogging of the MC-ODS precolumn and/or the analytical column were not observed even after the repetitive injection of plasma sample up to 40 mL. Our results indicated that the MC-ODS precolumn could be used in pharmacodynamic and clinical studies.

Determination of d- and l-aspartate in cell culturing medium, within cells of MPT1 cell line and in rat blood by a column-switching high-performance liquid chromatographic method

HPLC fluorometric methods have been used to analyze trace amounts of d-amino acids in biological samples. In this study, we established an expedient column-switching fluorometric HPLC system that would improve the analysis of d-amino acids, in particular d-aspartate (Asp). Our system consists of the fluorogenic derivatization of amino acids with NBD-F and two chromatographic steps, one that separates individual amino acids in reverse phase mode and another that separates the chiral forms of each amino acid in normal-phase mode. The two separation steps are linked through a trapping column by an automated column-switching system. In addition, sample preparation is simplified and improved, where trichloroacetic acid is used for deproteinization, and borate buffer, pH 9.5 is employed for the fluorescent derivatization. The detection limit for d-Asp in culturing medium is 5 n M. The resulting peak heights correlated well with concentrations that ranged from 12.5 to 250 n M for both d- and l-Asp. The present method was applied to determine d- and l-Asp levels in cell culturing medium, and within cells of MPT1 cell line. The detected cellular levels of d- and l-Asp agree with those detected by our previous method. In addition, this method was used to measure d- and l-Asp levels in rat blood samples, and the results are consistent with the reported values.

Simultaneous determination of byak-angelicin and oxypeucedanin hydrate in rat plasma by column-switching high-performance liquid chromatography with ultraviolet detection

A simple and sensitive column-switching HPLC method was developed for the simultaneous determination of two furocoumarin compounds, byak-angelicin and oxypeucedanin hydrate, which are the main components of hot water extract of Angelica dahurica root (AE), in rat plasma. Plasma sample was simply deproteinated with perchloric acid. After centrifugation, the supernatant was injected into a column-switching HPLC system consisting of a clean-up column (Symmetry Shield RP 8, 20×3.9 mm I.D.) and analytical column (Symmetry C 18, 75×4.6 mm I.D.) which were connected with a six-port switching valve. The flow-rate of the mobile phase (acetonitrile–water, 20:80) was maintained at 1 ml/min. Detection was carried out at wavelength 260 nm with a UV detector. The column temperature was maintained at 40°C. The calibration curves of byak-angelicin and oxypeucedanin hydrate were linear over the ranges 19.6 to 980 ng/ml ( r 2>0.997). The accuracy of these analytes was less than 4.4%. The intra- and inter-day relative standard deviations of byak-angelicin and oxypeucedanin hydrate were within 12.0% and 12.7%, respectively. The present method was applied for the analysis of plasma concentration from rats after administration of AE.

Calpain inhibitor, SJA6017, reduces the rate of formation of selenite cataract in rats

Purposes. 1) To measure the amount of calpain inhibitor SJA6017 taken up by lenses of young rats after administration; and 2) To test efficacy of SJA6017 against selenite cataract in regard to amelioration of proteolysis of lens protein and prevention of lens nuclear opacity. Methods. Selenite nuclear cataracts were produced by subcutaneous injection of an overdose of sodium selenite to 16-day-old rats. SJA6017 was administered daily using intraperitoneal injections at 100 mg/kg body weight/day for 4 days. Lenses were observed and photographed by slit lamp biomicroscopy, and scored into one of three stages. Enucleated lenses were also scored into one of four stages and lens opacities in the nuclear region were quantified by image analysis. Proteolysis of crystallins was detected by SDS-PAGE. The amount of SJA6017 taken up by the lens was detected with a column switching HPLC system. Results. Nuclear cataracts were visible in 31% of the animals receiving only selenite, while the frequency of nuclear cataract in the Se+SJA6017 group was reduced to only 16%. This effect of SJA6017 was confirmed by densitometric analysis as a reduction in the density of the nucleus. Similar proteolytic changes of crystallins occurred at all stages of selenite cataract formation. The amount of SJA6017 in the lens was detected at the level of 0.03 µM. Conclusions. Systemic SJA6017 was taken up by the lens, and SJA6017 ameliorated in vivo selenite cataract formation. These studies are important because they partially validate the biochemical rationale for developing non-surgical, drug treatments for cataract prevention in man.

Simultaneous determination of NK-104 and its lactone in biological samples by column-switching high-performance liquid chromatography with ultraviolet detection

A simple and sensitive column-switching HPLC method has been developed for the simultaneous determination of NK-104 (HMG–CoA reductase inhibitor) and its lactone in human and dog plasma. Plasma sample was extracted with methyl tert-butyl ether and then the extract was subjected to methylation with diazomethane to prevent the mutual conversion between NK-104 and its lactone. The extract was injected into the column-switching HPLC system. The calibration curves of NK-104 and NK-104 lactone were linear over the ranges 0.5 to 100 ng/ml for human plasma samples and 0.5 to 500 ng/ml for dog plasma, respectively. The intra-day and inter-day C.V. values of these analytes were less than 13.3%. The intra-day and inter-day accuracies of these analytes were between −14.0 and 6.5%. The proposed method has been applied to plasma samples obtained after oral administration of a single 2 mg dose of NK-104 to volunteers.

Prevention of co-elution of steroid sulfates with serum proteins from pre-column in column-switching HPLC system.

A method to prevent co-elution of steroid sulfates with proteins in serum from the pre-column in column-switching HPLC was developed. The pre-column, a polymer-coated mixed function column, was used for ion-pair chromatography with 5 mM tetra-n-butylammonium (TBA) ion. As steroid sulfates, estriol 3-sulfate, dehydroepiandrosterone 3-sulfate and pregnenolone 3-sulfate were used. Human serum (25 microl) was diluted with mobile phases including 5, 100 and 500 mM TBA ion, and then injected directly into the pre-column. The peak areas of the steroid sulfates in serum samples were compared with those of the steroid standards without serum. When 25/microl of serum was diluted with mobile phase including 100 or 500 mM TBA ion, the steroid sulfates in serum were retained in the pre-column; however, the steroid sulfates from the same sample diluted with mobile phase containing 5 mM TBA ion were not retained in the pre-column. Addition of an excess amount of counter ion (TBA ion) into the serum sample made it possible to retain the steroid sulfates in the pre-column. This method was applied to column-switching HPLC for measurement of steroid sulfates in serum using a semi-microcolumn as the analytical column.

Stereoselective determination of unchanged and glucuroconjugated eliprodil, a new anti-ischaemic drug, in human plasma and urine by precolumn derivatization and column-switching high-performance liquid chromatography with fluorescence detection

An HPLC method was developed and validated for the determination in human plasma and urine of the enantiomers of eliprodil, (±)-α-(4-chlorophenyl-4[(4-fluorophenyl)methyl]piperidine-1-ethanol hydrochloride, a new anti-ischaemic agent administered as a racemate. Both enantiomers are present in human plasma in unchanged and glucuroconjugated form, whereas only the glucuroconjugated form is excreted into urine; as a consequence, such metabolites in human plasma and urine should be submitted to enzymatic deconjugation with β-glucuronidase (Escherichia coli) before being extracted. The general method involves a liquid-liquid extraction of eliprodil and internal standard from alkalinized plasma or urine with n-hexane, evaporation of the organic phase and derivatization with ( S)-(+)-naphthylethyl isocyanate to give carbamate diastereoisomeric derivatives of ( S)-(+)- and ( R)-(−)-eliprodil and internal standard; after evaporation of the derivatizing mixture and dissolution of the residue in a small volume of phosphate buffer-acetonitrile (60:40, v/v), an aliqout is injected into a column-switching HPLC system. The derivatized sample extract is purified on a precolumn filled with C 8-bonded silica material, which is flushed with acetonitrile-water, then diastereoisomers of eliprodil and the internal standard are automatically transferred by the mobile phase to the analytical column. The analytical column is a C 8 type, specially deactivated for basic compounds, the mobile phase is 0.025 M phosphate buffer (pH 2.6)-methanol-acetonitrile (42:2:56) at a flow-rate of 1.2 ml min −1 and a fluorimetric detector operating at λ ex = 275 nm and λ em = 336 nm is used. The retention times, under these conditions, are about 16 and 17 min for ( S)-(+)- and ( R)-(−)-eliprodil diastereoisomers, respectively, and about 19 min for the first-eluted diastereoisomer of the internal standard. During the analysis time, the precolumn, reset in a different path from that of the analytical column, is back-flushed with different solvents, then re-equilibrated with acetonitrile-water before the next injection. Linearity in plasma, for unchanged eliprodil enantiomers, was assessed in the range 0.15–10 ng ml −1 and for total eliprodil enantiomers (unchanged + conjugated) in the range 0.75–500 ng ml −1; the limit of quantitation (LOQ) is 0.15 ng ml −1 for each unchanged enantiomer and 0.75 ng ml −1 for each total enantiomer. Linearity was also assessed in urine for total (conjugated) eliprodil enantiomers in the range 50–25 000 ng ml −1; the LOQ is 50 ng ml −1 for each enantiomer. The intra- and inter-day precision and accuracy of the method were investigated in plasma and urine and found to be satisfactory for pharmacokinetic studies. The method has been extensively used in pharmacokinetic studies in man treated with a 20-mg dose of eliprodil racemate and some results of this application are reported.

Determination of Proguanil, Cycloguanil and 4-Chlorophenyl-biguanide in Saliva and Plasma by Ion-Pairing Column Switching HPLC

Abstract The application of a column switching HPLC system to the analysis of the antimalarial proguanil and its two metabolites in plasma and saliva is described. The assay is based on a previously developed hydrophobic ion-pairing separation using sodium dodecylsulphate on ODS Hypersil. The use of a precolumn to concentrate and allow pre-treatment of these drugs in the two matrices is shown. This is followed by the transfer and subsequent separation of the biguanides by the analytical phase containing a high concentration of hydrophobic pairing ion. It is found that such on-line sample pre-treatment can yield comparable analytical characteristics to off-line solid phase extraction procedures if the precolumn is conditioned between sample injections. The columns can be used for large numbers of saliva assays but when plasma is the biological matrix components of the plasma cause rapid deterioration of the precolumn. The column switching assay described is used to determine the steady state concentrations...

Direct injection of large volumes of plasma in a column-switching system for the analysis of local anaesthetics I. Optimization of semi-permeable surface precolumns in the system and characterization of some interference peaks

Possibilities for accomplishing direct injection of large volumes (500 μl) of plasma samples into a column-switching HPLC system were investigated. A new format of precolumn containing a semi-permeable surface (SPS) support (1 cm × 1 cm) was used for the sample clean-up and trace enrichment and was combined with a Kromasil C 18 column for the final separation. A stable chromatographic system with respect to the separation selectivity and separation time was constructed and evaluated. The main parameters were the hydrophobicity of the SPS column, pH of the eluents, concentration of the organic modifier in the eluents and the detection wavelenght. Two main interference peaks that were eluted in front of the ropivacaine peak were systematically characterized by varying the loading conditions for the SPS precolumn. The SPS column could tolerate large volumes (≤500 μl) of plasma injections with a total volume of more than 50 ml. The developed system is stable, which permits the detection of 30 ng/ml ropivacaine in human plasma.

Simultaneous determination of human neutrophil elastase inhibitor (ONO-5046) and its metabolite in plasma and urine by direct injection column-switching HPLC

The direct injection method for the simultaneous determination of a human neutrophil elastase inhibitor (ONO-5046) and its metabolite (ONO-EI-601) in plasma and urine has been developed using a column-switching HPLC system. The system was set up with a pre-treatment column, a pre-concentration column and an analytical column which were connected in series via two automatic switching valves. The calibration lines showed a good linearity for concentrations of ONO-5046 and ONO-EI-601 in a range of 156–1000 ng ml −1 in plasma and 100–156 μg ml −1 in urine. all correlation coefficients being greater than 0.9999. The limit of quantitation of ONO-5046 was 156 ng μg ml −1 in plasma, that of ONO-EI-601 was 313 μg ml −1 in plasma, and those of ONO-5046 and ONO-EI-601 were 1.56 μg ml −1 in urine. The developed method is rapid and sensitive with automated operation, allowing untreated samples to be analysed every 45 min. The application of the present method to the real plasma and urine samples proved to be useful for pharmacokinetic, toxicological and clinical studies.

Regulation of acetylcholine release in vivo from rat hippocampus by monoamines as revealed by novel column-switching HPLC with electrochemical detection

To clarify monoaminergic regulation of acetylcholine (ACh) release in the rat hippocampus, the effects of administration of monoamines through a dialysis probe on extracellular ACh levels were examined using in vivo brain microdialyssi combined with a novel column-switching HPLC system. Infusion of dopamine or 5-hydeoxytryptamine, but not noradrenaline, increased ACh levels. The ACh levels also increased following infusion of apomorphine and 5-methoxy- N,N-dimethyltryptamine. These results demonstrate that hippocampal ACh release is regulated by dopamine and 5-hydroxytryptamine.

Quantification of midodrine and its active metabolite in plasma using a high performance liquid chromatography column switching technique

An automated column-switching HPLC system is described for the simultaneous determination of midodrine, an alpha-adrenergic stimulating drug, and its active metabolite, ST-1059. Serum or plasma (850 microliters) is directly injected onto a RP18 (30 micrograms particle size) pre-column (9 x 4 mm ID) which acts as an on-line liquid-solid extractor and analyte enrichment system. The injection is followed by washing steps. The fraction containing the analytes is transferred onto an analytical RP18 column via step gradient elution where the final analysis is performed. Fluorescence detection is used (lambda ex 290 nm and lambda em 322 nm), and method detection limits of 0.8 ng/mL plasma were reached. These were sufficiently low to determine the plasma concentration-time profiles for both compounds following oral administration of 2.5 mg and 5 mg midodrine hydrochloride. The assay in serum or plasma was linear in the range of 1 to 15 ng analyte/mL, the recovery was greater than 95%, and the reproducibility was sufficient. The assay was rugged and was maintained by routinely changing the home-made, dry packed pre-column every 20th serum injection.

High-performance liquid chromatography of 5-fluorouracil after derivatization with 4-bromomethyl-7-methoxycoumarin. Characterization of the derivative and the use of column switching for the improvement of resolution and the enhancement of sensitivity

The derivatization of 5-fluorouracil with 4-bromomethyl-7-methoxycoumarin has been reported previously; however, the structure of the derivative was not confirmed. The synthesis and purification of the 5-FU derivative is described along with the spectroscopic (MS and NMR) determination that it is labelled at both heterocyclic nitrogens as expected. A column switching HPLC system is also presented which consists of primary separation on a cyanopropyl column followed by a final separation on an ODS column with fluorescence detection. This system removes all interferences from the derivatization system and has a limit of detection for the pure derivative of <50 fmol (injection volume = 100 μl).

Trace Analysis by Microbore HPLC

Abstract The potential and problems of trace analysis by microbore HPLC with on-column concentration and the benefits of additional column-switching in several modes are discussed. An example of the performance of an on-column concentration column-switching microbore HPLC system is shown.