Chemo-affinity of titania for the column-switching HPLC analysis of phosphopeptides.

A column-switching high-performance liquid chromatography (HPLC) system for the determination of phosphopeptides has been developed. The method is based on the selective adsorption of phosphopeptides on a titania (TiO2) precolumn and successive HPLC separation of the phosphopeptides on an anion-exchange column with a UV detector (215 nm). The recoveries of phosphopeptides were tested using authentic phosphopeptides [Gln-Ile-Ser(p)-Val-Arg, Ile-Ser(p)-Val-Arg and Lys-Gln-Ile-Ser(p)-Val-Arg] at an injection amount of 1 microg. The recoveries were 74.3, 79.6, and 82.6%, respectively, while the corresponding dephosphopeptides were not retained on the titania precolumn.     

Titania as a chemo-affinity support for the column-switching HPLC analysis of phosphopeptides: application to the characterization of phosphorylation sites in proteins by combination with protease digestion and electrospray ionization mass spectrometry.

A method for the determination of phosphorylation sites in phosphoproteins based on column-switching high-performance liquid chromatography (HPLC) has been developed. The HPLC system consisted of a titania precolumn for the selective adsorption of phosphopeptides, an anion-exchange analytical column and a UV detector (215 nm). Rabbit muscle phosphorylase a (RPa) and porcine stomach pepsin (PSP) were tested as model phosphoproteins. After protease digestion, the resulting phosphopeptides were successfully isolated by column-switching HPLC. The phosphopeptide fractions were analyzed by electrospray ionization mass spectrometry with a positive or negative ion mode after purification by reversed-phase HPLC. Pseudo-molecular ion peaks corresponding to Gln-Ile-Ser(p)-Val-Arg (MW 681.7) and Glu-Ala-Thr-Ser(p)-Gln-Glu-Leu (MW 856.8) were detected from the tryptic digest of RPa and chymotryptic digest of PSP, respectively, which agreed with the theoretically expected phosphopeptide fragments.     

Application of an internal surface reversed-phase column for the automated determination of flucycloxuron residues

A liquid chromatographic column-switching system for the automated determination of flucycloxuron, a benzoylphenylurea pesticide, in crop and environmental matrices is described. The system consists of an internal surface reversed-phase (ISRP) column, a phenyl-bonded precolumn and an analytical reversed-phase (RP) C₁₈ column. Sample extracts are evaporated to dryness and dissolved in the mobile phase of the ISRP column. An aliquot of this solution is injected into the column-switching system. Clean-up, with regard to removal of large molecules, is performed on the ISRP column. The flucycloxuron fraction from the ISRP column is concentrated on the phenyl-bonded precolumn. Additional clean-up can be performed by washing the precolumn. Finally, the compound is desorbed from the precolumn and separation and determination of the Z- and E-isomers of flucycloxuron are performed with the analytical RP-C₁₈ column using UV detection at 254 nm. The total analysis time required is 40 min. The reproducibility of the method obtained with the column-switching system, expressed as relative standard deviation, varies between 3.7 and 10% for apple, strawberry, citrus and soil samples for flucycloxuron levels between 0.04 and 0.33 mg/kg. The system showed no loss of analytical performance after more than 300 analyses.     

High-performance liquid chromatographic column-switching technique for the determination of intermediates of anaerobic degradation of toluene in ground water microcosm.

A reversed-phase liquid chromatographic column-switching system was used for the determination of phenol, benzoic acid and cresol (PBC) in the presence of toluene in ground water microcosm. A precolumn was connected in series with an analytical column via a column-switching valve. After the injection, as soon as PBC were eluted from the precolumn to the analytical column, the valve was switched so that the precolumn was between the analytical column and the UV detector. Toluene and other non-polar compounds were eluted from the precolumn in a very short time and detected along with the solvent front. Subsequently, PBC were separated on the analytical column and passed through the precolumn one more time before being detected by the UV detector. The total analysis time was 15 min. This technique facilitated the study of the basic mechanism and path way of anaerobic degradation of toluene in ground water aquifer.     

HPLC determination of cis-diamminedichloroplatinum(II) in plasma and urine with UV detection and column-switching

A method for determining cis-diamminedichloroplatinum(II) (CDDP), an anticancer drug, in plasma and urine by HPLC with UV detection and column-switching has been developed. Typical conditions were as follows. An apparatus was composed of two columns, two pumps, a UV detector, a sample injector with a 100 microL loop, a switching valve, a column oven and a recorder. A Rheodyne model 7125 sample injector was used as the switching valve. A precolumn (4.6 mm ID x 25 cm) was packed with MCI GEL CK10S (a strong cation exchanger), and an analytical column (4.6 mm ID x 5 cm) was packed with MCI GEL CDR10 (a strong anion exchanger). Both columns were connected in series via the switching valve. The CDDP-containing fraction of the effluent from the precolumn was loaded to the analytical column by column-switching and the effluent from the analytical column was monitored at 210 nm. An eluent of 0.3 M sodium dihydrogen phosphate was pumped at a flow rate of 1 mL/min and the columns were maintained at 40 degrees C. CDDP was eluted at about 11 min and the identity of the peak of CDDP on the chromatogram was confirmed by its 3-dimensional chromatogram and analysis of platinum in the column effluent. Under the conditions described above, a linear relationship was obtained between peak height and concentration of CDDP up to 100 microM. Correlation efficients were 0.998 for plasma and 0.999 for urine. The detection limit was 0.1 microM for CDDP in both plasma and urine (S/N = 3,0.005 AUFS). The reproducibility was within 3% for 10 determinations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Application of an anhydrotrypsin-immobilized precolumn for selective separation of peptides having arginine or lysine at their C-termini by column-switching high-performance liquid chromatography

A column-switching high-performance liquid chromatographic (CS-HPLC) system which consisted of an anhydrotrypsin (AHT)-immobilized diol-silica precolumn and a reversed-phase analytical column was developed for the selective separation of peptides having Arg or Lys at their C-termini. Tuftsin (Thr-Lys-Pro-Arg) could be enriched almost quantitatively on the precolumn when loaded with water as a carrier solvent and the precolumn was washed with 10-30 mM acetate buffer (pH 5.0). An investigation of the affinity characteristics of 55 peptides to the AHT precolumn showed that among twelve peptides having Arg or ArgNH2 at their C-termini and more than four amino acid residues, ten were retained almost quantitatively on the precolumn, and eight out of nine peptides having Lys at their C-termini were less retained. The peptide having D-Arg at its C-termini was not retained. However, twelve out of thirty peptides having no Arg or Lys at their C-termini were also retained, but the retention was greatly decreased, in contrast to the Arg peptides, when the precolumn was washed with 20 mM calcium chloride solution. The results indicate that the CS-HPLC system equipped with an AHT precolumn offers new selectivity in the HPLC selectivity in the HPLC separation of peptides.     

Characterization of the precolumn bio trap 500 C18 for direct injection of plasma samples in a column-switching system

Summary A new restricted access media (RAM) type of precolumn, Bio Trap 500 C₁₈, for direct injection of plasma samples in column-switching systems was evaluated with respect to the elution of plasma proteins in different mobile phases, the loading capacity of plasma samples, the chromatographic behavior during plasma injections and protein contamination of the packing and sealings. More than 95% of plasma proteins could be excluded from the precolumn within three minutes for all selected mobile phases. Quantitative analyte recoveries could be obtained by injecting plasma samples ranging from 5 to 500 μL with the analyte mass>150 ng onto a BioTrap 500 C₁₈ column (20×4 mm I.D.). One precolumn tolerated about 15 mL of plasma injection without out noticeable change in retention and pressure. Clogging of the precolumn was encountered (≥45 mL of plasma) due mainly to the adsorption of proteins on the packing. The performance of the analytical column (Kromasil C₁₈) was also examined. The column efficiency decreased by 60% after processing 45 mL plasma in total.     

Determination of the N-methyl-D-aspartate receptor NR2B subunit blocker Ro 63-1908 in rat, cynomolgus monkey and human plasma by high-performance liquid chromatography with column switching and fluorescence detection

A HPLC method with automated column switching was developed and validated for the determination of Ro 63-1908 in rat and cynomolgus monkey plasma. Human plasma was used for calibration and was also included in the validation process. Ro 63-1908 belongs to a class of neuroprotective N-methyl-D-aspartate (NMDA) receptor blockers which were in development for the treatment of stroke and traumatic brain injury. The method involves deproteinisation of plasma samples with ethanol and direct injection of the supernatant (1.4 ml) into the HPLC column-switching system. To prevent a breakthrough of the analyte and the internal standard on the precolumn (Purospher RP-18, 75x4 mm) due to the high ethanol content, the injection solution was diluted, on-line, using an additional pump and a T-piece. 1% ammonium acetate-ethanol (100:2, v/v) was used as mobile phase for injection, as well as for on-line dilution, resulting in pre-concentration of the analyte and the internal standard on the precolumn. As Purospher RP-18 is a non-endcapped stationary phase with a special selectivity for amines, the analyte and the internal standard could then be selectively eluted with 30% acetonitrile (without any buffer in the mobile phase) and transferred to the analytical column [consisting of two coupled columns (125+250x4 mm) packed with Superspher 60 RP-select B], where they were separated by gradient elution and detected by fluorescence detection. Compared to the use of a 125 mm long precolumn and dilution of the supernatant with ammonium acetate prior to injection, the 75 mm precolumn and the on-line dilution procedure allowed about one third shorter run times (21 min) and, therefore, a higher sample throughput. The limit of quantification was 1 ng/ml using 0.4 ml plasma. The method was applied to more than 670 plasma samples from pharmacokinetic and toxicokinetic studies and is also suitable for other matrices and NMDA receptor blockers.     

Column-switching high-performance liquid chromatographic determination of clarithromycin in human plasma with electrochemical detection

A column-switching HPLC method was described for the direct analysis of clarithromycin in human plasma using electrochemical detector without sample pre-purification step. Plasma samples were diluted with washing solvent, i.e. acetonirile-methanol-0.05 M potassium phosphate buffer (pH 7.0) (5:2:93, v/v) and then, injected to the precolumn. After plasma proteins had flowed out from the precolumn, clarithromycin and internal standard (roxithromycin) were eluted to a Luna 2 C(18) column and separated with acetonitrile-methanol-0.05 M potassium phosphate buffer (pH 7.0) (41:6:53, v/v). Electrochemical oxidation of clarithromycin occurred at 0.87 V vs. Ag/AgCl reference electrode with glassy carbon electrode. The calibration curve was linear in the concentration range 0.1-4 mug ml(-1) with correlation coefficient of 0.998. This method showed excellent precision (RSD 3.8% at 0.1 mug ml(-1)) and accuracy (+/-2%) with the total analysis time per sample of 30 min. The present method was successfully applied to the pharmacokinetic study of clarithromycin in volunteers receiving a single oral administration of clarithromycin.     

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.     

Automated column-switching high-performance liquid chromatographic determination of flumequine and oxolinic acid in extracts from fish

Two methods for automated analysis of extracts from edible muscle tissue of Atlantic salmon are described. Oxolinic acid and flumequine are extracted with phosphate buffer pH 9, and the extracts are analysed by high-performance liquid chromatography using a column-switching system. One method applies on-line concentration and clean-up of the extracts on a precolumn packed with polystyrene-divinylbenzene. This method was useful for the analysis of oxolinic acid and flumequine in the microgram/g range. The other method was based on on-line dialysis and concentration of the dialysate on the polymeric precolumn. This method was shown to be a reliable method for residue analysis, and the limit of detection was 2 ng/g for oxolinic acid and 3 ng/g for flumequine with fluorescence detection.     

Determination of aflatoxin M1 using a dialysis-based immunoaffinity sample pretreatment system coupled on-line to liquid chromatography. Reusable immunoaffinity columns.

A liquid chromatographic column-switching system containing a dialysis unit and an anti-aflatoxin immunoaffinity precolumn (immuno precolumn) is described for the automated determination of aflatoxin M1 in milk samples. Both a flat membrane dialysis unit working according to the flowing donor-flowing acceptor principle and a laboratory made hollow-fibre dialysis unit working according to the stagnant donor-flowing acceptor principle were evaluated. The hollow-fibre unit is superior with respect to repeatability (3% relative standard deviation) and detection limit (10 ng/l for aflatoxin M1 in milk), in spite of the fact that the overall recovery is only 6%. Interfering compounds, which would destroy the activity of the immuno precolumn, are efficiently removed from the system by the dialysis step; a single immuno precolumn can then be used for over 70 milk analyses. No decrease in the performance of either the immuno precolumn or the hollow-fibre dialysis unit is observed.     

Rapid determination of clenbuterol residues in urine by high-performance liquid chromatography with on-line automated sample processing using immunoaffinity chromatography

A liquid chromatographic column-switching system for automated sample pretreatment and determination of clenbuterol in calf urine, using an immunoaffinity precolumn with Sepharose-immobilized polyclonal antibodies against clenbuterol, is described. A second precolumn packed with C18-bonded silica was used for the reconcentration of desorbed clenbuterol prior to the analytical separation. Urine, after 2-fold dilution with buffer (pH 7.4), was loaded directly onto the immuno precolumn, where clenbuterol was trapped by the immobilized antibodies. This immuno precolumn has been used for more than 200 runs with standard solutions and samples. Bound analyte was desorbed with 0.01 M acetic acid and transferred, via the second precolumn, to the analytical column. The total runtime per sample was 35 min. Using a sample load of 27 ml of dilute urine and UV detection at 244 nm, the detection limit was 0.5 ng/ml. The mean recovery of clenbuterol added to a blank urine sample at the 5 ng/ml level was 82 +/- 2% (n = 5) as determined with standard solutions loaded onto the same system. Urine samples from treated animals were analysed and the clenbuterol concentrations were comparable to those obtained by high-performance liquid chromatography using solid-phase extraction for sample clean-up.     

Determination of the active metabolites of sibutramine in rat serum using column-switching HPLC

A simple and direct analysis using column-switching HPLC method was developed and validated for the quantification of active metabolites of sibutramine, N-mono-desmethyl metabolite (metabolite 1, M1) and N-di-desmethyl metabolite (metabolite 2, M2) in the serum of rats administered sibutramine HCl (5.0 mg/kg, p.o.). Rat serum was directly injected onto the precolumn without sample prepreparation step following dilution with mobile phase A, i. e., methanol-ACN-20 mM ammonium phosphate buffer (pH 6.0 with phosphoric acid) (8.3:4.5:87.2 by volume). After the endogenous serum components were eluted to waste, the system was switched and the analytes were eluted to the trap column. Active metabolites M1 and M2 were then back-flushed to the analytical column for separation with mobile phase B, i. e., methanol-ACN-20 mM ammonium phosphate buffer (pH 6.0 with phosphoric acid) (35.8:19.2:45 by volume) and detected at 223 nm. The calibration curves of active metabolites M1 and M2 were linear in the range of 0.1-1.0 microg/mL and 0.15-1.8 microg/mL. This method was fully validated and shown to be specific, accurate (10.4-10.7% error), and precise (1.97-8.79% CV). This simple and rapid analytical method using column-switching appears to be useful for the pharmacokinetic study of active metabolites (M1 and M2) of sibutramine.     

Determination of serum cortisol by reversed-phase liquid chromatography using precolumn sulphuric acid-ethanol fluorescence derivatization and column switching.

An assay method for serum cortisol, using precolumn sulphuric acid-ethanol fluorescence derivatization and reversed-phase liquid chromatography with a column-switching technique, has been developed. The crude precolumn fluorescence cortisol derivative was prepared by the addition of sulphuric acid to serum deproteinized with ethanol, and directly injected onto an octadecylsilane-bonded silica gel (ODS) precolumn for concentration and purification. After switching columns the samples were separated using an ODS analytical column and monitored fluorimetrically. When the pH of the mobile phase in the analytical separator decreased to 1.85, the emission wavelength of the cortisol derivative changed to 520 nm (excitation of 365 nm) and the fluorescence intensity increased. Among the sulphuric acid-ethanol derivatives of various steroids, cortisol, corticosterone and testosterone emitted fluorescence. However, their retention times differed from those of the cortisol derivatives (12.5 min). The detection limit of cortisol was 0.3 micrograms/dl (signal-to-noise ratio of 3). Use of the fully automated column-switching system contributed to good reproducibility and recovery.     

Weak cation-exchange restricted-access material for on-line purification of basic drugs in plasma

A methylcellulose-immobilized weak cation-exchange (MC-WCX) silica-based restricted-access material (RAM) was developed. The MC-WCX consists of an MC outer surface and 2-carboxyethyl phase internal surface, allowing for direct analysis of basic drugs in plasma. The retention properties of the MC-WCX were evaluated for sulpiride, quinidine, ranitidine, and desipramine. The MC-WCX retained model drugs by cation-exchange, and retained drugs were eluted with the mobile phase containing small amount of acids or salts compared with the MC strong cation-exchanger (MC-SCX). These results indicated the ease of use of the MC-WCX solid-phase extraction (SPE) column when coupled to a reversed-phase analytical column in column-switching high-performance liquid chromatography (HPLC), and various detection principals. Further direct analysis of model drugs in plasma using the MC-WCX SPE column in a column-switching HPLC system successfully performed with sufficient recovery. It is concluded that the MC-WCX is useful for the analysis of basic drugs in plasma.     

High-performance liquid chromatographic determination of peptides in biological fluids by automated pre-column fluorescence derivatization with fluorescamine

Peptides containing a free alpha- or epsilon-amino group react with fluorescamine under mild alkaline conditions to generate a highly fluorescent but unstable reaction product and, consequently, practical high-performance liquid chromatographic (HPLC) approaches to analysis have typically involved the use of postcolumn derivatization. An automated precolumn approach is reported in which peptides are reacted with fluorescamine just prior to HPLC analysis by a commercially available autoinjector with derivatization capabilities. The autoinjector added base and fluorescamine reagent solutions to a sample vial containing peptide analytes, and the derivatization reaction was allowed to proceed for 5 min at room temperature prior to injection into the HPLC system. The derivatized peptides were analyzed by reversed-phase HPLC with fluorescence detection (excitation at 390 nm; emission 470-nm cut-off filter) on an octylsilica column. Optimization of the precolumn reaction conditions and the use of narrower HPLC columns (2 mm I.D.) resulted in a typical on-column detection limit of 30-50 fmol of peptide, which was substantially lower than that in previously reported post-column methods. This approach was applied to the HPLC of several naturally occurring and synthetic peptides containing alpha- and epsilon-amino groups. In combination with solid-phase extraction, prior to automated precolumn fluorescence derivatization and chromatographic analysis, the methodology was used for the determination of a synthetic growth hormone-releasing peptide in plasma samples.     

Determination of the N-methyl- -aspartate receptor NR2B subunit blocker Ro 63-1908 in rat, cynomolgus monkey and human plasma by high-performance liquid chromatography with column switching and fluorescence detection

A HPLC method with automated column switching was developed and validated for the determination of Ro 63-1908 in rat and cynomolgus monkey plasma. Human plasma was used for calibration and was also included in the validation process. Ro 63-1908 belongs to a class of neuroprotective N-methyl- -aspartate (NMDA) receptor blockers which were in development for the treatment of stroke and traumatic brain injury. The method involves deproteinisation of plasma samples with ethanol and direct injection of the supernatant (1.4 ml) into the HPLC column-switching system. To prevent a breakthrough of the analyte and the internal standard on the precolumn (Purospher RP-18, 75×4 mm) due to the high ethanol content, the injection solution was diluted, on-line, using an additional pump and a T-piece. 1% ammonium acetate–ethanol (100:2, v/v) was used as mobile phase for injection, as well as for on-line dilution, resulting in pre-concentration of the analyte and the internal standard on the precolumn. As Purospher RP-18 is a non-endcapped stationary phase with a special selectivity for amines, the analyte and the internal standard could then be selectively eluted with 30% acetonitrile (without any buffer in the mobile phase) and transferred to the analytical column [consisting of two coupled columns (125+250×4 mm) packed with Superspher 60 RP-select B], where they were separated by gradient elution and detected by fluorescence detection. Compared to the use of a 125 mm long precolumn and dilution of the supernatant with ammonium acetate prior to injection, the 75 mm precolumn and the on-line dilution procedure allowed about one third shorter run times (21 min) and, therefore, a higher sample throughput. The limit of quantification was 1 ng/ml using 0.4 ml plasma. The method was applied to more than 670 plasma samples from pharmacokinetic and toxicokinetic studies and is also suitable for other matrices and NMDA receptor blockers.     

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.     

HPLC柱切换技术用于同时分析鱼腥草中3种黄酮类成分

利用柱切换技术解决了鱼腥草中难分离组分的分离和低含量组分的定量问题。以Synergi Fusion-RP柱(250 mm×4.6 mm i.d.,4μm)作为预柱,甲醇-0.05%磷酸溶液作流动相,以Acclaim120 C18柱(250mm×4.6 mm i.d.,5μm)作为分析柱,乙腈-0.05%磷酸溶液作流动相,对鱼腥草中的3种黄酮类成分进行分析。该方法在10.0～1 000.0 mg.L-1范围内线性关系良好,相关系数(r)不低于0.998,回收率为95%～99%,RSD值均小于3%。该方法操作简便、快速,可作为分析鱼腥草中黄酮类组分的有效手段。