Determination of urinary free cortisol by high performance liquid chromatography with sulphuric acid-ethanol derivatization and column switching.

A rapid and highly sensitive determination method for urinary free cortisol has been developed using reversed phase high performance liquid chromatography (HPLC) with a precolumn for sulphuric acid-ethanol fluorescence derivatization and column switching. Urinary cortisol, eluted from the octadecylsilane-bonded silica (ODS) minicolumn with 90% aqueous ethanol, was derivatized with the addition of sulphuric acid only at ambient temperature. Cortisol derivatives injected directly onto the ODS precolumn were purified on-line. After switching the columns, the cortisol derivative was separated on an ODS analytical column with a retention time of 15.3 min and monitored at an emission wavelength of 520 nm (exitation wavelength of 365 nm) to decrease the detection limit to 0.26 microgram/dL (signal-to-noise ratio = 3). The automated HPLC operation resulted in good reproducibility and recovery of the stable cortisol derivative at 5 degrees C.     

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)     

Column switching-back flushing technique for the analysis of aromatic compounds in gasoline

A simple method, based on the technique of capillary column switching-back flushing, has been developed for the detailed analysis of aromatic compounds in gasoline. The sample was first separated on a 30-m long OV-2330 polar precolumn and then backflushed onto a nonpolar analytical column. The early eluting components from the precolumn and the components of interest (aromatic compounds plus heavier compounds) eluting from the analytical column are all directed to the same flame ionization detection system through a T piece, which permits the quantitative analysis of aromatic hydrocarbons in gasoline by a normalization method using correcting factors. The switching time window of the method is +/-5 s, resulting in easier operation and higher reliability. The reproducibility of the quantitative analysis was < or = 3% RSD for real gasoline samples.     

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.     

Chiral analysis of baclofen by alpha-cyclodextrin-modified capillary electrophoresis and laser-induced fluorescence detection

An enantioselective method for baclofen (4-amino-3-p-chlorophenylbutyric acid) based on capillary electrophoresis (CE) separation and laser-induced fluorescence (LIF) detection has been developed. Naphthalene-2,3-dicarboxaldehyde (NDA) was used for precolumn derivatization of the nonfluorescent drug. alpha-Cyclodextrin (alpha-CD) was included in the buffer as a chiral selector for the separation of NDA-labeled S-(+)- and R-(-)-baclofen. Optimal resolution and detection were obtained with an electrophoretic buffer of 50 mM sodium borate (pH 9.5) containing 7 mM alpha-CD and a He-Cd laser (lambda ex = 442 nm, lambda em = 500 nm). Combined with a simple cleanup procedure, this method can be applied to the analysis of baclofen enantiomers in human plasma. The relative standard deviation (RSD) values on peak areas of a plasma sample containing 1.0 microM racemic baclofen were 6.4 and 4.9% (n = 8) for the S-(+)- and R-(-)-enantiomer, respectively. The RSD value on migration times of both enantiomers was 0.5% (n = 8). Calibration graphs for S-(+)- and R-(-)-baclofen in plasma showed a good linearity (r > or = 0.999) in the concentration range of 0.1-2.0 microM. The limit of detection of baclofen in plasma was about 10 ng/mL.     

Rapid and sensitive on-line precolumn purification and high-performance liquid chromatographic assay for bile acids in serum

A simple and rapid technique for the simultaneous isolation and analysis of fifteen kinds of bile acid was developed using reversed-phase high-performance liquid chromatography with an automatic dual-precolumn switching system. The serum samples were directly injected onto a first precolumn (hydroxyapatite), which was flushed with 1 mM phosphate buffer. Serum proteins were strongly retained on the hydroxyapatite column, but bile acids were unretained. The bile acids were absorbed on a second precolumn (Serumout-25) and eluted onto the analytical column with solvent B (acetonitrile-methanol-30 mM ammonium acetate, 20:20:60, v/v/v). For the separation of each bile acid, the gradient elution technique was used (solvent A was acetonitrile-methanol-30 mM ammonium acetate, 30:30:40). After separation of the bile acids, NADH was produced by use of immobilized 3 alpha-hydroxysteroid dehydrogenase column and then determined fluorimetrically (gamma em = 460 nm, gamma ex = 350 nm). The recoveries of bile acids in serum generally approached 100%.     

Determination of paraquat in human blood plasma using reversed-phase ion-pair high-performance liquid chromatography with direct sample injection

This report describes the determination of paraquat (PQ) in human blood plasma samples by a direct-injection reversed-phase ion-pair chromatographic method. Blood plasma filtrate was injected directly into the LiChrospher® RP-18 alkyl-diol silica (ADS) precolumn integrated in a column switching system using a mixture of 3% 2-propanol and 10 mM sodium octane sulfonate (SOS) in a 0.05 M phosphate buffer (pH 2.8). After washing with this phase, the ADS precolumn was back-flushed with the analytical mobile phase consisting of 40% of methanol and 10 mM SOS in a 0.05 M phosphate buffer (pH 2.8) at a flow rate of 1.0 ml min⁻¹, in order to carry the analyte to a conventional reversed-phase analytical column, where the separation of PQ was achieved and finally detected by UV at 258 nm. The recoveries of PQ from human blood plasma samples ranged between 95.0 and 99.5% at nine different concentrations (from 0.05 to 3.00 μg of PQ ml⁻¹) with coefficients of variation <2.5% (n=3). The precision expressed as relative standard deviation was below 3.5% for between-day and below 4.3% for within-day measurements (n=5). The detection limit (signal-to-noise ratio, S/N>3) was 0.005 μg ml⁻¹ with an injection volume of 200 μl. The proposed method is promising for the identification and quantification of PQ at low concentration levels and is suitable for its analysis in human blood plasma samples from intentional or accidental poisonings cases with a sample throughput of 5 samples per hour.     

Chiral separation of the β2‐sympathomimetic fenoterol by HPLC and capillary zone electrophoresis for pharmacokinetic studies

The development of methods for the separation of the enantiomers of fenoterol by chiral HPLC and capillary zone electrophoresis (CZE) is described. For the HPLC separation precolumn fluorescence derivatization with naphthyl isocyanate was applied. The resulting urea derivatives were resolved on a cellulose tris(3,5‐dimethylphenylcarbamate)‐coated silica gel column employing a column switching procedure. Detection was carried out fluorimetrically with a detection limit in the low ng/mL range. The method was adapted to the determination of fenoterol enantiomers in rat heart perfusates using liquid–liquid extraction. As an alternative a CE method was used for the direct separation of fenoterol enantiomers comparing different cyclodextrin derivatives as chiral selectors. Copyright © 2010 John Wiley & Sons, Ltd.     

High performance liquid chromatographic determination of Picumast and two active metabolites in plasma using on-line sample preparation.

A method for determining Picumast, an antiallergic drug, in plasma by HPLC and column switching has been developed. The system consisted of two precolumns, an analytical column, three pumps, an autosampler and a fluorescence detector. The precolumns (17 x 4.6 mm i.d.) were packed with LiChroprep RPR (a moderately polar reversed phase) and the analytical column with Nucleosil ODS (RP 18, 5 microns). The columns were connected according to the alternating precolumn technique. The mobile phase consisted of 30% CH3CN/70% 0.05 M KH2PO4, pH 2.5, with a flow gradient. Detection wavelengths were 333 nm for excitation and 383 nm for emission. The retention times of Picumast, M1 and M2 were 12, 3.6 and 4.0 min, respectively. Total run time was 15 min. The limit of detection was 3 ng/mL for M1 and 1 ng/mL for M2 and Picumast using an injection volume of 150 microL. The recoveries vary between 89% and 97% with standard deviations between 2.4 and 3.3%.     

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.     

Enantioselective high-performance liquid chromatography with fluorescence detection of methamphetamine and its metabolites in human urine.

A simple, rapid and highly sensitive high-performance liquid chromatographic method with fluorescence detection for determining the enantiomers of methamphetamine and its major metabolites, amphetamine and p-hydroxymethamphetamine, in urine samples was developed. Using a newly developed reagent for amines, namely, 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride, six enantiomers were derivatized under mild conditions (i.e., 10 min at room temperature, pH 9.0) and separated isocratically on a cellulose tris(3,5-dimethylphenylcarbamate) coated silica gel column following a pre-separation on an ODS column within 42 min, and the effluent was monitored at 440 nm (lambda ex 330 nm). Calibration curves for these derivatives using spiked human urine were linear in the range 0.05-100 mumol dm-3 with correlation coefficients > or = 0.999. The detection limits at a signal-to-noise ratio of 3 were 2.8-8.8 fmol per 5 microliters injection. The relative standard deviations of within- (n = 6) and between-day (n = 5) variations were < or = 7.4%. The method was successfully applied to discriminate between (S)-(+)-methamphetamine and its corresponding metabolites found in abusers' urine and their antipodes in a sample taken from a Parkinsonian patient on selegiline (Deprenyl) therapy.     

Direct injection of plasma to determine pseudoephedrine by high performance liquid chromatography with column switching

An HPLC method has been developed for the determination of pseudoephedrine in plasma using column switching. Preparation of the sample was simple in that only 1000 microL of water was added to 200 microL of plasma before injection. A 900 microL aliquot was injected onto the precolumn. Double distilled water was used to elute and remove proteins and polar components in the sample. The components retained on the precolumn were flushed forward onto the analytical column by the mobile phase (acetonitrile-0.2 mol/L ammonium sulphate, 10:90 v/v) with automated column switching. The limit of determination of pseudoephedrine in plasma was 12 ng/mL. The relative standard deviations of intra- and inter-assay for the determination of pseudoephedrine in plasma were 1.2-9.8% over the concentration range 1020-21.8 ng/mL. The mean recovery by on-line solid phase extraction was 94.76% (RSD = 1.1%).     

Determination of the enantiomers of mefloquine in plasma and whole blood using a coupled achiral-chiral high-performance liquid chromatographic system

A coupled achiral-chiral high-performance liquid chromatographic system has been developed for the determination of the enantiomers of mefloquine, (+)-MFQ and (-)-MFQ, in plasma and whole blood. The MFQ was separated from the interfering components in the biological matrix and quantified on a cyano-bonded phase, and the enantiomeric composition was determined on an (S)-naphthylurea chiral stationary phase. The two columns were connected by a switching valve equipped with a silica precolumn. The precolumn was used to concentrate the MFQ in the eluent from the achiral column before backflushing onto the chiral phase. The coupled-column system was validated and applied to the analysis of a pilot study of the pharmacokinetics of (+)- and (-)-MFQ in plasma and whole blood.     

Use of direct injection precolumn techniques for the high-performance liquid chromatographic determination of the retinoids acitretin and 13-cis-acitretin in plasma.

A previously developed highly sensitive high-performance liquid chromatographic method for the determination of retinoids, using direct injection of large plasma volumes, on-line solid-phase extraction and ultraviolet detection, was improved and fully validated for the determination of acitretin and 13-cis-acitretin in plasma samples. The addition of acetonitrile to improve the recovery was performed on-line by a T-piece, avoiding any cis-trans isomerization which could occur when acetonitrile was added prior to storage in the autosampler. About 30 injections could be made onto one precolumn despite the large injection volume (1 ml of plasma containing the internal standard). Full automation was attained by the use of automated precolumn replacement. In addition, forward- and back-flush purging of the precolumn enhanced the longevity of the analytical column. This consisted of three coupled C18 columns of 125 mm length each. The quantification limit was 0.3 ng/ml, using ultraviolet detection at 360 nm, and the mean inter-assay precision was 3.8% for the two compounds.     

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.     

Reversed-phase high-performance liquid chromatographic analysis of atenolol enantiomers in plasma after chiral derivatization with (+)-1-(9-fluorenyl)ethyl chloroformate.

A sensitive high-performance liquid chromatographic method for the determination of the enantiomers of atenolol in rat plasma has been developed. Racemic atenolol and practolol (internal standard) were extracted from alkalinized plasma (pH 12) into dichloromethane containing 3% (v/v) heptafluoro-1-butanol, and the organic layer was evaporated. The samples were derivatized with (+)-1-(9-fluorenyl)ethyl chloroformate at pH 8.5 for 30 min. After removal of excess reagent, the diastereomers were extracted into dichloromethane. The diastereomers were separated on a Microspher C18 column (3 microns) with a mobile phase of acetonitrile-sodium acetate buffer (0.01 M, pH 7) (50:50, v/v) at a flow-rate of 0.8 ml/min. Fluorescence detection (lambda ex = 227 nm, lambda em = 310 nm) was used. When 100 microliters of plasma were used, the quantitation limit was 10 ng/ml for the atenolol enantiomers. The assay was applied to measure concentrations of atenolol enantiomers in plasma after intravenous administration of racemic atenolol to rats.     

Direct determination of non-steroidal anti-inflammatory drugs by column-switching LC-MS

A method for determination of 16 non-steroidal anti-inflammatory drugs (NSAIDs) in human plasma samples without time-consuming sample pre-treatments was developed. The system consisted of two pumps for mobile phase delivery, a six-port switching valve, a pre-column (Oasis HLB Cartridge Column), and a reversed phase analytical column (COSMOSIL 3C18-MS-II). The analytes were trapped on the precolumn and subsequently separated on the analytical column. The present method allowed on-line sample clean-up and enrichment, leading to improved sensitivity without any tedious sample preparation. The recoveries of NSAIDs from human plasma by column-switching were greater than 72.6%. The total analysis time for a single analytical run was approximately 11 min. The detection limits of NSAIDs were 0.0025 to 0.2 microg/mL using the selected ion monitoring mode.     

Quantitative analysis of retinoids in biological fluids by high-performance liquid chromatography using column switching. I. Determination of isotretinoin and tretinoin and their 4-oxo metabolites in plasma

A fully automated gradient high-performance liquid chromatographic method for the determination of isotretinoin, tretinoin and their 4-oxo metabolites in plasma was developed, using the column-switching technique. After dilution with an internal standard solution containing 20% acetonitrile, 0.5 ml of the sample was injected onto a precolumn (17 X 4.6 mm I.D.), filled with C18 Corasil 37-53 micron. Proteins and polar plasma components were washed out using 1% ammonium acetate-acetonitrile (9:1, v/v) as mobile phase 1. After valve switching, the retained components were transferred to the analytical column in the backflush mode, separated by gradient elution and detected at 360 nm by UV detection. Using two coupled reversed-phase columns (125 mm long), the separation of cis and trans isomers was possible, and all four compounds could be quantified down to 2 ng/ml of plasma. The inter-assay precision in the concentration range 20-100 ng/ml was between 1.0 and 4.7% for all compounds.     

Automated determination of disopyramide and N-monodealkyldisopyramide in plasma by reversed-phase liquid chromatography with a column switching system.

A rapid and simple column liquid chromatographic method involving a column switching system for the determination of disopyramide and its N-monodealkyl metabolite (NMD) in plasma is described. The deproteinized plasma is applied to an automated system. Purification and concentration were performed using a precolumn connected to a six-position valve; analytical separation was done on-line using a cyano reversed-phase column with a mobile phase consisting of 10 mmol/l trimethylamine (pH 2.5, adjusted with phosphoric acid)-acetonitrile-tetrahydrofuran (78:20:2, v/v/v). Absorbance was measured at 265 nm, with a minimum detectable amount of disopyramide and NMD of 0.1 micrograms/ml. The method can be applied to drug monitoring and pharmacokinetic studies.     

Determination of rotenone in river water utilizing packed capillary column switching liquid chromatography with UV and time-of-flight mass spectrometric detection

Fast and sensitive packed capillary column switching liquid chromatography methodology has been developed for the determination of the pesticide rotenone in river water. Sample volumes of up to 1 ml are loaded onto a 23 x 0.25 mm, 5 microm Kromasil C18 packed capillary precolumn using a noneluting solvent composition of water-acetonitrile (99:1, v/v) at flow-rates up to 100 microl/min prior to solute backflushing onto a 200 x 0.32 mm, 3.5 microm Kromasil C18 packed capillary analytical column using a mobile phase of water-acetonitrile (30:70, v/v) at a flow-rate of 5 microl/min. The method was evaluated using river water samples spiked with rotenone in the concentration range 0.5-50 ng/ml using UV detection. The within-assay precision was between 5.0 and 7.7% relative standard deviation (RSD, n = 6) and the between assay precision was between 7.5 and 8.9% RSD (n = 6). The method was linear within the investigated mass range displaying a calibration curve correlation factor of 0.997. The mass limit of detection was 10 pg corresponding to a concentration limit of detection of 10 pg/ml, using time-of-flight mass spectrometry.