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.     

Multidimensional high-performance liquid chromatography on Pinkerton ISRP and RP18 columns: direct serum injection to quantify creatinine.

A two-dimensional high-performance liquid chromatographic method for the determination of creatinine with direct serum injection without sample pretreatment has been developed. The column-switching technique allowed a switch from columns packed with internal surface reversed-phase (ISRP) material to columns of almost any other material, even if the eluents necessary in a particular case do not appear to be directly compatible. A Pinkerton ISRP column, which stands out because of its very good stability when loaded with undiluted serum samples, was used as precolumn. The creatinine-containing fraction was switched to a reversed-phase Shandon RP18 column and was focused there by alteration of the eluent from pH 6.5 to phosphoric acid-ion-pair reagent. The separation occurs via a pH gradient, with ultraviolet detection at 234 nm. This method stands out particularly for its good long-term stability, simple sample handling without pretreatment, high selectivity, a broad linearity (0.3-30 mg/dl creatinine), good reproducibility (inter-assay coefficient of variation less than 3%) and high recovery (97-100%) relative to values obtained with gas chromatography-mass spectrometry.     

Evaluation of an on-line coupled continuous flow liquid membrane extraction and precolumn system as trace enrichment technique by liquid chromatographic determination of bisphenol A

An on-line coupled continuous flow liquid membrane extraction (CFLME) and C₁₈ precolumn system was developed for sample preconcentration in liquid chromatography determination. After preconcentration by CFLME, which is based on the combination of continuous flow liquid–liquid extraction and supported liquid membrane, bisphenol A (BPA) was enriched in 960 μl of 1 mol l⁻¹ NaOH used as acceptor. This acceptor was on-line neutralized and transported onto the C₁₈ precolumn where analytes were absorbed and focused. Then the focused analytes were injected onto a C₁₈ analytical column for separation and detected at 220 nm with a diode array detector. CFLME related parameters such as flow rates, pH of donor and acceptor, and enrichment time were optimized. The proposed method presents a detection limit of 0.03 μg l⁻¹ (S/N=3) when 60 ml samples was enriched with an enrichment time of 30 min. Compared with C₁₈ based column-switching procedure, this proposed procedure presents similar sample throughput and lower detection limits. The proposed method was successfully applied to determine BPA in tap water, river water, and municipal sewage effluent 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.     

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.     

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.     

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.     

Separation and determination of polyethylene glycol fatty acid esters in cosmetics by a reversed-phase HPLC/ELSD

A comprehensive analytical method was established for the separation of polyethylene glycol (PEG) stearates according to the distribution of ethylene oxide (EO) and subsequent determination of the surfactants in cosmetic samples by using a high-performance liquid chromatography–evaporative light scattering detection. Separation of the PEG stearates comprising approximately up to 82 EO adducts was performed on a reversed-phase YMC-Pack C₈ column using water–acetonitrile gradient elution. The PEG oligomers were separated in order of the increasing number of EO adducts. Quantitation of the PEG fatty acid esters, which was separated as single peak per each component, was performed by chromatography on a reversed-phase Wakosil 10 C₁₈ column using water–methanol gradient elution. The standard curve to quantify the PEG stearates was constructed by the log–log plot, which showed good linearity with the correlation coefficients (R²) 0.998 and more. Working range, repeatability, limit of detection and recovery were acceptable for analysis of the surfactants in cosmetic products. The analytical methods were applied to characterize the PEG stearates according to the EO distributions, then to quantify the surfactants in cosmetic products.     

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.     

Examination of some reversed-phase high-performance liquid chromatography systems for the determination of lipophilicity

Three reversed-phase systems [based on the divinylbenzene-styrene copolymer (PRP-1), the C₁₈-derivatized divinylbenzene-styrene copolymer (ACT-1), and the Nucleosil C₈ columns] were studied for their suitability in lipophilicity determination. Acetonitrile-water was selected as the mobile phase. Correlation between log k′ and log P_cyc for both the PRP-1 and Nucleosil C₈ systems was superior to the correlation between log k′ and either log P_oct or log P_cyc (oct = octanol; CYC = cyclohexane) on the ACT-1 column. On the PRP-1 and Nucleosil columns, correlation between log k′ and log P_oct was much improved when test compounds were grouped into classifications of non-H bonding, single amphiprotics (alcohols, phenols, amides) or double amphiproties. Although the PRP-1 system gave broad peaks with lipophilic substrates, there was good correlation between log k′ values on the Nucleosil silica-based reversed-phase system and the polymer PRP-1 system, indicating that either is suitable for the determination of lipophilicity.     

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.     

Trace-level determination of polar phenolic compounds in aqueous samples by high-performance liquid chromatography and on-line preconcentration on porous graphitic carbon

The use of porous graphitic carbon (PGC) was investigated for the trace enrichment and the on-line liquid chromatographic separation of polar phenolic compounds (phenol, di- and trihydroxybenzenes, aminophenols, etc.) from aqueous samples. Comparison between retentions obtained with PGC and with the copolymer-based sorbent PRP-1 showed similar variations of the capacity factors with the mobile phase composition, but an inverse retention order. The capacity factor of a very polar analyte, such as 1,3,5-trihydroxybenzene (phloroglucinol), is 1000 in pure water, whereas this analyte is not retained by C₁₈-silica and is poorly retained by PRP-1 (k′ = 3 in water). A precolumn packed with PGC can be coupled to a PGC analytical column for simple separation in the reversed-phase mode. This methodology has been applied to the direct determination of pyrocatechol, resorcinol and phloroglucinol below the 0.1 μg/1 level in a 50-ml sample.     

Reversed-phase high-performance liquid chromatographic method for the quantitative determination of alkylbis(2-benzothiazolylsulfen)amides

An isocratic, reversed-phase HPLC procedure was developed for the simultaneous determination of isopropyl-, tert.-butyl-, tert.-amyl-, cyclohexylbis(2-benzothiazolylsulfen)amides. Quantitation is performed on a C₁₈ bonded-phase column (Separon SGX C₁₈ 5 μm) using N-dicyclohexyl-2-benzothiazolesulfenamide as internal standard followed by UV photodiode-array detection. The precision (n = 7) for all derivatives of alkylbis(2-benzothiazolylsulfen)amides is within 1.5%. Identification of the compounds also in the mixtures was done by NMR spectroscopy.     

Simple high-performance liquid chromatographic method for the determination of a new phytochemical drug, fellavine, and its metabolites in human and rat plasma and urine.

The use of a small precolumn instead of an injection loop for the determination of a new phytochemical drug, fellavine, and its metabolites is described. The method combines the direct injection of plasma and urine into the reversed-phase precolumn with separation on a Spheri-5 RP-18 analytical column. Different sorbents in the precolumn were compared. A recovery of fellavine and its metabolites from biological fluids except rat plasma of almost 100% was achieved on Chrompack RP (30-40 microns) and LiChrosorb RP-18 (7 microns). For rat plasma only the last sorbent gave 80% fellavine recovery. The influence of the protein binding on the fellavine recovery was examined. The limit of detection was equal to 0.05 micrograms/ml fellavine for plasma and 0.02 micrograms/ml for urine. To enhance the limit of detection longer precolumns were perferred.     

Ion chromatographic separation of alkali metal and ammonium cations on a C18 reversed-phase column

The separation of alkali metal (Li⁺, Na⁺, K⁺, Rb⁺ and Cs⁺) and ammonium cations on a C₁₈ reversed-phase column using three anionic surfactants [sodium 1-eicosyl sulphate, sodium dodecyl benzenesulphonate and sodium dodecyl sulphate (SDS)] is described. Two methods were examined: (a) “permanent” coating, with the use of a C₁₈ reversed-phase column previously coated with the surfactants; and (b) dynamic coating, with addition of the surfactants to the mobile phase. With method (a) the separation of the six cations was achieved with SDS. However, the retention times gradually decreased owing to dissolution of the SDS coating. Good separation was obtained with method (b), where 10 mM HNO₃ containing 0.1 mM SDS was used as the mobile phase with conductivity detection, and it was applied satisfactorily to real samples. The effect of system peaks on determination is also discussed.     

Automated analysis of fluvoxamine in rat plasma using a column-switching system and ion-pair high-performance liquid chromatography

We have established a robust, fully automated analytical method for the analysis of fluvoxamine in rat plasma using a column-switching ion-pair high-performance chromatography system. The plasma sample was injected onto a precolumn packed with Shim-pack MAYI-ODS (50 microm), where the drug was automatically purified and enriched by on-line solid-phase extraction. After elution of the plasma proteins, the analyte was back-flushed from the precolumn and then separated isocratically on a reversed-phase C18 column (L-column ODS) with a mobile phase (acetonitrile-0.1% phosphoric acid, 36:64, v/v) containing 2 mM sodium 1-octanesulfonate. The analyte was monitored by a UV detector at a wavelength of 254 nm. The calibration line for fluvoxamine showed good linearity in the range of 5-5000 ng/mL (r > 0.999) with the limit of quantification of 5 ng/mL (RSD = 6.51%). Accuracy ranged from -2.94 to 4.82%, and the within- and between-day precision of the assay was better than 8% across the calibration range. The analytical sensitivity and accuracy of this assay is suitable for characterization of the pharmacokinetics of orally-administered fluvoxamine in rats.     

Determination of eight water- and fat-soluble vitamins in multi-vitamin pharmaceutical formulations by high-performance liquid chromatography

In the present work, a reversed-phase high-performance liquid chromatographic procedure has been developed for the determination of water-soluble vitamins (thiamine hydrochloride, pyridoxine hydrochloride, nicotinamide, riboflavin phosphoric ester and cyanocobalamine) and fat-soluble vitamins (retinol palmitate, cholecalciferol, -tocopherol acetate) in multi-vitamin pharmaceutical formulations. The sample treatment proposed consists of a solid-phase extraction with C₁₈ AR cartridges that allow the separation of fat-soluble vitamins, which were retained on the sorbent, from water-soluble vitamins. Afterwards, the water-soluble vitamins were analysed by HPLC on a Nova-Pack C₁₈ (150×3.9 mm, 4 μm) analytical column, using CH₃OH–0.05 M CH₃COONH₄ as mobile phase The chromatographic analysis of the fat-soluble vitamins was carried out after their sequential elution with methanol and chloroform from C₁₈ sorbent, on the above column. The mobile phase employed was MeOH–CH₃CN (95:5, v/v) working at a flow-rate of 2 ml min⁻¹ in isocratic mode. The solid-phase extraction for these vitamins had been previously optimised. The experimental variables studied were: application volume, elution solvents and cleaning solutions. The UV–Vis detection of vitamins was made at 270 nm for all the water-soluble vitamins (362 nm for B₁₂) and 285 nm for the water-soluble and fat-soluble vitamins present in real samples at different concentration levels. The accuracy of the method was tested obtaining an average recovery ranging between 78 and 116%.     

Solid-phase extraction and high-performance liquid chromatographic determination of flumequine and oxolinic acid in salmon plasma

Two methods for determination of oxolinic acid and flumequine in salmon plasma are described. The first method applies sample pretreatment on C2 disposable solid-phase extraction columns. The second method is based on direct plasma injection and on-line sample clean-up on a polystyrene-divinylbenzene precolumn. After column-switching, the analytes are separated on a polystyrene-divinylbenzene analytical column and detected with a fluorescence detector. Validation of the methods showed good sensitivity, precision and reproducibility. Both methods are well suited for determination of plasma levels of the drugs in pharmacokinetic studies in Atlantic salmon.     

Determination of traces of pyrethrins and piperonyl butoxide in biological material by high-performance liquid chromatography

A liquid chromatographic method for the determination of pyrethrins and of the synergist piperonyl butoxide in human plasma after C₁₈ solid-phase extraction is described. UV detection was found to be sensitive enough to determine concentrations far below the limit of toxicity. With respect to future investigations concerning studies in biological materials, a column-switching system for sample preparation was developed and compared with solid-phase extraction. Both methods show comparable limits of detection, but the column-switching technique has the advantage of fully automating the system.