Rapid and sensitive on-line precolumn purification and high-performance liquid chromatographic assay for disulfiram and its metabolites

The disulfiram (Antabus) metabolites diethyldithiocarbamate, diethyldithiocarbamate methyl ester, carbon disulphide and bis(diethyldithiocarbamato) copper complex were quantitatively analysed from directly injected heparin plasma by reversed-phase high-performance liquid chromatography. The highly volatile metabolite, carbon disulphide, was converted to the methyl ester of dimethyldithiocarbamate before chromatography. The analytical procedure is simple and does not require sample preparation or addition of an internal standard, and the compounds are eluted from the columns in 15 min. After automated on-line precolumn enrichment, the parent compound and biotransformation products could be back-flushed and chromatographed on an ordinary reversed-phase column. The influence of plasma protein binding on the constituents in the precolumn enrichment step was also investigated. Dissociation and partition of constituents from plasma proteins gave a complete retardation on the precolumn. The time courses of diethyldithiocarbamate and its methyl ester were followed in patients receiving therapeutic doses of disulfiram.     

Comparative study on the determination of the anti-neoplastic drug teniposide in plasma using micellar liquid chromatography and surfactant-mediated plasma clean-up

The potential of micellar liquid chromatography and of an on-line surfactant-mediated sample cleanup, which involves column-switching prior to conventional reversed-phase high-performance liquid chromatography, has been evaluated for the determination of the anti-neoplastic drug teniposide in plasma by using electrochemical detection. A major advantage of surfactant-mediated techniques is that they allow fully automated processing of plasma samples, because protein precipitation is prevented by the addition of the surfactant sodium dodecylsulphate. With the automated column-switching technique, a degree of sample enrichment and of selectivity can be attained, which is similar to that for the conventional procedure which, however, involves a labour-intensive off-line isolation of teniposide, using liquid-liquid extraction prior to chromatography. An inherent drawback of automated micellar liquid chromatography is that no sample clean-up or preconcentration can be carried out, which results in only a moderate detection limit and selectivity. The linearity, reproducibility and recovery of the surfactant-mediated techniques are similar to those of the conventional procedure. Based on the presented results, it was concluded that the surfactant-mediated column-switching technique is a highly attractive sample enrichment technique with respect to simplicity, speed and cost.     

Simple automated high-performance liquid chromatographic column-switching technique for the measurement of dopa and 3-O-methyldopa in plasma

A reliable assay procedure for the determination of 3,4-dihydroxyphenyl-L-alanine (dopa) and its O-methylated metabolite 3-O-methyldopa (3-OMD) is described. Supernatants from deproteinized plasma samples were directly injected into a column-switching system, allowing on-line prepurification of the samples by cation-exchange chromatography followed by reversed-phase high-performance liquid chromatography with electrochemical detection. With this method, the detector response was linear from endogenous levels of dopa and 3-OMD up to microgram concentrations. This technique combines the simplicity of direct injection methods with advantages of procedures using a sample clean-up. It represents a valid tool for the rapid and accurate measurement of large numbers of plasma samples in animals treated with levodopa and in clinical trials with new levodopa formulations.     

On-line turbulent-flow chromatography-high-performance liquid chromatography-mass spectrometry for fast sample preparation and quantitation

A sensitive and selective liquid chromatography-mass spectrometry method has been developed for the simultaneous identification and quantitation of drug substances and metabolites in rat plasma. The method combines on-line turbulent-flow chromatography, high-performance liquid chromatography and mass spectrometry. This combination is considered to be a new approach suitable for fast bio-analysis in drug discovery. Dextromethorphan, and its two metabolites, dextrorphan and 3-methoxymorphinan served as model substances. The analytes present in plasma were collected on a Cyclone column using turbulent-flow chromatography and were subsequently transferred on-line to and focused on an X-Terra MS C8 column. The analytes were eluted by a linear gradient and detected by a fast scanning mass spectrometer. The detector response was quadratic and the dynamic range was estimated to be 0.5-100 ng/ml plasma or 12.5 pg to 2.50 ng injected into the system.     

Electrodialytic sample treatment coupled on-line with high-performance liquid chromatography

Summary An electrodialytic sample treatment method coupled on-line with high-performance liquid chromatography (EDIST-HPLC) is discussed in this paper. The performance of EDIST as a function of the donor-phase (sample solution) flow rate, the voltage applied over the electrodialysis block, and the time of dialysis has been studied using the basic drug ephedrine as a model compound. Enrichment of the analyte by a factor of 10–20 was possible. The determination of human plasma spiked with ephedrine is briefly discussed.     

Bioanalysis of the neuropeptide des-enkephalin-gamma-endorphin by high-performance liquid chromatography with on-line sample pretreatment using gel permeation and solid-phase isolation.

A bioanalytical method is described that allows the determination of a number of beta-endorphin-related peptides. The method is based on the application of fluorescence detection after high-performance liquid chromatography followed by post-column derivatization with o-phthaldialdehyde. Concentrations exceeding 10-25 ng/ml could be determined by using conventional fluorescence detection, whereas lower concentrations demand the use of laser-induced fluorescence detection. The sample pretreatment includes the use of on-line gel permeation, on-line solid-phase isolation and heart cutting of a peak from reversed-phase gradient elution. The sample pretreatment procedure does not discriminate between the dodecapeptide des-enkaphalin-gamma-endorphin (DE gamma E) and its metabolites in order to obtain similar recoveries for all components. The final chromatographic phase system is based on ion-pair formation, which permits the separation of DE gamma E from its metabolites and degradation products. The optimized procedure allows the determination of these peptides in plasma at concentration levels down to about 1 ng/ml, demanding a sample volume of 1 ml.     

Determination of free phenytoin in plasma by ultrafiltration and high-performance liquid chromatography

A process for determining the free concentration of the highly protein-bound anti-convulsant drug phenytoin (5,5-diphenylhydantoin) is described. The procedure involves rapid isolation of the unbound drug from the drug/protein complex by ultrafiltration followed by high-performance liquid chromatography. Total time for a free phenytoin determination is about 15 min. The procedure is used to examine the protein-binding of phenytoin, as well as the effects of a displacer, salicylic acid. Commonly prescribed anticonvulsant drugs are resolved under the selected chromatographic conditions.     

Direct analysis of food samples by high-performance liquid chromatography

A short review on the sample analysis of food samples by high-performance liquid chromatography is presented. The paper is focused on direct injection of liquid samples, automated solid-phase extraction and column switching techniques, on-line dialysis and application of restricted-access media.     

Determination of femtomole/milliliter concentrations of enprostil acid in human plasma using high-performance liquid chromatography-laser-induced fluorescence detection

This paper describes the use of multiple-column high-performance liquid chromatography (HPLC) combined with laser-induced fluorescence for the determination of femtomole/milliliter concentrations of enprostil acid, a prostaglandin analogue, in human plasma. The drug is isolated from plasma by phenyl solid-phase extraction and fluorescently labeled at its carboxyl functional group with a large excess of 2-bromoacetyl-6-methoxynaphthalene. A multi-column method using both normal- and reversed-phase chromatography is necessary to separate the labeled drug from the unreacted reagent. Post-column dilution of the mobile phase with water after the reversed-phase chromatography allows on-line concentration of the labeled analyte onto a guard column prior to the microbore HPLC. A loop guard column device provides a simple way to inject up to 1.0 ml of sample solution onto a microbore column without significantly reducing the column efficiency. A 325-nm He-Cd laser is used to excite the labeled drug, and fluorescence emission is monitored at 450 nm. Using this system, we are able to derivatize, detect, and quantify 5 pg of the prostaglandin analogue in 1.0 ml of plasma.     

Analysis of protein denaturation by high-performance continuous differential viscometry

The intrinsic viscosity of protein solutions changes when denaturation occurs. Therefore, viscosity measurement using traditional glass capillary viscometers has been a key method to study the process of protein denaturation. Such measurements are laborious, time consuming and need at least 10 ml of sample. The Viscotek differential viscometer can be used as an on-line detector for high-performance liquid chromatography to monitor the viscosity of column effluent. In this study, samples were injected using an autosampler onto a "delay" column containing glass beads in place of the high-performance liquid chromatography columns. Results indicate guanidine hydrochloride, heat and pH act as denaturing agents and changes the intrinsic viscosities of serum albumin, turkey egg albumin, and ovalbumin solutions. The differential viscometer is sensitive and provides accurate measurements of minor changes in viscosities of very dilute protein solutions undergoing denaturation. The advantage of using the differential viscometer instead of conventional glass capillary viscometer is the increased sensitivity, precision, speed and operational ease that permits measurements of solution viscosity of low sample concentrations up to 1.2 micrograms of pure proteins.     

Fully automated sample handling system for liquid chromatography based on pre-column technology and automated cartridge exchange

The design of an automated cartridge exchange module for on-line sample handling in liquid chromatography is described. When combined with a low-cost purge pump, a solvent selection valve and an auto-sampler, a fully automated sample handling system is obtained. Samples are sorbed on a disposable cartridge packed with 40 micron octyl-bonded silica, purged for clean-up and eluted on-line to the analytical column. Unattended operation of the system is demonstrated for various examples, i.e., the determination of anti-epileptic drugs in serum, an anti-cancer drug in plasma, barbiturates in urine, phenylurea herbicides in river water and caffeine in a soft drink.     

Improvement of selectivity and sensitivity by column switching in the determination of glycyrrhizin and glycyrrhetic acid in human plasma by high-performance liquid chromatography

A sensitive method is described for the simultaneous determination of glycyrrhizin and glycyrrhetic acid in human plasma. Quantitation is by high-performance liquid chromatography using ion-pair chromatography on a reversed-phase column. Variable-wavelength ultraviolet detection is employed. For the extraction of both compounds from plasma, a new solid-phase ion-pair extraction procedure using octadecylsilane columns was developed. Because of the strong forces involved in the protein binding of glycyrrhizin, quantitative extraction of this compound from plasma was possible only after an alkaline pH shift. A considerable improvement in selectivity and sensitivity was obtained by automated column switching involving on-line preseparation of the solid-phase extract on a short precolumn and chromatographic analysis of a heart-cut from the precolumn eluate. The limit of detection of both glycyrrhizin and glycyrrhetic acid was 0.1 mg/l in 0.5 ml of plasma. From a preliminary study in human volunteers, it was concluded that glycyrrhetic acid rather than glycyrrhizin is preferred in a study in human volunteers to assess the zero effect level of glycyrrhizin.     

Application of column switching in high-performance liquid chromatographic analysis of medroxalol in plasma

An automated high-performance liquid chromatographic (HPLC) column-switching system is described for the analysis of medroxalol, a potential antihypertensive agent, in plasma. The HPLC system uses two six-port switching valves with a Corasil C18 short pre-column for an on-line sample clean-up and an SGE ODS analytical column for separation. Plasma samples were diluted with a phosphate buffer (pH 7.2) containing an internal standard and aliquots were injected directly on the HPLC system. The column-switching system was applicable to continuous analysis of hundreds of plasma samples since this technique provided very efficient on-line sample clean-up and regenerated the pre-column effectively. Results were in good agreement and the total analysis time was one third that of an alternative method.     

Direct analysis of plasma samples for drug discovery compounds using mixed-function column liquid chromatography tandem mass spectrometry

A sensitive, efficient, high throughput, direct injection bioanalytical method based on a single column and high-performance liquid chromatography (HPLC) with tandem mass spectrometry (MS/MS) was developed for pharmacokinetic analysis of early drug discovery compounds in plasma samples. After mixing with a working solution containing an internal standard each plasma sample was directly injected into a polymer-coated mixed-function column for sample cleanup, enrichment and chromatographic separation. The stationary phase incorporates hydrophilic polyoxyethylene groups and hydrophobic groups to the polymer-coated silica. This allows proteins and macromolecules to pass through the column due to restricted access to the surface of the packing while retaining the drug molecules on the bonded hydrophobic phase. The analytes retained in the column with a largely aqueous liquid mobile phase were then chemically separated by switching to a strong organic mobile phase. The column effluent was diverted from waste to the mass spectrometer for analyte detection. Within 200 plasma sample injections the response ratio (analyte vs. internal standard, %CV = 4.6) and the retention times for analyte and internal standard were found consistent and no column deterioration was observed. The recoveries of test compound in various plasma samples were greater than 90%. The total analysis time was 相似文献   

Sensitive procedure for the determination of reboxetine enantiomers in human plasma by reversed-phase high-performance liquid chromatography with fluorimetric detection after chiral derivatization with (+)-1-(9-fluorenyl)ethyl chloroformate

A sensitive and selective high-performance liquid chromatographic method for the determination of reboxetine enantiomers in human plasma was developed. Although two chiral centres are present in reboxetine, its stereospecific synthesis leads to two rather than four possible enantiomers. After extraction from plasma and reaction with (+)-1-(9-fluorenyl)ethyl chloroformate, reboxetine enantiomers were separated as diastereoisomeric derivatives by reversed-phase high-performance liquid chromatography (HPLC) and determined by fluorimetric detection. The HPLC analysis time was about 90 min. The linearity, precision, accuracy and limit of quantification of the method were evaluated. No interference from blank plasma sample was observed. The suitability of the method for in vivo samples was assessed by the analysis of plasma samples obtained from a healthy male volunteer who had received a single oral dose of 4 mg of reboxetine in tablet form.     

Quantification of sifuvirtide in monkey plasma by an on-line solid-phase extraction procedure combined with liquid chromatography/electrospray ionization tandem mass spectrometry

A simple, automated and rapid method has been developed for the determination of a novel antiviral peptide sifuvirtide in monkey plasma. Raw plasma samples were directly loaded onto an on-line solid-phase extraction (SPE) column, which removes the time-consuming and laborious sample pretreatment. Following a timed valve-switching event, the analyte was eluted on-line to a reversed-phase high-performance liquid chromatography (RP-HPLC) column and subsequently introduced into a linear ion trap mass spectrometer, LTQ-MS, via an electrospray ionization (ESI) interface. The multiply charged peptides were specified and quantitatively analyzed using selective reaction monitoring (SRM). A highly pure four iodine-sifuvirtide was synthesized using an optimized iodogen method and proved to be a suitable internal standard (IS). A single analysis run takes about 18 min. Validation of the method demonstrated that the linear calibration curves covered the range of 4.88-5000 ng/mL, and the correlation coefficients were above 0.9923. The limit of detection (LOD) with the signal-to-noise (S/N) ratio higher than 12 was calculated as 1.22 ng/mL. The intra- and inter-batch precisions were less than 12.7% and 9.1%, and the mean accuracy ranged from -5.2% to 3.6%, respectively. Any carry-over effect from the system was negligible. In a pharmacokinetic (PK) study of sifuvirtide after a single intravenous or subcutaneous dose in monkeys, the on-line SPE-LC/MS/MS system was successfully utilized to determine hundreds of samples with only one extraction column, which indicated the feasibility and the reliability of this method for application in preclinical and clinical PK studies of peptide drugs.     

The direct combination of HPLC solute focusing with supercritical fluid chromatography and mass spectrometry to enhance sensitivity and improve identification of trace components in polar solvent extracts

A system for combining normal and reversed phase high-performance liquid chromatography solute focusing with packed column supercritical fluid chromatography and supercritical fluid chromatography–mass spectrometry has been developed. The technique has been used to identify paracetamol and stanozolol in spiked plasma extracts and its utility for the analysis of a sample from a process waste stream is illustrated.     

Quantitative 252Cf plasma desorption mass spectrometry for pharmaceuticals: A new approach to coupling liquid chromatography with mass spectrometry

A ²⁵²Cf plasma desorption mass spectrometer has been set up to analyse thin layers of non-volatile organic samples. Molecular and fragment ions were produced and their mass was determined by a time-of-flight measurement. A novel interface combines a high-performance liquid chromatograph with the ²⁵²Cf plasma desorption mass spectrometer in a twofold way: introducing the effluent continuously through a capillary inlet in the on-line liquid chromatography mass spectrometry mode or transferring already prepared samples through a vacuum lock into the mass spectrometer in the off-line liquid chromatography+mass spectrometry mode. The off-line mode has been applied for the quantitative analysis of pharmaceuticals in blood using stable isotope labelled standards.     

Piroxicam quantitation in human plasma by high-performance liquid chromatography with on- and off-line solid-phase extraction.

A comparative study of two analytical methodologies for piroxicam quantitation in plasma by off-line and on-line solid-phase extraction (SPE) and high-performance liquid chromatography (HPLC) is described. The SPE cartridges contained C8 for both extraction methods. The analytes piroxicam and tenoxican (internal standard) were separated on a C18 column with a mobile phase consisting of acetonitrile:20 mM phosphate buffer pH 3.1 (50:50, v/v) followed by UV detection at 360 nm. The validation of the methods demonstrated good recoveries (over 90%), sensitivity (limits of quantification of 0.05 microgram/ml with on-line SPE and 0.1 microgram/ml with off-line SPE, based on a 100 microliters and 200 microliters sample volume, respectively), accuracy and precision (better than 9.5%). Both methodologies have been used for bioequivalence studies.     

Rapid narrow band elution for on-line SPE using a novel solvent plug injection technique

Determination of trace constituents in biological and environmental samples usually requires a pre-concentration step. While solid-phase extraction (SPE) has been widely used, it is slow, labor intensive and adversely affected by analytical errors from handling. On-line SPE eliminates some of the flaws but often suffers from solvent compatibility problems with the subsequent chromatography separation. In this study, we are presenting a technical solution for overcoming some of these compatibility issues, by utilizing a fully automated, focused SPE sample transfer technique utilizing narrow-band solvent plugs, for seamless hyphenation with high-performance liquid chromatography (HPLC) or flow injection mass spectrometry (MS). A wide range of pharmaceutical compounds was studied in different sample matrices. Short plugs of high elution strength solvent were generated by means of an electrically actuated sample loop and enrichment and transfer steps monitored using on-line SPE-MS. The impact of the solvent plugs on chromatographic separation was studied using hyphenated SPE-LC-MS. By carefully examining elution profiles of solvent plugs of different compositions, optimum conditions for quantitative elution within well-defined volumes were found for all substances. In addition, the highly focused elution bands resulted in excellent retention time and peak area reproducibilities when injected on-line onto HPLC columns. Finally, to demonstrate proof-of-principle, the fully integrated on-line SPE-LC-MS system was applied to the analysis of spiked urine and river water samples.