Systematic approach to the determination of cephalosporins in biological fluids by reversed-phase liquid chromatography

The chromatographic behaviour of some cephalosporins as a function of pH and ionic strength of the mobile phase was studied on 10-microns LiChrosorb RP-18. Acidic cephalosporins were retained longest in their neutral form with an acidic eluent. Amphoteric cephalosporins were retained longest in their protonated form with an acidic eluent of low ionic strength. Cefotiam was retained longer with an alkaline mobile phase. LiChrosorb RP-18, Nucleosil C18 and muBondapak C18 gave rise to different selectivities when an acidic eluent, methanol-water (25:75) containing 0.2% of 1.8 M H2SO4 was used. This may be related to interactions with residual silanol groups. The studied cephalosporins (with the exception of cefotiam and cefsulodin) were separated from compounds present in biological fluids on 5-microns LiChrosorb RP-18 using the mobile phase 0.2% of 1.8 M H2SO4 in a mixture of methanol and water with various methanol contents. The determination of cefotiam in biological fluids was performed with an alkaline mobile phase. The preparation of the sample was simple and rapid: precipitation of plasma proteins or dilution of urine. The method was applied to the determination of ceftizoxime in human plasma and urine. Concentrations down to 0.2 micrograms/ml of plasma and 25 micrograms/ml of urine could be determined with good reproducibility and accuracy.     

Simultaneous determination of uric acid and creatinine in biological fluids by column-switching liquid chromatography with ultraviolet detection

A column-switching liquid chromatographic method for the simultaneous determination of uric acid and creatinine in human serum and urine was developed. Creatinine and uric acid were separated by size-exclusion chromatography on a hydrophilic gel column (C1) and creatinine eluted from Cl was separated from proteins by filtration through a longer hydrophilic gel column (C2). The creatinine fraction eluted from C2 was transferred to a weakly acidic cation-exchange column (C3) and then to a strongly acidic cation-exchange column (C4). Uric acid eluted from Cl after creatinine was transferred to an anion-exchange column (C5) and then to a hydrophilic gel column (C6). The mobile phase was a mixed buffer of pH 5.1 (propionic acid-succinic acid-NaOH, 60:15:60 mmol/1 in water). Diluted serum and urine could be injected onto C1, and Cl was backflushed after the transfer of uric acid from Cl to C5.

Creatinine and uric acid in the eluate were determined by measuring their ultraviolet absorption at 234 and 290 nm, respectively. The recovery of uric acid and creatinine added to diluted serum (20-fold dilution, concentration 20 and 5 μmol/1, respectively) was 98.9±0.56% and 100.9±1.29%, respectively. The recovery of uric acid and creatinine added to diluted urine (100-fold dilution, concentration 50 and 100 μmol/l, respectively) was 99.4±0.72% and 98.7±1.45%, respectively (mean±R.S.D., n=6).     

A reversed-phase high-performance liquid chromatography method with pulsed amperometric detection for the determination of glycosides

We have developed a reversed-phase high-performance liquid chromatography pulsed amperometric detection (RP-HPLC-PAD) method for the determination of glycosides. It is sensitive, repeatable, and selective without the pretreatment step. Ginsenosides were separated completely in 50 min using an water-acetonitrile gradient as the eluent and detected by PAD under NaOH alkaline conditions. The ginsenoside detection limit (S/N=3) was 0.02-0.07 ng and the quantification limit (S/N=10) was 0.1-0.2 ng. The coefficient of linear regression was 0.9984-0.9998 for concentrations between 1 and 50 microg/mL. The intra- and inter-day precision (RSD) was less than 6.35% in Ginseng Radix and Shy-jiun-tzyy-tang extracts. The average recoveries from Ginseng Radix and Shy-jiun-tzyy-tang extracts were 98.19-105.45% and 96.89-102.22%, respectively.     

Liquid chromatographic determination of ethylenethiourea using pulsed amperometric detection.

A liquid chromatographic method was developed using pulsed amperometric detection at a gold working electrode to measure residue levels of ethylenethiourea (ETU) in crops and groundwater. Use of the sequential pulsing program eliminates electrode fouling while preserving the sensitive and selective detection of ETU. Minimum detection limits in crops were 5-10 ppb (1.25-2.5 ng on-column) and 5 ppb (0.5 ng) in groundwater. The commercial availability of the pulsed electrochemical detector and its gold working electrode that remains functional with a minimum of conditioning is an improvement in method simplicity.     

Determination of saccharides in biological materials by high-performance anion-exchange chromatography with pulsed amperometric detection

High-performance anion-exchange chromatography (HPAEC) coupled with pulsed amperometric detection (PAD) under alkaline conditions (pH 9-13) separates aminosaccharides, neutral saccharides and glycuronic acids based upon their molecular size, saccharide composition and glycosidic linkages. Carbohydrates were extracted by utilizing 0.5 M H2SO4 (neutral monosaccharides), 0.25 M H2SO4 coupled with enzyme catalysis (glycuronic acids) and 3 M H2SO4 (aminosaccharides). Solid-phase extraction with strong cation and strong anion resins was used to partition the cationic aminosaccharides and anionic glycuronic acids and to deionize acid extracts for neutral saccharides. Separation was conducted on a medium-capacity anion-exchange column (36 mequiv.) utilizing sodium hydroxide (5-200 mM and sodium acetate (0-250 mM) as the mobile phase. The saccharides were detected by oxidation at a gold working electrode with triple-pulsed amperometry. HPAEC-PAD was found superior to high-performance liquid chromatography with refractive index (RI) detection for neutral monosaccharides and aminosaccharides and to low-wavelength UV detection for glycuronic acids in terms of resolution and sensitivity. HPAEC-PAD was not subject to interferences as was the case for low UV detection (210 nm) or RI analyses and was highly selective for mono- and aminosaccharides and glycuronic acids. The use of HPAEC-PAD was applied for the determination of the saccharide composition of organic materials (plant residues, animal wastes and sewage sludge), microbial polymers and soil.     

Capillary electrophoresis with contactless conductivity detection for uric acid determination in biological fluids

The suitability of capillary electrophoresis (CE) with capacitively coupled contactless conductivity detection (C⁴D) for the direct determination of uric acid in human plasma and urine was investigated. It was found that a careful optimization of the buffer composition and pH was necessary to achieve selective determination in the complex sample matrices. An electrolyte solution consisting of 10 mM 2-morpholinoethanesulfonic acid (MES), 10 mM histidine and 0.1 mM hexadecyltrimethylammonium bromide (CTAB), pH 6.0, was finally found suitable for use as running buffer for both sample matrices. The limit of detection (3 S/N) was determined as 3.3 μM. The linearity of the response was tested for the range between 10 and 500 μM and a correlation coefficient of 0.9996 was obtained. Intra- and inter-day variabilities were <10%. Quantitative analysis of urine and plasma samples showed a good correlation with the routine enzymatic method currently used at the University Hospital of Basel.     

Simultaneous determination of codeine, norcodeine and morphine in biological fluids by high-performance liquid chromatography with fluorescence detection

A novel high-performance liquid chromatographic method for the determination of codeine, norcodeine and morphine in plasma and urine has been developed. The compounds were separated on a cyano column (15 cm x 4.6 mm, 5 microns particle size) using a mobile phase of acetonitrile-triethylamine-distilled water (4:0.1:95.9, v/v) pH 3.1 and then determined by fluorescence detection. Calibration curves in the range 5-200 ng/ml for plasma and 0.1-10 micrograms/ml for urine were linear and passed through the origin. The imprecision and inaccuracy of the assay were less than 10% and the limits of detection were 2 ng/ml for all three compounds in human plasma.     

Analysis of erythromycin in biological fluids by high-performance liquid chromatography with electrochemical detection

A simple and sensitive high-performance liquid chromatographic assay was developed for the quantitative determination of major erythromycin components and their potential metabolites or degradation products in plasma and urine. An ether extract of alkalized plasma sample was chromatographed on a reversed-phase column and the components in the column effluent were monitored by an electrochemical detector. The recovery of the drug from extraction was virtually 100%. The detection limits for erythromycin A in plasma were 5-10 ng/ml and 30 ng/ml using 1 and 0.2 ml of sample, respectively. For urine samples, a simple one-step deproteinization with two volumes of acetonitrile was satisfactory for analysis. The method has been evaluated in plasma and urine from dogs receiving oral or intravenous erythromycin A. The standard curves for potential metabolites or degradation products were not constructed due to the lack of sufficient samples.     

Membrane sampler for interference-free flow injection NO determination in biological fluids with chemiluminescence detection

The development of a chemiluminescence (CL) method based on the perm-selective properties of a Nafion–cellulose acetate (CA) composite membrane for the monitoring of nitric oxide (NO) in biological fluids is described. Horseradish peroxidase (HRP) was used as NO trapping solution, forming the stable compound HRP–NO. The HRP was denatured and the trapped NO was released and detected by using the luminol–H₂O₂ system. Using a mixed (size-exclusion and polar-based) transport control, the interference effects of various compounds were minimized. The method was used for NO monitoring in simulated samples, by using a blood specimen as sample matrix. The 3σ detection limit is 0.9×10⁻⁶ mol and linear semi-log calibration plot in the range 1.8×10⁻⁶ to 2.7×10⁻³ mol NO was constructed. The applied methodology was further used to prolong the NO lifetime in order to increase the sensitivity of its determination. This was based on the increase of the response in the presence of certain reductive species, which act as NO preservatives in biological fluid samples.     

Voltammetric/amperometric detection for liquid chromatography

A voltammetric/amperometric detector based on a dual-electrode electrochemical detector is described for liquid chromatography. The detector combines the advantages of both voltammetric and amperometric detection. A three-dimensional data array of current response as a function of both time (chromatographic domain) and potential (electrochemical domain) is obtained. From the chromatographic point of view, this allows post-experimental choice of the optimal detection potential. Different detection potentials can even be chosen for each chromatographic peak. Having the voltammetric data as well as the chromatographic data provides ready identification of chromatographically unresolved compounds and the ability to resolve such co-eluting compounds voltammetrically. The voltammetric data also provide a second method of peak identification for greater certainty in peak assignments. Voltammetric detection limits of less than 10 pmol of material injected on the column were achieved with this detection method. From the electrochemical perspective, voltammetric/amperometric detection provides a technique for obtaining hydrodynamic voltammograms with small amounts or small volumes of sample. Voltammograms can also be obtained for the individual components of complex mixtures without the need for isolation steps.     

Direct determination of free cyanide in drinking water by ion chromatography with pulsed amperometric detection

Cyanide is a regulated contaminant in drinking water in the United States. This paper describes an ion chromatography method with pulsed amperometric detection (PAD) that directly determines free cyanide in drinking water. Samples are treated with sodium hydroxide to stabilize cyanide and with a cation-exchange cartridge to remove transition metals. Cyanide is separated by anion-exchange chromatography and detected by PAD with a waveform optimized for cyanide and used with a disposable silver working electrode. The recovery of cyanide spiked into five water samples was >80%. With an MDL of 1.0 microg/L, this method determines cyanide concentrations well below the reporting limits for free cyanide in drinking water.     

Automated high performance liquid chromatography with on-line reduction of disulfides and chemiluminescence detection for determination of thiols and disulfides in biological fluids

In general, the reduction of disulfide bonds with tris(2-carboxyethyl)phosphine (TCEP) is performed using off-line operation, which is not only time-consuming but also vulnerable to the spontaneous re-oxidation of thiols during sample preparation and subsequent analysis procedures. To the best of our knowledge, there has been not any case on the on-line reduction for biological disulfides coupled with high performance liquid chromatography (HPLC). In this study, these obstacles are overcome by packing Zn(II)-TCEP complexes into a home-made column. The as-synthesized Zn(II)-TCEP complexes enable efficient reduction of disulfide bonds at pH 3.0. This acidic pH value was compatible with that of the mobile phase for HPLC separation of thiols and disulfides. Therefore, using fluorosurfactant-prepared triangular gold nanoparticles as HPLC postcolumn specific chemiluminescence (CL) reagents for thiols, the feasibility of the established on-line reduction column has been confirmed for the direct identification of both thiols and disulfides by incorporating this reduction column into a single chromatographic separation. Detection limits for these analytes range from 8.3 to 25.4 nM and the linear range in a log–log plot can comprise three orders of magnitude. Finally, the utility of this automated on-line reduction of disulfides-HPLC-CL system has been demonstrated for the reliable determination of thiols and disulfides in human urine and plasma samples.     

Liquid chromatography amperometric detection of carboxylic acids and phenolic acids with a copper-based chemically-modified electrode

A copper-based chemically-modified electrode has been constructed and characterized by various experimental parameters in flow-through amperometric detection of carboxylic acids and phenolic acids. Novel hydrodynamic voltamperograms were first obtained in flow-through amperometric detection with the Cu-based CME and subsequently negative and positive peaks were observed in a single chromatogram. This unique and flexible potential dependence could be of great benefit in chromatographic speciation and quantification. These observations suggest that the detector response was governed by the complexation reaction of copper ions with the solutes.     

New automatized method with amperometric detection for the determination of azithromycin

A FIA-amperometric method for azithromycin determination was developed. A working glassy carbon electrode and a Ag/AgCl/NaCl (3 M) reference electrode were used. The determination is based on the electrochemical oxidation of the azithromycin at 0.9 V in Britton-Robinson buffer solution (pH 8.0). Due to the adsorption of the reaction products on the electrode surface, an effective cleaner cycle was implemented. By using the optimum chemical and FIA conditions, a concentration linear range of 1.0-10.0 mg L⁻¹ and a detection limit (LOD) of 0.76 mg L⁻¹ are obtained. The method was validated and satisfactorily applied to the determination of azithromycin in pharmaceutical formulations.