Amperometric detection of simple alcohols in aqueous solutions by application of a triple-pulse potential waveform at platinum electrodes

Application of a triple-pulse waveform is described for the anodic detection of methanol, ethanol, and ethylene glycol. The execution of the waveform incorporates the cleaning and reactivation of the platinum electrode, by alternate anodic and cathodic polarization, with measurement of the faradaic signal for the analyte at the reduced electrode surface. Some results for formic acid are also presented. The waveform is completed within approximately 2 s and the faradaic signal exhibits no decay with time as would be the case for amperometric detection at a constant applied potential. Calibration curves made by plotting —1/I vs. 1/C are linear. This is consistent with a reaction mechanism in which the analyte is adsorbed prior to anodic detection. The technique is applicable for detection in chromatographic and flow-injection systems.     

Sensitive liquid chromatographic-tandem mass spectrometric method for the determination of fluoxetine and its primary active metabolite norfluoxetine in human plasma

A sensitive method for the simultaneous determination of fluoxetine and its major active metabolite norfluoxetine in plasma was developed, using high-performance liquid chromatographic separation with tandem mass spectrometric detection. The samples were extracted from alkalised plasma with hexane-isoamyl alcohol (98:2, v/v) followed by back-extraction into formic acid (2%). Chromatography was performed on a Phenomenex Luna C18 (2) 5 microm, 150x2 mm column with a mobile phase consisting of acetonitrile-0.02% formic acid (340:660, v/v) at a flow-rate of 0.35 ml/min. Detection was achieved by a Perkin-Elmer Sciex API 2000 mass spectrometer (LC-MS-MS) set at unit resolution in the multiple reaction monitoring mode. TurbolonSpray ionisation was used for ion production. The mean recoveries for fluoxetine and norfluoxetine were 98 and 97%, respectively, with a lower limit of quantification set at 0.15 ng/ml for the analyte and its metabolite. This assay method makes use of the increased sensitivity and selectivity of mass spectrometric (MS-MS) detection to allow for a more rapid (extraction and chromatography) and sensitive method for the simultaneous determination of fluoxetine and norfluoxetine in human plasma than has previously been described.     

Rapid and sensitive RP-LC method with amperometric detection for pharmacokinetic assessment of propafenone in human serum of healthy volunteers

A rapid and sensitive liquid chromatography method with amperometric detection has been developed for the determination of propafenone in serum. Sample preparation based on single extraction with dichloromethane using bupivacaine hydrochloride as internal standard. The compounds were separated on C-18 reversed-phase analytical column with the mobile phase composed of methanol-acetonitrile-10 mM K₂HPO₄ (45: 25: 30, v/v/v). Analytes were detected electrochemically with the use of amperometric detector. The quantification limit for propafenone in serum was 10 ng/mL. Linearity of the method was confirmed in the range of 10–500 ng/mL with correlation coefficient of 0.9998. Inter-day relative standard deviations (RSD) ranged from 0.27 to 11.9% and intra-day RSD equalled from 1.1 to 9.7%. The newly developed method was applied for the monitoring of the drug in blood levels with 18 healthy volunteers taking tablet with propafenone.     

Improved high-performance liquid chromatographic determination with amperometric detection of alpha-amanitin in human plasma based on its voltammetric study

A sensitive and simple method is described for the selective determination in human plasma of alpha-amanitin, the most poisonous and prevalent toxin in the lethal fungi of species Amanita, using high-performance liquid chromatography with amperometric detection. After an extraction of plasma with disposable C18 silica cartridges, the extracts were separated by isocratic reversed-phase chromatography using a macroporous poly(styrene-divinylbenzene) column and a mobile phase of 0.05 M phosphate buffer-acetonitrile (91:9) at the apparent pH of 9.5. Amperometric detection was performed by applying an oxidation potential as low as +350 mV (vs. Ag/AgCl) to a glassy carbon electrode, in a thin-layer flow-cell. The linear range for alpha-amanitin was 3-200 ng/ml, and the relative limit of detection in plasma was 2 ng/ml at a signal-to-noise ratio of 2. The intra-assay precision was evaluated at levels of 10 and 200 ng/ml; the coefficients of variation were 4.5 and 2.6% (n = 5), respectively. Inter-assay coefficients of variation were 6.5 and 4.2% (n = 5) for the same concentrations of toxin. These analytical conditions have been chosen on the basis of a preliminary in batch cyclic voltammetric investigation of alpha-, beta- and gamma-amanitins, which has allowed their oxidation process to be clarified and the pH dependence of their oxidation potentials to be determined. All three amanitins are oxidized at the same potential values, and adsorption onto the electrode surface of both reactant and products was found in all cases. This adsorption did not affect the signal recorded for alpha- and gamma-amanitins at the amperometric detector, and for beta-amanitin a stronger adsorption for the anodic product was found, which leads to a marked positive shift of the potential required for the oxidation of this isomer in the amperometric detector cell.     

LC-amperometric determination of tert-butylcatechol in N-methyl-2-pyrrolidone stream containing polar and nonpolar impurities

Amperometric detection is shown to be highly selective and sensitive for the determination of tert-butylcatechol (TBC) added as an antioxidant in the circulating N-methyl-2-pyrrolidone (NMP) used for the purification of 1,3-butadiene by extractive distillation. The separation of TBC from numerous polar and nonpolar species present in the analyte is achieved on a 5-cm reversed-phase C-18 column using acetonitrile-water gradient conditions, both solvent components containing tetrabutyl ammonium dihydrogen phosphate as the amphiphilic modifier. The separation process is optimized for the resolution of TBC from nonpolar hydrocarbons as well as polar compounds, and a complete elution of all species from the column. An applied potential of 0.6 V relative to the Ag/AgCI electrode is found to provide an optimum sensitivity by the amperometric detector. It is also shown that the hydrocarbon content in NMP can be monitored simultaneously by coupling a spectrophotometric detector in series.     

Analysis of phenolic xenoestrogens by pressurized CEC with amperometric detection

A new method, pressurized CEC with end‐column amperometric detection using carbon paste electrode, has been developed for the separation and determination of five phenolic xenoestrogens in chicken eggs and milk powder samples. Efficient separation of five analytes was performed by pressurized CEC using a mobile phase consisting of 60% v/v ACN and 40% v/v Tris buffer (5 mmol/L, pH 8.0), +6 kV of applied voltage and 7.0 MPa of supplementary pressure. Detection limits of 50, 5, 2, 10 and 20 ng/mL for pentachlorophenol, bisphenol‐A, 2,4‐dichlorophenol, 4‐tert‐octylphenol and 4‐nonylphenol, respectively, were achieved using carbon paste electrode as working electrode and +0.8 V as detection potential. Matrix solid phase dispersion extraction method had been employed during sample preparation procedure, and mean recoveries ranged from 79.2 to 102.6% at different concentrations of phenolic xenoestrogens for spiked egg and milk powder samples were obtained.     

Determination of ranolazine in human plasma by liquid chromatographic-tandem mass spectrometric assay

A highly sensitive liquid chromatographic-tandem mass spectrometric method (LC-MS-MS) is developed to quantitate ranolazine in human plasma. The analyte and internal standard tramadol are extracted from plasma by liquid-liquid extraction using diethyl ether-dichloromethane (60:40 v/v), and separated on a Zorbax extend C(18) column using methanol-10mM ammonium acetate (60:40 v/v, pH 4.0) at a flow of 1.0 mL/min. Detection is carried out by multiple reaction monitoring on a QtrapTM LC-MS-MS system with an electrospray ionization interface. The assay is linear over the range 10-5000 ng/mL with a limit of quantitation of 10 ng/mL and a lower limit of detection (S/N > 3) of 1 ng/mL. Intra- and inter-day precision are < 3.1% and < 2.8%, respectively, and the accuracy is in the range 96.7-101.6%. The validated method is successfully used to analyze the drug in samples of human plasma for pharmacokinetic studies.     

The Direct Electrochemical Detection of Amino Acids at a Platinum Electrode in an Alkaline Chromatographic Effluent

Abstract

It is the general experience that most organic compounds including amino acids do not produce reversible or even quasi-reversible anodic waves at a Pt electrode under conditions of conventional cyclic voltammetry. Furthermore, amperometric detection of these compounds at a constant electrode potential is not successful because of the accumulation of adsorbed reaction products and/or an oxide film at the electrode surface. However, it is observed that a Pt electrode surface is cleaned quite effectively of adsorbed organic molecles and radicals simultanaeously with the anodic formation of the oxide layer. This oxidation of adsorbed organic species is concluded to be electrocatalyzed by PtOH formed as the first step in the production of the oxide layer (PtO). A pulsed-potential waveform applied at a frequency of ca. 1 Hz is demonstrated to provide direct amperometric detection of adsorbed amino acids at a Pt electrode. Satisfactory analytical precision (i.e., < 3% rel. std. dev.) results because the waveform reproducibly generates the catalytically active surface state at the Pt electrode. Both primary and secondary amino acids are determined with satisfactory detection limits: e.g., ca. 13 ng for glycine, 7 ng for phenylamine and 23 ng for hydroxyproline in 50-μL samples. Analytical response is concluded to depend on the adsorption isotherm of the amino acid being detected. Hence, the calibration plot of I/I_peak vs. 1/C^D is linear for low surface coverages. Results are shown for amperometric detection of a synthetic mixture of amino acids by anion-exchange chromatography using NaOH as the eluent and supporting electrolyte.     

Determination of Vitamin D3 and 25-Hydroxyvitamin D3 Using Liquid Chromatography with Amperometric Detection

Abstract

The electrochemical behaviour of vitamin D₃ and 25-hydroxyvitamin D₃ (25-OH D₃) in a high performance liquid chromatography system using amperometric detection is described. Separation is carried out using a C18 reversed-phase column and the optimum mobile phase was a 0.1 M LiClO₄ solution in methanol-water (97:3, v/v) at a flow rate of 1.25 ml/min. 25-OH D₃ and vitamin D₃ were eluted with good resolution at retention times of 3 and 6 minutes respectively, and determined by amperometric detection with a glassy carbon electrode at + 1.050 V (vs Ag/AgCl). Calibration graphs for both substances showed good linearity when amounts of vitamin D₃ between 18 and 312 ng and 27 and 412 ng of 25-OH D₃ were injected. Detection limits of 8 ng (vitamin D₃) and 25 ng (25-OH D₃); relative standard deviations of 3.2% (vitamin D₃) and 5.8% (25-OH D₃) were obtained.     

Comparative Study of the Determination of Parabens in Shampoos by Liquid Chromatography with Amperometric and Coulometric Detection

The simultaneous determination of four para‐hydroxybenzoic acid esters (parabens) in shampoos was studied by liquid chromatography (LC) with amperometric (LC‐AD) and coulometric (LC‐CD) detection. The parabens were separated on an ODS C18 reversed column by isocratic elution with a mobile phase based on methanol‐0.1 M acetic acid (60 : 40%, v/v) with 0.02 M NaClO₄ at a flow rate of 0.8 mL min^?1. The limit of detection (S/N>3) for the analytes was in the 15–25 pg (injected mass) range at an applied potential of 1.20 V vs. Ag/AgCl using the LC‐AD and in the 2–3 pg range at a potential of 0.790 V vs. Pd using the LC‐CD. The peak ratio of the internal standard peak (IS: 4‐hydroxybenzoic acid sec‐butyl ester) versus the analyte peak was found to be related to the amount injected from 0.1 ng to 100ng (r=0.996–0.999) with the LC‐AD and from 0.050 ng to 100 ng range (r=0.999–1.000) with the LC‐CD. The relative standard deviation (RSD, n=10) was comprised between 1.8 to 3.5% by LC‐AD ( 5 ng injected) and between 2.0 to 2.4% by LC‐CD (0.5 ng injected). The determination of four most used parabens in ten different shampoos was successfully realized.     

Analysis of Cimetidine in Biological Fluids by High Performance Liquid Chromatography

Abstract

The clean-up and analysis of cimetidine in human urine and blood is described. Samples were prepared by adsorption on Waters Sep-pak C-18 disposable pre-columns in basic solution followed by elution in 1 ml methanol. For blood samples, the eluate was concentrated under a stream of nitrogen; urine samples required no further concentration. The separation was performed on a reversed phase column using a mixture of methanol-1 mM sodium dodecyl sulphate in a 10 mM phosphate buffer of pH 3.0 (35:65) as mobile phase. Procaine was used as internal standard. Detection was by UV at 228 nm yielding a minimum detectable quantity of 20 ng with linearity over three decades of concentration.     

Determination of halofuginone in bovine plasma by competing-ion high performance liquid chromatography after solid phase extraction

A high performance liquid chromatography (HPLC) method for the determination of the anticoccidial and antitheilerial drug halofuginone in bovine plasma was developed. Samples were diluted with acetic acid (10%, v/v) and cleaned up on a Bond Elut C8 column. The analyte was eluted from the extraction column and chromatographed by reversed-phase HPLC using decylamine as a competing-ion reagent. Detection was by UV at 243 nm. Recovery from plasma was 75%, and within-day and between-day coefficients of variation were 5.23 and 6.35% respectively. The specificity and sensitivity of this method (limit of detection in plasma, 1 ng/mL) were sufficiently high to enable us to characterize the time course of the drug in plasma after oral administration of therapeutic doses to cattle.     

Determination of the anticancer drug 5-fluorouracil added to blood serum by liquid chromatography with anodic amperometric detection

Liquid chromatography with on-line anodic amperometric detection is used after a liquid/solid extraction step for the determination of the anticancer drug 5-fluorouracil added to blood serum. A detection limit of 15 ng ml^?1 can be achieved. The anodic electrochemical behaviour of the drug at a mercury electrode, which is the basis of the detection principle, is also described. Anodic amperometric detection at a mercury electrode is compared with detection at a glassy carbon electrode in terms of sensitivity, linearity and selectivity.     

A Unique Solid Phase Extraction Column for Isolation of 11-Nor-Δ-9-Tetrahydrocannabinol-9-Carboxylic Acid in Human Urine

Abstract

A sensitive, specific, qualitative, and quantitative extraction procedure followed by an hplc assay of 11-nor-Δ-9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH) from human urine is developed. Using a new, “mixed mode”, bonded silica gel solid phase extraction (SPE) column, the analyte was selectively isolated from the urine component. Following extraction, the presence of THC-COOH was confirmed and quantitated by a UV detector on a Varian 15cm C18 column using 35:65 v/v 50 mM phosphoric acid:acetonitrile at a flow rate of 1.5 mL/min. The limit of detection was 10 ng/mL at a signal to noise ratio of 2.5. The method showed linearity in the 10–300 ng/mL range (r=0.999) with good precision (RSD 1.4%) and accuracy (87% absolute recovery).     

On-line sample cleanup and enrichment chromatographic technique for the determination of ambroxol in human serum

A sensitive and efficient on-line clean up and pre-concentration method has been developed using column-switching technique and protein-coated μ-Bondapak CN silica pre-column for quantification of ambroxol (AM) in human serum. The method is performed by direct injection of serum sample onto a protein-coated μ-Bondapak CN silica pre-column, where AM is pre-concentrated and retained, while proteins and very polar constituents are washed to waste using a phosphate buffer saline (pH 7.4). The retained analyte on the pre-column is directed onto a C(18) analytical column for separation, with a mobile phase consisting of a mixture of methanol and distilled deionized water (containing 1% triethylamine adjusted to pH 3.5 with ortho-phosphoric acid) in the ratio of 50:50 (v/v). Detection is performed at 254 nm. The calibration curve is linear over the concentration range of 12-120 ng/mL (r(2) = 0.9995). The recovery, selectivity, linearity, precision, and accuracy of the method are convenient for pharmacokinetic studies or routine assays.     

Quality by Design enabled enhanced bioanalytical extraction and UFLC determination of vilazodone from rat serum

An ultrafast liquid chromatographic bioanalytical method was developed and validated for the determination of vilazodone in Wistar rat serum. Principles of quality by design were implemented for enhancing the bioanalytical liquid–liquid extraction of vilazodone from rat serum. A Box–Behnken design was utilized in the studies by selecting extraction time, centrifugation speed, and vortex time as the critical method variables for evaluating their effect on the analytical attribute, i.e., %recovery of vilazodone. Chromatographic separation was achieved within a run time of 10?min using a C-18 column and mobile phase comprising of methanol:phosphate buffer of pH 7 (85:15 v/v) flowing at 1.5?mL/min. Photodiode array detection was performed at 242?nm. Results of validation studies were satisfactory. The method was linear over a concentration of 100–2,000?ng/mL with acceptable accuracy and precision. Limits of detection and quantitation for the developed method were 50 and 100?ng/mL, respectively. This QbD-based approach was found suitable for routine bioanalysis of vilazodone in the biological matrix.     

Integrated quality by design (QbD) and design of experiments (DoE) approach for UFLC determination of telaprevir in rat serum

An ultrafast liquid chromatographic bioanalytical method was developed and validated for the determination of telaprevir in Wistar albino rat serum. Principles of quality by design (QbD) were implemented for enhancing the bioanalytical liquid–liquid extraction of telaprevir from rat serum. A Box–Behnken design was utilized in the studies by selecting extraction time, centrifugation speed, and vortex time as the critical method variables for evaluating their effect on the critical analytical attribute, i.e., %recovery of telaprevir. Chromatographic separation was achieved within a run time of 10?min using a C-18 column and mobile phase comprising of methanol:borate buffer of pH 9 (90:10 v/v) flowing at 1.2?mL/min. Photodiode array detection was performed at 270?nm. Results of validation studies were satisfactory. The method was linear over a concentration of 25–10,000?ng/mL. Limit of detection for the developed method was 10?ng/mL. Further, design of experiments (DoE) used for inter-day accuracy and precision study suggested superior method reliability. This integrated QbD- and DoE-based approach ensured the development of a validated and reliable analytical method for optimum bioanalysis of telaprevir in biological matrix.     

Retention Characteristics of Co+3, Fe+3, and Cu+2 4-(2-Pyridylazo)Resorcinol (PAR) Complexes on C-18 and Amino Silica Packings

Abstract

Chloroform extraction of Co-PAR, Fe-PAR, and Cu-PAR complexes in a pH 6.5 phosphate buffer implied the first two species were primarily monoanions, but the latter was a dianion. Examination of the literature and retention data of these complexes on C-18 and amino columns confirmed the more anionic nature of Cu-PAR. The Co⁺³ and Cu⁺² complexes were slightly resolved from each other, but the Fe⁺³ complex was retained longer on the C-18 silica. In contrast, the Co⁺³ and Fe⁺³ complexes were not resolved, but the Cu⁺² complex was well-retained on the weak anion exchange amino silica column. Use of short amino and C-18 columns in series provided a good separation of all three complexes. Detection of the metal complexes at 546 nm instead of 254 nm avoided interference by PAR and good detection limits were still maintained.     

Determination of fat-soluble vitamins in yogurt by HPLC with electrochemical detection

A method employing HPLC with electrochemical detection for the rapid and simultaneous determination of vitamins A, D(3) and E is described. The method uses a C-18 reverse phase column and 2.5 mM HAcO-NaAcO in methanol-water (99:1, v/v) solution as the mobile phase. The compounds are quantified using amperometric detection with a glassy carbon electrode at a potential of + 1300 mV (vs. Ag/AgCl) and the results are compared with those obtained using UV detection at a wavelength of 280 nm. The method was successfully applied to the analysis of vitamins A, D(3) and E in yogurt samples. After saponification, fat-soluble vitamins were extracted and the methanolic solution of the extracts was injected directly into the chromatographic system, avoiding the clean-up step which is necessary when no electrochemical detection is used. Good recovery percentages were obtained.     

Evaluation of an automated solid-phase extraction system for the enrichment of organochlorine pesticides from waters

An automatic method for the determination of organochlorine pesticides (OCPs) in water samples was developed. The analytes are preconcentrated onto a customized RP-C(18) column and subsequently eluted with 80 mul of ethyl acetate. Gas chromatography with electron capture detection is used for their separation and selective detection. Detection limits for 10 ml sample volumes range from 0.01 to 0.1 ng ml(-1), with standard deviations between 4 and 6%. The average recovery at a fortification level of 2 ng ml(-1) is 92%. The method was used to screen OCPs in natural waters collected near agricultural areas and also to tap waters. Positive findings were confirmed by gas chromatography with mass spectrometric detection. Finally, the adsorption/degradation of OCPs in natural pond water containing a high level of suspended matter was examined; some OCPs were found to be degraded and irreversibly adsorbed on suspended matter present in the water.