Determination of Levodopa and Benserazide Hydrochloride in Pharmaceutical Formulations by CZE with Amperometric Detection

A novel method, capillary electrophoresis with amperometric detection, has been established for rapid and effective measurement of levodopa (L-dopa), and benserazide (BS) and its impurity (R,S)-2-amino-3-hydroxypropanohydrazide (Ro-04-1419) in co-beneldopa pharmaceutical formulations. Suitable separation and amperometric detection conditions were investigated and optimized. The optimum conditions of CZE detection were 40 mm phosphate solution at pH 5.3 as running buffer, 17 kV separation voltage, carbon-disk working electrode, 0.95 V (relative to Ag/AgCl) as detection potential, and sample injection for 8 s at 17 kV. The linear ranges were from 1.25 to 50 g mL^–1 for L-dopa, 1.2 × 10^–1 to 25.5 g mL^–1 for BS, and 1.0 × 10^–2 to 4.4 × 10^–1 g mL^–1 for Ro-04-1419, with correlation coefficients of 0.9994, 0.9951, and 0.9933, respectively. The detection limits for L-dopa, BS, and Ro-04-1419 were 0.38, 0.02, and 0.004 g mL^–1, respectively. Average recoveries were 100.2% for L-dopa, 102.4% for BS, and 90.8% for Ro-04-1419. This method was successfully applied to co-beneldopa granules and tablets.Revised: 30 November and 22 December 2004     

Application of l-cysteine-capped nano-ZnS as a fluorescence probe for the determination of proteins

Nanometer-sized l-cysteine-capped ZnS particles have been synthesized and used as a fluorescence probe to investigate the effect of proteins on fluorescent intensity. With =190 nm, maximum and constant synchronous fluorescence enhancement was produced at 267 nm and pH 5.12 in the presence of proteins. A highly sensitive synchronous fluorescence method for the rapid determination of proteins has been developed. Under optimum conditions, calibration graphs are linear over the range 0.03–8.0 g mL^–1 for bovine serum albumin (BSA), 0.01–6.0 g mL^–1 for human serum albumin (HSA), 0.05–8.0 g mL^–1 for -globulin (-G), and 0.04–4.0 g mL^–1 for ovalbumin, respectively. The relative standard deviations of seven replicate measurements were 1.75% for 1.0 g mL^–1 BSA, 1.90% for 1.0 g mL^–1 HSA, 1.65% for 1.0 g mL^–1 -G, and 2.32% for 1.0 g mL^–1 ovalbumin.     

Flow injection chemiluminescence determination of l-cysteine in amino acid mixture and human urine with the BrO3 −–quinine system

In this paper, a novel flow injection chemiluminescence (CL) determination of l-cysteine is proposed. The method is based on the CL reaction of l-cysteine and KBrO₃ in acidic medium. The CL intensity was greatly enhanced in the presence of quinine. The CL intensity was linear with l-cysteine concentration in the range of 0.2–80 g L^–1, and the detection limit was 0.1 g L^–1 (3). A complete analysis, including sampling and injecting, could be performed in 1 min, giving a throughput of about 60 h^–1. The relative standard deviation was 1.6% for 0.8 g L^–1 l-cysteine (n=11). The proposed method was satisfactorily applied to the determination of cysteine in an amino acid mixture and human urine. The mechanism of the CL reaction is also discussed.     

Determination of 6-amino-2,2-dimethyl-1,3-dioxepan-5-ol by postcolumn derivatization witho-phthalaldehyde/2-mercapto-ethanol after ion-pair chromatography

Summary A high-performance liquid chromatographic method is discribed for the determination of 6-amino-2,2-dimethyl-1,3-dioxepan-5-ol using Spherisorb ODS II stationary phase and mobile phase 30:70 (v/v) methanol: aqueous 1-octane sulfonic acid. Detection was fluorimetric following postcolumn derivatization with o-phthaladehyde/2-mercaptoethanol. The procedure was applied to the analysis of aqueous solutions and microcrystalline suspensions in liquid paraffin, prepared for investigation of the toxicological profile. The method was validated for selectivity, linearity of detector response, repeatability, limit of detection and quantitation. The HPLC method was selective. The instrumental limit of detection was 0.5 ng per injection (0.05 g mL^–1). The method detection limits were 0.5 g mL^–1 aqueous solution and 5 g mL^–1 liquid paraffin suspension, the quantitation limit 0.05 mg mL^–1 aqueous solution and 1.0 mg mL^–1 liquid paraffin. Linearity was within 0.94–47.1 g mL^–1. Intra-assay accuracy accounted for 99–100% in the range 0.05–226 mg mL^–1 aqueous solution, intra-assay precision for 2% (C.V.). For microcrystalline liquid paraffin suspensions with 1 and 250 mg mL^–1 99 and 109% was found for intra-assay accuracy. Intra-assay precision was 5% (C.V.). Reliable results over a wide concentration range can be obtained. The procedure is considered valid for determination of the analyte in aqueous solution or microcrystalline paraffin oil suspensions.     

Detection of 2,4,6-trichloroanisole in chlorinated water at nanogram per litre levels by SPME–GC–ECD

A method involving solid-phase micro extraction (SPME) and gas chromatography with electron capture detection (SPME–GC–ECD) has been optimised for identification and quantification of 2,4,6-trichloroanisole (TCA) at ng L^–1 concentrations in disinfected (chlorinated) water samples. A central composite design was used for factorial analysis of four factors, three factors related to the SPME (PDMS fibre) procedure (adsorption time, temperature of the sample during headspace sampling, and desorption time) and one related to the GC operation (the rate of increase of the temperature of the GC oven). Good linearity (linear correlation coefficient greater than 0.999) was observed for TCA concentrations up to 50 ng L^–1, limits of detection and quantification of 0.7 and 2.3 ng L^–1, respectively, and good precision (relative standard deviation 2.8% and 3.4% for 5 and 30 ng L^–1 of TCA, respectively). Besides TCA, this system also enables the detection and quantification of the four trihalomethanes in the g L^–1 concentration range with limits of detection and quantification of approximately 0.3 g L^–1 and 1 g L^–1, respectively.     

Simultaneous capillary GC-MS determination of triazines and amides in water

Summary A simultaneous capillary Gas Chromatographic-Mass Spectrometric (GC-MS) method is described for the determination of thirteen pesticides belonging to the triazine and amide families in water. The sample is extracted in liquid-liquid mode (dichloromethane) and then the determination of the residues is carried out by capillary gas chromatography with mass spectrometric detection in the Selected-Ion Monitoring mode (SIM). The average recoveries of spiked compounds are in the 78.4–135.4% range between the relative low level (0.100 g L^–1) and the relative high level (10.0 g L^–1). The limits of detection (LOD) are in the 0.009–0.128 g L^–1 range.     

Development and validation of a novel HPLC/ELSD method for the direct determination of tobramycin in pharmaceuticals,plasma, and urine

A novel method for the direct determination of the aminoglycoside tobramycin was developed and validated based on reversed-phase high-performance liquid chromatography (RP-HPLC) with evaporative light scattering detector (ELSD). Using a Waters ODS-2 C18 Spherisorb column with an evaporation temperature of 45°C and nitrogen pressure of 3.5 bar, the selected mobile phase consisted of water/acetonitrile 55:45 containing 1.5 mL L^–1 HFBA (11.6 mM) in an isocratic mode at a rate of 1.0 mL min^–1. Tobramycins retention time was 4.3 min with an asymmetry factor of 1.7. A logarithmic calibration curve was obtained from 1 to 38 g mL^–1 (r > 0.9998). LOD was 0.3 g mL^–1; within-day %RSD was 1.0 (n = 3, 4.7 g mL^–1) and between-day %RSD was 1.1 (3 days within a week). The developed method was applied to the determination of tobramycin in a pharmaceutical crude substance and formulations (eye drops and ointments). Dilution experiments revealed the absence of interference from excipients (no constant and proportional errors); recovery from spiked samples was 99–103% with %RSD < 2.2 (n = 3×3). The developed HPLC/ELSD method was also found to be applicable in the determination of tobramycin in human plasma (0.6–12.5 g mL^–1) and urine (1.5–12.5 g mL^–1) after solid-phase extraction using carboxylate cartridges followed by solvent evaporation (×2 preconcentration). A mean recovery of 86% for plasma and 91% for urine was obtained.     

Determination of rofecoxib,in the presence of its photodegradation product,in pharmaceutical preparations by micellar electrokinetic capillary chromatography

A simple and rapid micellar electrokinetic capillary chromatographic (MEKC) method for analysis of rofecoxib (ROF) and its photodegradation product (PDP) in pharmaceutical preparations has been developed and validated. Analyses were conducted in a fused silica capillary (72 cm effective length, 50 m i.d.) with a background electrolyte consisting of 25 mmol L^–1 borate buffer at pH 7.0 containing 15 mmol L^–1 sodium dodecyl sulfate (SDS) and 10% acetonitrile (ACN). The separation was performed by voltage-controlled system, applying 30 kV at 30 °C, detecting at 225 nm; injection was hydrodynamic at 50 mbar for 2 s. Nifedipine was used as internal standard (IS). Under the optimum conditions ROF, PDP, and IS were well separated with in 10 min. The method was validated with regard to linearity, limit of detection and quantitation, precision, accuracy, specificity, and robustness. The detection limit of the method was low, 0.8 g mL^–1, and the linearity range was wide, 2.5 to 125 g mL^–1. The method was highly efficient—5×10⁵ plates m^–1 for ROF. The method was applied to the tablet form of ROF-containing pharmaceutical preparations. The data were compared with those from the voltammetric method described in literature. No statistically significant difference was found.     

Simultaneous determination of lomefloxacin,fenbufen and felbinac in human plasma using high performance liquid chromatography

Summary A simple, specific, and sensitive high-performance liquid chromatography method has been developed for simultaneous determination of the lomefloxacin, febufen, and felbinac in human plasma. Plasma-spiked with internal standard, was vortex-mixed for 1 min with a mixture of dichloromethane-diethylether (80:20, v/v). The evaporated extract was dissolved in 0.02 M NaOH. The extracts were chromatographed on an Supelcosil LCSAX column (5 m 250×4.6 mm I.D.) equipped with a guard column with a mibile phase composed of acetonitrile and phosphate buffer, with ultraviolet detection. Drugs were resolved at ambient temperature on a flow rate was 1.2 mL min^–1, and monitoring was performed at 280 nm. The detection limits for lomefloxacin was 0.05 g mL^–1, 0.02 g mL^–1 for fenbufen and 0.03 g mL^–1 for felbinac. No interference from other commonly administered drugs or endogenous substances was observed. The method is fast since it involves two extraction steps followed by evaporation of organic solvent and chromatography of the residue. This method was found to be applicable to pharmacokinetic and pharmacodynamic studies of each drug after the concomitant administration of lomefloxacin and febufen.     

Microdialysis sampling and high-performance liquid chromatography with chemiluminescence detection for in-vivo on-line determination and study of the pharmacokinetics of levodopa in blood

A simple, reliable, and reproducible method for in-vivo on-line separation and determination of levodopa has been based on microdialysis then high-performance liquid chromatography with chemiluminescence detection. The perfusate is perfused at a flow rate of 5 L min^–1. The concentration of levodopa in the dialysate is determined on line with a chemiluminescence system. The dialysate sample volume is approximately 20 L. The response of the system is linearly related to the concentration of levodopa in the range 1×10^–8 to 1×10^–6 g mL^–1 (r²=0.9995) with a detection limit (3) of 3×10^–9 g mL^–1 and sample throughput of 12 h^–1; RSD is 2.8% (n=11). The method has been successfully used to study the pharmacokinetics of levodopa in vivo; the values of the pharmacokinetics parameters C_max, AUC_0–t and T_max were 16.60, 20.92 ng mL^–1, and 90 min, respectively.     

Sample introduction assisted by compressed air in flame furnace AAS: a simple and sensitive method for the determination of traces of toxic elements

The power of detection of flame AAS for the toxic elements Cd, Hg, Pb and Tl can be improved by 1–2 orders of magnitude by using flame furnace AAS. In flame-furnace AAS, liquid samples are introduced directly into a nickel tube located in the flame, in the simplest case through a ceramic thermospray capillary. Transportation of the samples is achieved by using compressed air only. Comparatively low detection limits are achieved by both beam injection flame furnace (BIFF-AAS) and thermospray flame furnace AAS (TS-FF-AAS). For TS-FF-AAS, a pressure of less than 20 kPa (<80 in. water) is required. The TS-FF-AAS technique is very simple, robust and cheap. The detection limits were 0.2–0.4 g L^–1 (Cd), 40–100 g L^–1 (Hg), 5–9 g L^–1 (Pb) and 4–14 g L^–1 (Tl), respectively, depending on the method, flow rate and sample volume used. Pb and Cd were found at concentrations of 0.1–2 and 0.005–0.3 g g^–1, respectively, in samples of various spices.Dedicated to the memory of Wilhelm Fresenius.     

Performance of a new nebulizer system for simultaneous determination of Sb,Sn (hydride generation), V,and Zn by ICP–OES

A new nebulizer system is described that extends the analytical capability of the inductively coupled plasma technique to include the simultaneous determination of two elements Sb and Sn (hydride-forming), with two conventional elements, V and Zn. The main advantage of this system is its simultaneous determination of elements that form volatile hydrides and elements that do not, without any instrumental changes. Optimization of reaction and instrumental conditions was performed to characterize the new system. The performance of the new nebulizer system was evaluated by studying the effect of some transition metals (Ni, Cu, Co, and Fe, 1–1000 mg L^–1) on the Sb, Sn, V, and Zn emission signals (1 mg L^–1). Interferences from transition metal ions were found to be insignificant for determination of the four elements in presence of L-cysteine. Long-term and short-term stability was also evaluated. The precision, expressed as RSD for 15 replicate measurements was 0.7% for Sb, 1.7% for Sn, 2.5% for V, and 2.3% for Zn at 200 g L^–1 of each analyte. The detection limits obtained were 0.52, 1.3, 3.2, and 4.7 g L^–1 for Sb, Sn, V, and Zn, respectively. Spike and recovery experiments were performed on the NIST 1643c trace metals in water standard reference material and results were in agreement with the certified values.     

Determination of Dextromethorphan and Dextrorphan in Human Urine by High Performance Liquid Chromatography for Pharmacogenetic Investigations

The aim of the present study was to develop a sensitive method to measure dextromethorphan and dextrorphan in urine by HPLC to support pharmacogenetic studies in ethnic groups. Linearity was assessed in the range: 0.015–10 g mL^–1 for dextromethorphan and 1-10 g mL^–1 for dextrorphan. Inter and intra-day coefficients of variation were < 10%. Limits of detection and quantitation were 0.003 g mL^–1 and 0.015 g mL^–1 for dextromethorphan and 0.24 g mL^–1 and 1.0 g mL^–1 for dextrorphan, respectively. The method is reliable in helping determine the phenotype of Mexican ethnic groups using model drugs such as dextromethorphan.     

Solid-phase reactor flow-injection on-line oxidizing spectrofluorimetry for determination and dissolution studies of folic acid

A simple, sensitive and specific flow-injection spectrofluorimetric method has been developed for the determination of folic acid in pharmaceuticals. The method is based on use of a lead dioxide solid-phase reactor for on-line oxidation of folic acid into a strongly fluorescent compound with a maximum excitation wavelength of 281 nm and an emission wavelength of 450 nm. Under optimum conditions the fluorescence intensity of oxidation product is proportional to the concentration of folic acid over the range 0.008–2.5 g mL^–1. The detection limit is 0.0001 g mL^–1, the relative standard deviation is 0.85% for 11 replicate determinations of 0.05 g mL^–1 folic acid, and the sample throughput is 20 h^–1. In combination with an on-line filter and dilution, an automated drug-dissolution system was established. The proposed method has been successfully applied to the determination of folic acid in pharmaceutical preparations and dissolution testing.     

Determination of fosfomycin in human urine by capillary gas chromatography: Application to clinical pharmacokinetic studies

Summary A capillary gas chromatographic method for the determination of fosfomycin in human urine is described. After dilution of the sample and derivatization, analysis was on a HP-1 capillary column and a flame ionization detector was used to determine the bistrimethylsilyl derivative of fosfomycin. Response was linear in the range 50–5000 g mL^–1. The detection limit was about 10 g mL^–1. The within and between day coefficients of variation did not exceed 6%. The method was applied to the determination of fosfomycin in urine samples collected during clinical pharmacokinetic studies.     

Determination of Benzimidazole Fungicides by HPLC with Fluorescence Detection After Micellar Extraction

An analytical method was developed to determine the benzimidazole fungicides and their residues (benomyl, carbendazim, thiabendazole and fuberidazole) in real water samples. Analyses were performed by reverse phase (RP) HPLC with direct fluorescence detection with mobile phase methanol:water, 40:60 (v/v) with 0.6% (v/v) ammonia. The extraction of analytes from water samples was performed with the use of micellar systems. Specifically, oligoethylene glycol monoalkyl ether (Genapol X-080) and polyoxyethylene 10 lauryl ether (POLE) were used as extractants. The recoveries of fungicides obtained in spiked water samples ranged from 68% to 94% for Genapol and from 68% to 96% for POLE. The limit of detection (LOD) was lower than 6 g L^–1 for carbendazim, 7 g L^–1 benomyl, 0.15 g L^–1 for thiabendazole and 0.01 g L^–1 for fuberidazole in both surfactants.     

Capacitive chemical sensor for fenvalerate assay based on electropolymerized molecularly imprinted polymer as the sensitive layer

A capacitive chemical sensor for fenvalerate is reported. By using ac impedance measurements the sensor has been based on the decrease in capacitance caused by the analyte used as the template in the formulation of an electropolymerized molecularly imprinted polymer as receptor layer. Improvement of the insulating properties of the sensor was investigated in detail. The capacitive sensor was prepared by a deposition of a self-assembled monolayer of 2-mercaptobenzimidazole (2-MBI) before electropolymerization of 2-MBI and subsequent treatment with n-dodecanethiol to eliminate pinholes and defects in the polymerized 2-MBI film. From the calibration curve concentrations of fenvalerate up to 9 g mL^–1 could be detected with a linear determination range up to 5 g mL^–1 and a detection limit of 0.36 g mL^–1. No significant interference was observed from common pyrethroid insecticides.     

Determination of Endocrine Disruptors in Environmental Water Samples by Stir Bar Sorptive Extraction-Liquid Desorption - Large Volume Injection-Gas Chromatography

A method based on stir bar sorptive extraction with liquid desorption combined with gas chromatography and mass spectrometry detection has been developed to determine a group of endocrine disruptors in water samples. Large volume injection was used with a programmed temperature vaporiser injector in gas chromatography to enhance sensitivity. The parameters affecting stir bar sorptive extraction and large volume injection were optimised. The limits of quantification in the full scan mode were between 0.02 and 0.2 g L^–1 and the limits of detection were between 0.005 and 0.02 g L^–1 for river water samples. The reproducibility between days of the method (n=3) for river water samples spiked at 0.2 g L^–1 was below 15%. The performance of the method was checked with several water samples from the sea, and effluent and influent sewage treatment plants. We found 4-tert-octylphenol, benzylbutyl phthalate and bis(2-ethylhexyl) adipate in all the water samples analysed at levels between 0.02–14.04 g L^–1. Diazinon was found in only one effluent wastewater sample at 0.16 g L^–1.Acknowledgements This work was supported by the Ministerio de Ciencia y Tecnologia (projects AMB1999-0875 and PPQ2001-1805-CO3). We would like to thank Dr P. Sandra for kindly providing the stir bars.     

Rapid analysis of pyrethroids in whole urine by high-performance liquid chromatography using a monolithic column and off-line preconcentration in a restricted access material cartridge

A rapid high-performance liquid chromatography (HPLC) method using a monolithic column with UV detection at 238 nm was developed for the determination of fenpropathrin, betacyfluthrin, deltamethrin, and permethrin (cis and trans isomers) in whole urine. The method is based on the use of a monolithic chromatographic column and a restricted access material (RAM) cartridge for sample preparation. The mobile phase was water/acetonitrile (42:58 v/v), the flow rate was 3 mL min^–1, and chromatographic separation was carried out in 10 min. The separation of cis and trans isomers of permethrin was also possible under the above-mentioned conditions. Detection limits in reconstituted whole urine samples were between 0.9 g L^–1 for betacyfluthrin and 4.4 g L^–1 for fenpropathrin and trans-permethrin. Recoveries for urine samples spiked with different amounts of pyrethroids (between 19 g L^–1 and 75 g L^–1) were in the 70±6 to 90±7% range.     

Determination of biogenic amines in wine after clean-up by solid-phase extraction

Summary A suitable method for the determination of 16 biogenic amines in wine has been developed. The method involves clean-up of wine samples using ion-exchange cartridges and a preconcentration step, under controlled vacuum, before derivatization of the amines by treatment with phthalaldehyde (PA) and reversedphase HPLC with gradient elution and fluorimetric detection. Linearity of response was obtained for all the biogenic amines from 100 g L^–1 to mg L^–1. Limits of detection for the amines were similar for all PA-derivatives (25–50 g L^–1) and the quantitation limits were about 0.1 mg L^–1. After clean-up and preconcentration, the concentration levels increased 10-fold for all amines except putrescine and cadaverine, which gave poor recovery by this method unlike the rest which gave recoveries of almost 90%. The overall process was successfully applied to identify and quantify biogenic amines in several red wines from the Tarragona region.