Reagentless chemiluminescence flow sensor for the determination of riboflavin in pharmaceutical preparations and human urine

A novel continuous-flow sensor based on chemiluminescence (CL) detection was developed for the determination of riboflavin at pg ml(-1) levels by the immobilization of the reagents. It was found that the CL intensity from the oxidation between luminol and periodate could be enhanced in the presence of riboflavin. The increase of CL emission was correlated with the riboflavin concentration in the range from 0.04 to 200 ng ml(-1), and the detection limit was 0.02 ng ml(-1) (3s). Considering the effective reaction ions, luminol and IO4- was immobilized on anion-exchange resin. The system could produce an evident CL signal by water as eluant and it was also shown that the flow sensor could greatly improve the selectivity and sensitivity for determination of riboflavin with a high signal-to-noise ratio. A complete analysis, including sampling and washing, could be performed in 0.5 min with a relative standard deviation of less than 3.0%. The flow sensor was applied successfully to the determination of riboflavin in pharmaceutical preparations and human urine samples.     

流动注射-抑制化学发光法测定岩白菜素

本文基于岩白菜素对鲁米诺-牛血清白蛋白体系化学发光信号显著的抑制作用,建立了快速灵敏测定岩白菜素的流动注射-化学发光分析新方法。实验发现,化学发光强度的降低值与岩白菜素质量浓度对数值呈良好的线性关系,方法的线性范围为3.0~5.0×10~5 pg/mL,检出限(3σ)为1.0pg/mL。当溶液流速为2.0mL/min时,完成一次分析过程仅需30s,采样频率120/h。本法用于片剂、人血清和尿液中岩白菜素的含量测定,回收率为98.1%~102.7%,相对标准偏差小于2.0%(n=7)。同时对化学发光反应机理进行了探讨。     

A new analytical procedure for assay of lysozyme in human tear and saliva with immobilized reagents in flow injection chemiluminescence system.

A novel analytical procedure based on chemiluminescence (CL) detection was described for the determination of lysozyme at ng ml(-1) level by using controlled-reagent-release technology in a flow injection system. The analytical reagents involved in the CL reaction, including luminol and periodate, were both immobilized on the anion-exchange resins in the flow injection system. Through water injection, luminol and periodate were eluted from the anion-exchange column to generate the chemiluminescence, which was inhibited in the presence of lysozyme. By measuring the decrease of CL intensity, one could analyze the lysozyme quantitatively. The decrement of CL emission was linear over the logarithm of lysozyme concentration in the range of 30-1000 ng ml(-1). A typical analytical procedure, including sampling and washing, could be performed in 0.5 min at a flow rate of 2.0 ml min(-1), giving a throughput of 120 h(-1), with a relative standard deviation of less than 3.0%. The proposed method was applied successfully to the determination of lysozyme in human tear and saliva samples, and the recovery was from 92.0% to 105.7%.     

Sensitive determination of sub-nanogram amounts of rutin by its inhibition on chemiluminescence with immobilized reagents

An interesting inhibitory effect of rutin on the chemiluminescence (CL) reaction between luminol and periodate was reported, and this effect was used for the determination of rutin in medicine and human urine. The CL reagents, luminol and periodate, were both immobilized on an anion-exchange column. The CL signal produced by the reaction between luminol and periodate, which were eluted from the column through water injection, was decreased in the presence of rutin. Rutin was sensed by measuring the decrement of CL intensity, and which was observed to be linear over the logarithm of 0.1-30 ngml(-1) rutin concentration range, and the limit of detection was 0.03 ngml(-1) (3sigma). At a flow rate of 2.0 mlmin(-1), both sampling and washing could be performed in 0.5 min with a relative standard deviation of less than 3.0%. The method proposed offered reagent-less procedures and remarkable stability in the determination of rutin, and could be easily reused over 80 h. The method proposed was applied successfully in the determination of rutin in pharmaceutical preparations and monitoring the excretion of rutin in human urine.     

In Vitro Determination of Dobesilate in Medicine and Human Urine with Chemiluminescence Detection

A unique flow injection chemiluminescence (CL) method for the determination of calcium dobesilate in pharmaceutical preparations and human urine is presented in this paper. The analytical reagents involved in the CL reaction, luminol and ferricyanide, were both immobilized on an anion-exchange column in an FI system. The CL signal produced by the reaction of luminol with ferricyanide (the reagents had been eluted from the column through sodium phosphate injection) decreased in the presence of dobesilate. The decreased CL intensity was linear to the dobesilate concentration in the range 0.2100.0ngmL^–1. At a flow rate of 2.0mLmin^–1, one analytical cycle can be completed in 1.5min, including sampling and washing, resulting in a throughput of 40 cycles per hour. The proposed method was applied successfully to the determination of dobesilate in pharmaceutical preparations and human urine without any pre-treatment. It was found that, after oral administration, the dobesilate concentration reached its maximum after three hours, and the dobesilate metabolism ratio in 24 hours was 57.1% in the bodies of volunteers.Received September 14, 2002; accepted March 11, 2003 Published online July 16, 2003     

The study of a chemiluminescence immunoassay using the peroxyoxalate chemiluminescent reaction and its application

The paper presented a novel chemiluminescence (CL) immunoassay method, which combines the advantages of traditional enzyme-linked immunosorbent assays (ELISA) and bis (2,4,6-trichlorophenyl) oxalate (TCPO)-hydrogen peroxide CL detection system. A fluorescent product 2,3-diaminophenazine (DAPN) was produced by reaction between o-phenylenediamine (OPDA, 1,2-diaminobenzene) and H₂O₂ catalyzed by horseradish peroxidase (HRP). DAPN was excited by the reactive intermediate of TCPO-H₂O₂ chemiluminescent reaction, and led to CL. The dependence of the CL intensity on the concentrations of antigen was studied. As analytical application, the proposed method was used for determination of recombinant human interleukin 6 (rHu IL-6) and β-human chorionic gonadotropin (β-HCG). Under the selected experimental conditions, a linear relationship was obtained between the CL intensity and the concentration of rHu IL-6 in the range of 4.0-625.0 pg/ml, and β-HCG in the range of 12.5-400.0 mIU/ml. The detection limits were 0.5 pg/ml for rHu IL-6 and 3 mIU/ml for β-HCG with relative standard deviation of 2.3 for 78.0 pg/ml rHu IL-6, and 3.9 for 50.0 mIU/ml β-HCG. This method has been applied to the determination of rHu IL-6 in human serum and β-HCG in urine with satisfactory results.     

Chemiluminescence flow sensor for folic acid with immobilized reagents.

A novel chemiluminescence (CL) sensor for folic acid combined flow-injection (FI) technology was presented in this paper. The analytical reagents involved in the CL reaction, including luminol and hexacyanoferrate(III), were both immobilized on an anion-exchange column in FI system. The CL signal produced by the reaction between luminol and hexacyanoferrate(III), which were eluted from the column through sodium phosphate injection, was decreased in the presence of folic acid. The CL emission was correlated with the folic acid concentration in the range from 0.01 to 15 microg ml(-1), and the detection limit was 3.5 ng ml(-1) folic acid (3sigma). At a flow rate of 2.0 ml min(-1), including sampling and washing, could be performed in 2 min with a relative standard deviation of < 2.5%. The flow sensor could be reused more than 300 times and has been applied to the analysis of folic acid in pharmaceutical preparations. and the recovery was from 97.4% to 100.4%.     

Determination of rotenone in river water utilizing packed capillary column switching liquid chromatography with UV and time-of-flight mass spectrometric detection

Fast and sensitive packed capillary column switching liquid chromatography methodology has been developed for the determination of the pesticide rotenone in river water. Sample volumes of up to 1 ml are loaded onto a 23 x 0.25 mm, 5 microm Kromasil C18 packed capillary precolumn using a noneluting solvent composition of water-acetonitrile (99:1, v/v) at flow-rates up to 100 microl/min prior to solute backflushing onto a 200 x 0.32 mm, 3.5 microm Kromasil C18 packed capillary analytical column using a mobile phase of water-acetonitrile (30:70, v/v) at a flow-rate of 5 microl/min. The method was evaluated using river water samples spiked with rotenone in the concentration range 0.5-50 ng/ml using UV detection. The within-assay precision was between 5.0 and 7.7% relative standard deviation (RSD, n = 6) and the between assay precision was between 7.5 and 8.9% RSD (n = 6). The method was linear within the investigated mass range displaying a calibration curve correlation factor of 0.997. The mass limit of detection was 10 pg corresponding to a concentration limit of detection of 10 pg/ml, using time-of-flight mass spectrometry.     

Selective clean-up method using an immunoaffinity column following radioimmunoassay of prostaglandin F2 alpha in biological fluids.

A selective clean-up method using an immunoaffinity column followed by radioimmunoassay (RIA) was developed for determining prostaglandin F2 alpha (PGF2 alpha) in human urine and plasma. Polyclonal antibody raised against PGF2 alpha, obtained from rabbits, was coupled to a tresyl-activated support based on a synthetic hydrophilic resin, TSKgel Tresyl-Toyopearl 650M, and used as the stationary phase for the immunoaffinity column. A human urine or plasma sample was introduced to this column, and PGF2 alpha was eluted with methanol-water (50:50, v/v) after the column had been washed. The eluate was subjected to competitive RIA for PGF2 alpha. The cross-reactivities of the RIA to a number of endogenous prostanoids, except PGD2, were negligible and the sensitivity was 4 pg/tube (p less than 0.05), giving a detection limit of 40 pg/ml when 1 ml of plasma or urine was available. The recoveries of plasma and urine samples were 98-108% and 96-106%, respectively, and their assay variances were 7-23%. The concentrations of endogenous PGF2 alpha in plasma and urine used here were estimated to be 72 and 98 pg/ml, respectively. This method should be very useful for various biological samples because of its good specificity, sensitivity, reliability and reproducibility.     

Quantitative Monitoring of Rutin in Human Urine by Flow Injection‐Chemiluminescence Analysis

In this paper, a rapid and sensitive flow injection‐chemiluminescence (FI‐CL ) method is proposed for the quantitative determination of rutin based on the inhibitory effect of rutin on the chemiluminescence intensity from the luminol–chymotrypsin (CT ) system. The decrease of CL intensity was found to be proportional to the logarithm of rutin concentration in the range 0.1–30.0 ng/mL . A method for the quantification of rutin is proposed, with the limit of detection (LOD ) of 0.03 ng/mL (3σ). A complete analytical process including sampling and washing for rutin determination, which was conducted at a flow rate of 2.0 mL /min, could be performed completely within 30 s, yielding a sample efficiency of 120 h⁻¹. The proposed procedure was successfully applied for the determination of rutin in human urine after oral intake, with recoveries varying from 93.9 to 108.1% and relative standard derivation <4.0% (n = 5). Results showed that urine reached the maximum concentration at ~2.5 h, and the total excretion ratios were (83.5 ± 0.6) and (86.8 ± 0.7)%, respectively, for two volunteers in 8 h. The pharmacokinetic parameters, including the half‐life (1.05 ± 0.02 h), absorption rate constant (1.18 ± 0.01 h⁻¹), and elimination rate constant (0.70 ± 0.01 h⁻¹), were obtained. The possible CL mechanism of the luminol–CT –rutin reaction is discussed by FI‐CL , fluorescence, and molecular docking methods.     

Determination of benzo[a]pyrene tetrols by column-switching capillary liquid chromatography with fluorescence and micro-electrospray ionization mass spectrometric detection

The present work displays capillary liquid chromatographic column switching methodology tailored for determination of benzo[a]pyrene tetrol isomers in biological matrices using on-line fluorescence and micro-electrospray ionization mass spectrometric detection. A well-established off-line crude solid phase extraction procedure was used in order to make the method compatible with several biological matrices. The solid phase extraction eluates were evaporated to dryness, redissolved in 1.0 ml methanol:water (10:90, v/v), loaded onto a 0.32 mm I.D. x 40 mm 5 microm Kromasil C(18) pre-column for analyte enrichment and back-flushed elution onto a 0.30 mm I.D. x 150 mm 3.5 microm Kromasil C(18) analytical column. The samples were loaded with a flow rate of 50 microl min(-1) and the tetrols were separated at a flow rate of 4 microl min(-1) with an acetonitrile:10 mM ammonium acetate gradient from 10 to 90%. A sample loading flow rate up to 50 microl min(-1) was allowed. The fluorescence excitation and emission were set to 342 and 385 nm, respectively, while mass spectrometric detection of the benzo[a]pyrene tetrols was obtained by monitoring their [M - H](-) molecular ions at m/z 319. The method was validated over the concentration range 0.1-50 ng ml(-1) benzo[a]pyrene tetrols in a cell culture medium with 100 microl injection volume, fluorescence detection and the first eluting tetrol isomer as model compound, resulting in a correlation coefficient of 0.993. The within-assay (n= 6) and between-assay (n= 6) precisions were determined to 2.6-8.6% and 3.8-9.6%, respectively, and the recoveries were determined to 97.9-102.4% within the investigated concentration range. The mass limit of detection (by fluorescence) was 3 pg for all the tetrol isomers, corresponding to a concentration limit of detection of 30 pg ml(-1) cell culture medium. The corresponding mass spectrometric mass limits of detection were 4-10 pg, corresponding to concentration limits of detection of 40-100 pg ml(-1) cell culture medium.     

Chemiluminescence assay for uric acid in human serum and urine using flow-injection with immobilized reagents technology

A novel chemiluminescence (CL) flow sensor for the determination of uric acid in human urine and serum has been developed by using controlled-reagent-release technology. The reagents involved in the chemiluminescence (CL) reaction, luminol and periodate, are immobilized on anion-exchange resin packed in a column. After injection of water, chemiluminescence generated by released luminol and periodate in alkaline media is inhibited in presence of uric acid. By measuring the decreased chemiluminescence (CL) intensity the uric acid is sensed. The decreased response is linear in the 5.0-500.0 ng mL(-1) range, with a detection limit of 1.8 ng mL(-1). The flow sensor showed remarkable operational stability and could be easily reused for over 80 h with sampling frequency of 100 h(-1). The proposed sensor was applied to the determination of uric acid in human urine and serum, and monitoring metabolic uric acid in human urine with RSD less than 3.0%.     

Ion chromatographic preconcentration of Cu and Cd from ultra-high-purity water and determination by electrothermal atomic absorption spectrometry

A method based on preconcentration of Cu and Cd from ultra-high-purity water by ion chromatography (IC) and determination by electrothermal atomic absorption spectrometry is described. A small low-capacity ion-exchange concentrator Dionex HPIC-CG5 and mobile phase of 3 mM pyridine-2,6-dicarboxylic acid (PDCA) are used. Water samples are loaded onto the preconcentration column at a flow-rate ranging from 1 to 3.5 ml min(-1). Large sample volumes (up to 200 ml) can be loaded onto the concentrator without losing metal ions. Elution is carried out in the reverse direction of sample loading and the volumes of effluent are as small as 0.150 and 0.200 ml for copper and cadmium, respectively. Under these conditions the preconcentrated ions coelute. The detection limits, based on the Hubaux-Vos method, for Cu using a 1300-fold preconcentration in the IC step was found to be 1 pg ml(-1), and was limited due to impurity in PDCA, while the detection limit found for Cd using a 1000-fold preconcentration was 0.02 pg ml(-1). Ultra-high-purity water produced by a Millipore system is successfully analysed by the proposed method and the content of Cu and Cd are found to lie in the range 1-10 pg ml(-1).     

Flow-injection chemiluminescence determination of quinine using on-line electrogenerated cobalt(III) as oxidant

A novel chemiluminescence (CL) flow system for the determination of quinine is described. It is based on the direct chemiluminescence reaction of quinine and cobalt(III) in sulfuric acid medium. The unstable Co(III) was on-line electrogenerated by constant-current electrolysis. The chemiluminescence intensity was linear with a quinine concentration in the range of 0.1-100 mug ml(-1). The determination limit was 3.3x10(-8) g ml(-1). The whole process could be completed in 1 min. The proposed method is suitable for automatic and continuous analysis, and has been applied successfully to the analysis of quinine in pharmaceutical preparation.     

Flow injection chemiluminescence determination of thiamine based on its enhancing effect on the luminol-hydrogen peroxide system

A new flow injection chemiluminescence (CL) method is proposed for the determination of thiamine, based upon its enhancing effect on the CL reaction of luminol with hydrogen peroxide in alkaline solution. The method allows the determination of thiamine within 0.05-8 mug ml(-1) range with a detection limit (3sigma) of 0.01 mug ml(-1). The relative standard deviation is 1.4% (n=11, 0.5 mug ml(-1) thiamine) and the sample throughput is about 90 samples h(-1). The method was successfully applied to the determination of thiamine in pharmaceutical preparations.     

Rapid determination of subnanogram urapidil using flow injection enhancement chemiluminescence

A sensitive flow-injection (FI) chemiluminescence (CL) for the determination of urapidil is described in this paper. It is based on the enhancement effect of urapidil on the CL reaction between luminol and hydrogen peroxide. The increment of CL intensity is proportional to the concentration of urapidil in the range 0.1−10 ng/mL (R ²=0.9986), with a detection limit (3σ) of 0.03 ng/mL. The whole process, at a flow rate of 2.0 mL/min, including sampling and washing, could be completed in 0.5 min, and the relative standard deviation (RSD) at the concentration of 0.1, 1.0, and 10.0 ng/mL was less than 3.0% (n = 5). The proposed method has been successfully applied for the determination of urapidil in pharmaceutical preparation, human urine, and serum. The text was submitted by the authors in English.     

Development and validation of a liquid chromatographic/tandem mass spectrometric method for determination of tetracycline in human plasma: application to bioequivalence study

A fast, sensitive, and specific liquid chromatographic/tandem mass spectrometric method was developed and validated for determination of tetracycline in human plasma. Tetracycline and oxytetracycline [internal standard (IS)] were extracted from the plasma by protein precipitation. The mobile phase consisted of acetonitrile-formic acid 0.1% (48 + 52, v/v), run at a flow rate of 1 ml/min (split 1:5). Detection was performed by positive electrospray ionization in multiple reaction monitoring mode, monitoring the transitions 444.8 > 410.0 and 461.0 > 426.0 for tetracycline and IS, respectively. The analysis was performed in 3.5 min and the method was linear in the plasma concentration range of 50-6000 ng/mL. The mean extraction recoveries for tetracycline and IS from plasma were 92.14 and 94.04%, respectively. Method validation investigated parameters such as the linearity, precision, accuracy, specificity, and stability, giving results within the acceptable range. The proposed method was successfully applied for determination of tetracycline in human plasma samples to support bioequivalence studies.     

A novel capillary microextraction on ordered mesoporous titania coating combined with electrothermal vaporization inductively coupled plasma mass spectrometry for the determination of V, Cr and Cu in environmental and biological samples

In this work, an ordered mesoporous titania film was introduced to coat a capillary by means of sol-gel technique. Sol-gel titania coating was developed for the preconcentration/separation of trace V, Cr and Cu by capillary microextraction (CME), and the adsorbed analytes were eluted for electrothermal vaporization inductively coupled plasma mass spectrometry (ETV-ICP-MS) detection. By immobilizing sol-gel titania on the inner surface of a fused-silica microextraction capillary, the sol-gel titania coating was prepared easily. Its adsorption properties, stability and the factors affecting the adsorption behaviors of V, Cr and Cu were investigated in detail. At pH range of 7 to 9, the titania-coated capillary (50 cm x 0.25 mm) is selective towards V, Cr and Cu, and the target analytes could be desorbed quantitatively with 50 microl of 1.0 mol l(-1) HNO3 at the rate of 0.05 ml min(-1). With a consumption of 2 ml sample solution, an enrichment factor of 33.3, and a detection limit (3 s) of 1.1 pg ml(-1) (10.5 fg) for V; 3.3 pg ml(-1) (33.0 fg) for Cr and 6.3 pg ml(-1) (63.1 fg) for Cu respectively were obtained. The precisions Relative Standard Deviations (RSDs) for nine replicate measurements of 1 ng ml(-1) V, Cr and Cu were 3.4, 5.1 and 6.4%, respectively. The proposed method has been applied to the determination of V, Cr and Cu in human urine and lake water, and the recoveries for these elements were 89.2 approximately 105%. The developed method was also applied to the determination of the target elements in NIES No. 10-a (rice flour-unpolished) and NIES No. 9 (sargasso) certified reference materials, and the results found are in good agreement with the certified values.     

Chemiluminescence of Luminol–Graphene Oxide for the Sensitive Detection of Puerarin in Biological Fluid and Chinese Gegen

Graphene oxide (GO ), one of the water‐soluble derivatives of graphene, could initiate strong chemiluminescence (CL ) emission of luminol in the absence of any oxidant, and then the CL intensity was inhibited by puerarin (PUE ), a main component in the traditional Chinese medicine Gegen. Based on this, a novel CL method was established for the determination of PUE . This method showed a linear relationship between the CL signal and the logarithm of PUE concentration in the range 0.01–6.00 μM . The limit of detection was 2.83 nM and the relative standard deviation (RSD ) was 1.94% for 11 determinations of 0.4 μM PUE . This method was successfully used for the determination of PUE in Gegen and human urine samples. The possible CL reaction mechanism was investigated by UV –vis, fluorescence, and CL spectra, as well as by some chemical experiments.     

流动注射-电化学发光法测定煤灰中痕量钼(Ⅵ)

基于钼(Ⅵ)在-0.60V(vs.Ag/AgCl)电位下在线还原为钼(Ⅲ),且在碱性条件下钼(Ⅲ)与鲁米诺发生化学发光反应,据此提出了流动注射-电化学发光法测定煤灰中痕量钼(Ⅵ)的方法。钼(Ⅵ)的质量浓度与化学发光强度的增加值在5.0×10-7～5.0×10-4g.L-1范围内呈线性关系,检出限(3s/k)为5×10-8g.L-1。对1.0×10-6g.L-1钼(Ⅵ)标准溶液进行11次测定,测定值的相对标准偏差为2.6%。方法可用于煤灰中痕量钼(Ⅵ)的测定,测定值与国标方法测定值相符。