Chromatographic and mass spectrometric fingerprinting analyses of Angelica sinensis (Oliv.) Diels-derived dietary supplements.

Angelica sinensis (Oliv.) Diels ("Danggui" in Chinese) is one of the most commonly used traditional Chinese medicines. It has been used to invigorate blood circulation for the treatment of anemia, hypertension, chronic bronchitis, asthma, rheumatism, and cardiovascular diseases. There are a number of A. sinensis-derived dietary supplements in the US markets. However, no study have been conducted to investigate the quality of these dietary supplements. In this paper, high-performance liquid chromatographic and flow-injection mass spectrometric fingerprints were both evaluated to assess the consistency of A. sinensis-derived dietary supplements. Similarity analysis was carried out on the high-performance liquid chromatographic (HPLC) fingerprints. Meanwhile, principal component analysis (PCA) was performed on the data obtained from flow-injection mass spectrometric (FIMS) fingerprints, which can analyze each sample in 2 min, compared with 30 min required for the chromatographic fingerprint. Both methods show significant chemical differences between samples that may be due to differences in growing locations, growing conditions, harvesting times, and/or botanical processing. The loading plots obtained from PCA singled out the discriminatory ions that were responsible for chemical differences of A. sinensis-derived dietary supplements.

High-precision isothermal titration calorimetry with automated peak-shape analysis.

Isothermal titration calorimetry (ITC) is a powerful classical method that enables researchers in many fields to study the thermodynamics of molecular interactions. Primary ITC data comprise the temporal evolution of differential power reporting the heat of reaction during a series of injections of aliquots of a reactant into a sample cell. By integration of each injection peak, an isotherm can be constructed of total changes in enthalpy as a function of changes in solution composition, which is rich in thermodynamic information on the reaction. However, the signals from the injection peaks are superimposed by the stochastically varying time-course of the instrumental baseline power, limiting the precision of ITC isotherms. Here, we describe a method for automated peak assignment based on peak-shape analysis via singular value decomposition in combination with detailed least-squares modeling of local pre- and postinjection baselines. This approach can effectively filter out contributions of short-term noise and adventitious events in the power trace. This method also provides, for the first time, statistical error estimates for the individual isotherm data points. In turn, this results in improved detection limits for high-affinity or low-enthalpy binding reactions and significantly higher precision of the derived thermodynamic parameters.

Dual-signal analysis eliminates requirement for milk sample pretreatment.

Detection of analytes in complex biological samples, such as milk and blood, normally requires sample pretreatment. These pretreatment regimes reduce assay throughput and increase testing costs. Technologies that make it possible to eliminate sample pretreatment are of great industrial interest. Here we report the development of a dual-signal flow injected analysis device which eliminates the need for sample pretreatment. The device employs thermal traducers to measure the signal from an enzyme and a reference column. This makes it possible to independently monitor and correct for non-specifically generated heat, thereby eliminating the need for sample pretreatment. The ability of the dual-signal device to determine urea and lactate in milk samples without any prior treatment was evaluated. The spiked milk samples, the urea assay had a linear range from 0.1 to 50mM (R=0.996), and the lactate assay had a linear range from 0.025 to 5.0mM (R=0.9998). The linear regression values for urea and lactate for 0.5%, 1.5% and 3.0% fat milk were at least 0.990. The dual-signal design improves assay reproducibility, accuracy and sensitivity. Addition benefits are shorter assay times and lowers costs, as well as reducing equipment and training requirements. The potential application of the technology for multi-analyte analysis in point of care and decentralized diagnostic testing in healthcare, agriculture and environmental areas is discussed.

Flow injection analysis biosensor for urea analysis in adulterated milk using enzyme thermistor.

Urea in adulterated milk is one of the major health concern, it is especially harmful to pregnant women, children, and the sick. A sophisticated and reliable detection system is needed to replace current diagnostic tools for the urea in the milk. In this work, we report a flow injection analysis-enzyme thermistor (FIA-ET) bio-sensing system for monitoring of urea in adulterated milk. This biosensor was made of the covalently immobilized enzyme urease (Jack bean) on controlled pore glass (CPG) and packed into a column inside thermistor, which selectively hydrolysed the urea present in the sample. The specific heat registered from the hydrolysis of urea was found proportional to the concentration of urea present in the milk sample. The biosensor showed a linear range 1-200 mM, with % R.S.D. 0.96 for urea in 100 mM phosphate buffer, pH 7.2. Good recoveries were obtained (97.56-108.7%) for urea up to 200 mM in the spiked milk samples with % R.S.D. 0.95. In the adulterated milk, a simple filtration strategy and matrix matching technique was used to analyse urea. The response time of the sensor was evaluated for urea, which was 2 min, and it gives satisfactory output. A good comparison was observed between the urea concentrations measured through FIA-ET and the colorimetric method. These results indicate that utilizing this system could be very effective to detect low and high level of urea in adulterated milk. The immobilized urease column exhibited a good operational stability up to 180 days when used continuously at room temperature.

Trace-level detection of atrazine using immuno-chemiluminescence: dipstick and automated flow injection analyses formats.

A sensitive chemiluminescence (CL)-based immunoassay technique based on both dipstick and flow injection analytical formats is reported for the detection of atrazine. In the dipstick-based immunoassay technique, antibody (anti-atrazine) was first immobilized on the nitrocellulose membranes. The dipstick was then treated with atrazine and atrazine-horseradish peroxidase conjugate (atra-HRP) to facilitate the competitive binding. The dipstick was further treated with urea-hydrogen peroxide (U-H202) and luminol to generate photons. The number of photons generated was inversely proportional to the atrazine concentration. In the flow injection analysis (FIA) format, the antibody was immobilized on protein-A sepharose matrix and packed in a glass capillary column, which functioned as an immunoreactor. Competitive binding of antigen and antibody occurred. The CL signals generated during the biochemical reactions were correlated with atrazine concentrations in the analytical samples. By using dipstick technique, it was possible to detect atrazine concentration down to 0.1 ng/mL; with the FIA format, the detection of atrazine was down to 0.01 ng/mL.

Chemiluminescence flow sensor with immobilized reagent for the determination of pyrogallol based on potassium hexacyanoferrate(III) oxidation.

A novel chemiluminescence (CL) flow-through sensor for the determination of pyrogallol has been developed. The method is based on the reaction between pyrogallol and potassium hexacyanoferrate(III) in sodium hydroxide solution. Potassium hexacyanoferrate(III) involved in the CL reaction was electrostatically immobilized on anion-exchange resin packed in a column. Pyrogallol was sensed by the CL reaction between pyrogallol and potassium hexacyanoferrate(III) which was eluted from the ion-exchange column through sodium phosphate injection. The CL emission allows quantitation of pyrogallol concentration in the range 0.01-3.8 microg/mL with a detection limit (3 sigma) of 0.003 microg/mL and a sample throughput of 118 h(-1). The relative standard deviation (n=7) was 2.2% for 0.2 microg/mL of pyrogallol. The influence of foreign compounds was tested.

Resolution of ofloxacin-ciprofloxacin and ofloxacin-norfloxacin binary mixtures by flow-injection chemiluminescence in combination with partial least squares multivariate calibration.

A flow-injection chemiluminescence (CL) method is described for the determination of ciprofloxacin (CIP), norfloxacin (NOR) and ofloxacin (OFL), commonly used antibiotics of the fluoroquinolones family. The method is based on the CL reaction of the fluoroquinolones with tris(2,2'-bipyridyl) ruthenium(II) and Ce (IV), in sulfuric acid medium. The maximum CL emission, given at 0.45 min for CIP, at 0.35 min for NOR and at 0.04 min for OFL, respectively, were measured, allowing the simple application of the proposed method to the routine analysis of the antibiotics. The methods were applied to the determination of CIP, NOR and OFL, in several pharmaceutical preparations, with very satisfactory results, and validated by a previously reported HPLC method. The time-resolved equipment allowed the measurement of the kinetic evolution of the chemiluminescence signals. In base to the differences in the kinetic behaviour of ofloxacin with respect to ciprofloxacin and norfloxacin, binary mixtures of the drugs were resolved by using the time-resolved chemiluminescence signals, in combination with first-order partial least-squares (PLS) multivariate calibration.

A simple flow-injection spectrofluorimetric method for the determination of mercury.

A highly sensitive flow-injection spectrofluorimetric method is presented for the rapid and simple determination of Hg (II) in environmental and pharmaceutical samples. Murexide (ammonium purpurate) was used as the fluorescence reagent in the carrier stream. An emission peak of murexide, which is decreased linearly by addition of Hg (II), occurs at 435 nm in aqueous solution with excitation at 335 nm. A linear calibration was obtained for 5-200 ng ml(-1) Hg (II) with the relative standard deviation 2.5% (n = 5) for a 20 microl injection volume Hg (II). The limit of the detection was 1 ng ml(-1) and the sampling rate was 80 h(-1). No significant interference was found by the ions commonly found in the most environmental samples. The proposed method was successfully applied for the determination of trace mercury in real samples and the validation of the proposed methodology is provided.

Highly selective spectrophotometric flow-injection determination of trace amounts of bromide by catalytic effect on the oxidation of m-cresolsulfonephthalein by periodate.

A new flow-injection method is reported for the determination of bromide. The method is based on catalytic effect of bromide on the oxidation of m-cresolsulfonephthalein by periodate in acidic media. The reaction was followed spectrophotometrically by measuring the decrease in absorbance at 528 nm. The influence of pH, reagent concentration and manifold variables on the sensitivity was studied. Under optimum conditions, a calibration graph was obtained in the range of 0.160-20.00 microg ml(-1) bromides with a limit of detection of 0.150 microg ml(-1) bromide. The relative standard deviation for ten replicate measurement of 1.0 microg ml(-1) bromide was 2.1%. The influence of potential interfering ions on the selectivity was studied. The method successes to measure bromide in the presence of other halide ions. The method was used to measure bromide in river water and tap water.

A flow-through optosensing device with fluorimetric transduction for rapid and sensitive determination of dipyridamole in pharmaceuticals and human plasma.

A flow-through optosensor with fluorimetric transduction has been prepared for the sensitive and selective determination of dipyridamole in aqueous solutions and biological fluids. The method is based on a monochannel flow-injection analysis system using Sephadex QAE A-25 resin, placed into a Hellma 176-QS fluorimetric flow-through cell, as an active sorbing substrate. The native fluorescence of dipyridamole fixed on the solid sorbent is continuously monitored at wavelengths of 305 and 490 nm for excitation and emission, respectively. After obtaining the maximum fluorescence intensity, the eluent solution (KH(2)PO(4)/NaOH buffer solution, c(T)=0.05 mol l(-1), pH 6.0) is allowed to reach the flow cell, the analyte is removed, and the resin support is regenerated. When an NaOH (10(-4) mol l(-1))/NaCl (0.1 mol l(-1)) solution is used as carrier solution, at a flow-rate of 1.56 ml min(-1), the sensor responds linearly in the measuring range of 10-500 microg l(-1) with a detection limit of 0.94 microg l(-1) and a throughput of 22 samples per hour (300 microl of sample volume). The relative standard deviation for ten independent determinations (200 microg l(-1)) is less than 0.82%. The method was satisfactorily applied to the determination of dipyridamole in pharmaceutical preparations and human plasma.

Determination of Henry's law constants of phenols by pervaporation-flow injection analysis.

A novel dynamic nonequilibrium technique for the determination of Henry's law constant (HLC) of phenols based on pervaporation-flow injection (PFI) is described. A linear relationship between HLC and the amount of phenol measured by a detector in the acceptor line of a PFI system was demonstrated. This relationship was constructed using five frequently encountered phenols (phenol, 2,4-dimethylphenol, 2,4-dichlorophenol, 2-chlorophenol, and 2,3-dimethylphenol) and used for the determination of the HLC of three other phenols (2,4,6-trichlorophenol, 2-methylphenol, and 3-methylphenol). The HLC of all eight phenols were also determined by the single equilibrium static technique (SEST). Fairly good agreementwas observed between both techniques regarding the HLC of 2,4,6-trichlorophenol, 2-methylphenol, and 3-methylphenol. On the basis of the results obtained, it was concluded that the PFI technique offers considerable advantages over SEST in terms of precision, speed, labor intensity, and possibilities for automation.

Semiautomatic flow-injection method for determination of volatile acidity in wines.

A flow-injection (FI) method based on analytical pervaporation was assessed for its routine use in the determination of volatile acidity in winery laboratories. The new method was compared with both the official method and the Mathieu method, which is most often used in Spanish wineries, by testing 30 different wines, including young and aged, and sweet and dry wines from Montilla-Moriles appellation d'origine. The robustness of the new method was established, and then all 3 methods were studied in terms of range of linearity and regression of the calibration curve, repeatability, reproducibility, sensitivity, detection and quantitation limits (LOD and LOQ, respectively), and time of analysis. The FI method surpassed the Mathieu method in reproducibility and both the Mathieu and official methods in LOD and LOQ and sensitivity; it also required less personnel involvement and shorter analysis time. The statistical criteria established by the Office International de la Vigne et du Vin were applied to the data and the results obtained indicated that the differences between the analytical parameters of the 3 methods are not significant and can be applied indistinctly. The correlation of the methods was studied by taking them 2 by 2, and the corresponding equations, coefficients, and deviations confirmed the statistical results. Thus, the new method can be used in winery laboratories with clear advantages over its 2 counterparts (the routine and official methods).

Superporous agarose monoliths as mini-reactors in flow injection systems. On-line monitoring of metabolites and intracellular enzymes in microbial cultivation processes.

A new type of agarose material, superporous agarose, was used as a support material in an analytical system designed for monitoring of bioprocesses with respect to metabolites and intracellular enzymes. The superporous agarose was used in the form of miniaturised gel plug columns (15 x 5.0 mM I.D. monolithic gel bed). The gel plugs were designed to have one set of very large pores (about 50 microm in diameter) through which cells, cell debris and other particulate contaminants from the bioreactor could easily pass. The material also had normal diffusion pores (300 A) characteristic of all agarose materials, providing ample surface for covalent attachment of antibodies and enzymes used in the analytical sequence. The superporous agarose gel plug columns were characterised with respect to flow properties and handling of heavy cell loads as well as dispersion of injected samples (a Bodenstein number of about 40 was observed with acetone tracer at a flow rate of 1 ml min(-1)). To evaluate the practical performance of the superporous gel plug columns, two applications were studied: (1) on-line determination of glucose in cultivation broth (gel plug with immobilized glucose oxidase) and (2) immunochemical quantification of intracellular beta-galactosidase in E. coli (gel plug with lysozyme to achieve cell lysis and gel plug with antibodies against beta-galactosidase).

Automated determination of microbial peroxidase activity in fermentation samples using hydrogen peroxide as the substrate and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) as the electron donor in a flow injection system.

An automated flow injection method has been developed for the determination of microbial peroxidase activity. The substrate used was hydrogen peroxide and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate (ABTS) was used as the electron donor. In the presence of hydrogen peroxide, peroxidase catalyses the dehydrogenation of ABTS, resulting in the formation of a resonance-stabilized radical cation of ABTS. The green-blue colour formed, recorded at 418 nm, is taken as a measure of the peroxidase activity. The general technical conditions and the general enzymic kinetics have been optimized. Conditions for activation and stabilization of the enzyme were found, e.g., ammonium sulfate acts as a peroxidase activator. The resulting method has a good precision, sensitivity and speed.

Flow injection spectrofluorimetric method for the determination of magnesium in blood serum.

A flow injection configuration for the spectrofluorimetric determination of magnesium is proposed. The method is based on the formation of a strongly fluorescent complex of magnesium ion with 2-hydroxy-1-naphthaldehyde salicyloylhydrazone in alkaline, aqueous-ethanolic (50% v/v) solutions. The optimal chemical conditions for complex formation in the mixed solvent were studied and the flow injection manifold was optimized and used for the determination of magnesium in 1000-fold diluted serum samples without any other pre-treatment. The measurement throughput was 120 h-1. The detection limit of the method was 2 micrograms l-1 (2 mg l-1 for serum). The concentration range of application is 4.8-120 micrograms l-1 (0.2-5.0 mumol l-1). Within-run relative standard deviations for the method at 4.8, 24 and 120 micrograms l-1 of magnesium were 3.5, 1.2 and 0.7%, respectively. Analytical recoveries range from 96.2 to 102% (mean 98.8%).

Continuous-flow assay with immobilized enzymes for determining of inorganic phosphate in serum.

An automated method for the determination of inorganic phosphate based on flow-injection analysis and on the use of immobilized enzymes is reported. The method features a linear range between 0.1 and 20 mumol/L with a CV < 2.1% and 3.4% for the within-run and between-run studies, respectively, and a sampling throughput of 40 h-1. The sensitivity of the method makes a 1:250 dilution of the serum samples feasible, thus making undetectable the interferences from analytes commonly present in serum. The method shows an excellent correlation with conventional automated analyzers based on the same enzymatic reaction (Hitachi, r = 0.988) but with the catalyst in solution, and with the Kodak Ektachem method (r = 0.974) based on the use of dry reagents and formation of the phosphomolybdo heteropolyacid.

Fully automated flow-injection system for quantifying 1,5-anhydro-D-glucitol in serum.

We have developed a flow-injection system with colorimetric detection to measure 1,5-anhydro-D-glucitol in serum. Serum samples are directly and serially injected into a clean-up column every 3.5 min to remove interferences before the enzymatic reaction. 1,5-Anhydro-D-glucitol, after being passed through the column, is oxidized by immobilized pyranose oxidase (EC 1.1.3.10), and the hydrogen peroxide produced reacts with the chromogen substrate in the presence of immobilized horseradish peroxidase (EC 1.11.1.7) to form Bindshedler's Green. The detection limit was 1.2 mumol/L (1.2 pmol). The correlation between results obtained with the present system (y) and gas chromatography-mass spectrometry (GC-MS) (x) in samples containing < 30 mumol/L 1,5-anhydro-D-glucitol, including many samples from patients with diabetes mellitus, was y = 0.975x-0.111 mumol/L (r = 0.997), which was superior to that obtained between the enzymatic and GC-MS methods. Our system needs only to be set up; it runs without any manual pretreatment, assays 17 samples/h, and shows imprecision (CV) of < 2%.

Determination of zinc in serum, blood, and ultrafiltrate fluid from patients on hemofiltration by graphite furnace/atomic absorption spectroscopy or flow injection analysis/atomic absorption spectroscopy.

Two methods were optimized for the determination of zinc in samples of blood, serum, and ultrafiltrate fluid from patients with chronic renal impairment undergoing hemofiltration. In the first procedure, after acid digestion of the samples, Zn in blood and serum is determined by a system coupled to flow injection analysis and atomic absorption spectroscopy. The method is rapid, automated, simple, needs small amounts of sample, and has acceptable analytical characteristics. The analytical characteristics obtained were as follows: determination range of method, 0.05-2.0 ppm of Zn; precision as coefficient of variation (CV), 5.3%; recovery, 95-105%; and detection limit (DL), 0.02 ppm. The second method is optimized for ultrafiltrate fluid because the sensitivity of the first procedure is not suitable for the levels of Zn (ppb or ng/mL) in these samples. The technique chosen was atomic absorption spectroscopy with electrothermal atomization in a graphite furnace. The analytical characteristics obtained were as follows: determination range of method, 0.3-2.0 ppb Zn; CV, 5.7%; recovery, 93-107%; and DL, 0.12 ppb. The methods were used to determine zinc in samples of blood, serum, and ultrafiltrate fluid from 5 patients with chronic renal impairment undergoing hemofiltration to discover whether there were significant differences in the zinc contents of blood, serum, and ultrafiltrate fluid after the hemofiltration process. An analysis of variance of the experimental data obtained from a randomly selected group of 5 patients showed that zinc concentrations in the ultrafiltrate fluid, venous blood, and venous serum do not vary during hemofiltration (p < 0.05), whereas in arterial blood and serum, the time factor has a significant effect.

A flow-injection biosensor system for the amperometric determination of creatinine: simultaneous compensation of endogenous interferents.

A flow-injection biosensor system was developed for the amperometric determination of creatinine based on coupled reactions of three sequentially aligned enzyme reactors, creatinine deiminase, glutamate dehydrogenase, and glutamate oxidase, using an oxygen electrode as the detector. To overcome the problem of endogenous ammonia and glutamate, the flow was split into two channels after the injector and rejoined before the glutamate dehydrogenase reactor. Double peak recording was obtained by setting a delay coil and a reference column in one of the two channels. The first peak gave the sum response of creatinine, endogenous ammonia, and glutamate, and the second that of endogenous ammonia and glutamate. By this method compensation for endogenous ammonia and glutamate, as well as for interfering ascorbic acid, was achieved simultaneously. The system gave linear calibrations up to 2 mM for the first peak and 3 mM for the second one. The lower detection limits were 0.1 and 0.02 mM for 35- and 100-microliters injection of sample, respectively. One run was completed within 2 min. The system showed good reproducibility (< 3%) and long operational stability (> 1300 runs). The assay results of creatinine in urine showed good correlation with those obtained from the chemical method of Jaffe.