Development of a quantitative method for the analysis of total L-ascorbic acid in foods by high-performance liquid chromatography

A new method has been developed for the determination of total vitamin C in foods. The method requires less time than the traditional methodologies and uses a radical oxidation of L-ascorbic acid (AA) to obtain dehydro-L-ascorbic acid (DHAA) by means of a peroxyl radical generated in situ by thermal decomposition of an azo-compound, 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH). The dehydro-L-ascorbic acid is condensed with benzene-1,2-diamine (o-phenylenediamine, OPDA) to form its highly fluorescent quinoxaline derivative, 3-(1,2-dihydroxyethyl)furo[3,4-b]quinoxaline-1-one (DFQ), which is then separated on a C(18) column eluted with a mobile phase of 80 mM phosphate buffer and methanol at pH=7.8 and detected fluorometrically at lambda(ex)=355 nm and lambda(em)=425 nm. The reaction conditions for the complete conversion of AA to DFQ were 56 degrees C, 36 min and a mumol AAPH/AA ratio of 60. The sample, extracted with an aqueous metaphosphoric acid solution, was analyzed after being filtered through a 0.45 microm membrane. The method has shown good repeatability, sensitivity and accuracy compared to the results obtained with the reference method. The response of the detection system was linear within a range of 0.5-8.0 microg/mL with a correlation coefficient of 0.9997. The limit of detection was 0.27 microg/mL and the limit of quantification was 0.83 microg/mL. The AA contents of some selected foods were analyzed.     

Determination of proteins with tetracarboxy manganese(II) phthalocyanine by resonance light scattering technique

A novel method for the determination of proteins by using tetracarboxy manganese(II) phthalocyanine (MnC4Pc) as a resonance light scattering (RLS) probe has been developed. At pH 3.0 Britton-Robinson (B-R) buffer solution, the RLS intensity of MnC4Pc at 385 nm is greatly enhanced in the presence of proteins. The effects of pH, reaction time, concentration of MnC4Pc and interfering substances on the enhanced RLS intensity are investigated, respectively. Under optimal conditions, the linear ranges of the calibration curves are 0-2.00 microg mL(-1) for bovine serum albumin (BSA) and human serum albumin (HSA), 0.0-1.75 microg mL(-1) for human-IgG and ovalbumin, with a detection limit of 16.37 ng mL(-1) BSA, 17.62 ng mL(-1) HSA, 19.41 ng mL(-1) human-IgG and 20.72 ng mL(-1) ovalbumin. The method has been applied to the determination of total proteins in human serum samples collected from a hospital and the results are in good agreement with those reported by the hospital.     

Fluorescence enhancement method for the determination of nucleic acids using cationic cyanine as a fluorescence probe

A fluorescence enhancement method with a cationic cyanine as a probe was developed for the determination of nucleic acids. Under the experimental conditions, the fluorescence enhancement of cyanine (lambda(ex)/lambda(em)= 524/591.5 nm) was observed in the presence of DNA. The calibration graphs were linear over the range of 0.01-15 microg mL(-1) for both calf thymus DNA (CT DNA) and fish sperm DNA (FS DNA). The limits of detection were 0.005 and 0.007 microg mL(-1) for CT DNA and FS DNA, respectively. The method was applied to the determination of DNA in synthetic and real samples and satisfactory results were obtained. A possible fluorescence enhancement mechanism was also studied.     

A flow injection sampling resonance light scattering system for total protein determination in human serum

A novel flow injection method with resonance light scattering detection was developed for the determination of total protein concentrations. This method is based on the enhancement of RLS signals from Methyl Blue (MB) by protein. The enhanced RLS intensities at 333 nm, in a pH 4.1 acidic aqueous solution, were proportional to the protein concentration over the range 2.0-37.3 and 1.0-36.0 microg ml-1 for human serum albumin (HSA) and bovine serum albumin (BSA), respectively. The corresponding limits of detection (3sigma) of 45 ng ml-1 for HSA and 80 ng ml-1 for BSA were attained. The method was successfully applied to the quantification of total proteins in human serum samples, the maximum relative error is less than 1% and the recovery is between 98% and 102%. The sample throughput was 60 h-1.     

A highly sensitive and rapid assay for protein determination in human urine and penicillin using a Rayleigh light-scattering technique with benzeneazo-8-acetylamino-1-naphthol-3,6-disulfonic acid sodium salt.

A simple and sensitive method was conducted for the determination of trace amounts of proteins with benzeneazo-8-acetylamino-1-naphthol-3,6-disulfonic acid sodium salt (azophloxine, AP) using a Rayleigh light-scattering (RLS) technique. At pH 2.60 and in the presence of an emulsifier OP microemulsion, the RLS of AP can be greatly enhanced by proteins, owing to the interaction between AP and protein. The enhanced intensity is proportional to the concentration of proteins. Four proteins, including bovine serum albumin (BSA), human serum albumin (HSA), lysozyme (Lys) and gamma globulin (gamma-G) have been tested. For example, the linear range of BSA was 0 - 0.06 microg mL(-1) with detection limits of 2.38 ng mL(-1). The method was applied to the analysis of protein in human urine and penicillin samples with satisfactory results. The relative standard deviation was in all instances less than 4.0%, and the recovery was in the range of 97.5 - 104%.     

Microdetermination of proteins with the arsenazo-DBN-Al(III) complex by Rayleigh light-scattering technique and application of the method

The determination of proteins with arsenazo-DBN and Al3+ by Rayleigh light-scattering (RLS) is described. The weak RLS of arsenazo-DBN and BSA can be enhanced greatly by addition of Al3+ in the pH range 5.3-7.0; this resulted in two enhanced RLS signals at 420-440 nm and 460-480 nm. The reaction between arsenazo-DBN, Al3+, and proteins was studied and a new method was developed for quantitative determination of proteins. This method is very sensitive (0.34-41.71 microg mL(-1) for bovine serum albumin, BSA, and 0.29-53.41 microg mL(-1) for human serum albumin, HSA), rapid (< 2 min), simple (one step), and tolerant of most interfering substances. The effects of different surfactants were also examined. When these proteins were determined in four human serum samples the maximum relative error was not more than 2% and the recovery was between 97 and 103%.     

Fast assay of glucosamine in pharmaceuticals and nutraceuticals by capillary zone electrophoresis with contactless conductivity detection

A novel capillary electrophoresis (CE) method with contactless conductivity detection suitable for the determination of glucosamine (GlAm) and K(+) in pharmaceuticals was devised. Under the optimum conditions (aqueous 30 mM acetate buffer of pH 5.2 as the background electrolyte; voltage 30 kV; 25 degrees C), GlAm (migrating as glucosaminium cation) was well separated from K(+) that could occur in the dosage forms as excipient. The CE analysis was performed in fused-silica capillaries (50 microm i.d., 75 cm total length, 27 cm to detector) and the separation took <3 min. The calibration graphs were linear for both GlAm (100-300 microg/mL; r(2)=0.997) and K(+) (15-75 microg/mL; r(2)=0.997) when using ethanolamine (100 microg/mL) as the internal standard. The LOD values (S/N=3) were 9.3 microg/mL for GlAm and 2.9 microg/mL for K(+). The method was applied to the assay of GlAm content in various dosage forms. Intermediate precision evaluated by determining the content of GlAm in a single formulation on 3 consecutive days was characterized by RSD 2.35% (n=15). Acceptable accuracy of the CE method was confirmed by the added/found GlAm recovery experiments (recoveries 94.6-103.3%) and by statistical comparison of the results attained by the proposed CE and a reference HPLC method.     

Resonance light scattering spectroscopy of beta-cyclodextrin-sodium dodecylsulfate-protein ternary system and its analytical applications.

A simple, highly sensitive and dye-less assay for proteins was reported using a resonance light-scattering (RLS) technique based on the enhanced RLS intensity of beta-cyclodextrin (beta-CD)-sodium dodecylsulfate (SDS)-protein system. Under the optimum conditions, the enhanced RLS intensity is in proportion to the concentration of proteins in the range of 0.01 to 2.3 microg ml(-1) for bovine serum albumin (BSA), 0.01 to 2.0 microg ml(-1) for human serum albumin (HSA), 0.015 to 5.0 microg ml(-1) for gamma-globulin (gamma-G), 0.02 to 3.5 microg ml(-1) for egg albumin (EA), 0.02 to 4.0 microg ml(-1) for pepsin (Pep), and 0.02 to 3.6 microg ml(-1) for alpha-chymotrypsin (Chy). Their detection limits (S/N = 3) are 1.1, 1.6, 2.4, 6.7, 5.4 and 4.2 ng ml(-1), respectively. Synthetic samples and human serum samples were determined satisfactorily, and the results were in reasonable agreement with those obtained by a documented spectrophotometric (Bradford) method.     

Synthesis of a novel fluorescent probe and investigation on its interaction with nucleic acid and analytical application

A novel fluorescent probe N-(N-(2-(4-morpholinyl)ethyl)-4-acridinecarboxamide)-alpha-alanine (N-(N-(ME)-4-ACA)-alpha-ALA) was synthesized. The structure was characterized by 1H NMR, MS, elemental analysis, fluorescent and ultraviolet spectra. This new compound exhibited high binding affinity to DNA, intense fluorescence and high water solubility. Experiment indicated that the fluorescent intensity was quenched when DNA was added. A method for DNA determination based on the quenching fluorescence (lambda(ex)=258nm, lambda(em)=451nm) of N-(N-(ME)-4-ACA)-alpha-ALA was established. Under optimal conditions (pH 7.2, CN-(N-(ME)-4-ACA)-alpha-ALA)=3 x 10(-6) mol L(-1)), the linear range is 0.1-4.0 microg mL(-1) for both fish semen (fsDNA) and calf thymus DNA (ct-DNA). The corresponding determination limits are 4.6 ng mL(-1) for fsDNA and 5.1 ng mL(-1) for ct-DNA, respectively. The relative standard deviation is 1.0%. Thus this compound can be used as a DNA fluorescent probe. The experiments proved that the interaction mode between N-(N-(ME)-4-ACA)-alpha-ALA and DNA was groove binding. The modified Rosenthal's graphical method gave the binding constant of 1.0 x 10(6) L mol(-1) and a binding size of 0.31 base pairs per bound drug molecule.     

High-performance liquid chromatographic method for determination of reversible isomers of SU5416

SU5416 shows light-induced reversible geometric isomerism. A simple, reliable, isocratic HPLC method using an UV-vis detector at lambda(425nm) was developed. The method provides efficient (R(S)=3.5) analysis of the two isomers with retention of the isomeric integrity. Additionally, the method has linearity over a wide range (50-1000microg/mL, r(2)=0.99), is accurate (99-102%, RSD <4%), and reproducible (RSD <0.8%). The method was used for analyzing pharmaceutical samples and understanding the kinetics of SU5416 isomers in methanol. In addition, this method can be used for quantifying the non-isolatable E-isomer.     

Immunoassay for B. globigii spores as a model for detecting B. anthracis spores in finished water

The 2001 anthrax alarm in the US raised concerns about the Nation's preparedness to the threat of bioterrorism, and the demand for early warning systems that might be used in the case of a biological attack continues to grow. Here we develop an ultra-sensitive rapid detection method for B. globigii(BG) spores, the simulant of B. anthracis(BA) spores. BG spores were detected by a bead-based sandwich immunoassay with fluorescence detection. Paramagnetic Dynal beads were used as a solid support, primary antibody was attached to the beads by streptavidin-biotin coupling and the secondary antibody had an alkaline phosphatase (AP) enzyme label. Enzymatic conversion of fluorescein diphosphate (FDP) to fluorescein by AP was measured in real time with lambda(ex)= 490 nm and lambda(em)= 520 nm. The assay was linear from 2.6 x 10(3)-5.6 x 10(5) BG spores mL(-1), and the detection limit was 2.6 x 10(3) spores mL(-1) or 78 spores. All reagent concentrations and incubation times were optimized. The assay time from the moment the spores were introduced to the system was 30 min, and real-time fluorescence detection was done in less than 1 min. Formation of the BG spores-capture beads complex was confirmed by environmental scanning electron microscopy (ESEM). BG spores were detected successfully when doped into Cincinnati tap water to demonstrate the applicability of the developed method to detect the spores in non-buffered media.     

Study on naringenin-CTMAB-DNA system by resonance light scattering technique and its analytical application

A new high-sensitivity determination method of deoxyribonucleic acid (DNA) with detection limit at nanogram levels was proposed. Based on the measurement of resonance light scattering (RLS), it was found DNA could combine with naringenin and cetyltrimethylammonium bromide (CTMAB) in basic Tris-HCl buffer and produce enhanced RLS signal. The optimum conditions for this system were studied in detail. The enhanced intensity of RLS of naringenin-CTMAB at 353 nm was directly proportional to the concentration of DNA in the range of 0.017-1.7 μg mL(-1). The detection limit was 5.06 ng mL(-1). Using the proposed method, the synthetic samples were analyzed with satisfactory results, the recovery was 99.3-105.0% and RSD was 0.7-3.7%.     

Non-protected fluid room-temperature phosphorimetric procedure for the direct determination of naftopidil in biological fluids

Non-protected fluid room temperature phosphorescence, NPRTP, has been applied to the determination of naftopidil in biological fluids. The proposed method is based on obtaining a phosphorescence signal from naftopidil using potassium iodide as heavy atom perturber and sodium sulfite as a deoxygenating reagent without a protected medium. Optimized conditions for the determination were 1.4 mol L= KI, 5.0 x l0(-3) mol L(-1) sodium sulfite, pH 6.5 (adjusted with sodium hydrogen phosphate-dihydrogen phosphate buffer solution, 5.0 x 10(-2) mol L(-1). The delay time, gate time, and time between flashes were 70 micros, 400 micros, and 5 ms, respectively. The maximum phosphorescence signal appeared instantly and the intensity was measured at lambda(ex)=287 nm and lambda(em)=525 nm. The response obtained was linearly dependent on concentration in the range 50 to 600 ng mL(-1). The detection limit, according to error-propagation theory, was 7.93 ng mL(-1) and the detection limit as proposed by Clayton was 11.12 ng mL(-1). The repeatability was studied by using ten solutions of 400 ng mL(-1) naftopidil; if the theory of error propagation is assumed the relative error is 0.88%. The standard deviation of replicates was found to be 3.5 ng mL(-1). This method was successfully applied to the analysis of naftopidil in human serum and urine with recoveries of 104.0 +/- 0.6% for serum and 106.0 +/- 1.0% for urine.     

Utility of oxidation-reduction reaction for the determination of ranitidine hydrochloride in pure form,in dosage forms and in the presence of its oxidative degradates

Three simple, accurate and sensitive colorimetric methods (A, B and C) for the determination of ranitidine HCl (RHCl) in bulk sample, in dosage forms and in the presence of its oxidative degradates are described. The first method A is based on the oxidation of the drug by N-bromosuccinimide (NBS) and determination of the unreacted NBS by measurement of the decrease in absorbance of amaranth dye (AM) at a suitable lambda(max)=520 nm. The methods B and C involve the addition of excess Ce(4+) and determination of the unreacted oxidant by decrease the red color of chromotrope 2R (C2R) at a suitable lambda(max)=528 nm for method B or decrease the orange pink color of rhodamine 6G (Rh6G) at a suitable lambda(max)=526 nm for method C. Regression analysis of Beer-Lambert plots showed good correlation in the concentration ranges 0.2-3.6, 0.1-2.8 and 0.1-2.6 microg ml(-1) for methods A, B and C, respectively. The apparent molar absorptivity. Sandell sensitivity, detection and quantitation limits were calculated. For more accurate results, Ringbom optimum concentration ranges were 0.3-3.4, 0.2-2.6 and 0.2-2.4 microg ml(-1) for methods A, B and C, respectively. Analyzing pure and dosage forms containing RHCl tested the validity of the proposed methods. The relative standard deviations were 相似文献   

Detection of avian influenza virus based on magnetic silica nanoparticles resonance light scattering system

A highly sensitive method for the detection of avian influenza virus (AIV) antigen by the resonance light scattering (RLS) technique has been developed in this paper. Magnetic silica nanoparticles (MNs) were modified with AIV antibody via covalent binding firstly, the MNs/AIV antibody-AIV antigen immunocomplex was formed after the addition of AIV antigen, which can increase the RLS signal at 545 nm. Under the optimized conditions, the enhanced intensities of RLS at 545 nm (ΔI(RLS)) were proportional to the concentrations of AIV antigen in the range of 0.5-50 ng mL(-1), with a detection limit of 0.15 ng mL(-1) and correlation coefficient of 0.995. This method was applied to the analysis of AIV antigen in spiked chicken serum samples and saliva samples with satisfactory results.     

Validated spectrophotometric and fluorimetric methods for analysis of clozapine in tablets and urine

Five spectrophotometric methods and one fluorimetric method have been developed and validated for the analysis of clozapine. The spectrophotometric methods were based on the charge-transfer complexation reaction between clozapine as electron donor and each of iodine as sigma-acceptor or 7,7,8,8-tetracyanoquinondimethane (TCNQ), 2,3-dichloro-5,6-dicyano-1,4-benzo-quinone (DDQ), tetracyanoethane (TCNE), and p-chloranilic acid (pCA) as pi-acceptors. The obtained complexes were measured spectrophotometrically at 365, 843, 460, 414, and 520 nm for iodine, TCNQ, DDQ, TCNE, and pCA, respectively. The fluorimetric method was based on the oxidation of clozapine in the presence of perchloric acid by cerium (IV), and subsequent measuring the fluorescence of the produced cerium (III) fluorimetrically at lambda(excitation) 260 and lambda(emission) 355 nm. Under the optimum assay conditions, Beer's law was obeyed at concentrations ranged from 4-200 microg mL(-1) for the spectrophotometric methods and from 24-250 ng mL(-1) for the fluorimetric method. The limits of detection for the spectrophotometric methods were 1.12, 1.76, 2.22, 0.95, and 13.26 microg mL(-1) for iodine, TCNQ, DDQ, TCNE, and pCA, respectively. The limit of detection for the fluorimetric method was 6.69 ng mL(-1). The proposed methods were successfully applied to the analysis of clozapine in tablets with good recoveries. The fluorimetric method could also be applied to the analysis of clozapine in spiked urine samples. The molar ratios and the reaction mechanisms were investigated.     

Resonance light scattering of 1-hydroxypyrene-ethyl violet-anionic surfactant system and its analytical application.

A novel method for the rapid and sensitive analysis of 1-hydroxypyrene (1-OHP) in human urine has been developed that uses a resonance light scattering (RLS) technique. The assay was based on the interaction of ethyl violet (EV) with 1-hydroxypyrene to form an ion-associate complex, which resulted in the enhancement of RLS intensity and the appearance of new RLS spectra. In the presence of anionic surfactant, the maximum RLS peak of the system was located at 396 nm at pH 8.0. Under the optimum conditions, it was found that the enhanced RLS intensity was directly proportional to the concentration of 1-hydroxypyrene in the range of 4.0 - 982 microg l(-1). The detection limit was 1.2 microg l(-1) and the recoveries of 1-hydroxypyrene were 92.8 - 102.3% (n = 6). The proposed method was successfully applied to the analysis of human urine samples. The results of 1-hydroxypyrene were in agreement with those obtained by the method of high-performance liquid chromatography.     

A sensitive rutin assay using a simple probe manganese sulfate based on its novel resonance light scattering decrease phenomenon

A novel resonance light scattering (RLS) decrease method was developed to determine rutin with a simple probe manganese sulfate. At pH 7.5, the strong RLS intensity of manganese sulfate was remarkably decreased by the addition of rutin with the maximum peak located at 389.0 nm. Under the optimum conditions, a good linear relationship between the changes of RLS intensities of manganese sulfate with and without rutin and the concentrations of rutin was obtained over the range of 0.49-24.4 microg ml(-1) and a low detection limit (3sigma) 0.42 microg ml(-1) was achieved in the mean time. Based on this approach, a novel method for quantitative analysis of rutin is proposed in this paper. The method proposed was also applied successfully to the determination of rutin in commercial pharmaceutical preparations of compound rutin tablets and human urine samples. The assay is sensitive, rapid, inexpensive, practical and relatively free from interference generated by coexisting substances.     

Spectrofluorimetric kinetic determination of selenium (IV) by flow injection analysis in cationic micellar medium

A sensitive catalytic kinetic spectrofluorimetric method for determining ng ml(-1) of selenium by flow injection analysis has been developed. The method, based on the catalytic effect of Se (IV) on the reduction of resorufin by sulphide, in the presence of cetylpyridinium chloride, is monitored spectrofluorimetrically (lambda(ex)=480 nm; lambda(em)=583 nm). The linearity range of the calibration graph is dependent on the concentration of sulphide. The variables affecting the rate of the reaction were investigated. The method is simple, rapid, precise, sensitive, and widely applicable. The limit of detection is 1 ng ml(-1) Se (IV), and the calibration range is 5-1000 ng ml(-1). Sampling rate is 60 samples h(-1), and the relative standard deviation of 12 determinations of 100 ng ml(-1) Se was 0.76%. The determination of Se (IV) in the presence of Se (VI) and total selenium is described. The method was applied to the determination of Se in selenium tablets, and several synthetic samples.     

Sensitive determination of bisphenol A and alkylphenols by high performance liquid chromatography with pre-column derivatization with 2-(4-carboxyphenyl)-5,6-dimethylbenzimidazole

A new and sensitive high-performance chromatographic method for the determination of bisphenol A and 8 alkylphenols with fluorescence detection is reported. Each phenol was derivatized by reaction with 2-(4-carboxyphenyl)-5,6-dimethylbenzimidazole at 40 degrees C for 60 min. The fluorescence derivatives were separated on a Wakosil 5C18 column (4.0 i.d. x 300 mm, 5 microm) with methanol:water (10:90) as mobile phase (detection wavelength: lambda(ex) 336 nm, lambda(em) 440 nm). The detection limits were in the range of 0.1-10.0 pg/mL in serum. The calibration graphs were linear to 1.0 microg/mL. The relative standard deviations were 7.2-8.9%, respectively. The proposed method was applied to the determination of bisphenol A in mother and infant rat serum.