6-[N-(3-Propionohydrazino)thioureido]benzo[g]-phthalazine-1,4(2H,3H)-dione was found to be a sensitive chemilumi-nescence (CL) derivatization reagent for fatty acids in high-performance liquid chromatography. The reagent readily reacted selectively with fatty acids at room temperature in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodi-imide and pyridine in aqueous solution to form the corresponding acid hydrazide derivatives, which produce chemiluminescence by reacting with hydrogen peroxide in the presence of potassium hexacyanoferrate(III) in an alkaline solution. The CL derivatives of seven fatty acids (myristic, linolenic, palmitoleic, linoleic, palmitic, oleic and stearic acids) were separated within 55 min by reversed-phase (C18) liquid chromatography with isocratic elution, and were detected with CL detection after mixing with the oxidizing reagents. Calibration curves were linear up to at least 66.7 pmol per 20 µl injection volume (r=0.996 to 0.999). The detection limits (signal-to-noise ratio=3) for the acids were 11.8 - 64.4 fmol for an injection volume of 20 µl.
We developed a sensitive and rapid PCR-RFLP ELISA using acetate kinase (AK) and firefly luciferase as a detection system. AK used as a label enzyme could sensitively be detected by bioluminescent assay using the firefly luciferase reac-tion. The detection limit was 10-20 mol/assay and the luminescence was stable for 48 h. FITC-labeled sense primer and biotin labeled anti sense primer were used for PCR amplification of the vitamin D receptor gene. After PCR, the products were digested with Taq I or Apa I enzyme. The reaction products were diluted with assay buffer and transferred to a plate coated with anti FITC IgG. After incubation for 2 h at 37°C, the plate was washed and reacted with avidin/biotinylated AK, the AK activity was detected by bioluminescence assay using the firefly luciferin/luciferase system. DNA polymorphism types (AA, Aa, aa, TT, Tt, tt) of the vitamin D receptor gene (VDR) could be clearly determined by measuring the bioluminescent intensity or by using photon imaging with a CCD camera.
A method for the quantitative determination of pregnenolone 3-sulfate (PS) in rat brains has been developed using liquid chromatography/electrospray ionization-mass spectrometry (LC/ESI-MS). The PS fraction was obtained from the rat brain homogenate by solid-phase extraction and ion-exchange chromatography. After the derivatization with 4-(N,N-dimethylaminosulfonyl)-7-hydrazino-2,1,3-benzoxadiazole, PS was determined using LC/ESI-tandem MS along with the standard addition method. This method was applied to the quantitative determination of this steroid in the brains of Wistar strain rats, most of which showed a much lower amount than that previously reported.
An indirect amperometric detection of underivatized amino acids has been developed based on the use of a plastic film ring-disk carbon electrode in liquid chromatography. Bromide present in the postcolumn addition reagent is oxidized to bromine at the upstream (disk) electrode, and is subsequently detected at the downstream (ring) electrode. Most of the underivatized amino acids that are electroinactive under conventional amperometric conditions can react rapidly with the electrogenerated bromine. The concentration of amino acids can therefore be indirectly determined by continuously mon-itoring the reduction current of bromine. The signal monitored at the downstream electrode was largely dependent on the pH of the mobile phase, the generation and collection potentials, and the bromide concentration in the mobile phase. Under the optimized conditions, the detection limits of the 0.5 µM level were obtained for a number of amino acids with a linear correlation coefficient of 0.990 - 0.998 over the concentration range 1 - 100 µM. The primary advantage of this approach lies in the capability that it affords a simple and sensitive determination of underivatized amino acids. In addition, it was almost unaffected by fouling effects normally arising from the anodic oxidation of the sulfur-containing amino acid.
A novel spectrofluorometric method for the determination of 2,6-dimethylphenol has been developed. It is based on detecting a fluorophore intermediate produced in the course of 2,6-dimethylphenol photo-degradation with TiO2 as a catalyst. A quartz glass reactor containing the required amount of analyte, buffer and TiO2 is irradiated with UV light (<380 nm). The formed intermediate is detectable with fluorescence and UV absorption. What is more, the fluorescence intensity of the intermediate is dramatically enhanced when extracted into the CHCl3 phase. A linear calibration graph was obtained over a 2,6-dimethylphenol concentration ranging from 0.002 µg ml-1 to 0.1 µg ml-1 with a detection limit of 0.0008 µg ml-1 . Other phenolic compounds as well as those common inorganic ions do not interfere. The kinetics of the photo-degradation reaction, which followed a successive first-order reaction kinetic model, were studied using an error-compensated algorithm.
The interaction between aluminium ion (Al) and quinolic acid (L) in acidic aqueous solution was investigated by poten-tiometry and 13C and 27Al NMR spectroscopy. In the 27Al NMR spectra, two peaks were observed at 8 and 15.6 ppm, which were assigned to AlL and AlL2 complexes, respectively. The 13C NMR spectra provide clear evidence that the L acts as a bidentate ligand and that the L coordinates with a nitrogen atom and an oxygen atom of the adjacent carboxylic group to form a chelate complex with a five-membered ring. From a potentiometric study, the formation constants (log β1 and log β2) for the AlL and AlL2 complexes were estimated to be 4.52±0.02 and 7.99±0.05, respectively.
The concentration of ascorbate in a raw leaf was determined by monitoring a nitroxyl radical reduction with X-band ESR. One piperidine nitroxyl derivative, 2,2,6,6-tetramethyl-4-hydroxypiperidine-1-oxyl (TEMPOL), was reduced according to first-order kinetics by more than 5-fold excess of ascorbate. The observed reduction rate was found to be proportional to ascorbate concentration in phosphate buffer (0.1 mol/dm3 , pH 7.4) at 25°C. The reduction of TEMPOL in leaf homogenate of sprouts of radish was measured; it was found to obey first-order kinetics. The ascorbate concentration in leaf was estimated from observed reduction rate and found to be virtually identical with that from a visible absorption spectrophotometric analysis. The features of our ESR method for sensitivity, procedure and measuring time were discussed with comparisons from other methods. The results indicated that the determination of ascorbate in a raw leaf by ESR spectroscopy was simple and effective.
A simple and sensitive spectrophotometric method for the determination of sulfonamides in bulk, in pharmaceutical dosage forms and in biological fluids was developed. The method is based on coupling the diazotized sulfa with the anti-malarial ingredient, primaquine phosphate, in an acid medium. The resulting orange color is stable for several weeks and has an absorption maximum between 468 - 474 nm, depending on the sulfa drug. The effect of different variables on color development was studied. Calibration graphs were established in the concentration range of 0.1 to 12 µg/ml with a minimum detection limit of 0.05 µg/ml. The molar absorptivities were in the range of 47589 - 66414 l mol-1 cm-1 , depending upon the sulfa drug. The percentage recoveries of sulfa drugs in dosage forms were in the range of 97.4 to 104.0%. The method was also utilized in the determination of sulfamethoxazole and N-acetyl sulfamethoxazole in serum and urine samples.
A flow-through sensor for the determination of diclofenac sodium was developed, based on retention of the analyte on a Sephadex QAE A-25 anion-exchange resin packed in a flow-cell of 1 mm of optical path length, and monitoring of its intrinsic absorbance by UV-spectrophotometry at 281 nm. Diclofenac could be determined in the concentration ranges 2.0 - 40.0, 1.0 - 22.0 and 0.5 - 14.0 µg ml-1 with RSD (%) ranging from 1.05 to 1.53 for sample volumes of 300, 600 and 1200 µl, respectively. The proposed sensor was satisfactorily applied to the rapid determination of diclofenac in commercial pharmaceutical preparations and in semi-synthetic pharmaceuticals containing diclofenac and paracetamol.
This paper presents the use of poly(vinylpyrrolidone) (PVP), which is a low-cost polymer for lead preconcentration in natural-water samples. The weak granular structure of this polymer imposed the use of a stainless-steel filter holder for its conditioning in a thin filtering layer. This unit was inserted into a sequential injection analysis system coupled to a flame atomic absorption spectrophotometer (SIA/FAAS). The developed system allows sampling rates of 16 samples/h with a 60-fold enrichment factor and a detection limit of less than 5 µg/l for a 13 ml sample volume. The performance of the present system was also assessed by carrying out recovery trials, which provided results of between 96% and 101%. The precision of the methodology developed has never been over 3% regarding RSD and 5% for the deviations to the conventional procedure.
The concentrations of normal saturated hydrocarbons (C10 - C24) were determined in water at the stations of Postagolla, Sadarghat and Sowarighat of the Buriganga river, Bangladesh. Samples were collected from different water depths (5 and 25 cm) of the sampling stations. Hydrocarbons were extracted from these water samples (500 ml) into 70 ml hexane which were further preconcentrated by evaporation to a volume of 10 ml solution. Constituents of hydrocarbons were analyzed qualitatively and quantitatively by gas liquid chromatography (GLC) using flame ionization detection (FID). Different concentrations of hydrocarbon constituents were obtained in river water at the various sampling stations. Irrespective of the locations and depths of the sampled water, few lower hydrocarbons (C10 - C13) were detected, but higher hydrocarbons (C14 - C24) were found to occur. However, the average concentrations of the components e.g., C22 and C24 at Postagolla, C16 and C22 at Sadarghat and C16 at Sowarighat (5 cm depth) were determined to be higher than the other hydrocarbons. The concentrations of individual constituents were found in the range of 1.04 to 6135 ppb. This method could permit the analysis of water for hydrocarbons with a detection limit as low as 1.04 ppb level.