Fluorimetric methods

O Neill and Sakamoto reported an enzymatic fluorimetric method for the determination of acetylcholine in biological extracts [41]. Nanomolar amounts of acetylcholine were determined in perchloric acid extracts of biological materials (brain tissues) by use of a system containing acetylcholineesterase, acetyl CoA synthetase, maleate dehydrogenase, and citrate synthase. The production of NADH2 was stoichiometrically related to the amount of acetylcholine in the system, and was followed fluorimetrically. Interfering fluorescent substances in the brain extracts were removed with acid-washed Florisil. 

Sutoo et al. developed a high sensitivity and high linearity fluorescence microphotometry system for distribution analysis of neurotransmitter and related substances in the small brain regions [42]. The method makes use of the fluorescence intensity of not more than 10,000 points in animal brain slices, which are immunohistochemically and histochemically stained. 

A fluorimetric assay method for the determination of acetylcholine with picomole sensitivity was reported by MacDonald [44]. The method is based on the hydrolysis of acetylcholine to choline and acetate, catalyzed in the presence of acetylcholineesterase, oxidation of choline to betaine, and H202 in the presence of choline oxidase, and oxidation of 4-hydroxyphenylacetic acid by H202 to a fluorescent product, catalyzed by peroxidase. The interference in the analysis of brain homogenates was discussed. 

Roulier et al. reported a sensitive and specific method for the measurement of choline and hydrogen peroxide in sea-water [45]. Choline was oxidized by choline oxidase to produce betaine and H202. The latter was used with horse-radish peroxidase to oxidize hydro-xyphenyl-propionic acid to produce a fluorescent diphenol end product. The resulting fluorescence at 410 nm (excitation at 320 nm) was proportional to the amount of H202, and could thus be used to measure the amount of choline present in the sample. Only 2-dimethyl aminoethanol interfered. The method was optimized, and used to determine 0 15 nM choline in coastal sea-water. 

Brennan et al. used a method to detect the reaction of acetylcholineesterase with acetylcholine [46]. The method was based on the use of a monolayer, consisting of fatty acids having Ci6 chain lengths, which were covalently attached to quartz wafers and which contained a small amount of nitrobenzoxadiazole dipalmitoyl phosphatidylethanolamine (NBD-PE) (partitioned from water into the membrane). The enzyme substrate reaction produced a decrease in fluorescence intensity from the monolayer, and the detection system was sensitive to the changes in bulk concentration of as small as 0.1 pM, with a limit of detection of 2 pM of acetylcholine. The mechanism of transduction of the enzymatic reaction was investigated using spectrofluorimetric methods and fluorescence microscopy. 

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Wang, F., The sensitive fluorimetric method for the determination of curcumin using the enhancement of mixed micelle, J. Fluoresc., 16, 53, 2006. 

There are methods available to quantify the total mass of americium in environmental samples. Mass spectrometric methods provide total mass measurements of americium isotopes (Dacheux and Aupiais 1997, 1998 Halverson 1984 Harvey et al. 1993) however, these detection methods have not gained the same popularity as is found for the radiochemical detection methods. This may relate to the higher purchase price of a MS system, the increased knowledge required to operate the equipment, and the selection by EPA of a-spectrometry for use in its standard analytical methods. Fluorimetric methods, which are commonly used to determine the total mass of uranium and curium in environmental samples, have limited utility to quantify americium, due to the low quantum yield of fluorescence for americium (Thouvenout et al. 1993). 

Fluorimetric methods of analysis make use of the natural fluorescence of the analyte, the formation of a fluorescent derivative or the quenching of the fluorescence of a suitable compound by the analyte. Fluorescence cannot occur unless there is light absorption, so that all fluorescent molecules absorb, but the reverse is not true only a small fraction of all absorbing compounds exhibits fluorescence. The types of molecule most likely to show useful fluorescence are those with delocalised ji-orbital systems. Often, the more rigid the molecule the stronger the fluorescence intensity. Naturally fluorescent compounds include Vitamin A, E (tocopherol). 

Applications A TLC-fluorimetric method has been described for the determination of 2,4- and 2,6-diaminotoluene in methanol extracts of flexible polyurethane foams [736]. The precision of this method at the... 

Tabuchi T, Okayama A, Ogawa Y, et al. 1989. A new HPLC fluorimetric method to monitor urinary delta-aminolevulinic acid (ALA-U) levels in workers exposed to lead. Int Arch Occup Environ Health 61 297-302. 

Using picosecond flash spectroscopy Gupta et al. 2k) reported for 2-hydroxyphenylbenzotriazole in ethanol a short-lived transient (6 ps) followed by a transient absorption whose lifetime is estimated to be 600 ps. The authors assigned the short-lived transient to the "vertical singlet" while the long-lived transient is presumably the "proton transferred species". These measurements of transient absorptions with the picosecond flash method confirm our results derived from the fluorescence emission using the phase fluorimetric method. 

Byeon et al. [23] described a fluorimetric method for (z>)-penicillamine using 9-fluorenylmethyl pentafluorophenyl carbonate and acetonitrile. Capsules containing penicillamine were extracted with water and then filtered. The solution was incubated at 70 °C for 40 min with borate buffer solution. After cooling, the mixture was extracted with diethyl ether and the fluorescence of the aqueous phase measured at (excitation = 260 nm, emission = 313 nm). The calibration graph was linear for 0.4-5.0 pM of penicillamine with a coefficient of variation of 0.4%. 

A specific and sensitive fluorimetric method was proposed by Al-Majed for the determination of (7))-penicillamine in its pure state and in its dosage forms [24], The method is based on the coupling between (/))-penicillamine and 4-fluoro-7-nitroben-zo-2-oxa-1,3-diazole, and analysis of the fluorescent product was measured at an excitation wavelength of 465 nm and an emission wavelength of 530 nm. The fluorescence intensity was found to be a linear function of the drug concentration over the range of 0.6-3 pg/mL, and the detection limit was 2 ng/mL (13 nM). 

Aaron et al. [28] described a photochemical fluorimetric method for the determination of primaquine absorbed on silica gel chromatoplates. 

Ibrahim et al. [30] described a fluorimetric method for the determination primaquine and two other aminoquinoline antimalarial drugs using eosin. Powdered tablets or ampule contents containing the equivalent of 50 mg of the drug was extracted with or dissolved in water (100 mL). A 10 mL aliquot was mixed with 10 mL of aqueous ammonia, 1 mL of 0.001% eosin (C.I. acid red 87) in dichloro-ethane, and dichloroethane was added to volume. Primaquine was determined fluorimetrically at 450 nm (excitation at 368 nm). Calibration graphs were rectilinear for 0.1-5 pg/mL of primaquine. Recoveries were quantitative. The method could be readily adapted for determination of the drug in biological fluids. 

Cheng et al. reported the use of a synchronous fluorimetric method for the determination of primaquine in two-component antimalarial tablets [31]. Ground tablets were dissolved in water and the mixture was filtered. The fluorescence intensities of chloroquine phosphate and primaquine phosphate, in the filtrate, were measured at 380 nm (excitation at 355 nm) and 505 nm (excitation at 480 nm), respectively. The calibration graphs were linear from 1 to 8 pg/mL of chloroquine phosphate and 10 to 110 pg/mL of primaquine phosphate. The mean recoveries were 98.2-101.49% and the relative standard deviations were 2.23%. 

Three anticonvulsant drugs including valproic acid were determined using different dyes as ion-pair reagents. Gentian violet was used for the spectrophotometric detection at 588 nm and acridine orange for the fluorimetric detection at 470 nm after excitation at 297 nm. Calibration graphs were linear for 5-50 pg/mL 2.5 0.50 pg/mL for the spectrophotometric and fluorimetric methods, respectively [15]. 

Extraction of nalidixic acid with chloroform from utine has also been reported.(40) Another fluorimetric method for chicken liver and muscle containing not less than 100 ppb nalidixic acid was reported by Browning(9) using an ethyl-acetate extraction and alumina column to retain the nalidixic acid. The fluorescence was measured at 325/408 nm. 

The concentration of neomycin in urine has been determined with a fluorimetric method 15. Interfering amines were separated by chromatography on SE Sephadex C-25 before reacting the isolated neomycin with fluorescamine. 

Before seeing a (future ) whole integrated system, mixing UV-visible-infrared and fluorimetric methods, the first route is the development of UV-based microsystems, including some relevant spectral exploitation techniques such as the semi-deterministic one [69,70]. 

In order to select the operating conditions for any fluorimetric method, the excitation and emission spectra of the analyte must be determined. Figure 2.9 illustrates the fact that an emission spectrum is an approximate mirror image of the excitation spectrum, the latter being similar to the absorption spectrum of the compound. 

Table 8.7 Examples of fluorimetric methods of enzyme assay...

Fluorimetric methods are useful for monitoring reactions involving the nucleotide coenzymes. The natural fluorescence of the reduced forms in the region of 460 nm can be used in kinetic assays. However, this fluorescence is destroyed at pH values below 2.0, whereas any oxidized forms of the coenzymes present are stable. If the pH of the solution is then raised above 10.5 and heated, the oxidized forms are themselves converted to fluorescent derivatives. This latter procedure lends itself to fixed time assays such as is illustrated in Procedure 8.6. 

There are several compounds that will react with amino acids to give coloured or fluorescent products and as a result can be used in qualitative or quantitative methods. Fluorimetric methods are gaining in popularity and offer some important advantages over absorption spectrophotometry for amino acid analysis. 

The fluorimetric methods often offer improved specificity and sensitivity over colorimetric procedures and the quantitative assays for the aromatic amino acids tyrosine and phenylalanine illustrate this point. 

Derivatization of primary amino acids with o-phthalaldehyde (OPA) is simple and the poor reproducibility due to the instability of the reaction product can be improved by automation and the use of alternative thiols, e.g. ethanthiol in place of the 2-mercaptoethanol originally used. An alternative fluorimetric method using 9-fluoroenylmethylchloroformate (FMOC-CL) requires the removal of excess unreacted reagent prior to column chromatography. This procedure is more difficult to automate fully and results are less reproducible. However, sensitivity is comparable with the OPA method with detection at the low picomole or femtomole level, and it has the added advantage that both primary and secondary amino acids can be determined. 

Fluorimetric methods have proven useful for the assay of metal ions in solution 1 e.g., in vivo studies of calcium-selective fluorescence probes have been reported by... 

C. T. Yuen, P. R. N. Kind, R. G. Price, P. F. G. Praill, and A. C. Richardson, Colorimetric assay for N-acetyl-P-D-glucosaminidase (NAG) in pathological urine using the oo-nitrostyryl substrate the development of a kit and the comparison of the manual procedure with the automated fluorimetric method, Ann. Clin. Biochem., 21 (1984) 295-300. 

Dichloroacetamide present in chlorinated and chloraminated drinking waters was determined (at detection level of 23 pg L ) through an LC/fluorimetric method and a post-column derivatization reaction with o-phthaldialdehyde in the presence of sulfite at pH 11.5, that leads to the formation of a highly fluorescent isoindole fluorophore [225]. 

H.B. Li, F. Chen, A highly sensitive fluorimetric method for the determination of fluoride in biological material with AP -calcein complex, Fresenius J. Anal. Chem. 368 (2000) 501-504. 

Colorimetric Methods Fluorimetric Methods of Analysis Chromatographic Methods of Analysis... 

A fluorimetric method for the determination of chlorpromazine after extraction from blood and urine samples (protected from light) has been described [134]. The fluorimetric determination of chlorpromazine with tetrabromosulphone fluorescein was also reported [135],... 

Individual and simultaneous stopped-flow fluorimetric methods for the determination of chlorpromazine and perphenazine were reported, with the fluorescence being monitored at 380 ran after excitation at 340 nm [141]. The technique was used to determine chlorpromazine and perphenazine in mixture, tablet, and liquid formulations. 

One fluorimetric method for the determination of chlorpromazine hydrochloride in bulk and in various dosage forms begins with the mixing of 0.5 mL of a 60 pg/mL solution with 1 ml of pH 7.2 buffer solution. To this is also added 1 mL of 0.2 mg/mL A/-bromosuccinimide, whereupon the mixture is diluted to 10 mL with methanol. After 30 minutes of reaction time, the fluorescence intensity is measured at 378 nm (excitation at 280 nm). The method was found to be linear over the range of 0.005 -10 pg/mL [145]. 

The 2,6-di-tertbutyl-p-cresol reaction also produces fluorescent products with certain corticosteroids, which allows the development of fluorimetric methods for their determination. These will be discussed in a subsequent section. 

Fluorimetric Methods of Analysis 4.4.1 Reaction with Sulfuric Acid... 

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