Sensitivity fluorimetric analysis

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). 

The oxidation detector for the fluorimetric analysis of carbohydrates in effluents from liquid chromatography columns provides a sensitive method of analysis in blood and urine [110]. The principle involves the reduction of cerium(IV) to cerium(III) by oxidizable compounds such as organic acids and many carbohydrates. The fluorescence... 

Fluorescence spectrometry has become established as a routine technique in many specialized applications due to its high sensitivity. Examples of the current applications of fluorescence range from simple fluorimetric analysis for biomolecules, metal ions, and organic compounds to identification of specific DNA and/or RNA sequences in tissues. In addition, much of the current research in biochemistry, medicine, and molecular biology involves fluorescence spectroscopy of either intrinsic molecular fluorescence (from tyrosine or tryptophan residues) or exogenous fluorescent probes. 

The method was further extended for analysis of d- and L-amino acids in mouse kidney in a similar manner. HPLC methods, for the resolution of amino acid enantiomers that utilized a chiral stationary phase or a chiral mobile phase additive were considered expensive and less satisfactory due to the high retention results in broad peaks. The separation of FDAA derivatives followed by HPLC, as described above, was found to be much more successful as there was neither needed a post column derivatization nor fluorimetric analysis and moreover a subnanomolar sensitivity was attained [33], d- and L-enantiomers of glutamate, aspartate, asparagine, serine, threonine, alanine, proline, tyrosine, valine, methionine, isoleucine, leucine, phenylalanine, and histidine were derivatized with FDAA and the diastereomers were separated by 2D TLC. Each was separated except for the two spots comprising Tyr and Val, and He, Leu, and Phe. Only histidine was separated further into D- and L-diastereomers by TLC. The excess hydrolyzed FDAA moved to the front... 

A drawback to conventional amino analysis by chromatography is the need for pre- or post-column derivatization to improve sensitivity. Ninhy-drin, the reagent originally applied for detection, has been increasingly displaced by other reagents such as phenylisothiocyanate,71 9-fluorenylethyl chloroformate,72 and o-phthaldialdehyde (OPA). OPA allows fluorimetric detection, which offers the potential for greater sensitivity.73 A limitation of OPA is that it doesn t derivatize secondary amines, so an additional reaction must be added for proline detection. And, as noted for amine analysis in section A5.4.2, such derivatization adds to the analysis time and may yield unstable products. 

Wagner J, Hirth Y, Claverie N, Danzin C (1986) A sensitive high-performance liquid chromatographic procedure with fluorimetric detection for the analysis of decarboxylated S-ad-enosylmethionine and analogues in urine samples. Anal. Biochem 154 604-617... 

Diaminopyrimidines are used for antibacterial purposes. Although many of these compounds have some fluorescence, direct analysis is not useful because of the lack of sensitivity. The enhancement of the fluorescence of 2,4-diaminopyrimidines has been accomplished by treatment of the separated components with a solution of ammonium bisulfate [133], A fluorimetric method has also been developed for trimethoprim [2.4-diamino-5-(3,4, 5 -trimethoxybenzyl)pyrimidine] and its metabolites [134] which involves leaving the developed plates to stand for several days and then examining the increase in fluorescence. Amounts of ca. 10 ng could be detected after this time. 

Fluorimetric methods for the determination of amino acids are generally more sensitive than colorimetric methods. Fluorescamine (4-phenyl-spiro[furan-2(3H),l -phthalan]-3,3 -dione) and o-phthaldialdehyde (OPA) substances are used for protein analysis. Fluorescamine reacts with amino groups to form fluorophores that excite at 390 nm and emit at 475 nm (Weigele et al., 1972). Applications of fluorescamine include monitoring the hydrolysis of K-casein (Beeby, 1980 Pearce, 1979) and quantification of proteins, peptides, amino acids in extracts (Creamer et al., 1985). OPA produces fluorescence on reaction with 2-mercaptoethanol and primary amines, with strong absorption at 340 nm. Lemieux et al. (1990) claimed that this method was more accurate, convenient, and simple for estimating free amino acids than the TNBS, ninhydrin, or fluorescamine methods. 

The main advantage of using a fluorimetric detector is its inherently high sensitivity for pharmaceutical analysis, but very often application of this method to nonfluorescent drug substances requires derivatization with a strong fluorophore. The derivatization process can be performed either precolumn or postcolumn to increase the detection sensitivity [14]. 

A highly sensitive, accurate and improved fluorimetric method has been developed for analysis of chloroquine in biological samples (31). The chloroquine is extracted with heptane or methylene dichloride from an alkali medium buffered with borate, pH 9.5. Chloroquine is reextracted into 0.1 N HC1 which is then mixed with an equal volume of 0.2 M alcoholic NaOH and fluorescence is read at activation and emission wavelengths of 335 and 400 nm respectively. 

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. 

Analytical absorption spectroscopy in the ultraviolet and visible regions of the elechomagnetic spectrum has been widely used in pharmaceutical and biomedical analysis for quantitative purposes and, with certain limitations, for the characterisation of drugs, impurities, metabolites, and related substances. By contrast, luminescence methods, and fluorescence spectroscopy in particular, have been less widely exploited, despite the undoubted advantages of greater specificity and sensitivity commonly observed for fluorescent species. However, the wider availability of spectrofluorimeters capable of presenting corrected excitation and emission spectra, coupled with the fact that reliable fluorogenic reactions now permit non-fluorescent species to be examined fluorimetrically, has led to a renaissance of interest in fluorimetric methods in biomedical analysis. 

Chandrashekhar, T.G. Rao, P.S.N. Smrita, K. Vyas, S.K. Dutt, C. Analysis of amlodipine besylate by HPTLC with fluorimetric detection a sensitive method for assay of tablets. J. Planar Chromatogr.-Mod. TLC 1994, 7, 458-460. 

There has been a recent trend towards the use of high pressure systems and reagents other than ninhydrin to increase sensitivity and speed (e.g. see Moore 1972). Although not yet in general use, such systems hold considerable promise for the future. Expanded scales and one-column systems of analysis have also been used to boost sensitivity. However, in most of these more sensitive systems, the baseline is usually not as straight and free of drift as in the two-column systems, and there are often more problems encountered with interfering substances. At present we would recommend the use of the common commercial analyzers without special modifications (except for the possible inclusion of expanded scale modes) for the greatest accuracy in routine work. However, for situations where samples are obtained in limited amounts or where many samples need to be analyzed quickly, some of the newer systems may well be worthy of consideration. The application of fluorescamine to the quantitative fluorimetric determination of picomole quantities of amino acids, peptides and proteins is of particular interest in this context (Udenfriend et al. 1972). 

Postcolumn labeling is a characteristic feature of carbohydrate analysis in which no direct physical methods are available for sensitive detection. Many labeling methods have hitherto been developed. The methods with phenol in sulfuric acid [16], orcinol in sulfuric acid [17], anthrone in sulfuric acid [18], tetrazolium blue in alkali [19], copper(II)-2-2 -bicin-chonitate [20], and 2-cyanoacetamide [21] are used for photometric detection. The methods with 2-cyanoacetamide [6], ethylenediamine [22], ethanolamine [23], taurine [24], and arginine [25] are used for fluorimetric detection. Some labeling methods for electrochemical detection were reported by Honda and Suzuki in 1984 [26,27],... 

Sams oxidized reserpine prior to its HPLC-analysis. The 3-dehydro derivative formed was detected fluorimetrically (excitation 390 nm, emission 470 nm). This allowed a more specific and sensitive detection. 

In order to optimise the manifold design of the flow system, the lifetimes of the excited states of the molecules should be considered, because the processed sample is in motion. In the situation of too high a flow rate and too small a flow cell, a fraction of the excited molecules could exit the flow cell without emitting light. On the other hand, if the flow rate is too low, a significant fraction of the molecules may emit radiation before reaching the flow cell. Both situations can lead to a decrease in the analytical sensitivity. This feature can be considered as a "time window" in flow analysis and the effect is more pronounced in phosphorimetric methods where light emission is slow relative to fluorimetric methods [65]. This is also true for chemiluminescence and bioluminescence, as discussed in the next section. 

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