Spectrofluorimetric detection

As regards the aflatoxin detection, UV spectrophotometry has been definitively abandoned, because of the lack of selectivity and the very poor analytical response, resulting in a very high detection limit. Since aflatoxins show fluorescence under UV light, spectrofluorimetric detection has been adopted in the last years. 

Hernandez-Jover et al. (72) derived an improved analytical method from the HPLC procedure setup they developed in 1995 (73) for the determination of BAs in fish. The method consists of the extensive extraction with HC104, ion-pair (with sodium octanesulfonate) RP-HPLC separation, postcolumn derivatization with PA/ME, and spectrofluorimetric detection. Determination limits were up to 1.5 mg/kg. In particular, His, Tyr, Phe, Ser (serotonine), Cre (creatinine), Try, Oct (octopamine), Dop (dopamine), Cad, Put, Agm (agmatine), Spm, and Spd were studied in pork and beef meat, fresh, cooked, or ripened. Tyramine, His, Put, Cad, and Try levels were... 

Used to derivatize primary amines to yield isothiocyanates Used for derivatization of tripeptides provides high sensitivity toward spectrofluorimetric detection... 

Schmid et al. described a rapid and sensitive high-performance liquid chromatographic method for the determination of dipyridamole in human plasma [69], The column used was a 12.5 cm x 4.6 mm filled with Lichrosorb RP 18, 5 pm. The mobile phase consisted of methanol-0.2 M Tris HC1 buffer (80 20), eluted at a flow rate of 1 mL/min. Spectrofluorimetric detection at an emission wavelength of 478 nm (excitation at 415 nm) was used for detection. The results were confirmed by re-chromatographing the eluate from the column on silica gel G thin-layer plates, which were developed using a solvent system composed of toluene-isopropanol-ethanol-ammonia (70 15 15 1) and ra-butanol-methyl ethyl ketone (80 20). The Rf values of dipyridamole for the two solvent systems are 0.60 and 0.80, respectively. The plates were dried in a stream of cold air, and then inspected under ultraviolet light at 254 nm. 

Pentosidine is determined by HPLC with spectrofluorimetric detection (excitation and emission wavelengths of 335 and 385 nm, respectively) (S14), although immunochemical and ELISA assays for determination of various protein oxidative modification products have become increasingly popular (08). Protein-aldehyde adducts can be estimated using adduct-specific antibodies (U2, Wl). Another approach requires stabilization of adducts, producing derivatives resistant to conditions used in protein acid hydrolysis and quantification of hydrolysis products by gas chromatography-mass spectrometry (R7). 

Kinetic determination of ultratrace amou spectrofluorimetric detection. 

Mfllart H, Lamiable D. Determination of pyridoxal 5 -phosphate in human serum by reversed phase high performance fiquid chromatography combined with spectrofluorimetric detection of 4-pyridoxic acid 5 -phosphate as a derivative. Analyst 1989 114 1225-8. 

According to Nau and Biemann [511], samples between 2 nmol and 12 /xmol of the original peptides could be safely analyzed. The high-sensitivity aspect of GC methods may also have some importance for a variety of clinically important peptides, although the current approaches using HPLC and spectrofluorimetric detection are likely to become more popular. 

Surfactants are usually determined as a group of compovmds, so that sophisticated chromatographic procedures are not needed. Ion-pair formation between both anionic and cationic surfactants with polar dyes, such as methylene blue and brom-ocresol purple, respectively, with or without subsequent inline solvent extraction has been assessed using spectrophotometric or spectrofluorimetric detection as shown in Table 1. Polyoxyethylene and amine ethoxylate-based nonionic surfactants in environmental waters have been determined by spectrophotometry following polar interactions with chromogenic agents, and by CL detection in the presence of sensitizers, respectively. 

Flow systems with gas diffusion separation and spectrofluorimetric detection have been proposed for the determination of cyanide anion in aqueous samples (Figure 7.14). The cyanide transferred... 

A FI system with gas diffusion separation and spectrofluorimetric detection has been recommended for the determination of acid dissociable cyanide in waters [36]. Cyanide diffuses through a microporous PTFE membrane from an acidic donor stream into a sodium hydroxide acceptor stream. The cyanide transferred reacts with o-phthalaldehyde and glycine to form a highly fluorescent isoindole derivative. Complete recovery of cyanide was found for Zn(CN) , Cu(CN), Cd(CN) , Hg(CN) -, Hg(CN)2, and Ag(CN)2 complexes and low recovery from Ni(CN)4. No recovery was obtained from the species that are considered as nonfree cyanide producing, viz., Fe(CN)g, Fe(CN)g, and Co(CN)g. The sampling frequency was 10 Hz h and the detection limit was 0.5 pg L . ... 

JuUien, V. Tr61uyer, J.-M. Pons, G. Key, E. Determination of tenofovir in human plasma by high-performance liquid chromatography with spectrofluorimetric detection, J.Chromatogr.B, 2003, 785, 377-381. 

Determination of AGEs is based on the classic spectrofluorimetric detection 18-19. Fluorescence intensity was recorded at the emission maximum 440 nm upon excitation at 370 nm and at emission maximum 385 nm upon excitation at 335 nm for pentosidine fluorescence Fluorescence intensity is expressed in arbitrary units (AU) and corrected for dilutions. We employ a SPECTRAmax Gemini XPS spectrofluorometer with SOFTmax PRO software (Molecular Devices, Sunnyvale, CA). 

Analytical procedures for foods are generally based on extraction with purified solvents following saponification with alcoholic potash. Purification of the extracts by chromatography on column or plates is followed by analysis of the purified extract using TLC, GLC or HPLC. Spectrophotometric or spectrofluorimetric methods may be used for quantitation of the hydrocarbons a collaborative study of a spectrophotometric method showed it to be applicable at the 2 fig/kg level. HPLC techniques using spectrofluorimetric detection have been described for which improved levels of detection are claimed. Benzo(a)pyrene produces substitution products with nucleic acids. Hydrocarbon deoxyribonucleoside adducts may be isolated from DNA by gel permeation chromatography, and the formation of hydrocarbon epoxides by mammalian enzyme reaction has also been demonstrated . The limit of detection of spectrophotometric and fluorimetric methods has been improved by three orders of magnitude by the use of laser-induced fluorescence procedures for the... 

Parmentier, C. Leroy, P Wellman, M. Nicolas, A. Determination of cellular thiols and glutathione-related enzyme activities versatility of high-performance liquid chromatography-spectrofluorimetric detection. J. Chromatogr, B Biomed. Sci. Appl. 1998, 719, 37 6. 

Cholerton, S., Idle, M E., Vas, A., Gonzalez, F.J. Idle, J.R. (1992) Comparison of a novel thin-layer chromatographic-fluorescence detection method with a spectrofluorimetric method for the determination of 7-hydroxycoumarin in human urine. J. Chromatogr., 575, 325-330 Cohen, A.J. (1979) Critical review of the toxicology of coumarin with special reference to interspecies differences in metabolism and hepatotoxic response and their significance to man. Food chem. Toxicol, 17, 277-289... 

A simple, rapid, sensitive, and selective spectrofluorimetric method (2ex/ lem = 345/455nm) has been developed for the determination of zaleplon. Tang et al. have studied the influence of micellar medium on the absorption, fluorescent excitation, and emission spectra character of zaleplon The nonionic surfactant of Triton X-100 showed a strong sensitizing effect for the fluorescence of zaleplon in a pH 5.0 buffer. The possible enhancement mechanism was discussed. Based on the optimum conditions, the linear range was 1.32 x 10 8-1.00 x 10 mol/1. The detection limit was 4.0 x 10 mol/1 with a relative standard deviation (RSD) of 0.06%. The proposed method was successfully applied to the determination of zaleplon in tablets, serum, and urine. 

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. 

A variety of analytical methods has been used for determining trace concentrations of PAHs in environmental samples (Table 6-2). These include GC with various detectors, HPLC with various detectors, and TLC with fluorimetric detectors. Various detection devices used for GC quantification include FID, MS, Fourier transform infrared spectrometer (FT-IR), laser induced molecular fluorescence detector (LIMF), diode array detector (DAD), and gas phase fluorescence detector (GPFDA). GC/MS and HPLC with UV or spectrofluorimetric detectors are perhaps the most prevalent analytical methods for determining concentrations of PAHs in environmental samples. 

Fluorescence Spectroscopy. - A method for the spectrofluorimetric determination of six organophosphorus pesticides has been established. The fluorescence of the solutions was measured at excitation wavelength, 380 nm and emission wavelength, 488 nm. The optimum pH for measurement is 9.3 - 9.6 and transition metal ions interfere but can be removed by complexation. Detection limits varied between 10and 10 g/ml, depending on the substance. There are... 

The UV methods reported minimum detectable limits of q)proximately 0.5 pg/mL. The development of a spectrofluorimetric method 305 nm, 505 nm) lowered this limit to 0.1 pg/mL [36]. 

A spectrofluorimetric procedure is applied to detect an analyte based on its characteristic emission band and to analyse the concentration of a target analyte from its emission intensity. 

Hossu et al. (2009) presented spectrofluorimetric methods for the determination of fat-soluble vitamin E in multivitamin pharmaceutical products. In method I, M-hexane was used as solvent for a-tocopherol assay, while in method II, ethanol was used as a carrier between the aqueous solution and the n-hexane fluorescent solution of a-tocopherol. At 290/306 nm excitation and emission wavelengths, method I was linear for a-tocopherol over the range 1-100 p,g/mL R = 0.97687), having a limit of detection 1 p,g/mL and a limit of quantification 2 p,g/mL, and method II was linear over the range 2-50 p,g/mL R = 0.9709) with a limit of detection 0.68 J.g/mL and a limit of quantification 2.27 j,g/mL. 

In the analysis of PAHs, chromatographic techniques, mainly gas chromatography (GC) and liquid chromatography (LC), are often the preferred approach for separation, identification and quantification. Spectrofluorimetric techniques have also been used with screening purposes. In general, capillary GC is preferred over LC for the determination of PAHs mainly because of favorable combination of greater selectivity and resolution [49-51]. On the other hand, HPLC with fluorescence detection (FD) presents the best detection limits for PAHs [15],... 

Titrimetric (or volumetric) methods depend on the detection of an end point of the stoichiometry of a reaction. Strictly interpreted, the classical methods are limited to visual indicators of the end point. There are, however, other instrumental modes that include a variety of electrometric, spectrophotometric, spectrofluorimetric, and enthalpic techniques. These latter methods are also amenable to... 

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