Determination Of Fluorescein Research Articles

Determination of fluorescein and brominated fluoresceins in the color additive D&C Orange No. 5 and its lakes using high-performance liquid chromatography

ABSTRACTThe colour additives D&C Orange No. 5 (O5) and its lakes (O5L) are subject to batch certification by the U.S. Food and Drug Administration (FDA) to ensure compliance with specifications in the Code of Federal Regulations (CFR). The present study reports the development of a high-performance liquid chromatography (HPLC) method for the quantitative determination of seven CFR-specified components in O5 and O5L – fluorescein and six brominated fluoresceins. The analytes were quantified using six-point calibration curves with data points (w/w) that ranged as follows: 20.0–70.0% for 4ʹ,5ʹ-dibromofluorescein; 9.8–44.1% for 2ʹ,4ʹ,5ʹ-tribromofluorescein; 1.01–15.2% for 2ʹ,4ʹ,5ʹ,7ʹ-tetrabromofluorescein; 0.10–3.12% for 2',4ʹ-dibromofluorescein; 0.10–3.06% for 2ʹ,5ʹ-dibromofluorescein; 0.11–2.85% for 4ʹ-bromofluorescein; and 0.10–2.02% for fluorescein. For all seven analytes, the HPLC instrument response was linear (R2 > 0.999) over the tested concentration ranges and the limits of detection (0.01–1.55%) were well below the CFR-specified levels. Other validation data showed good analyte recovery (87.91–101.73%) as well as method precision measured by the relative standard deviation (0.53–1.56%). The new method was applied to the analysis of test portions from 15 batches of O5 and eight batches of O5L submitted to FDA for certification by domestic and foreign manufacturers. Compared to the thin-layer chromatography/spectrophotometric procedure currently used for routine batch-certification analyses, the new method was found to be more sensitive, simpler to implement, and significantly faster, requiring 25 minutes rather than six hours to analyse one sample.

Quantitative Photoacoustic Spectroscopy: Determination of Fluorescein and Bromocresol Green Adsorbed on Modified Silica Gel Samples

A series of standards was prepared by mixing the fluorescein or bromocresol green adsorbed on silica gel modified with N,N-diethylaminopro-pyltriethoxysilane with varying amounts of unmodified silica. Photoacoustic magnitude and phase values were obtained at their respective absorption maxima, 470 and 600 nm. Calibration graphs were prepared by combining the magnitude and phase into the response function. Two-component samples were prepared by mixing the different ratios of the standard samples. The concentration of the dyes in the two-component samples was determined by referring to the respective calibration graphs and solving a pair of simultaneous equations. The relative error in the determination ranged from −5.6% to 5.1% for fluorescein and −16.8% to 38% for bromocresol green when compared to their concentration determined by spectrophotometry.

Use of time resolution to eliminate bilirubin interference in the determination of fluorescein.

Use of time resolution to eliminate bilirubin interference in the determination of fluorescein.

Minimisation of bilirubin interference in the determination of fluorescein using first-derivative synchronous excitation fluorescence spectroscopy.

The use of first-derivative synchronous excitation fluorescence is described for the minimisation of bilirubin interference in the determination of fluorescein. The limits of detection and the standard errors of estimates are better using the synchronous first-derivative approach than with conventional subtraction of the bilirubin blank. Results are shown for the quantitation of fluorescein in the nanomole concentration range.

Flow injection analysis with chemiluminescence detection in the determination of fluorescein- and fluorescamine-labelled species

Many fluorescence immunoassays have indifferent limits of detection because of the background signals from biological samples. Scattered light contributes to this background, but can be eliminated by exciting conventional fluorescent labels via chemiluminescence reactions involving bis(2,4,6-trichlorophenyl)oxalate. This reaction, whose rate can be controlled, is conveniently used in a flow injection system with a luminometer as a detector. Such a system is applied to study fluorescein- and fluorescamine-labelled species at concentrations as low as 10 −11 M (ca. 0.5 pg in a 100 μl sample). The effects of antibodies on the luminescence signals from labelled antigens are discussed.