Accurate anisotropy recovery from fluorophore mixtures using Multivariate Curve Resolution (MCR).

Anisotropy resolved multidimensional emission spectroscopy (ARMES) provides valuable insights into multi-fluorophore systems like proteins that have complex overlapping emission bands. The method combines multidimensional fluorescence, anisotropy, and chemometrics to facilitate the differentiation of fluorophores with very similar emission properties. Here, we address the critical issue of standardizing the chemometric methods required to accurately extract spectral and anisotropy information from fluorophore mixtures using two standard sample sets: perylene in glycerol, and a mixture of Erythrosin B and Phloxine B with overlapping emission but different anisotropies. We show for the first time how to accurately model component anisotropy using Multivariate Curve Resolution (MCR) from data collected using total synchronous fluorescence scan (TSFS) and Excitation Emission Matrix (EEM) measurement methods. These datasets were selected to avoid the presence of inner filter effects (IFE) or Förster resonance energy transfer (FRET) that would depolarize fluorescence emission or reduce data tri-linearity. This allowed the non-trilinear TSFS data to yield accurate component anisotropy data once modelled using the correct data augmentation strategy, however, the EEM data proved to be more accurate once optimal constraints (non-negativity and correspondence among species) were employed. For perylene (S2) and Phloxine B which both have very weak anisotropy (<0.06), while the spectral recovery was excellent, the modelled anisotropy values were reasonably accurate (±20% of the real value) because of large relative noise contributions. However, for perylene (S1) and Erythrosin B which have large (>0.2) anisotropies, bilinear and trilinear EEM models built using a total tri-linearity constraint, yielded solutions without any rotational ambiguities and very accurate (±4% of real value) anisotropy values. These sample systems thus provide simple and robust test systems for validating the spectral measurement and chemometric data analysis elements of ARMES.

Molecularly imprinted coated graphene oxide solid-phase extraction monolithic capillary column for selective extraction and sensitive determination of phloxine B in coffee bean.

A method was developed to sensitively determine phloxine B in coffee bean by molecularly imprinted polymers (MIPs) coated graphene oxide (GO) solid-phase extraction (GO-MISPE) coupled with high-performance liquid chromatography and laser-induced fluorescence detection (HPLC-LIF). The GO-MISPE capillary monolithic column was prepared by water-bath in situ polymerization, using GO as supporting material, phloxine B, methacrylic acid (MAA), and ethylene dimethacrylate (EDMA) as template, functional monomer, and cross-linker, respectively. The properties of the homemade GO-MISPE capillary monolithic column, including capacity and specificity, were investigated under optimized conditions. The GO-MIPs were characterized by scanning electron microscopy (SEM) and Fourier transform-infrared spectroscopy (FT-IR). The mean recoveries of phloxine B in coffee bean ranged from 89.5% to 91.4% and the intra-day and inter-day relative standard deviation (RSD) values all ranged from 3.6% to 4.7%. Good linearity was obtained over 0.001-2.0 µg mL(-1) (r=0.9995) with the detection limit (S/N=3) of 0.075 ng mL(-1). Under the selected conditions, enrichment factors of over 90-fold were obtained and extraction on the monolithic column effectively cleaned up the coffee bean matrix. The results demonstrated that the proposed GO-MISPE HPLC-LIF method can be applied to sensitively determine phloxine B in coffee bean.

Determination of synthetic by-products and an intermediate in the colour additives D&C Red Nos 27 and 28 (phloxine B) and their lakes using conventional HPLC.

Specifications in the US Code of Federal Regulations (CFR) for the colour additives D&C Red No. 27 (R27) and D&C Red No. 28 (R28) limit the levels of the synthetic by-products 2,3,4,5-tetrachloro-6-(3´,5´-dibromo-2´,4´-dihydroxybenzoyl)benzoic acid (SBBA) and "brominated resorcinol" and of the intermediate 3,4,5,6-tetrachlorophthalic acid (TCPA). The present study reports the development and application of a conventional HPLC method for the quantitative determination of these impurities in R27, R28 and their lakes. Because the CFR-listed "brominated resorcinol" refers to a group of synthetic by-products, six variously brominated resorcinols were examined as possible impurities. Due to their rapid decomposition in the presence of the dye, the existence of any brominated resorcinols in R27 and R28 is highly unlikely, and this supposition is supported by the results obtained in this study. SBBA, 2,4,6-tribromoresorcinol (Br3R, the brominated resorcinol most likely to be found as an impurity) and TCPA were quantified by using five-point calibration curves with data points (w/w) that ranged from 0.066% to 0.820% for SBBA, from 0.050% to 0.499% for Br3R, and from 0.061% to 1.410% for TCPA. The HPLC method was applied to the analysis of test portions from batches of R27, R28 and their lakes submitted to the USFDA for certification by domestic and foreign manufacturers.

Absorption and elimination of D and C Red No. 28 in male F-344 rats.

D and C Red No. 28 (Red 28) is a US certified color additive used in drugs and cosmetics. Little is known about the extent of systemic absorption and pharmacokinetic behavior of Red 28. Therefore, these studies were performed to determine oral bioavailability and pharmacokinetic parameters of Red 28 in male F-344 rats following single and repeated oral dosing. Rats were administered either a single i.v. dose (50 mg/kg), a low oral gavage dose (50 mg/kg), or a high oral gavage dose (500 mg/kg) of Red 28. Plasma, urine and feces samples were subjected to solid phase extraction (SPE) and analyzed by HPLC for Red 28. Regardless of the dose or route of administration, the terminal t(1/2) of Red 28 was 2.5 h. The major route of elimination was fecal excretion, with 88% (i.v.) and 98% (50 mg/kg p.o.) of the dose recovered by 96 h. Urinary excretion of Red 28 accounted for 1% of the dose following i.v. administration. No Red 28 was detected in urine after p.o. administration. Biliary excretion was determined experimentally to be the primary route of elimination for systemically available Red 28. Bioavailability following p.o. administration was very low (1-2%) and was not altered significantly by 14 days of dietary pretreatment with Red 28.

Method for determination of xanthene dyes in guava fruits and its application in a field dissipation study.

Xanthene dyes, i.e., phloxine B and uranine or phloxine B alone, are phototoxic to tephritid fruit flies infesting guava fruits. An analytical method was developed for determination of residues of these dyes used in bait solutions for suppression of the tephritid fruit fly population in guava fruits. The procedure involved solvent extraction, anion-exchange cleanup, and determination by liquid chromatography or capillary zone electrophoresis. The dyes were extracted from 50 g guava fruit at 45 degrees degrees with 400 mL methanol-acetonitrile (1 + 1) and 5 g magnesium oxide added as an alkaline and clarifying agent. The guava extract was adjusted to pH 8.5 and subjected to an amino column cleanup. Average recoveries of xanthene dyes added to guava purees ranged from 77 to 99% for phloxine B and from 79 to 102% for uranine at spiking levels of 0.05-1.00 microg/g. The method was applied to the determination of phloxine B residues in guava fruits collected from a dye-sprayed orchard. After phloxine B was applied at a rate of 62.5 g/ha for 14 weekly sprayings, it was found on guava fruits at an average concentration of 111 +/- 18 ng/g 4 h after the llth spraying. The concentration of phloxine B was 426 +/- 94 ng/g in selected fruits with high deposits of the dye 4 h after spraying. Average concentrations of phloxine B 5 days after the 7th and 14th sprayings were 29 +/- 7 and 19 +/- 8 ng/g, respectively.

Rapid quantification of hexachlorobenzene in the color additives D&C Red Nos. 27 and 28 (phloxine B) using solid-phase microextraction and gas chromatography-mass spectrometry.

The present paper describes the development of a method for the quantification of hexachlorobenzene (HCB) in the color additives D&C Red Nos. 27 and 28 (phloxine B) using solid-phase microextraction followed by gas chromatography-mass spectrometry (GC-MS) analysis. The method is simple and fast (1 h for each analysis), generates little solvent waste, and does not involve a solid matrix, thus permitting a more efficient extraction than does a previously developed Soxhlet extraction-GC-MS method. Test portions from 30 batches of US-certified color additives D&C Red Nos. 27 and 28 were analyzed for HCB using the new method. Those batches represent domestic (five) and foreign (one) manufacturers that requested certification for the colors during the past four years. All the samples contained HCB, ranging from 0.2 ppm to 244.3 ppm. The analyses revealed significant differences in the levels of HCB across batches from the same manufacturer as well as among different manufacturers. The range of HCB levels found in the analyzed batches (0.2-244.3 ppm) suggest that the contamination with HCB may be decreased by avoiding use of starting material (tetrachlorophthalic anhydride) heavily contaminated with HCB.

Determination of phloxine B and uranine in water by capillary zone electrophoresis.

Phloxine B and uranine are color additives in drugs and cosmetics as well as potential photoactive insecticides. A capillary zone electrophoretic (CZE) method is developed to determine phloxine B and uranine in water. A fused-silica capillary (67 cm, 75-micron i.d.) and borate buffer are used. Migration of phloxine B and uranine increases slightly as the pH of the running buffer increases between the range of 8-9. Although there are only slight effects of ionic strength on the analyte migration in the range of 0-20 mM NaCl in the running buffer, the migration of phloxine B and uranine increases as the percentage of methanol in the samples increases. Methanol shows little effect on the quantitation of phloxine B and uranine. The CZE procedure is applied to determine phloxine B and uranine fortified in tap and stream water samples. Solid-phase extraction is employed to recover the analytes spiked in the water samples. Recoveries range from 87-112% for phloxine B spiked at 10-200 ppb in the tap and stream water. Uranine recoveries are 86-91% at fortification levels of 10-50 ppb in the water samples.

Complementary use of counter-current chromatography and preparative reversed-phase high-performance liquid chromatography in the separation of a synthetic mixture of brominated tetrachlorofluoresceins.

A synthetically prepared mixture of brominated 4,5,6,7-tetrachlorofloresceins was separated by a combination of preparative reversed-phase high-performance liquid chromatography and high-speed counter-current chromatography. Two new lower-brominated subsidiary colors of D&C Red Nos. 27 and 28 (phloxine B), 4',5'-dibromo-4, 5,6,7-tetrachlorofluorescein and 2',4',5'-tribromo-4,5,6, 7-tetrachlorofluorescein, were isolated and characterized by 1H NMR and chemical ionization mass spectrometry.