Identification, separation by spiral high-speed counter-current chromatography, and quantification of 7-chloro-5-methyl-2H-1,4-benzothiazin-3(4H)-one, an impurity in the thioindigoid color additive D&C Red No. 30 and its lakes.

An impurity in the color additives D&C Red No. 30 (R30) and D&C Red No. 30 lakes (R30L) was newly identified and characterized as 7-chloro-5-methyl-2H-1,4-benzothiazin-3(4H)-one (BTZ), and its extent and level in certified batches of these color additives was determined. BTZ was extracted from the dye with ethanol, resulting in a crude extract enriched to a concentration of over 60%. BTZ was then separated from a portion of the enriched extract by high-speed counter-current chromatography using a spiral-tube assembly column with intermittently pressed tubing of 60 ml capacity. It was the first reported use of such a column to separate a small, moderately hydrophobic compound. The two-phase solvent system was also moderately hydrophobic, consisting of hexane-ethyl acetate-methanol-water (5:2:5:2), and the retention of the organic stationary phase measured after the separation was 83.3%. The separation yielded BTZ of two purity grades, the higher of which (~95.5%) was used as a standard to quantify the impurity in 37 batches of R30 and R30L using an HPLC method developed and validated for that purpose. Analyses revealed a wide range of BTZ levels across batches, <0.05 - 0.84%, and suggested that BTZ contamination could be reduced by appropriate adjustments in the manufacturing process. An explanation of the likely source of BTZ - as a side-reaction product in a particular step of the manufacturing process - was also presented.

Determination of sulphonated quinophthalones in Quinoline Yellow and its lakes using high-performance liquid chromatography.

Quinoline Yellow (QY, Colour Index No. 47005) is internationally used as a colour additive in foods, drugs, and cosmetics. The manufacture of QY requires sulphonating quinophthalone, and depending on the degree of sulphonation, various forms of QY result, containing different proportions of quinophthalone mono-, di-, and trisulfonic acid sodium salts (monoSA, diSA, and triSA, respectively). Regulations on the specific composition and uses of QY differ across countries with associated differences in names for QY. The QY form certified for use in the U.S. in drugs and cosmetics is known as D&C Yellow No. 10 (Y10). The Code of Federal Regulations (CFR) specifies that Y10 and its lakes consist of predominantly monoSA's, the sum of whose levels is ≥ 75%, and that the sum level of diSA's is ≤ 15%, with one of them (6'8'diSA) at ≤ 3%. The present work reports the development of an HPLC method for determining those CFR-specified values and the level of a non-CFR-specified component, 6'8'5triSA. The selected analytes, 6'SA, 6'5diSA, 6'8'diSA, and 6'8'5triSA, were quantified by using five-point-calibration curves (R2 > 0.999) with data-point ranges of 9.96-96.53%, 0.54-21.69%, 0.10-5.00%, and 0.11-5.53% by weight, respectively. The method was found to be precise (relative standard deviation values, 0.55-0.80%) and accurate (recovery values, 91.07-99.45%). LOD and LOQ values, respectively, were as follows: 1.23 and 3.70%, 6'SA; 0.42 and 1.26%, 6'5diSA; 0.11 and 0.34%, 6'8'diSA; and 0.01 and 0.04%, 6'8'5triSA. The HPLC method was applied successfully to the analysis of 20 Y10 and eight Y10 lake samples. It can be extended to other QY forms such as E104 and Yellow 203 because it enables analysis of 6'8'5triSA. This paper also addresses the implications of the varying structure depictions and CAS numbers of the QY components that are due to the existence of three tautomeric forms of quinophthalone.

Determination of Seven Manufacturing Impurities in FD&C Red No. 40 by Ultra-High-Performance Liquid Chromatography.

BACKGROUND: The U.S. Food and Drug Administration batch-certifies color additives to ensure that each lot meets published specifications for coloring food, drugs, and cosmetics. OBJECTIVE: An ultra-high-performance LC (UHPLC) method was developed to determine seven manufacturing impurities in the monoazo color additive FD&C Red No. 40 (R40). The analytes consist of two intermediates, an impurity originating from one intermediate, a reaction by-product, and three subsidiary colors. The intermediates are 4-amino-5-methoxy-2-methylbenzenesulfonic acid [cresidine-p-sulfonic acid (CSA)] and 6-hydroxy-2-naphthalene sulfonic acid sodium salt (SS). The impurity originating from the intermediate SS is 6,6'-oxybis[2-naphthalenesulfonic acid] disodium salt. The reaction by-product is 4,4'-(diazoamino)bis[5-methoxy-2-methylbenzenesulfonic acid disodium salt. The subsidiary colors are sodium salts of CSA coupled with 2-naphthol-3,6-disulfonic acid, 2-naphthol-6,8-disulfonic acid, or 2-naphthol. METHODS: Samples of R40 were dissolved in an ammonium acetate buffer modified to pH 9.2, filtered, and analyzed by UHPLC. Quantitation of the analytes was performed by calibration in the presence of the color additive matrix. RESULTS: UHPLC validation studies showed linear calibration curves (R2 = 0.9999), good recovery (95-121%) and precision (RSDs = 1.0-6.3%), and LOQs ranging from 0.002 to 0.030%. Survey analyses of 31 samples from 11 manufacturers yielded results by the new UHPLC method and a previously used HPLC method that are consistent within experimental error. CONCLUSIONS: The new UHPLC method provides faster analysis time, improved separation, and similar sensitivity compared to the HPLC method. HIGHLIGHTS: An UHPLC method was developed and validated to determine seven manufacturing impurities in R40 submitted to the FDA for batch certification.

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

The 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 4',5'-dibromofluorescein; 9.8-44.1% for 2',4',5'-tribromofluorescein; 1.01-15.2% for 2',4',5',7'-tetrabromofluorescein; 0.10-3.12% for 2',4'-dibromofluorescein; 0.10-3.06% for 2',5'-dibromofluorescein; 0.11-2.85% for 4'-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.

Separation using high-speed counter-current chromatography and identification of 1,3-bis(4-phenylazophenyl)triazene, an impurity in the color additive D&C Red No. 17 (Sudan III).

The present work describes the application of high-speed counter-current chromatography to the preparative separation of a previously unreported impurity in the color additive D&C Red No. 17 (R17, Colour Index No. 26100, Sudan III). Due to the hydrophobic nature of the impurity, a hydrophobic two-phase solvent system (hexane-ethanol-water, 5:4:1) was used for its separation. The separated impurity was chemically characterized by spectroscopic methods as a disazo triazene, 1,3-bis(4-phenylazophenyl)triazene (PAPT). This impurity was synthesized and used as a reference material to quantify it in 15 batches of the color additive produced by various domestic and foreign manufacturers and certified by the U.S. Food and Drug Administration (FDA). Analysis of test portions by high-performance liquid chromatography showed a range of PAPT levels, from "not detected" (<0.006%) to 0.70%, across batches. The variability suggests that contamination by PAPT can be decreased or eliminated through manufacturing modifications. A chemical pathway for PAPT formation and an associated adjustment to minimize it during the process of manufacturing R17 are proposed.

Identification and quantification of the decarboxylated analogue of Pigments Red 57 and 57:1 in the color additives D&C Red No. 6, D&C Red No. 7, and their lakes, using a chelating agent and UHPLC.

Pigment Red 57 (Colour Index No. 15850, PR57) and Pigment Red 57:1 (Colour Index No. 15850:1, PR57:1) are certifiable in the USA as the color additives D&C Red No. 6 (R6) and D&C Red No. 7 (R7) for use in drugs and cosmetics. In the EU, PR57:1 is permitted in cosmetics and also as a food additive (E180) for colouring edible cheese rinds. The USFDA batch-certifies R6, R7, and their lakes in accordance with limiting specifications for impurities stated in the Code of Federal Regulations (CFR). In the current work, an impurity not specified in the CFR was studied because of its consistent presence in samples of R6 and R7 submitted for certification. Using spectroscopic methods, the impurity was tentatively identified as 4-[(4-methyl-2-sulfophenyl)azo]-3-naphthalenol (DPR57), the decarboxylated analogue of PR57 and PR57:1. Its identity was confirmed by synthesising DPR57 and determining that the UHPLC retention time, UV/visible spectrum and mass spectrum of the synthetic material were identical to those of the impurity. Using the synthesised DPR57 as a reference material, the impurity was quantified in 43 batches of R6, R7, and lakes produced by eight different manufacturers. Calibration curves ranging from 0.02% to 1.00% (w/w) were prepared by plotting the UHPLC area of DPR57 at 485 nm against its concentration. DPR57 levels ranged from < 0.02% to 0.50%. To facilitate dissolution of the color additive samples for DPR57 analysis, a relatively simple procedure was developed by adapting a previously published method that involves use of a basic solution of the chelating agent EDTA and the organic solvent N,N'-dimethylformamide. A source for DPR57 contamination of the color additives is also proposed.

Ultra-Performance Liquid Chromatographic Determination of Manufacturing Intermediates and Subsidiary Colors in D&C Red No. 6, D&C Red No. 7, and Their Lakes.

An ultra-performance LC (UPLC) method was developed to determine the manufacturing intermediates and subsidiary colors in the monosulfo monoazo color additives D&C Red No. 6 and D&C Red No. 7 and their lakes. This method is intended for use in batch certification of the color additives by the U. S. Food and Drug Administration to ensure that each lot meets published specifications for coloring drugs and cosmetics. The intermediates are 2-amino-5-methylbenzenesulfonic acid (PTMS) and 3-hydroxy-2-naphthalenecarboxylic acid (3-hydroxy-2-naphthoic acid). The subsidiary colors are 3-hydroxy-4-[(4-methylphenyl)azo]-2-naphthalenecarboxylic acid (unsulfonated subsidiary color) and 1-[(4-methylphenyl) azo]-2-naphthalenol (4-methyl Sudan I). The analytes were identified by comparing their UPLC retention times and UV-Vis absorption spectra with those of standards. Validation studies showed that calibration curves were linear (average R2=0.9994), and recoveries were 96-106%. Average LOD was 0.0014-0.0061% and average LOQ was 0.0047-0.020%. Results for RSD at the specification levels ranged from 0.67 to 5.79%. Survey analyses of 42 samples from 14 domestic and foreign manufacturers yielded results by the new UPLC method and a previously reported HPLC method that were consistent within experimental error. The new UPLC method provided increased sensitivity, faster analysis times, and improved separations compared to the HPLC method.

Quality Characteristics and Shelf-Life of Ultra-High Pressure Homogenized (UHPH) Almond Beverage.

The effects of ultra-high-pressure homogenization (UHPH) at 200 MPa, in combination with different inlet temperatures (55 or 75 °C) during storage at 4 °C were studied and compared with pasteurized (90 °C, 90 s) almond beverage. Microbiological analysis of the physical (particle sedimentation and color) and volatile profile of the most relevant compound in almond beverages was performed at days 1, 7, 14, and 21 of cold storage. UHPH treatment 200 at 75 °C led to higher microbiological reduction after treatment and higher stability during cold storage in almond beverages than pasteurization or UHPH 200 at 55 °C. Physical characteristics of UHPH-treated samples exhibited a high stability during storage with a stable color. Volatile compounds extracted by solid-phase microextraction were identified by gas chromatography coupled with mass spectrometry. The effect of UHPH treatment significantly (p < 0.05) affected the volatile profile compared with pasteurized beverages, although UHPH conditions applied produced similar effects in almond beverages. Benzaldehyde was the most abundant compound detected in all treatments. Hexanal was more abundant in UHPH-treated samples, indicating a higher lipid oxidation compared to pasteurized almond beverages.