Identification of 2-bromo-3,4,5,6-tetrachloroaniline and its quantification in the color additives D&C Red Nos. 27 and 28 (phloxine B) using solid-phase microextraction and gas chromatography-mass spectrometry

The present work describes (a) the identification and characterization of a contaminant, 2-bromo-3,4,5,6-tetrachloroaniline (2BTCA), in the color additives D&C Red Nos. 27 and 28 (phloxine B) and (b) the determination of the extent and level of 2BTCA contamination in certified lots of these colors. For these purposes, 2BTCA (a compound not previously reported in the literature) and its positional isomer 4-bromo-2,3,5,6-tetrachloroaniline (4BTCA) were synthetically prepared. 4BTCA was used as the internal standard for the quantification of 2BTCA in the colors. Test portions from 35 certified lots of D&C Red Nos. 27 and 28 were analyzed for 2BTCA using a solid-phase microextraction-GC-MS method. Those lots were submitted for certification by both domestic (seven) and foreign (four) manufacturers during the past 4 years. Of the test portions analyzed, 22 (62.9%) contained 2BTCA in amounts ranging from 0.15 to 435.7 ppm with an average value of approximately 131.7 ppm. The remaining 13 (37.1%) test portions contained no detectable 2BTCA or less than 0.01 ppm, which is the limit of quantification of the present method. The analyses revealed substantial differences in the level of 2BTCA across lots from the same manufacturer as well as among different manufacturers. The wide range of 2BTCA levels found in the analyzed lots suggests that the presence of 2BTCA in D&C Red Nos. 27 and 28 may be avoided or significantly reduced during the manufacturing process. A direct correlation was observed between the presence of 2BTCA and that of 3,4,5,6-tetrachlorophthalic acid in analyzed batches of D&C Red Nos. 27 and 28. A chemical pathway that could explain the presence of 2BTCA in these color additives, and ways to avoid its formation, are also proposed.     

Identification of alkyl carbazoles and alkyl benzocarbazoles in Brazilian petroleum derivatives

The present work describes (a) the identification and characterization of an impurity, 2,4,5,7-tetrabromo-6-hydroxy-9-(2,3,4,5-tetrachlorophenyl)-3H-xanthen-3-one (BCPX), in the color additives D&C Red Nos. 27 and 28 (phloxine B) and (b) the determination of the extent and level of BCPX contamination in certified lots of these colors. For these purposes, BCPX (a compound not previously reported in the literature) was synthetically prepared. Test portions from 42 certified lots of D&C Red Nos. 27, 28 and 27 lakes were analyzed for BCPX using an HPLC method that included gradient elution and UV-vis photodiode array detection. Those lots were submitted for certification by both domestic (six) and foreign (six) manufacturers during the past 4 years. Of the test portions analyzed, 32 (76.2%) contained BCPX in amounts ranging from 0.01 to 3.21%. The remaining 10 test portions (23.8%) contained no detectable BCPX or less than 0.008%, which is the limit of quantification for the present method. The analyses revealed substantial differences in the level of BCPX across different manufacturers. The wide range of BCPX levels found in the analyzed lots suggests that the presence of BCPX in D&C Red Nos. 27 and 28 may be avoided or significantly reduced during the manufacturing process.     

Direct method for determination of Sudan I in FD&C Yellow No. 6 and D&C Orange No. 4 by reversed-phase liquid chromatography

A reversed-phase liquid chromatographic method was developed to determine parts-per-million and higher levels of Sudan 1, 1-(phenylazo)-2-naphthalenol, in the disulfo monoazo color additive FD&C Yellow No. 6 and in a related monosulfo monoazo color additive, D&C Orange No. 4. Sudan I, the corresponding unsulfonated monoazo dye, is a known impurity in these color additives. The color additives are dissolved in water and methanol, and the filtered solutions are directly chromatographed, without extraction or concentration, by using gradient elution at 0.25 mL/min. Calibrations from peak areas at 485 nm were linear. At a 99% confidence level, the limits of determination were 0.008 microg Sudan I/mL (0.4 ppm) in FD&C Yellow No. 6 and 0.011 microg Sudan I/mL (0.00011%) in D&C Orange No. 4. The confidence intervals were 0.202 +/- 0.002 microg Sudan I/mL (10.1 +/- 0.1 ppm) near the specification level for Sudan I in FD&C Yellow No. 6 and 20.0 +/- 0.2 microg Sudan I/mL (0.200 +/- 0.002%) near the highest concentration of Sudan I found in D&C Orange No. 4. A survey was conducted to determine Sudan I in 28 samples of FD&C Yellow No. 6 from 17 international manufacturers over 3 years, and in a pharmacology-tested sample. These samples were found to contain undetected levels (16 samples), 0.5-9.7 ppm Sudan I (0.01-0.194 microg Sudan I/mL in analyzed solutions; 11 samples including the pharmacology sample), and > or =10 ppm Sudan I (> or = 0.2 microg Sudan I/mL; 2 samples). Analyses of 21 samples of D&C Orange No. 4 from 8 international manufacturers over 4 years found Sudan I at undetected levels (8 samples), 0.0005 to < 0.005% Sudan I (0.05 to < 0.5 microg Sudan I/mL in analyzed solutions; 3 samples, including a pharmacology batch), 0.005 to <0.05% Sudan I (0.5 to <5 microg Sudan I/mL; 9 samples), and 0.18% Sudan I (18 microg Sudan I/mL; 1 sample).     

Preparative purification of tetrabromotetrachlorofluorescein and phloxine B by centrifugal counter-current chromatography.

A centrifugal counter-current chromatographic method for preparative purification of commercial tetrabromotetrachlorofluorescein and Phloxine B (D&C Red Nos. 27 and 28, respectively) was developed. Ethyl acetate-n-butanol-0.01 M ammonium acetate (1:1:2) was used as the two-phase solvent system. Each purification trial involved 50 mg of sample and yielded 22 mg (+/- 2 mg) of pure dye. The purity of the product was measured by high-performance liquid and thin-layer chromatography and was found to be 99.9%. The partition coefficients of these compounds were found to be highly concentration-dependent in the two-phase solvent system used. If this problem can be circumvented, then the counter-current chromatographic method can be extended for use with gram quantities of dye.     

Determination of 1,3-diphenyltriazene and azobenzene in D&C Red No. 33 by solvent extraction and reversed-phase high-performance liquid chromatography

Data are presented for the determination of trace levels of 1,3-diphenyltriazene (DPT) and azobenzene (AB) in D&C Red No. 33. The contaminants are extracted with chloroform from an aqueous solution of the color, and the chloroform is removed under vacuum. The residue is dissolved in acetonitrile and the solution is analyzed by reversed-phase high-performance liquid chromatography (HPLC) with detection at 365 nm. DPT and AB were determined at levels of less than or equal to 439 ppb and less than or equal to 2.2 ppm, respectively, in samples of commercial D&C Red No. 33. Method precision for the DPT determination (five analyses) was 6.8% relative standard deviation. The UV-Vis spectra of the HPLC analytes, obtained by using a rapid-scan diode-array spectrophotometer, were used to confirm the identity of the DPT and AB responses produced during analysis of the D&C Red No. 33 extracts. The method is also useful for the separation of cis- and trans-AB.     

Ultra-performance liquid chromatographic determination of manufacturing intermediates and subsidiary colors in D&C Red No. 34 and its lakes

An ultra-performance liquid chromatography (UPLC) method was developed to determine the manufacturing intermediates and subsidiary colors in the monosulfo monoazo color additive D&C Red No. 34 and its lakes. This method is currently used for 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 new UPLC method has replaced an HPLC method for determining the intermediates and a TLC method for determining the subsidiary colors. The intermediates are 2-amino-1-naphthalenesulfonic acid (Tobias acid) and 3-hydroxy-2-naphthalenecarboxylic acid (3-hydroxy-2-naphthoic acid). Subsidiary colors are positional isomers of the major dye component or related compounds containing lower numbers of substituent groups. The analytes are identified by comparison of their UPLC retention times and UV or visible absorption spectra with those of standards. Validation studies showed that peak area calibrations for the analytes were generally linear (R > 0.999), and recoveries were 98-103%. The LODs were 0.002-0.02%, and the RSDs at the specification levels were 0.7-2.2%. Survey analyses of 12 samples of certified D&C Red No. 34 straight colors and lakes from six domestic and foreign manufacturers yielded results for the intermediates by UPLC and HPLC that were consistent within experimental error. The UPLC analyses yielded results for the subsidiary colors that were consistently lower than results previously obtained by TLC, which we attribute to limitations of the TLC method. The new UPLC method provides sharper peaks, better peak separation, and faster analysis times than the formerly used HPLC method and is more accurate, much faster, and much less labor-intensive than the formerly used TLC method.     

Automated microwave digestion of certifiable color additives for determination of mercury by cold vapor atomic absorption spectrometry

A procedure using an automated microwave flow digestion technique was developed and validated for the digestion of samples of certifiable color additives before mercury determination by cold vapor atomic absorption spectrometry. Recovery studies were performed by spiking most of the color additives subject to batch certification by the U.S. Food and Drug Administration with inorganic mercury (HgNO3) and with organic mercury (CH3HgCl). Successful recoveries of 72-113% Hg added at the 1 microg/g level were obtained. A method detection limit of 0.2 microg Hg/g was estimated from a Hg-spiked FD&C Yellow No. 6 sample. At the specification level of 1 ppm Hg (1 microg Hg/g), the 95% confidence interval was +/- 0.2 ppm (0.2 microg Hg/g).     

Determination of mercury in carbon black by cold vapor atomic absorption spectrometry

Recently, a new color additive, D&C Black No. 2, a high-purity furnace black in the general category of carbon blacks, was listed as a color subject to batch certification by the U.S. Food and Drug Administration. A simple procedure was developed to determine mercury (Hg) in D&C Black No. 2, which is limited by specification to not more than 1 ppm Hg. The method uses partial acid digestion followed by cold vapor atomic absorption and was developed by modifying a method used for other color additives. The carbon black samples are treated with a mixture of nitric and hydrochloric acids and heated by microwave in sealed Teflon vessels. The resulting solutions, which are stable to Hg loss for at least 1 week, are diluted and analyzed for Hg using cold vapor atomic absorption spectrometry. Validation was performed by spiking carbon black samples with inorganic Hg (HgNO3) at levels from 0.1 to 1.5 microg/g, and by analyzing 2 standard reference materials. At the specification level of 1 ppm Hg (1 microg Hg/g), the 95% confidence interval was +/-0.01 ppm Hg (0.01 microg Hg/g). The method developed in this study gave good results for very difficult-to-analyze materials, such as coal standard reference materials and carbon black. By eliminating volatility and adsorption factors through the formation of HgCl4(-2) complexes, one can avoid using extremely hazardous acids such as HF and HClO4.     

Arsenic determination in certifiable color additives by dry ashing followed by hydride generation atomic absorption spectrometry

The preparations of digested samples of certifiable color additives by dry ashing and wet digestion for arsenic analysis by hydride generation atomic absorption spectrometry (AAS) were compared. The dry ashing technique was based on the preparation used in ASTM D4606-86 for determination of As and Se in coal. The acid digestion method used nitric and sulfuric acids heated by microwaves in sealed vessels. The digested color additives were analyzed for As by using hydride generated from sodium borohydride mixed with the acidified solution on a flow injection system leading to an atomic absorption spectrometer. Dry ashing was preferable to wet digestion because wet digestion yielded poor recoveries of added As. Dry ashing followed by hydride generation AAS gave determination limits of 0.5 ppm As in the color additives. At a specification level of 3 ppm As, the precision of the method using dry ashing was +/- 0.4 ppm (95% confidence interval).     

Isolation, characterization and determination of trace organic impurities in FD&C red no. 40

The unsulfonated aromatic amine 4-nitro-p-cresidine (2-methoxy-5-methyl-4-nitrobenzenamine) was identified as an impurity in the regulated color additive FD&C Red. No. 40. The compound was isolated from the water-soluble color by extraction with chloroform, followed by transfer of the free amines to acid solution and subsequent separation by reversed-phase high-performance liquid chromatography. The 4-nitro-p-cresidine was collected and then identified by gas chromatography-mass spectrometry. The levels of 4-nitro-p-cresidine as well as p-cresidine and aniline were determined in commercial batches of FD&C Red. No. 40.     

Spectrophotometric determination of germaninm(IV) with bromopyrogallol red

A method is proposed for the spectrophotometric determination of germanium with Bromopyrogallol Red. A red-violet coloured complex is formed at pH 2-3, with a stoichiometry equivalent to Ge(BPR)(2), and a molar absorptivity of 20.5 x 10(3) at 550 mmu. Beer's law is obeyed over the range 0.2-3 ppm. Germanium in cupriferous ores has been determined by the method.     

Determination of sulphonate dyes in water by ion-interaction high-performance liquid chromatography

An ion-interaction high-performance liquid chromatography method for quick separation and determination of the sulphonated dyeAcid Yellow 1, and the sulphonated azo dyes Acid Orange 7, Acid Orange 12, Acid Orange 52, Acid Red 2, Acid Red 26, Acid Red 27 and Acid Red 88 has been developed. An RP-ODS stationary phase is used, and the mobile phase contains an acetonitrile-phosphate buffer (27:73, v/v) mixture at pH 6.7, containing 2.4 mM butylamine as ion-interaction reagent. Good separations were obtained using isocratic elution and spectrophotometric detection at 460 nm. The detection limits for the eight dyes ranged from 7 to 28 microg/l for an injection volume of 100 microl. Spiked tap water samples (100 ml), containing different concentration levels (0.3-1.2 microg/l) of the dyes were analyzed after acidification (pH 3) and preconcentration in disposable solid-phase extraction C18 cartridges.     

Spectrophotometric determination of nickel with biacetyl bis(4-phenyl-3-thiosemicarbazone)

A simple method is described for the rapid spectrophotometric determination of nickel with biacetyl bis(4-phenyl-3-thiosemicarbazone). The yellow color is measured at 460 nm (? = 22,800 liters mol^?1 cm^?1) against a reagent blank at pH 2.5 in a solution containing 60% ($\text{v}\text{v}$) dimethylformamide. Beer's law is obeyed over the range 0.2–2 ppm of nickel.     

Qualitative and Quantitative Analysis for the Quality Control of Rhizoma Coptidis by HPLC-DAD and HPLC-ESI-MS

For quality control purpose, an approach of fingerprinting and simultaneous quantification of five major bioactive constituents of Rhizoma Coptidis was established via a high-performance liquid chromatograph coupled with a photodiode array UV detector(HPLC-DAD) and an electrospray ionization mass spectrometer(HPLC-ESI/MS). The compounds were identified on the basis of the comparison of their mass spectra with literature data and those of standard samples and quantified by the HPLC-DAD method. Baseline separation was achieved on an XTerra C 18 column(5 μm, 250 mm×4.6 mm i. d.) with linear gradient elution of formate buffer(consisting of 0.5% formic acid, adjusted to pH=4.5 with ammonia) and acetonitrile(consisting of 0.2% formic acid and 0.2% triethylamine). The me-thod was validated for linearity(r 2 0.9995), repeatability(RSD3.1%), intra-and inter-day precision(RSD1.8%) with recovery(99.9%―105.1%), limits of detection(0.15―0.35 μg/mL), and limits of quantification(0.53―0.82 μg/mL). The similarities of 32 batches of Rhizoma Coptidis and their classification according to their manufacturers were based on the retention time and peak areas of the characteristic compounds. The five compounds were selected for quality assessment of Rhizoma coptidis via partial least squares analysis(PLS).     

六味地黄丸的精细指纹图谱分析及模式识别分类研究

采用高效液相色谱建立了六味地黄丸的指纹图谱,对两个厂家的16批产品进行了测定,并结合中药相似度软件和主成分分析法对全指纹图谱和其精细指纹图谱进行了模式识别研究。结果表明,中药相似度软件能够对不同厂家的产品进行区分但也可能造成误判;在主成分分析法的投影图中,两个厂家的产品明显聚为两类,而且不同批次产品的差异也能够显示出来。     

A new method of analysis of peroxydisulfate using ion chromatography and its application to the simultaneous determination of peroxydisulfate and other common inorganic ions in a peroxydisulfate matrix

A new method for the determination of peroxydisulfate using ion chromatography has been developed. Elution of peroxydisulfate was effected by isocratic elution using 200 mM NaOH at 40°C. A modification of the method using gradient elution was able to simultaneously determine other common inorganic ions (nitrate, nitrite, sulfate and chloride) down to significantly low concentrations in a peroxydisulfate matrix. The relative standard deviations (RSD) were in the range of 0.5-5%, for peak areas and <0.2% for peak retention times. The recoveries were between 95% and 120% for a concentration range of about 0.5-42 ppm. The limit of detection for peroxydisulfate ion was 0.2 ppm and for the other ions were ≤2×10(-2) ppm. The calibration curves were linear with slope and intercepts close to 1 and 0, respectively.     

6S,8R-Stereochemistry of the C27- and C29-alkane-6,8-diols isolated from three compositae flowers

The Deltadelta (deltaS-deltaR) values for the C-1 methyl 1H signals in the 1H-NMR spectroscopy of the bis-MTPA esters of four synthetic stereoisomers of alkane-6,8-diols, viz., bis-MTPA esters of (6S,8R)-C27- (1a) and C29- (3a) (Deltadelta = -0.05 ppm), (6R,8S)-C27- (2a) and C29- (4a) (Deltadelta = +0.05 ppm), (6S,8S)-C27- (5a) (Deltadelta = -0.01 ppm), and (6R,8R)-C27- (6a) (Deltadelta = +0.01 ppm) alkane-6,8-diols, made it possible to differentiate unequivocally among the four stereoisomers. This allowed the determination of the (6S,8R)-stereochemistry (Deltadelta = -0.05 ppm for the bis-MTPA esters) for the natural C27- and C29-alkane-6,8-diols isolated from the flowers of three Compositae plants, Carthamus tinctorius, Cynara cardanclus, and Taraxacum platycarpum.     

A new reagent for the spectrophotometric microdetermination of cadmium

2,2′-Dipyridyl-2-pyridylhydrazone (DPPH) allows a simple, rapid, and sensitive spectrophotometric microdetermination of cadmium in aqueous solution. The yellow 1:2 metal-to-ligand complex formed has a molecular extinction coefficient of 5.5 × 10⁴ liters mole^?1 cm^?1 at the absorption maximum of 444 nm. The determination of cadmium is carried out at pH 12.3 ± 0.2. Beer's law is obeyed over the concentration range of 0.2 to 2 ppm and the Sandell sensitivity of the color reaction is 0.002 μg of cadmium/cm² for an absorbance of 0.001.     

Micellar electrokinetic chromatography fingerprinting combined with chemometrics as an efficient strategy for evaluating the quality consistency and predicting the antioxidant activity of Lianqiao Baidu pills

An approach combining micellar electrokinetic chromatography fingerprinting with chemometrics was developed to evaluate the quality consistency of Lianqiao Baidu pills, which are traditional Chinese patent medicines composed of 19 herbs used mainly to treat skin ulcers, common cold, rheumatism, herpes, and constipation. The triangle optimization method was employed to choose a satisfactory background electrolyte, with the information index, I , as an objective function for assessing the capillary electrophoresis conditions. Then, under the optimal conditions, the micellar electrokinetic chromatography fingerprints of 28 batches of samples were established, and five marker compounds were quantitatively determined simultaneously. A limited‐ratio quantified fingerprint method was introduced to evaluate the chromatographic fingerprints both qualitatively and quantitatively. Principle component analysis revealed that the 28 batches of samples can be clustered according to different manufacturers. Moreover, the relationship between the fingerprint and the antioxidant activity was explored by orthogonal partial least‐squares regression, which provided critical medicinal efficacy information for quality control. The present study establishes a powerful and reliable method for monitoring the quality consistency of Lianqiao Baidu pill.     

Development of a monitoring tape for phosgene in air

A porous cellulose tape impregnated with a processing solution that includes 4-p-nitroben-zylpyridine, N-benzylaniline and methanol is a highly sensitive means of detecting phosgene and maintains stable sensitivity for at least three months in air in a desiccator. When the sample including phosgene was passed through the tape, the color of tape changed to red. The degree of color change was proportional to the concentration of phosgene at a constant sampling time and flow rate. The degree of color change could be recorded by measuring the intensity of reflecting light (555 nm). The detection limit was 6 ppb for phosgene with a sampling time of 60 sec and a flow rate of 400 ml/min. Reproducibility tests showed that the relative standard deviation of response (n = 10) was 2.6% for 0.2 ppm phosgene. No interference was observed from ethanol (1 vol.%), trichloroethylene (1 vol.%), acetone (1 vol.%), carbon dioxide (4.9 vol.%), carbon monoxide (100 ppm), nitrogen dioxide (100 ppm), sulfur dioxide (50 ppm), hydrogen chloride gas (5 ppm), chlorine (3 ppm), acetic acid gas (24 ppm), ammonia (40 ppm), or benzyl chloride (20 ppm).