Accurate mass measurements for the confirmation of Sudan azo-dyes in hot chilli products by capillary liquid chromatography-electrospray tandem quadrupole orthogonal-acceleration time of flight mass spectrometry

The potential of capillary liquid chromatography (microLC)-quadrupole/time-of-flight mass spectrometry (Q-TOF MS) for the confirmation of Sudan I, II, III and IV azo-dyes as contaminants in hot-chilli food products was demonstrated. Using the microLC-electrospray ionization (ESI)-Q-TOF MS technique, accurate mass measurements of Sudan dyes were performed both on standard solutions and on matrices. Precision of exact mass measurements was calculated taking into account the ion statistics according to the number of ion sampled in the measurement. Accurate mass measurements by MS/MS experiments were performed to elucidate azo-dye fragmentation patterns. Selectivity of the microLC-Q-TOF MS method was assessed by evaluating matrix suppression effects by pre-column injection of blank hot chilli tomato sauce matrices. The results were compared with those obtained on a LC-triple quadrupole-MS system. Confirmation of Sudan I present in hot chill tomato sauce samples was obtained by accurate mass measurements. In real samples trueness of exact mass measurements was estimated to be 1.6 and 4.4 ppm when calculated for hot chilli tomato sauce and hot chilli tomato with cheese sauce samples, respectively; precision was calculated around 9.5 ppm.     

Development and optimization of an analytical method for the determination of Sudan dyes in hot chilli pepper by high-performance liquid chromatography with on-line electrogenerated BrO- -luminol chemiluminescence detection

The determination of four Sudan dyes by means of high-performance liquid chromatography (HPLC) with chemiluminescence (CL) detection was proposed. The method was based on the enhancement effect of Sudan dyes on the chemiluminescence reaction between luminol and BrO-, which was on-line electrogenerated by constant current electrolysis. The separation was carried out on Nucleosil RP-C18 column (250 mm x 4.6 mm i.d., 5 microm, pore size, 100 A) at 35 degrees C. The mobile phase consisted of a V (methanol): V (0.2% aqueous formic acid) = 90:10 solution. At a flow-rate of 1.0 ml min(-1), the total run time was 25 min. The effects of several parameters on the HPLC resolution and CL emission were studied systematically. For the four Sudan dyes, the limits of detection (LOD) at a signal-to-noise of 3 ranged from 4 to 8 microg kg(-1) and the limits of quantification (LOQ) at a signal-to-noise of 10 ranged from 13 to 27 microg kg(-1). The relative standard deviations (RSD) of intra-and inter-day precision were below 4.4%. The average recoveries for all four Sudan dyes (spiked at the levels of 1.0 and 1.5 mg kg(-1)) in chilli tomato sauce and hot chilli pepper ranged from 94% to 105%, and the relative standard deviations of the quantitative results were from 2.5 to 4.2%. The proposed method had been successfully applied to the determination of four Sudan dyes in hot chilli products.     

Development of a method for the analysis of seven banned azo-dyes in chilli and hot chilli food samples by pressurised liquid extraction and liquid chromatography with electrospray ionization-tandem mass spectrometry

An automated, confirmatory and sensitive procedure has been developed and validated for the determination of Sudan (I-IV), Sudan Orange G, Sudan Red 7B and Para Red in hot chilli food samples. The proposed method includes pressurised liquid extraction (PLE) with acetone, gel permeation chromatography (GPC) clean-up and detection by liquid chromatography (LC) coupled to electrospray ionization in positive mode tandem mass spectrometry (ESI-MS-MS). The main parameters affecting the performance of the different ionization sources and PLE parameters were previously optimised using statistical design of experiments (DOE). The method was in-house validated on chilli powder and chilli meat. Linear calibrations were obtained with correlation coefficients R² > 0.999. The limits of detection (LOD) and quantification (LOQ) of the method were in the ranges of 0.002-0.012 ng g⁻¹ and 0.006-0.036 ng g⁻¹, respectively for chilli powder. The decision limit and detection capability were between 0.005-0.022 ng g⁻¹ and 0.007-0.026 ng g⁻¹, respectively for chilli meat. Recoveries ranged from 94% to 105%. The applicability of the method to the determination of azo-dyes in hot chilli products was demonstrated.     

Determination of banned 10 azo-dyes in hot chili products by gel permeation chromatography-liquid chromatography-electrospray ionization-tandem mass spectrometry

An accurate method based on the use of gel permeation chromatography (GPC)-liquid chromatography-tandem mass spectrometry interfaced with electrospray ionization (GPC-LC-ESI-MS/MS) was devised for the simultaneous determination of Sudan (I-IV), Sudan Orange G, Sudan Red B, Sudan Red G, Sudan Red 7B, Butter Yellow and Para Red in hot chili products. A GPC clean-up procedure was developed for simultaneous quantification of 10 dyes in hot chili and hot chili products avoiding some interference and permitting multiple injections without damaging the column. A HPLC was performed on an Inertsil C18 column using a multistep gradient elution with 0.1% formic acid and methanol as the mobile phase. Mass spectral acquisition was done in positive ion mode. Linearity of around three orders in the magnitude of concentration was generally obtained with the correlation coefficients (r2) of 0.9984-0.9997. Limit of detection (LOD) and limit of quantification (LOQ) for the investigated dyes were in the ranges of 0.1-1.8 and 0.4-5.0 microg/kg depending on matrices, respectively. The recoveries of the 10 synthetic dyes in five matrices ranged from 81.7 to 92.9%. The intra- and inter-day precision (RSDs) was between 2.9-7.8 and 3.9-8.1%, respectively. This method has been applied successfully for the determination of the studied 10 banned dyes in hot chili products.     

Enzyme-linked immunosorbent assay (ELISA) using a specific monoclonal antibody as a new tool to detect Sudan dyes and Para red

To set up an immunoassay-based method to detect Sudan dyes and Para red, we generated a monoclonal antibody (Mab) using a specially designed carboxyl derivative of Sudan I (CSD I) as the immunogen. CSD I was synthesized by azocoupling reaction using 2-naphthol and diazotised 4-aminobenzoic acid. The antibody was obtained from a hybridoma, which was derived from the fusion of the mouse myeloma SP2/0 cells and the splenocytes from the mice immunized with the CSD I-bovine serum albumin (BSA) conjugate. In addition, we showed that the Mab was highly specific for Sudan I, III and Para red. The limit of detection was approximately 0.01 ng mL⁻¹ in phosphate-buffered saline (PBS) buffer and 0.5 ng g⁻¹ in chilli tomato sauce. The recoveries of Sudan I, III and Para red for the chilli tomato sauce were from 84% to 99% and coefficients of variation were from 14.9% to 33.3%. Thus, the enzyme-linked immunosorbent assay (ELISA) method is a rapid and high throughput screening tool to detect Sudan dyes and Para red in food products.     

Sensitive and fast determination of Sudan dyes in chili powder by partial-filling micellar electrokinetic chromatography-tandem mass spectrometry

A fast and sensitive method for the simultaneous determination of Sudan dyes (I, II, III, and IV) in food samples was developed for the first time using partial filling micellar electrokinectic chromatography-mass spectrometry (MEKC-MS). The use of MEKC was essential to achieve the separation of these neutral analytes, while the partial filling technique was necessary to avoid the contamination of the ion source with non-volatile micelles. MEKC separation and MS detection conditions were optimized in order to achieve a fast, efficient, and sensitive separation of the four dyes. Filling 25% of the capillary with an MEKC solution containing 40 mM ammonium bicarbonate, 25 mM SDS, and 32.5% (v/v) acetonitrile, a baseline separation of the four azo-dyes was obtained in 10 min. Tandem MS was investigated in order to improve the sensitivity and selectivity of the analysis. Limits of detection (LOD) values 5, 8, 15, and 29 times better were obtained for Sudan III, I, II, and IV, respectively, using partial filling MEKC-MS/MS instead of partial filling MEKC-MS. Under optimized conditions, LOD from 0.05 to 0.2 μg/mL were obtained. The suitability of the developed method was demonstrated through the fast and sensitive determination of Sudan I, II, III, and IV in spiked chilli powder samples. This determination could not be achieved by MEKC-UV due to the existence of several interfering compounds from the matrix.     

Rapid method for determination of chlormequat residues in tomato products by ion-exchange liquid chromatography/electrospray tandem mass spectrometry

A rapid method has been devised for the direct determination of chlormequat in tomato samples. No clean-up is required, and analysis uses ion-exchange liquid chromatography/tandem mass spectrometry interfaced with electrospray ionization (LC/ESI-MS/MS). A cation-exchange column was used with an aqueous ammonium acetate/acetonitrile mixture as the mobile phase under isocratic conditions. The method was validated in terms of detection limits (LOD), quantitation limits (LOQ), linearity, recovery, precision and accuracy. Good results in the low micro g kg(-1) level were obtained for the LOD and LOQ of chlormequat in tomato samples. Comparison of solvent and matrix-matched calibration curves demonstrated the absence of significant matrix effects and the feasibility of using external calibration. Linearity was established over two orders of magnitude by performing homoscedasticity and Mandel fitting statistical tests. The absence of both constant and proportional systematic errors was verified by evaluating the recovery function, demonstrating good method accuracy. Excellent precision in terms of intra-day repeatability was calculated (RSD% <3.4). Extraction recoveries from tomato products were calculated, by using a labelled internal standard (d(4)-chlormequat), to be in the 93 +/- 5-99 +/- 7% range. The applicability of the method to the determination of chlormequat residues in tomato products was demonstrated.     

A cloud point extraction approach using Triton X-100 for the separation and preconcentration of Sudan dyes in chilli powder

A CPE-HPLC (UV) method was developed for the determination of Sudan (I-IV) dyes, non-ionic surfactant Triton X-100 was used to extract and preconcentrate Sudan dyes from chilli powder prior to their determination by HPLC-UV. The separation and determination of Sudan dyes was then carried out in the HPLC-UV system with isocratic elution, and the detector was set at 500 nm. The parameters and variables that affect the extraction were investigated. Under optimum conditions: 3% of Triton X-100 (W/V), 10% of Na₂CO₃ (W/V), heat-assisted at 70 °C for 30 min. Recoveries of the Sudan dyes ranged from 80.70% to 85.45% in chilli powder by CPE method, with all the relative standard deviations of less than 3%. Limit of detection (LOD) and limit of quantification (LOQ) were in the range of 2.0-4.0 and 7.0-12.0 μg kg⁻¹ in chilli powder, respectively.     

Ionic liquids extraction of Para Red and Sudan dyes from chilli powder, chilli oil and food additive combined with high performance liquid chromatography

A simple analytical method, based on the coupling of ionic liquid-based extraction with high performance liquid chromatography (HPLC), is developed for the determination of Sudan dyes (I, II, III and IV) and Para Red in chilli powder, chilli oil and food additive samples. Two ionic liquids (ILs), 1-butyl-3-methylimidazolium hexafluorophosphate ([C₄mim][PF₆]) and 1-octyl-3-methylimidazolium hexafluorophosphate ([C₈mim][PF₆]), were compared as extraction solvents; experiments indicated that the latter possesses higher recoveries for each analyte. Parameters related to extraction of Sudan dyes and Para Red were also optimized. Under the optimal conditions, good reproducibility of extraction performance was obtained, with the relative standard deviation (RSD) values ranging from 2.0% to 3.5%. The detection limits of Sudan dyes and Para Red (LOD, S/N = 3) were in the range of 7.0-8.2 μg kg⁻¹ for chilli powder and 11.2-13.2 μg L⁻¹ for chilli oil and food additive. The recoveries were in the range of 76.8-109.5% for chilli powder samples and 70.7-107.8% for chilli oil and food additive samples.     

On-Line SPE Coupled with LC for Analysis of Traces of Sudan Dyes in Foods

We report a sensitive and simple method for analysis of traces of Sudan dyes in foods in which solid-phase extraction on activated silica gel for preconcentration was combined, on-line, with high-performance liquid chromatography. With a loading flow rate of 1.7 mL min^?1 for sampling 50 mL Sudan I–IV at pH 6.7, enrichment factors ranging from 196 to 991 were achieved. Detection limits (S/N = 3) of Sudan I–IV were in the range 1.4–7.0 ng L^?1, and the relative standard deviation for repeatability of peak areas in five replicate analyses of 0.01 μg L^?1 Sudan I–IV was 2.2–4.5%. When blank food samples (chilli powder, chilli sauce, and duck eggs) were spiked with Sudan III at two levels (0.25 and 0.50 μg L^?1) then analyzed by this method recovery ranged from 70.3 to 95.2%.     

Development of a new method for analysis of Sudan dyes by pressurized CEC with amperometric detection

A new analytical method, pressurized CEC (pCEC) with amperometric detection (AD) using 1.5 microm RP nonporous silica packed columns has been developed for the rapid separation and determination of four Sudan dyes in hot chilli. The influence of several experimental parameters on the retention behavior has been investigated. The electrochemical oxidation of Sudans I-IV separated by pCEC can be reliably monitored with a carbon electrode at +0.95 V (vs. Ag/AgCl). Fast and efficient separation of the analytes was achieved within 7 min by pCEC under the optimum conditions with an ACN/water (95:5%) mobile phase containing formic acid (pH 4.3), 5% acetone and 0.002% triethylamine using a separation voltage of 12 kV. The detection limits for four Sudan dyes ranged from 8.0 x 10(-7) to 1.2 x 10(-6) mol/L. To evaluate the feasibility and reliability of this method, the proposed pCEC-AD method was further demonstrated with hot chilli samples spiked with Sudan dyes.     

Analysis of illegal dyes in food by LC/TOF-MS

A sensitive and accurate methodology was developed for the analysis of seven illegal dyes (Sudan I, Sudan II, Sudan III, Sudan IV, Sudan Orange G, Sudan R and Para Red,) used as additives in food products, such as chilli powder and steak sauces. The analytical methodology consisted of solvent extraction with acetonitrile followed by liquid chromatography time-of-flight mass spectrometry detection. Accurate mass measurements were crucial in order to achieve a high degree of specificity for the target analytes in such complex samples. The dyes were effectively extracted from spice and sauce matrices achieving recoveries higher than 75%. Because of the excellent mass accuracy obtained for the target analytes (better than 2?ppm), no cleanup of the samples was required using this methodology, thus leading to a better precision and reproducibility of the results from the quantitative point of view. Calibration curves were linear and covered two orders of magnitude (from 0.01 to 1?mg?L^?1) for all the compounds studied with the exception of Para Red. A detailed study of matrix effects is also included in this work, showing a clear improvement when dilution of the extracts was carried out. Method detection limits were in the low mg?kg^?1 range, and the precision, calculated as the relative standard deviation, ranged from 5 to 15%. The methodology was successfully applied to market samples in a survey performed as part of a regional research programme organized by the Andalusian Health Service in Spain, and a positive confirmation for Sudan I was obtained in a chilli powder sample.     

Molecularly imprinted polymer coated solid-phase microextraction fiber prepared by surface reversible addition–fragmentation chain transfer polymerization for monitoring of Sudan dyes in chilli tomato sauce and chilli pepper samples

Surface reversible addition-fragmentation chain transfer (RAFT) polymerization method was firstly applied to the preparation of molecularly imprinted polymer (MIP) coated silicon solid-phase microextraction (SPME) fibers. With Sudan I as template, an ultra-thin MIP coating with about 0.55-μm thickness was obtained with homogeneous structure and controlled composition, due to the controllable radical growing and chain propagation in surface RAFT polymerization. The MIP-coated fibers were found with enhanced selectivity coefficients (3.0–6.5) to Sudan I–IV dyes in contrast with those reported in our previous work. Furthermore, the ultra-thin thickness of MIP coating was helpful to the effective elution of template and fast adsorption/desorption kinetics, so only about 18 min was needed for MIP-coated SPME operation. The detection limits of 21–55 ng L⁻¹ were achieved for four Sudan dyes, when MIP-coated SPME was coupled with liquid chromatography (LC) and mass spectrometry (MS) detection. The MIP-coated SPME–LC–MS/MS method was tested for the monitoring of ultra trace Sudan dyes in spiked chilli tomato sauce and chilli pepper samples, and high enrichment effect, remarkable matrix peaks-removing capability, and consequent high sensitivities were achieved to four Sudan dyes.     

Simultaneous determination of Sudan dyes and carotenoids in red pepper and tomato products by HPLC

To simultaneously detect Sudan dyes and carotenoids in red pepper and tomato products, high-performance liquid chromatography (HPLC) methods with photodiode-array detection are developed and validated. The methods include the use of end-capped and nonend-capped adsorbents with a gradient elution system starting with water containing methanol. Water content of 9% in the starting mobile phase is found to be necessary to ensure sufficient separation of Sudan dyes and to avoid overlapping or interference with the carotenoids of considerable content. The data of the validation reveal the accuracy and precision of the developed methods. A limit of Sudan dyes detection of 1-5 microg/g in red pepper or tomato sauce could be approached. The methods provide excellent separation of the carotenoids from the unsaponified extracts of red pepper and the tomato products.     

Enantioselective separation and determination of the dinotefuran enantiomers in rice,tomato and apple by HPLC

An effective chiral analytical method was developed for the resolution and determination of dinotefuran enantiomers in rice, tomato and apple samples. Dinotefuran enantiomers were baseline‐separated and determined on a novel chiral column, ChromegaChiral CCA, with n‐hexane–ethanol–methanol (85:5:10, v/v/v) as the mobile phase at a flow rate of 1.0 mL/min with UV detection at 270 nm. The resolution of dinotefuran enantiomers was about 1.8. The first eluted enantiomer was (+)‐dinotefuran and the second eluted one was (?)‐dinotefuran. The effects of mobile‐phase composition and column temperature on the enantioseparation were evaluated. The method was validated for linearity, repeatability, accuracy, LOD and LOQ. LOD was 0.15 mg/kg in rice and tomato, 0.05 mg/kg in apple, with an LOQ of 0.5 mg/kg in rice and tomato, 0.2 mg/kg in apple. The average recoveries of the pesticide from all matrices ranged from 75.8 to 92.9% for all fortification levels The precision values associated with the analytical method, expressed as RSD values, were <16.5% for the pesticide in all matrices. The methodology was successfully applied for the enantioselective analysis of dinotefuran enantiomers in real samples, indicating its efficiency in investigating the environmental stereochemistry of dinotefuran in food matrix.     

Rapid fluorescence assay for Sudan dyes using polyethyleneimine-coated copper nanoclusters

We report that the intensity of the blue fluorescence of copper nanoclusters coated with polyethyleneimine (PEI) is strongly reduced in the presence of the food dyestuffs Sudan I-IV. This finding was exploited in a label-free fluorescence assay for these Sudan dyes both in ethanol and aqueous solutions. The PEI-capped nanoclusters have an average diameter of 1.8 nm and are displaying, under 355 nm excitation, a blue emission at 480 nm that matches the absorption bands of the Sudan dyes. The clusters are stable in solution for at least 1 month. Under optimum conditions, this assay can be applied to the quantification of the dyes Sudan I, II, III, and IV, respectively, in the 0.1?30, 0.1–30, 0.1–25, and 0.1–25 μM concentration ranges, and the detection limits (3σ/slope) are 65, 70, 45, and 50 nM, respectively. The capability of reducing the fluorescence of the PEI-capped copper nanoclusters is directly related to the number of the functional groups in that Sudan III and IV give lower detection limits. This analytical scheme exhibits a remarkably high selectivity for the Sudan dyes over potentially interfering substances. The method was successfully applied to determine Sudan I, II, III, and IV in hot chilli powder.

Effective extraction and simultaneous determination of Sudan dyes from tomato sauce and chili‐containing foods using magnetite/reduced graphene oxide nanoparticles coupled with high‐performance liquid chromatography

A simple, effective, and robust magnetic solid‐phase extraction method was developed using magnetite/reduced graphene oxide nanoparticles as the adsorbent for the simultaneous determination of Sudan dyes (I, II, III, and IV) in foodstuffs. The magnetite/reduced graphene oxide nanoparticles were characterized by X‐ray diffraction, scanning electron microscopy, and vibrating sample magnetometry. The extraction parameters including extraction time, elution solution, and elution time and volume were investigated in detail. Such magnetite/reduced graphene oxide nanoparticles based magnetic solid‐phase extraction in combination with high‐performance liquid chromatography and variable wavelength detection gave the detection limits of 3–6 μg/kg for Sudan I–IV in chili sauce, tomato sauce, chili powder, and chili flake samples. The recoveries were 79.6–108% at three spiked levels with the intra‐ and inter‐day relative standard deviations of 1.2–8.6 and 4.5–9.6%, respectively. The feasibility was further performed by a comparison with commercial alumina‐N. This method is suitable for the routine analysis of Sudan dyes due to its sensitivity, simplicity, and low cost.     

腐殖酸键合硅胶固相萃取-液相色谱法测定辣椒粉及辣椒油中的苏丹红

以腐殖酸键合硅胶作为固相萃取介质,建立了固相萃取柱净化、高效液相色谱同时     

Interference-free determination of Sudan dyes in chilli foods using second-order calibration algorithms coupled with HPLC-DAD

This paper presents a new method for the determination of Sudan dyes contained in hot chilli samples. The method employs second-order calibration algorithms to handle the recorded data. The second-order calibration algorithms are based on the popular parallel factor analysis (PARAFAC), alternating trilinear decomposition (ATLD) and self-weighted alternating trilinear decomposition (SWATLD), respectively. These chemometric methodologies have the second-order advantage, which is the ability to get accurate concentration estimates of interested analytes even in the presence of uncalibrated interfering components. The results on a set of spiked chilli test shows that low contents of Sudan I and Sudan II in complex chilli mixtures can be accurately determined using the new method. The sample preparation was based on solvent extraction, and internal standard was not required. Quantification was carried out with simple mobile phase.     

Development and single-laboratory validation of a reversed-phase liquid chromatography-electrospray-tandem mass spectrometry method for identification and determination of acrylamide in foods

A rapid and accurate method using reversed-phase liquid chromatography-tandem mass spectrometry interfaced with electrospray was developed for determination of acrylamide in cooked food samples. A simplified sample treatment procedure using an extraction step with acidified water without cleanup was developed. A C18 column with an aqueous formic acid-methanol mixture as the mobile phase was used under isocratic conditions. The method was validated in-house for robustness, limits of detection (LOD) and quantitation (LOQ), linearity, recovery, and accuracy both on standard and baked-product and potato flour matrixes. Good results in the low ppb level were obtained for LOD (< 15 microg/kg) and LOQ (< 25 microg/kg) of acrylamide in samples. Excellent linearity (r2 = 0.999-1.000) was established over 2 orders of magnitude by performing statistical tests. The absence of both constant and proportional systematic errors demonstrated good method accuracy. Excellent results were obtained for intraday repeatability (RSD < 1.5%) and between-day precision (RSD < 5%). Extraction recoveries from food products were calculated in the 97 +/- 3-99 +/- 2% (n = 6) range with a labeled internal standard (13C3-acrylamide). The applicability of the method to determination of acrylamide in cooked food products was demonstrated.