Temperature-induced dispersive solid-phase extraction as a new approach for preconcentration of Sudan dyes in water and food samples prior to the determination by high-performance liquid chromatography.

This study introduced a new microextraction method named temperature-induced dispersive solid-phase extraction. The performance of the method was demonstrated with the determination of Sudan dyes in food and natural water samples. In this method, a low quantity of sorbent was added to the aqueous solution and the mixture was shaken manually for about one minute. Then, the solution was heated in an ultrasonic water bath, and the sorbent was dissolved. Subsequently, the solution was cooled down with ice water, and consequently, the solubility of the sorbent was reduced in the sample solution and became cloudy. The phase separation was accelerated by centrifugation. The upper liquid phase was picked up using a syringe, and the remainder was solved in methanol and introduced into the HPLC for analysis. Various parameters affecting the extraction yield were evaluated. Analytical parameters, including limits of detection (0.011-0.016 µg/L) and quantification (0.038-0.055 µg/L), relative standard deviations (2.3%-3.1%), and preconcentration factor (40) proved the high efficiency of the developed method for the analysis of Sudan dyes. The proposed method was used to measure Sudan dyes in water and food samples and showed good extraction recoveries (95.0%-103.5%).

Simultaneous Multiplexed Quantification of Banned Sudan Dyes Using Surface Enhanced Raman Scattering and Chemometrics.

Azo compounds such as the Sudan dyes I-IV are frequently used illegally as colorants and added to a wide range of foods. These compounds have been linked to a number of food safety hazards. Several methods have been proposed to detect food contamination by azo compounds and most of these are laboratory based; however, the development of reliable and portable methods for the detection and quantification of food contaminated by these chemicals in low concentration is still needed due to their potentially carcinogenic properties. In this study, we investigated the ability of surface enhanced Raman scattering (SERS) combined with chemometrics to quantify Sudan I-IV dyes. SERS spectra were acquired using a portable Raman device and gold nanoparticles were employed as the SERS substrate. As these dyes are hydrophobic, they were first dissolved in water: acetonitrile (1:10, v/v) as single Sudan dyes (I-IV) at varying concentrations. SERS was performed at 785 nm and the spectra were analyzed by using partial least squares regression (PLS-R) with double cross-validations. The coefficient of determination (Q2) were 0.9286, 0.9206, 0.8676 and 0.9705 for Sudan I to IV, respectively; the corresponding limits of detection (LOD) for these dyes were estimated to be 6.27 × 10-6, 5.35 × 10-5, 9.40 × 10-6 and 1.84 × 10-6 M. Next, quadruplex mixtures were made containing all four Sudan dyes. As the number of possible combinations needed to cover the full concentration range at 5% intervals would have meant collecting SERS spectra from 194,481 samples (214 combinations) we used a sustainable solution based on Latin hypercubic sampling and reduced the number of mixtures to be analyzed to just 90. After collecting SERS spectra from these mixture PLS-R models with bootstrapping validations were employed. The results were slightly worse in which the Q2 for Sudan I to IV were 0.8593, 0.7255, 0.5207 and 0.5940 when PLS1 models (i.e., one model for one dye) was employed and they changed to 0.8329, 0.7288, 0.5032 and 0.5459 when PLS2 models were employed (i.e., four dyes were modelled simultaneously). These results showed the potential of SERS to be used as a high-throughput, low-cost, and reliable methods for detecting and quantifying multiple Sudan dyes in low concentration from illegally adulterated samples.

Highly Selective and Sensitive Benzimidazole Based Bifunctional Sensor for Targeting Inedible Azo Dyes in Red Chilli, Red Food Color, Turmeric Powder, and Cu(Ii) in Coconut Water.

In this study, a simple Benzimidazole based bifunctional chemosensor 4-(2-(3,4-dimethoxyphenyl)-1H-benzo[d]imidazol-6-yl) benzene-1,2-diamine, L was synthesized and characterized. The sensor proved to be selective and sensitive towards detecting banned azo dyes Sudan Dye I, II, and Metanil Yellow via fluorescence turn-off response. The proposed mechanism of fluorescence quenching was the inner filter effect. LODs for Sudan I, II, and Metanil Yellow were found to be 0.009 µM, 0.012 µM, and 0.0073 µM, respectively. The developed chemosensor also showed a colorimetric response towards Cu (II) ions via an apparent color change from yellow to pink. LOD for Cu (II) ions was found to be 1.2 µM. The synthesized benzimidazole based bifunctional chemosensor was adequately tested to determine Sudan I in Red chili powder and red Food color samples, Metanil yellow in turmeric powder, and Cu(II) packaged coconut water.

Preparation of new hydrophobic deep eutectic solvents and their application in dispersive liquid-liquid microextraction of Sudan dyes from food samples.

In this work, a new generation of hydrophobic deep eutectic solvents (DESs) was prepared using eugenol (as hydrogen bond donor) and benzyltriethylammonium bromide, benzyltributylammonium bromide, benzyltriethylammonium chloride and benzyltributylammonium chloride (as hydrogen bond acceptor) in different molar ratios. These DESs were applied to vortex-assisted dispersive liquid-liquid microextraction of Sudan dyes from food samples, followed by high-performance liquid chromatographic determination. The influencing parameters, including the type of DES, amount of DES, extraction time, solution pH and salt addition, were investigated and optimized. Under the optimized conditions, a linear range of 2-1000 ng mL-1 with determination coefficients of <0.999 was obtained. Limits of detection and limits of quantification were in the range of 0.5 to 1 ng mL-1 and 2 to 3 ng mL-1, respectively. The proposed method was successfully used in the determination of Sudan dyes in chili sauce, chili powder and ketchup, and satisfactory recoveries of between 89.9 and 119.3% were obtained, with relative standard deviations in the range of 0.1-6.8%. The proposed method is simple, green and efficient, and can be applied to determine Sudan dyes in complex matrices.

Flower-like layered double hydroxide-modified stainless-steel fibers for online in-tube solid-phase microextraction of Sudan dyes.

Layered double hydroxides are a family of inorganic crystals that have gained a lot of attention due to its special structure and properties such as high porosity, large specific area, and excellent anion exchange ability. In this work, flower-like NiAl-layered double hydroxides with high specific area were in situ immobilized onto the stainless steel fibers by bioinspired polydopamine modification method and packed into poly (ether ether) ketone tube for online solid-phase microextraction with high performance liquid chromatography analysis. Thanks to the high specific surface area and excellent extraction ability of the NiAl-layered double hydroxides, the fibers showed excellent extraction performance to three Sudan dyes with enrichment factors between 260 to 650 folds. After optimization of the reaction and extraction conditions, an online solid-phase microextraction method was developed for determination of Sudan dyes in water samples and chili samples. The method has limits of detection of 0.01 to 0.02 ng/mL, good linearity and good reproducibility (≤1.45%).

Analysis of unauthorized Sudan dyes in food by high-performance thin-layer chromatography.

Food authenticity and food safety are of high importance to organizations as well as to the food industry to ensure an accurate labeling of food products. Respective analytical methods should provide a fast screening and a reliable cost-efficient quantitation. HPTLC was pointed out as key analytical technique in this field. A new HPTLC method applying caffeine-impregnated silica gel plates was developed for eight most frequently found fat-soluble azo dyes unauthorizedly added to spices, spice mixtures, pastes, sauces, and palm oils. A simple post-chromatographic UV irradiation provided an effective sample cleanup, which took 4 min for up to 46 samples in parallel. The method was trimmed to enable 23 simultaneous separations within 20 min for quantitation or 46 separations within 5 min for screening. Linear (4-40 ng/band) or polynomial (10-200 ng/band) calibrations of the eight azo dyes revealed high correlation coefficients and low standard deviations. Limits of detection and quantification were determined to be 2-3 and 6-9 ng/zone, respectively. After an easy sample extraction, recoveries of 70-120% were obtained from chili, paprika, and curcuma powder as well as from chili sauce, curry paste, and palm oil spiked at low (mainly 25-50 mg/kg) and high levels (150-300 mg/kg). For unequivocal identification, the compound in a suspect zone was eluted via a column into the mass spectrometer. This resulted in the hyphenation HPTLC-vis-HPLC-DAD-ESI-MS. Graphical abstract Simplified clean-up by UV irradiation for Sudan dye analysis in food by HPTLC-vis-HPLC-DAD-ESI-MS.

Electrochemiluminescence based competitive immunoassay for Sudan I by using gold-functionalized graphitic carbon nitride and Au/Cu alloy nanoflowers.

A flower-like Au/Cu alloy nanocomposite (Au/Cu NFs) was synthesized and used in an electrochemiluminescence (ECL) based method for sensitive determination of the dye Sudan I. The Au-g-C3N4 nanosheets as an ECL emitter were prepared by electrostatic adsorption between gold nanoparticles and g-C3N4. They form a film on a glassy carbon electrode (GCE) and then can be connected with Sudan I antigen via gold-nitrogen bond and amidation reactions. The Au/Cu NFs combined with Sudan I antibody also via the Au-N bond and was introduced into the modified GCE by specific recognition between the antibody and the antigen. The overlap between emission spectra of the Au-g-C3N4 nanosheets and absorption spectra of Au/Cu NFs enabled the appearance of ECL resonance energy transfer process. That is, when the Sudan I analyte not present, the ECL was weakened due to absorption by the gray Au/Cu NFs on applying voltages from -1.7 V to 0 V. Conversely, the Au/Cu NFs on the GCE are reduced due to the competition for the antibody between the analyte and the antigen. A strong green ECL emission was obtained. The ECL response is linear in the 0.5 pg mL-1 to 100 ng mL-1 Sudan I concentration range, and the detection limit is 0.17 pg mL-1. Graphical abstract An Au/Cu alloy flower-like nanocomposite (Au/Cu NFs) is firstly synthesized as an acceptor to constitute an electrochemiluminescence-resonance energy transfer (ECL-RET) system for sensitive measurement of Sudan I, while Au nanoparticles (Au NPs) functionalized graphitic carbon nitride (g-C3N4) acted as a donor.

Comparison of the toxicity of the dyes Sudan II and Sudan IV to catalase.

The mechanisms of the toxicity of Sudan dyes to the key antioxidant enzyme catalase (CAT) were investigated by spectroscopic methods, calorimetry techniques, enzyme activity assay, and molecular docking. Results showed that Sudan dyes bound to CAT through hydrophobic force, which changed the microenvironment of tryptophan and tyrosine residues, leading to a conformational alteration and shrinkage of the protein. Enzyme activity assay and molecular docking revealed that the activity of CAT was slightly inhibited in the presence of Sudan dyes. In comparison, the binding of Sudan II with CAT was slightly stronger than Sudan IV. Also, Sudan II and Sudan IV showed a different impact on the microenvironment of aromatic amino acid residues. But the dyes had very similar effects on conformation and activity of the protein. This work provides an essential reference for the evaluation of Sudan dyes' effects on body's antioxidant defense system and safe use of Sudan dyes.

Evaluation of a mixed anionic-nonionic surfactant modified eggshell membrane as an advantageous adsorbent for the solid-phase extraction of Sudan I-IV as model analytes.

The coadsorption of mixed anionic-nonionic surfactants, sodium dodecylbenzenesulfonate with Triton X-100, on the surface of eggshell membrane was investigated based on adsorption isotherms to improve the solid-phase extraction performance of eggshell membrane toward organic contaminants. Results showed that even though excess Triton X-100 might inhibit the adsorption of sodium dodecylbenzenesulfonate, a low dosage of Triton X-100 can significantly improve sodium dodecylbenzenesulfonate modification and enhance the extraction efficiency of eggshell membrane from 73.7 to 100.4% because of the formation of mixed hemimicelles. The highest recovery was achieved at 2:8 (Triton X-100/sodium dodecylbenzenesulfonate mass ratios), and multiple mechanisms involving π-π interactions, hydrophobic effect, and π-π electron donor-acceptor interactions contributed to the strong extraction affinity. When mixed, the Triton X-100 and sodium dodecylbenzenesulfonate modified eggshell membrane packed cartridge coupled with high-performance liquid chromatography was applied for the simultaneous determination of trace Sudan I-IV, and low detection limits (0.16-0.26 ng/L) were achieved with satisfactory linearity (R2  > 0.999) in 10-10 000 µg/L. For real samples, Sudan II and III in one chilli sauce sample were found at 4.3 and 1.7 µg/kg. Sudan I-IV recoveries at three spiked levels were 87.4-102.9% with precisions <6.8%. Comparison with commonly used solid-phase extraction adsorbents and methods further reflected the superiorities of the proposed adsorbent in sensitivity, retention ability, and applicability.

New approach applying a pet fish air pump in liquid-phase microextraction for the determination of Sudan dyes in food samples by HPLC.

A new approach applying a pet fish air pump is introduced to develop an extraction method, namely, air-pump-enhanced emulsion, followed by salt-assisted emulsion breaking based on solidified floating organic drop microextraction for the extraction and preconcentration of Sudan I-IV before high-performance liquid chromatography. The applicability of this method was successfully demonstrated by determination of these dyes in four chili products that include chili powder, chili oil, chili sauce, and chili paste. An enrichment factor of 62 was obtained only with a sample solution of 5 mL. A linear range of 0.5-2500 ng/mL was obtained with a limit of detection of 0.16-0.24 ng/mL and recovery of 90-110%. This method is superior to other liquid-liquid extraction methods, as is simple, rapid, environmental friendly, and its phase separation needs no centrifugation. It also needs no disperser solvent and requires less organic solvent, and satisfies the criteria to be called as a green extraction. Therefore, this facile extraction method can be successfully applied in the determination of Sudan dyes in food samples.

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.

Determination of Sudan dyes in chili pepper powder by online solid-phase extraction with a butyl methacrylate monolithic column coupled to liquid chromatography with tandem mass spectrometry.

A poly(butyl methacrylate-co-ethylene dimethacrylate) monolithic column was fabricated and used as a novel sorbent for online solid-phase extraction coupled to liquid chromatography with tandem mass spectrometry for the simultaneous determination of Sudan I-IV in chili pepper powder. The prepared columns were characterized by scanning electron microscopy, nitrogen adsorption-desorption, and pressure drop measurements. Online solid-phase extraction was performed on the synthesized monolithic column using 10 mM ammonium acetate solution as the loading solution with the aid of an online cleanup chromatography system. The desorption of Sudan I-IV was achieved with acetonitrile as the eluting solution at the flow rate of 0.5 mL/min. The extracted analytes were subsequently eluted into a C18 analytical column for chromatographic separation using a mixture of 10% acetonitrile/90% formic acid (0.5%) solution as the mobile phase. Under the optimized conditions, the developed method had linear range of 1.0-50 µg/kg, a detection limit of 0.3 µg/kg, and a quantification limit of 1.0 µg/kg for each analyte. The intraday and interday recoveries of Sudan I-IV in chili pepper powder samples ranged from 94.8 to 100.9% and 94.9 to 99.4%, respectively. The intraday and interday precision were between 3.37-7.01% and 5.01-7.68%, respectively.

In situ ionic-liquid-dispersive liquid-liquid microextraction of Sudan dyes from liquid samples.

In situ ionic-liquid-dispersive liquid-liquid microextraction was introduced for extracting Sudan dyes from different liquid samples followed by detection using ultrafast liquid chromatography. The extraction and metathesis reaction can be performed simultaneously, the extraction time was shortened notably and higher enrichment factors can be obtained compared with traditional dispersive liquid-liquid microextraction. When the extraction was coupled with ultrafast liquid chromatography, a green, convenient, cheap, and efficient method for the determination of Sudan dyes was developed. The effects of various experimental factors, including type of extraction solvent, amount of 1-hexyl-3-methylimidazolium chloride, ratio of ammonium hexafluorophosphate to 1-hexyl-3-methylimidazolium chloride, pH value, salt concentration in sample solution, extraction time and centrifugation time were investigated and optimized for the extraction of four kinds of Sudan dyes. The limits of detection for Sudan I, II, III, and IV were 0.324, 0.299, 0.390, and 0.655 ng/mL, respectively. Recoveries obtained by analyzing the seven spiked samples were between 65.95 and 112.82%. The consumption of organic solvent (120 µL acetonitrile per sample) was very low, so it could be considered as a green analytical method.

Highly sensitive fluoroprobe for detecting Sudan dyes in paprika utilizing carbon dot-embedded zeolitic imidazolate framework-8.

In this manuscript, we synthesized CDs@ZIF-8 through a one-step, in-situ method by integrating green-emitting carbon dots (CDs) with zeolitic imidazolate framework-8 (ZIF-8). The resulting CDs@ZIF-8 was utilized as an ultrasensitive probe for detection, leveraging the inner filter effect. The analysis demonstrated the capability to detect Sudan dyes. Sudan I, for example, could be detected within a concentration range spanning from 0.25 to 70 µM, achieving a remarkable detection limit of 76.56 nM. This established method was effectively employed for detecting Sudan I in paprika. Compared with CDs, CDs@ZIF-8 exhibited a 3.32-fold increase in sensitivity and a wider detection range. This enhanced performance was attributed to the porous ZIF-8, which allowed for the enrichment of targets around CDs and avoided the aggregation of CDs. Additionally, embedding the CDs in ZIF-8 improved their pH stability. Our study provides a new approach for using CDs under limited conditions by leveraging metal-organic frameworks.

Development of a γ-alumina- nanoparticle-functionalized porous polymer monolith for the enrichment of Sudan dyes in red wine samples.

Monolithic materials were synthesized in capillaries by in situ polymerization with N-isopropylacrylamide, glycidyl methacrylate, and ethylene dimethacrylate as the monomers, and methanol and PEG as the porogens. With γ-alumina nanoparticles attached to the surface of the porous monolithic column via epoxide groups, a novel polymer monolith microextraction (PMME) material was prepared with a good mechanical stability and a high extraction capacity. SEM and X-ray photoelectron spectroscopy were employed to characterize the modified monolithic column, demonstrating that γ-alumina nanoparticles were effectively functionalized onto the monolithic column. In addition, a new method was developed for the analysis of Sudan I-IV dyes using PMME coupled with HPLC. In order to obtain the optimum extraction efficiency, the PMME conditions including desorption solvent type, sample pH, sample volume, sample flow rate, and eluent flow rate were investigated. Under the optimum conditions, we obtained acceptable linearities, low LODs, and good intra- and interday RDSs. When applied to the determination of Sudan I-IV dyes in red wine samples, satisfactory recoveries were obtained in the range of 84.0-115.9%.

Ionic-liquid-based dispersive liquid-liquid microextraction for high-throughput multiple food contaminant screening.

This paper describes an innovation of dispersive liquid-liquid microextraction enabling multiple-component analysis of eight high-priority food contaminants in two chemically distinctive families: Sudan dyes and phthalate plasticizers. To provide convenient sample handling for solid and solid-containing matrices, a modified dispersive liquid-liquid microextraction procedure used an extractant precoated frit to perform simultaneous filtration, solvent mixing, and phase dispersion in one simple step. A binary ionic liquid extractant system was carefully tuned to deliver high quality analysis based only on affordable LC with diode array detector instrumentation. The method is comprehensively validated for robust quantification with good precision (6.9-9.8% RSD) in a linear 2-1000 µg/L range. Having accomplished enrichment factors up to 451, the treatment enables sensitive detection at 0.09-1.01 µg/L levels. Analysis of six high-risk solid condiments and sauces further verified its practical applicability within a 70-120% recovery range. Compared to other approaches, the current dispersive liquid-liquid microextraction treatment offers major advantages in terms of minimal solvent (1.5 mL) and sample (0.1 g) consumption, ultra-high analytical throughput (6 min), and the ability to handle complex solid matrices. The idea of performing simultaneous analysis for multiple contaminants presented here fosters a more effective mode of operation in food control routines.

Environmentally-friendly supramolecular solvent microextraction method for rapid identification of Sudan I-IV from food and beverages.

A new ultrasonically assisted supramolecular solvent-based microextraction (UA-SUPRAS-ME) method has been reported. This technique is one of the green methods for rapid microextraction and determination of Sudan I, II, III and IV dyes from food and beverage samples and the study investigated the effects of various parameters such as centrifugation time, 1-octanol volume, pH, supramolecular solvent type, THF volume, ultrasonication time on the optimization of the microextraction process. Addition and recovery of the method were carried out at two different concentrations (10 and 100 ng mL-1) to food and beverage samples and the accuracy of the method was determined. Sudan I dye was detected in red pepper and hot sauce from food samples. Extraction recovery values were found between 90.6 % and 102.5 % which are promising compared to many other methods.

Banned Sudan dyes in spices available at markets in Karachi, Pakistan.

Sudan dyes were investigated in branded and non-branded spices, commonly available in the markets of Karachi, Pakistan. High performance liquid chromatography (HPLC) with a variable wavelength detector (VWD) was applied to determine Sudan dyes I-IV. The non-branded samples had higher concentrations of Sudan dyes than the maximum limits of 0.1 mg/kg. The highest concentration of Sudan dye (I) was found in turmeric powder (8460 mg/kg) and the lowest concentration (1.50 mg/kg) of Sudan (IV) in Chaat Masala. This indicates that the use of non-branded spices is not safe, whereas no Sudan dye was found in the branded spice samples. Further studies regarding the higher carcinogenic risk posed by Sudan dye adulterated spices in Pakistan is strongly advised.

Novel Cu2+-based immobilized metal affinity magnetic nanoparticles for fast magnetic solid-phase extraction of trace Sudan dyes in food samples.

A novel type of Cu2+-based immobilized metal affinity magnetic nanoparticles (MHNTs@PDA@Cu2+) was designed and synthesized by a facile and green strategy, and served as an adsorbent for magnetic solid-phase extraction (MSPE) of trace Sudan dyes in foodstuffs. Owing to π-π stacking, hydrogen bonding, hydrophobic and Cu2+-azo coordination, MHNTs@PDA@Cu2+ possesses superior affinity and very fast adsorption capability to Sudan dyes (adsorption time of 1 min). The main parameters influencing the extraction efficiency of MSPE were investigated and optimized. By coupling MHNTs@PDA@Cu2+-based MSPE with HPLC-UV, a simple, fast and sensitive method for the determination of trace Sudan dyes in food samples were developed. Good linearity (R2 ≥ 0.9937) and low limits of detection (0.06-0.10 µg L-1) were obtained. The method was successfully applied to determine Sudan dyes in juice, soy sauce and red wine, and the recoveries for the spiked samples ranged from 78.1% to 112.4% with RSD less than 9.0%.

Deep eutectic solvents with low viscosity for automation of liquid-phase microextraction based on lab-in-syringe system: Separation of Sudan dyes.

In the work limitations of deep eutectic solvents in the flow-based analysis are discussed. Deep eutectic solvents based on terpenes and fatty acids with low viscosity were studied as extraction solvents for liquid-liquid microextraction into a lab-in-syringe system for the first time. As a result an automated deep eutectic solvent-based microextraction approach was proposed. The procedure involved aspiration of deep eutectic solvent (based on terpene and fatty acid) and aqueous sample solution followed by phases mixing by a magnetic stirrer inside a syringe of flow system. After phase separation the extract phase was transferred from the syringe into a vial followed by analysis by a high-performance liquid chromatography with diode-array detection. The determination of Sudan I, Sudan II and Sudan III in chili-based sauces was considered as an analytical task. The mass-transfer intensification performed by the magnetic stirring inside the syringe allowed to perform fast (2 min) and efficient (extraction recoveries 87-95%) extraction. The limits of detection, calculated from a blank test based on 3σ, were from 0.003 to 0.005 mg kg-1, RSD was <9%. The microextraction procedure did not involve the use of hazardous organic solvents, only 100 µL of natural deep eutectic solvent was required for dyes preconcentration.