MS using a volatile surfactant

Food Chemistry 310 (2020) 125963

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Determination of Sudan dyes in chili products by micellar electrokinetic chromatography-MS/MS using a volatile surfactant

T

David Moreno-González , Pavel Jáč, František Švec, Lucie Nováková ⁎

Department of Analytical Chemistry, Faculty of Pharmacy in Hradec Králové, Charles University, Akademika Heyrovského 1203, 500 05 Hradec Králové, Czech Republic

ARTICLE INFO

ABSTRACT

Keywords: Sudan dyes Chili products Micellar electrokinetic chromatography Mass spectrometry Sweeping Matrix effect

A new MEKC-MS/MS method was developed for the determination of four Sudan dyes in chili products. The separation and MS detection conditions were optimized to achieve fast, efficient, selective, and sensitive determination of Sudan I, Sudan II, Sudan III, and Sudan IV dyes. The target compounds were extracted from chili samples with acetonitrile and cleaned by freeze-out. This two-step sample preparation led to excellent extraction efficiency and minimal matrix effect. The analytical performance of the method was very good, with r2 ≥ 0.9914 and limits of quantification lower than 22 μg kg−1. The precision was below 15.7%. The recovery for spiked samples ranged from 84.4 to 99.6%, with relative standard deviations less than 8.0%. For all evaluated samples, the matrix effects did not exceed ± 10%. The applicability of the proposed method was demonstrated with 20 chili products, two of which were found to contain Sudan I and IV residues.

1. Introduction Sudan dyes are synthetic phenylazo derivatives commonly used as colorants due to their low price and better availability compared to natural pigments (Hunger et al., 2000). Azo dyes are employed in many industrial and scientific applications, such as the coloring of fuels and plastics and microscopy staining (Bafana, Devi, & Chakrabarti, 2011). Many azo dyes including Sudan I, Sudan II, Sudan III, and Sudan IV are classified as carcinogenic and mutagenic compounds by the International Agency for Research on Cancer (IARC, 1987), and thus their presence in foodstuff is unacceptable worldwide. Nevertheless, occasionally these colorants are illegally used in products such as chili powder, paste, and sauce to enhance the visual aesthetics and promote sales (Nisa, Zahra, & But, 2016). Specifically, the European Union has banned their use in chili products since 2004 (European Commission, 2004) to protect the consumers. The European Union Rapid Alert System for Food and Feed has reported the presence of Sudan I and IV in certain chili products. Most of the alerts concern chili products originating from non-European countries, with the detected levels of 2.8–3500 mg kg−1 (RASFF, 2019). Therefore, it is desirable to develop sensitive and selective methods for the determination of Sudan dyes in chili products. Several analytical approaches have been proposed for the separation of Sudan dyes, including high-performance liquid chromatography (HPLC) (Cornet, Govaert, Moens, Loco, & Degroodt, 2006; Qi, Zeng,

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Wen, Liang, & Zhang, 2011) and supercritical fluid chromatography (Khalikova, Šatínský, Solich, & Nováková, 2015). HPLC coupled with mass spectrometry (MS) is generally applied for the determination of Sudan dyes in chili products (Genualdi et al., 2016; Li et al., 2013; Schummer, Sassel, Bonenberger, & Moris, 2013; Zhao et al., 2012). Meanwhile, capillary electrophoresis (CE) is seldom used for the separation of Sudan dyes, even though it has several advantages over HPLC including high efficiency, low sample consumption, and reduced waste generation. Sudan dyes are weak acids with pKa values around 11.5. Thus, they can be considered neutral compounds in a wide range of pH (González, Gallego, & Valcárcel, 2003), and micellar electrokinetic chromatography (MEKC) is the best choice to achieve complete separation of these compounds. A MEKC method with UV detection has already been reported for the analysis of Sudan dyes (Mejia, Ding, Mora, & Garcia, 2007). Nevertheless, UV detection lacks adequate selectivity to enable the unequivocal identification of these dyes in complex matrices. While MS detection appears to be a better choice, the main obstacle in its coupling to MEKC systems is the low volatility of commonly used surfactants such as sodium dodecyl sulfate (SDS), causing ion-source contamination and low ionization efficiency that result in the loss of sensitivity. To overcome this problem, Fukuji et al. proposed a partialfilling approach to make SDS compatible with MS detection (Fukuji, Castro-Puyana, Tavares, & Cifuentes, 2011, 2012). In the partial-filling approach, a part of the capillary is filled with the micellar media, while

Corresponding author. E-mail address: [email protected] (D. Moreno-González).

https://doi.org/10.1016/j.foodchem.2019.125963 Received 12 June 2019; Received in revised form 4 November 2019; Accepted 26 November 2019 Available online 04 December 2019 0308-8146/ © 2019 Elsevier Ltd. All rights reserved.

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phenylazophenylazo)-2-naphthalenol, purity 90.0%), and Sudan IV (1[[2-methyl-4-[(2-methylphenyl)azo]phenyl]azo]-2-naphthalenol, purity 80.0%), which chemical structures are shown in Fig. SM1, were from Sigma-Aldrich. Sodium chloride (ACS reagent, purity ≥ 99.0%) and perfluorooctanoic acid (96%) were purchased from Sigma-Aldrich. Bulk C18 silica and primary secondary amine (PSA) sorbents were supplied by Agilent Technologies (Waldbronn, Germany). Supel™ QuE Z-Sep+ sorbent was from Supelco (Bellafonte, Palo Alto, USA). Nylon syringe filters (0.22 μm × 13 mm, Agilent Technologies) were used to filter sample extracts prior to injection into the MEKC-MS/MS system.

the rest accommodates the background electrolyte free of non-volatile micelles. However, the selectivity and the separation efficiency could be compromised due to migration of the analytes through the zone interface (Amini, Paulsen-Sörman, & Westerlund, 1999). A very promising approach for direct coupling of MEKC with MS is switching to a volatile surfactant such as ammonium perfluorooctanoate (APFO). This fluorocarbon-based surfactant allows researchers to overcome the main difficulties related to non-volatile surfactants without compromising the efficiency and selectivity (Moreno-González et al., 2017). This MSfriendly surfactant has already shown its benefits in the determination of pesticides (Moreno-González, Huertas-Pérez, García-Campaña, & Gámiz-Gracia, 2015), amino acids (Moreno-González et al., 2013), estrogenic compounds (D'Orazio, Asensio-Ramos, Hernández-Borges, Rodríguez-Delgado, & Fanali, 2015), and explosives (Brensinger et al., 2016) by MEKC‐MS. Sample treatment is another crucial point for obtaining reliable results. A wide variety of approaches were proposed to isolate these Sudan dyes from the matrix interferences (Rebane, Leito, Yurchenko, & Herodes, 2010). They include centrifugal sedimentation (Mejia et al., 2007), hollow fiber-liquid phase microextraction (Yu, Liu, Lan, & Hu, 2008), solid phase extraction (Li et al., 2013; Qi et al., 2011), gel permeation chromatography (Zhu et al., 2014), liquid-solid extraction (Calbiani, Careri, Elviri, Mangia, & Zagnoni, 2004; Cornet et al., 2006; Schwack, Pellissier, & Morlock, 2018), and ultrasound-assisted extraction (UAE) (Khalikova et al., 2015; Ma, Luo, Chen, Su, & Yao, 2006). The last technique represents several advantages over other sample treatments, including faster extraction, less solvent consumption, and better extraction efficiency of tightly bound compounds that are not easily released by conventional techniques (Pico, 2013). As a generic extraction method, it has the drawback of containing a relatively large number of co-extracted compounds in the final extract. As a consequence, significant matrix effect can be observed when using electrospray ionization, resulting in the need for additional sample cleanup. To this end, freeze-out clean-up represents an effective option to remove co-extracted lipids as well as other components with limited solubility in polar organic solvents (Liu et al., 2018). Lipids in the organic extract can be readily isolated from the target analytes due to their lower melting point compared to Sudan dyes. Therefore, freezeout clean-up allows a significant reduction of the matrix effect with only limited impact on the extraction efficiency. The aim of this work is to develop a fast, easy, and sensitive method for the simultaneous determination of Sudan dyes (I, II, III, and IV) in chili products such as powder, sauce, and paste by MEKC-MS/MS using APFO as the volatile surfactant. Moreover, the combination of UAE and freeze-out clean-up to isolate Sudan dyes from the matrix is proposed. To the best of our knowledge, this is the first application of the MEKCMS/MS method using APFO as surfactant for the quantitative analysis of Sudan dyes in chili products. Our new methodology was successfully applied to detect Sudan dyes in commercial chili products.

2.2. Preparation of solutions Individual stock standard solutions of each Sudan dye at 500 mg L−1 were prepared by dissolving accurately weighed amounts of each compound in THF. Intermediate stock standard solutions (100 mg L−1) were obtained by diluting the individual stock standard solutions with THF. All standard solutions were stored in dark vials at 4 °C. An aqueous solution of APFO (150 mmol·L−1) was obtained by titrating the appropriate amount of perfluorooctanoic acid with 5 mmol·L−1 ammonium hydroxide to pH 9.0. The background electrolyte (BGE) consisted of 60:20:10:10 (v/v/v/v) APFO solution/ACN/ MeOH/THF. The sample solvent was a mixture of 60:20:10:10 (v/v/v/ v) water/ACN/MeOH/THF. 2.3. Instrumentation MEKC-MS/MS experiments were carried out using a 7100 CE System (Agilent Technologies) coupled to a 6495 triple quadrupole mass spectrometer with iFunnel technology and jet stream electrospray ionization (AJS-ESI) (Agilent Technologies) using a nebulizer ESI source. The sheath liquid was delivered by an Agilent 1260 infinity II series isocratic pump equipped with a 1:100 flow splitter. Data acquisition and analysis were done by MassHunter workstation software (Version B.07.00, Agilent Technologies). 2.4. MEKC conditions Before first use, the new capillary was rinsed with 1 mol·L−1 NH4OH for 10 min, followed by deionized water for 10 min and then BGE for 20 min. At the beginning of each day, the capillary was preconditioned with 0.5 mol·L−1 NH4OH for 3 min, deionized water for 3 min, and finally the running buffer for 10 min. Before each run, the capillary was flushed with BGE for 5 min to reach adequate run-to-run repeatability. At the end of each day, the capillary was washed with water for 5 min, and dried with air for another 5 min. A voltage of +25 kV was applied for the electrophoretic separation. The temperature of the capillary was 25 °C. The sample was hydrodynamically injected for 50 s at 5000 Pa. 2.5. Mass spectrometry conditions

2. Experimental section

The mass spectrometer worked in the ESI positive mode using selected reaction monitoring (SRM) mode. The selected transitions and optimized collision energies are shown in the supplementary material (Table SM1). The sheath liquid consisted of 50:49.9:0.1 (v/v/v) IPA/ water/formic acid and was delivered at a flow rate of 10 µL min−1. The ESI parameters were: capillary voltage 3000 V, nebulizer pressure 206.8 kPa, gas flow 15 L min−1, gas temperature 250 °C, sheath gas temperature 100 °C, sheath gas flow 6 L min−1, nozzle voltage 2000 V, high pressure RF 200 V, and low pressure RF 80 V. To obtain a stable electric current, the steps of preconditioning and sample injection were carried out with the nebulizer pressure and the ESI voltage set at zero value.

2.1. Chemicals and reagents Acetonitrile (ACN), methanol (MeOH), isopropanol (IPA), formic acid, acetic acid, and ammonia solution (25%), all of LC-MS grade, were supplied by Sigma Aldrich (St. Louis, MO, USA). Ethanol, acetone, tetrahydrofuran (THF), and ethyl acetate, all CHROMASOLV™ HPLC 99.9% solvents, were also obtained from Sigma Aldrich. Ultrapure water produced by a Milli-Q plus system (Millipore, Bedford, MA, USA) was used throughout the work. Reference standards of Sudan I (1(phenylazo)-2-naphthalenol, purity 97.0%), Sudan II (1-[(2,4-dimethylphenyl)azo]-2-naphthalenol, purity 90.0%), Sudan III (1-(4-

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Fig. 1. Effect of the organic modifiers in the BGE: a) 60:40 (v/v) 150 mM APFO at pH 9.0 /ACN, b) 60:30:10 (v/v/v) 150 mM APFO at pH 9.0/ACN/THF, c) 60:30:10 (v/v/v) 150 mM APFO at pH 9.0 /ACN/MeOH, and d) 60:20:10:10 (v/v/v/v) 150 mM APFO at pH 9.0 /ACN/ MeOH/THF.

electroosmotic flow (EOF), the negatively charged APFO micelles migrate towards the cathode and enable the separation of neutral analytes from each other and the EOF. Also, Sudan dyes are very hydrophobic compounds with octanol/water partition coefficients higher than 5.5 (Abraham, Amin, & Zissimos, 2002). Thus, the addition of an organic solvent into the BGE was necessary to increase their solubility in the aqueous electrolyte. The literature shows that ACN can serve this purpose (Fukuji et al., 2011, 2012; Mejia et al., 2007). Therefore, BGE consisting of 60:40 (v/v) 100 mmol·L−1 APFO solution/ACN was selected as the starting point, and the MEKC separation parameters to be optimized were: pH of the aqueous component of BGE, APFO concentration, and the qualitative and quantitative composition of the organic modifier. The resolution, signal intensity, and analysis time were considered as response variables to select the optimal composition of BGE. The effect of pH was studied in the alkaline region between 8.5 and 10 to achieve sufficient magnitude and reproducibility of EOF (Viglio, Fumagalli, Ferrari, & Ladarola, 2010). However, the pH showed no significant effect on the selectivity in the studied range. When the pH values were higher than 9.0, a slight increase in the migration times was observed. This can be caused by the reduction of EOF as a consequence of the increased ionic strength of BGE. Additionally, since Sudan dyes are neutral in the studied pH range, their effective electrophoretic mobilities were not affected by the change in pH as they migrate only through their partitioning in the negatively charged pseudostationary phase. So, pH 9.0 was selected as a compromise between separation efficiency and analysis time (data not shown). Next, the effect of the APFO concentration between 25 and 200 mmol·L−1 was studied at pH 9.0. As expected, all compounds migrated without separation in a single zone when a concentration of 25 mmol·L−1 was used (Fig. SM2a). Since the critical micelle concentration (CMC) of APFO is 25 mmol·L−1 (Wang, Yan, Xing, Jin, & Xiao, 2010), the micelles were not fully formed at that concentration. However, similar behavior was also observed at 50 mmol·L−1 APFO (Fig. SM2b). Although this concentration exceeds the CMC, the use of 40% ACN as organic modifier in BGE caused disintegration of the micellar pseudostationary phase, resulting in a significant increase in CMC (Šteflová, Štefl, Walz, Knop, & Trapp, 2016). A concentration of 150 mmol·L−1 APFO was necessary to obtain a satisfactory separation (Fig. SM2c), while further increasing the APFO concentration to

2.6. Sample treatment Precisely 2.0 g of the tested material was weighed into a centrifuge tube followed by two-step ultrasound-assisted extraction. A sonication bath (Bandelin Sonorex Digitec, Berlin, Germany) was used at the fixed frequency of 35 kHz and power of 120 W. The first step of sonication was done in 5 mL ACN for 5 min. Then, the mixture was centrifuged at 4025×g for 5 min. The supernatant was transferred to a 15-mL centrifuge tube, and the solid residue was re-extracted by the same procedure. The supernatants from the two extractions were collected, combined, and then added with 2.5 mL of water and 1 g of sodium chloride. This mixture was placed at −20 °C for 3 h. Then, 100 µL of the ACN layer was diluted to 1:10 with 900 µL of 6:1:1:1 (v/v/v/v) water/ ACN/MeOH/THF mixture, leading to a final dilution of 1:50. Finally, this solution was filtered through a 0.22 μm nylon syringe filter and assayed by MEKC/MS/MS. 2.7. Matrix effect evaluation The matrix effect (ME; expressed in %) was evaluated by comparing the slopes of post-extraction spiked calibration curves prepared from blank sample extracts to those of external calibration curves of standard solutions at the same concentration levels, using the following equation (Matuszewski, Constanzer, & Chavez-Eng, 2003): ME = [(slope in post-extraction spiked calibration curve/slope in external calibration curve) − 1] × 100. The ME was classified into four groups: negligible (0–[ ± 10%]), soft ([ ± 10%]–[ ± 20%]), medium ([ ± 20%]–[ ± 50%]), and strong ([ ± 50%]) (Ferrer-Amate, Unterluggauer, Fischer, Fernández-Alba, & Masselter, 2010). 3. Results and discussion 3.1. Optimization of MEKC separation conditions Since Sudan dyes exist as neutral compounds in a wide range of pH, MEKC is the first choice for electrophoretic separation of these hydrophobic analytes. The MEKC separation is based on the partitioning of these neutral analytes between the pseudostationary phase formed by the APFO micelles and the bulk electrolyte. Due to the strong

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Table 1 Linearity parameters, LOQ, and matrix effect obtained with the proposed method.

Chili powder SUDAN I SUDAN II SUDAN III SUDAN IV Chili paste SUDAN I SUDAN II SUDAN III SUDAN IV Chili sauce SUDAN I SUDAN II SUDAN III SUDAN IV

Linear range (µg kg−1)

Slope ± SD

Intercept ± SD × 104

R2

LOQ (µg kg−1)

Matrix effect (%)

18–500 19–500 21–500 20–500

4475 ± 159 2864 ± 97 2309 ± 63 2456 ± 89

4.1 ± 1.2 2.8 ± 0.5 −3.1 ± 0.9 −5.2 ± 1.3

0.9974 0.9973 0.9964 0.9993

18 19 21 20

−5 0 −8 −9

17–500 18–500 20–500 22–500

4696 ± 210 2563 ± 123 2222 ± 68 2365 ± 74

5.2 ± 1.3 3.1 ± 0.7 −4.2 ± 1.6 −3.3 ± 1.2

0.9954 0.9982 0.9914 0.9954

17 18 20 22

2 −4 −7 −9

17–500 18–500 19–500 21–500

4789 ± 175 2456 ± 129 2136 ± 89 2136 ± 74

5.1 ± 1.7 3.6 ± 0.9 −4.9 ± 1.8 −3.2 ± 1.1

0.9984 0.9954 0.9963 0.9964

17 18 19 21

2 −7 −9 −10

200 mmol·L−1 did not lead to any improvement in the separation of Sudan dyes (Fig. SM2d). As stated before, 40% ACN was added to the BGE to improve the solubility of the hydrophobic dyes (Fig. 1a). Furthermore, several organic modifiers such as ACN, MeOH, and THF were tested as additives to explore how their nature and amount affect the final MEKC separation of Sudan dyes. From Fig. 1b, the addition of a 30:10 (v/v) ACN/ THF enhanced the sensitivity towards Sudan III and IV, demonstrating that THF increased the solubility of these compounds. However, the separation was not significantly improved. The use of a 30:10 (v/v) ACN/MeOH mixture achieved the best resolution of the peaks (Fig. 1c), confirming that MeOH has a stronger impact than other solvents on the partition coefficient of the analytes between the micelles and the bulk solution. Unfortunately, the solubility of the most hydrophobic analytes such as Sudan III and Sudan IV was insufficient, leading to a lower sensitivity and worse peak shape. Moreover, the analysis time was increased by the addition of THF and MeOH. All three organic modifiers differ in their viscosity (0.34, 0.46, and 0.55 mPa·s for ACN, THF, and MeOH, respectively). An increase in viscosity results in a decrease of EOF, prolonging the analysis time. Keeping in mind these results, a 20:10:10 (v/v/v) mixture of ACN/ MeOH/THF was selected to obtain the best sensitivity and resolution at an acceptable analysis time (Fig. 1d). The separation voltage and the temperature were also evaluated from + 20 to + 30 kV and from 20 to 30 °C, respectively. Only insignificant differences in separation selectivity were observed in these ranges (data not shown). To prevent excessive Joule heating and maintain a reasonable analysis time, the optimal values of temperature and separation voltage were + 25 °C and 25 kV, respectively. On-line preconcentration strategies can help improve the overall sensitivity of the method (Breadmore et al., 2015). Among others, sweeping is the most suitable approach for neutral and/or hydrophobic analytes in MEKC (Kitagawa & Otsuka, 2014). Sweeping is based on the interactions between APFO micelles in BGE and the analytes in a solvent with a similar composition to the BGE but free of micelles. Under these conditions, the analytes are focused in a narrow band within the capillary. As a consequence, a larger sample volume can be injected without any loss of resolution. Therefore, the sample solvent consisted of 60:20:10:10 (v/v/v/v) water/ACN/MeOH/THF that corresponds to the BGE composition without the APFO. The sweeping preconcentration was optimized by changing the hydrodynamic injection time from

Fig. 2. Extracted ion electropherogram of a chili powder spiked at the 100 µg kg−1 concentration level with each Sudan dye.

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20 to 100 s at a fixed pressure of 5 kPa. Injection times longer than 50 s were accompanied by a decrease in the separation efficiency for all compounds. So, an injection time of 50 s was selected as the optimal value. To estimate the gain in sensitivity, sensitivity enhancement factors based on peak heights (SEFheight) were calculated using the following equation:

in the following experiments. Then, the effect of the nozzle voltage was studied in a range of 0–2000 V, while keeping the sheath gas temperature and its flow rate at 100 °C and 8 L min−1, respectively (Fig. SM3b). An increase in the nozzle voltage to 2000 V improved the sensitivity. This behavior is also related to the insulating effect generated by the thermal confinement zone. As stated before, the efficiency of the capillary voltage to generate charged ions could be affected by the superheated sheath gas. The nozzle voltage allows the application of an additional potential on the tip of the capillary. Therefore, the insulating effect could be overcome to maintain the electric field in the thermal confinement zone. Note that the use of a nozzle voltage is critical for neutral compounds, while its effect on charged compounds is negligible since the formation of charged ions is mostly affected by the capillary voltage. Finally, the sheath gas flow was varied between 6 and 12 L min−1 while keeping the nozzle voltage and the sheath gas temperature at 2000 V and 100 °C, respectively. Fig. SM3c shows that the sheath gas flow affects MS sensitivity to a lesser extent than the other two parameters. The optimal sheath gas flow rate was determined to be 6 L min−1. This study demonstrated that a slower flow and a lower temperature of the sheath gas in combination with a higher nozzle voltage provide higher signal intensities for non-polar compounds in CE systems coupled to the AJS-ESI technology. Our parameters are different from the well-established values of 350 °C and 11 L min−1 recommended for AJS-ESI in LC-MS (Mordehai & Fjeldsted, 2009). Thus, the obtained results indicate that ion drying is not a critical point for common CE flow rates of 1–15 µL min−1 compared to the LC flow rates, which are typically two orders of magnitudes higher (e.g. 100 µL min−1). This is in agreement with the results of Kohler, Schappler, and Rudaz (2012). To optimize the sheath liquid composition, different organic solvents (ACN, MeOH, or IPA) were mixed with water and formic acid with a ratio of 50/49.9/0.1 (v/v/v). The best signal intensity was achieved with IPA as organic solvent. Then, IPA solutions in water were tested with 50%, 60%, 70%, and 80% IPA plus 0.1% formic acid. It was observed that higher percentages than 50% of organic solvent led to an unstable electric current, indicating a poor electrical contact between the CE and ESI electrical circuit. In summary, the optimized sheath liquid composition was 50:49.9:0.1 (v/v/v) IPA/water/formic acid. Finally, the following individual ESI parameters were studied as follows: drying gas flow 12–20 L min−1, drying gas temperature 150–200 °C, nebulizer pressure 69–207 kPa, sheath liquid flow rate 5–15 µL min−1, and capillary voltage 2,500–4,000 V. The effects of these parameters followed those clearly outlined elsewhere (TejadaCasado, Moreno-González, del Olmo-Iruela, García-Campaña, & Lara, 2017). The optimum values are described in Section 2.5.

SEFheight Peak height under sweeping injection/Analyte concentration =

in sweeping injection Peak height under hydrodynamic injection/Analyte concentration in hydrodynamic injection

Considering the sweeping (5 kPa for 50 s) in relation to the standard hydrodynamic injection (5 kPa for 10 s, sample solvent: BGE), SEFheight values of 200, 178, 160, and 147 were obtained for Sudan I, Sudan II, Sudan III, and Sudan IV, respectively under the optimal sweeping conditions. So, sweeping represents an excellent choice to improve the sensitivity in MEKC system based on APFO micelles without losing separation efficiency. 3.2. Optimization of MS/MS conditions SRM in the positive mode was chosen for the analysis. The molecular ion [M + H]+ was the most intense parent ion obtained for each Sudan dye. Two product ion transitions were selected for each of them. The most intense transition was used as the quantification ion (Q), and the other was considered as the qualifier ion (I). A dwell time of 150 ms was set for all SRM transitions, resulting in at least of 15 data points per peak. SRM parameters such as collision energy and collision accelerator voltage of the two most abundant transitions were optimized through individual direct infusion of each analyte to obtain the maximum MS response. Thus, we applied BGE containing 500 µg L−1 of each compound and +25 kV to enable SRM optimization under CE separation conditions. The optimized collision energy, collision accelerator voltage, and SRM transitions are summarized in the supplementary material (Table SM1). A fragmentor voltage of 380 V was set automatically during autotune in the triple quadrupole. The ion source parameters were also optimized by hydrodynamic injection in the MEKC-MS/MS system. The typical adjustable parameters in the ESI techniques are the drying gas temperature, drying gas flow rate, nebulizer pressure, and capillary voltage. As stated above, AJS-ESI is an additional feature that has been incorporated in all of the Agilent ESI sources. The superheated nitrogen sheath gas confines the nebulizer spray, in order to more effectively dry the ions and concentrate them in a thermal confinement zone to improve the sensitivity (Rodriguez-Aller, Gurny, Veuthey, & Guillarme, 2013). It should be noted that AJS-ESI is widely used in LC systems. However, its coupling to MEKC has not been discussed in detail yet. The temperature of superheated nitrogen sheath gas, its flow rate, and the nozzle voltage can influence the focusing effect. Thus, we optimized these parameters to estimate their effect on the ionization efficiency of the MEKC-MS/MS. First, the sheath gas temperature was varied between 100 and 350 °C while keeping the nozzle voltage and sheath gas flow rate at 2000 V and 8 L min−1, respectively. The sheath gas temperature had a remarkable effect on the MS sensitivity, with the best results obtained at lower temperatures (Fig. SM3a). The superheated gas produced a thermal confinement zone, which acted as an insulator for the electric field generated by the capillary voltage. Thus, the number of charged ions entering the MS was significantly decreased when this temperature was higher (Mordehai & Fjeldsted, 2009). A temperature of 100 °C was used

3.3. Sample treatment optimization According to the literature, UAE should be a suitable approach to extract target analytes from the chili samples (Rebane et al., 2010). We carried out extraction using 2 g of chili powder free of Sudan dyes spiked with 50 μg kg−1 of each compound as representative matrix. The matrix effect and extraction efficiency were considered as responses. The selection of a proper organic solvent that exhibits good solubility for non-polar dyes is essential for satisfactory recovery. Recoveries ranging from 90.1% to 97.3% were obtained when using ACN, THF, ethyl acetate, and acetone. However, when THF, ethyl acetate, and acetone were used, there were strong matrix effects, resulting in ion suppression higher than −50%. This suppression can result from the presence of undesirable impurities such as lipids and natural dyes in the extract. ACN as extraction solvent provided cleaner extracts with a lower content of co-extracted compounds and a medium matrix effect

5

6

1.1 1.0 1.3 1.2 11.0 13.3 9.7 14.9 1.2 1.3 1.4 1.5 11.2 14.3 10.1 12.3 1.2 1.4 1.3 1.4 13.9 15.0 9.5 15.7 1.1 0.9 1.0 0.9 4.1 3.9 4.0 3.9 1.0 1.2 1.1 1.2 0.8 0.9 0.8 0.9 5.1 5.3 5.5 4.8

4.2 3.9 4.2 3.8

8.6 8.5 6.4 10.8 1.1 1.1 1.3 1.5 8.7 8.9 6.1 10.9 1.0 0.9 1.2 1.4 8.5 6.2 13.1 11.8 0.7 0.9 1.0 0.9 3.8 4.0 3.2 3.6 0.8 0.9 0.9 1.0 0.7 0.7 0.8 0.8 6.5 5.6 5.4 4.0

3.7 4.2 3.0 3.8

1.2 1.1 1.4 1.5 9.1 11.5 12.3 9.1 1.3 1.3 1.5 1.6 9.9 12.2 14.6 8.8 1.2 1.2 1.4 1.5 14.2 14.7 8.4 9.7 0.6 0.8 1.1 0.8 4.1 2.8 2.8 3.2 0.7 0.9 1.0 1.1 4.4 2.7 3.0 3.3 0.6 0.7 0.8 0.9 4.7 5.7 7.3 4.2

Chili powder SUDAN I SUDAN II SUDAN III SUDAN IV Chili paste SUDAN I SUDAN II SUDAN III SUDAN IV Chili sauce SUDAN I SUDAN II SUDAN III SUDAN IV

Peak Area Peak Area Migration time Peak Area Peak Area

Migration time 25 µg kg

Peak Area

Migration time

50 µg kg

−1 −1

Intra-day RSD (%) (n = 9)

Table 2 Intra-day and inter-day precision of the proposed method for Sudan dyes.

250 µg kg

−1

Migration time

The proposed method was validated using several chili products including powder, paste, and sauce. The linear dynamic range, limit of quantification (LOQ), matrix effect, precision, selectivity, and trueness were determined. Procedural calibration curves were obtained by spiking blank chili powder, paste, and sauce samples with each Sudan dye at concentration levels of 25, 50, 125, 250, and 500 µg kg−1 before sample treatment. Two SRM transitions together with the retention time ensured adequate analyte identification. The validation results are shown in Table 1. The LOQs were calculated as the minimum analyte concentration yielding S/N = 10. The LOQ values were equal or lower than 22 µg kg−1 for all products studied, being lower than the recommended action limit of 500 µg kg−1 for Sudan dyes in foodstuff (European Commission, 2006). Therefore, our method is suitable for quantifying these residues at very low concentrations in the selected matrices. The determination of matrix effect was a key point in evaluating the sample clean-up, because the analytical response could be enhanced or decreased by co-extractants present in the final extract. As shown in Table 1, the matrix effects of the proposed method were negligible (0% to ± 10%) for all compound/commodity combinations, confirming that the sample treatment together with the final dilution step was successful in their reduction. An extracted ion electropherogram of chili powder spiked with each Sudan dye at a level of 100 µg kg−1 is shown in Fig. 2. Intra-day and inter-day precision were evaluated by applying our method for chili products spiked with each Sudan dye at three different concentration levels of 25, 50, and 250 μg kg−1. To evaluate the intraday precision, three samples at each level were prepared and injected in triplicate on the same day and assayed under the same conditions (n = 9). A similar procedure was applied for the evaluation of the interday precision: one sample at each concentration level was daily prepared and injected in triplicate for a total of five consecutive days (n = 15). The RSD of the peak areas and the migration times are shown in Table 2. Both the peak area and migration time had satisfactory precision, with the RSD lower than 15.7% and 1.6%, respectively.

Peak Area

3.4. Method validation

25 µg kg−1

Inter-day RSD (%) (n = 15)

50 µg kg−1

Migration time

250 µg kg−1

Migration time

ranging from −25% to −30%. Therefore, further optimization of the extraction procedure was aimed at the clean-up of ACN extract. Briefly, 100 mg of different dispersive solid phase extraction sorbents including Z-Sep, C18, primary secondary amine, and EMR-lipid were dispersed in 10 mL of organic extract. Matrix effect between 0% to −5% was obtained using all these sorbents. Unfortunately, the recoveries were reduced to 50%, because the hydrophobic synthetic dyes were also strongly retained by the sorbents. An interesting approach to remove co-extracted lipids as well as other components with limited solubility in ACN is the so-called freezeout step. From the analysis of pesticide residues, it is well known that good results could be obtained this way in terms of matrix effect and extraction efficiency (Liu et al., 2018). In our experiments, most of the lipophilic macromolecules, such as lipids and natural dyes (mainly capsanthin and capsorubin), precipitated when the extract was stored in the freezer at − 20 °C for 3 h. To improve the procedure, 2.5 mL of water and 1 g of sodium chloride were added to the extract before freezing to promote the phase separation of the matrix components and analytes (Zhao et al., 2012). Thus, the lipids and other compounds were separated in a layer between the water and ACN, while the target synthetic dyes remained in the ACN layer (Fig. SM4). Finally, 100 μL of the extract was diluted to 1:10 with 900 μL of 6:1:1:1 (v/v/v/v) water/ ACN/MeOH/THF to obtain a final extract dilution of 1:50. Matrix effects ranging from 2% to −10% were achieved without any significant loss of the Sudan dyes.

1.3 1.2 1.0 1.3

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The trueness of the method was tested with recovery experiments. If no Sudan dyes were detected above the LOD, the samples were spiked at three concentration levels equal to those used in the precision study. The trueness was between 84.4% and 99.6%, with excellent RSD values lower than 8.0% (Table 3).

(14 min) mean that 4.2 mL of waste was generated in a single run. In comparison, 40 runs using our MEKC method generated the same waste volume. Obviously, our method represents a remarkable reduction in waste generation compared to UHPLC-MS. To sum up, the need remains to further develop MEKC-based tech-

Table 3 Recovery for each sample type (n = 9). 25 µg kg−1

Chili powder SUDAN I SUDAN II SUDAN III SUDAN IV Chili paste SUDAN I SUDAN II SUDAN III SUDAN IV Chili sauce SUDAN I SUDAN II SUDAN III SUDAN IV

50 µg kg−1

250 µg kg−1

Recovery (%)

RSD (%)

Recovery (%)

RSD (%)

Recovery (%)

RSD (%)

93.8 94.2 92.8 89.7

6.7 6.9 6.6 7.4

95.5 98.1 94.6 91.2

3.3 4.3 5.6 4.6

95.6 98.3 95.2 92.2

3.5 4.1 5.8 4.7

95.2 94.9 92.2 91.8

8.0 6.1 6.3 5.8

99.4 95.2 97.5 91.7

3.2 4.9 4.7 4.2

98.3 96.1 96.9 93.2

3.3 4.8 4.9 4.3

99.6 94.7 90.1 84.4

6.9 6.0 6.8 6.9

98.2 95.8 91.8 89.8

3.6 4.3 4.8 3.6

97.5 96.1 92.3 90.0

3.8 4.2 4.6 3.9

Finally, the selectivity of the proposed method was evaluated. According to the European validation guideline for pesticide analysis, the Q/I ratio in the samples must be in a tolerance range of 30% with respect to that in the standard solution (European Commission, 2017). Our results here were always lower than 15% in all three types of products. Hence, our method provided adequate selectivity to conform to the current legislation.

niques for analyzing neutral hydrophobic compounds in complex matrices while maintaining compatibility with MS and environmental sustainability. However, this issue goes beyond the scope of this project. 3.5. Analysis of chili product samples To demonstrate the suitability of the developed MEKC/MS method, 20 chili products were purchased from supermarkets, food markets, and other local stores. They consist of 10 chili powders, four chili sauces, and six chili pastes originating from different countries such as the Czech Republic, Spain, Italy, Vietnam, and Thailand. All relevant information available from the sample labels is shown in Table SM3. Out of these 20 products, only two were found to contain Sudan dyes (Table SM4). Sudan I was detected in a chili powder from Thailand at a concentration of 125 ± 0.2 mg kg−1 (n = 3). Since this concentration was outside the linear dynamic range, we diluted the extract 1000 times to enable the concentration calculation. Another chili powder from Vietnam contained 0.095 ± 0.01 mg kg−1 (n = 3) of Sudan IV. The identity was confirmed by a variation of less than 10% between the Q/I ratio in the powder extracts and that in the standard solution. Extracted ion electropherograms for these positive samples and a negative sample are shown in Fig. 3. The sample with 125 mg kg−1 Sudan I is a clear case of adulteration. Generally, a dye concentration range from 100 to 1000 mg kg−1 is required to enhance the color of the foodstuff (Genualdi et al., 2016). However, the low Sudan IV concentration found in the other positive may be due to cross-contaminations, such as from the red bags used for drying, transport, and storage of the chili powder. To discern between accidental contamination and intentional addition, the European Union has recommended an action limit of 0.5 mg kg−1 for Sudan dyes in foodstuff (European Commission, 2006), which is much higher than the second positive here (0.095 ± 0.01 mg kg−1). None of the tested Sudan dyes were detected in the remaining 18 samples.

3.4.1. Comparison with other methods Table SM2 compares the LOQ, recovery, precision, and matrix effects of our method with other ones reported for the determination of Sudan dyes in chili products by using separation methods in conjunction with MS/MS detection. The sensitivity of our MEKC method is approximately 2–10 times better than the MEKC method based on partial filling (Fukuji et al., 2012). The LOQ values and precision were similar to those previously reported for LC-MS methods. The matrix effects were also similar to the LC-MS method, in which isotopically labeled internal standard was used to correct for the matrix effects for Sudan III and IV (Schummer et al., 2013). However, our MEKC-MS/MS method showed a negligible matrix effect without the need for such correction. In terms of environmental impact, CE can be considered a green method that fulfills two principles of green analytical chemistry (GAC) since it is a miniaturized technique (GAC principle No. 5) with a small volume of waste generation (GAC principle No. 7) (Gałuszka, Migaszewski, & Namieśnik, 2013). However, APFO is regarded a persistent organic pollutant (POP) that has substantial bioaccumulating and biomagnifying properties (European Commission, 2015). Hence its use does not comply with GAC principle No. 11 (“toxic reagents should be eliminated or replaced”) (Gałuszka et al., 2013). Nevertheless, we have to point out that the alternative method LCMS produces a large amount of chemical waste even though it does not generally use POPs. As an example, Zhao et al. (2012) proposed a UHPLC-MS method using a mixture of ACN and water as the mobile phase. The proposed flow rate (0.3 mL·min−1) and analysis time

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Acknowledgment This work was supported by the STARSS project (Reg. No. CZ.02.1.01/0.0/0.0/15_003/ 0000465) co-funded by ERDF. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.foodchem.2019.125963. References Abraham, M. H., Amin, M., & Zissimos, A. M. (2002). The lipophilicity of Sudan I and its tautomeric forms. Physical Chemistry Chemical Physics, 4, 5748–5752. Amini, A., Paulsen-Sörman, U., & Westerlund, D. (1999). Principle and applications of the partial filling technique in capillary electrophoresis. Chromatographia, 50, 497–506. Bafana, A., Devi, S. S., & Chakrabarti, T. (2011). Azo dyes: Past, present and the future. Environmental Reviews, 19, 350–371. Breadmore, M. C., Tubaon, R. M., Shallan, A. I., Phung, S. C., Abdul-Keyon, A. S., Gstoettenmayr, D., ... Quirino, J. P. (2015). Recent advances in enhancing the sensitivity of electrophoresis and electrochromatography in capillaries and microchips (2012–2014). Electrophoresis, 36, 36–61. Brensinger, K., Rollman, C., Copper, C., Genzman, A., Rine, J., Lurie, I., & Moini, M. (2016). Novel CE-MS technique for detection of high explosives using perfluorooctanoic acid as a MEKC and mass spectrometric complexation reagent. Forensic Science International, 258, 74–79. Calbiani, F., Careri, M., Elviri, L., Mangia, A., & Zagnoni, I. (2004). 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. Journal of Chromatography A, 1058, 127–135. Cornet, V., Govaert, Y., Moens, G., Loco, J. V., & Degroodt, J. M. (2006). Development of a fast analytical method for the determination of Sudan dyes in chili- and currycontaining foodstuffs by high-performance liquid chromatography-photodiode array detection. Journal of Agricultural and Food Chemistry, 54, 639–644. D'Orazio, G., Asensio-Ramos, M., Hernández-Borges, J., Rodríguez-Delgado, M.Á., & Fanali, S. (2015). Evaluation of the combination of a dispersive liquid–liquid microextraction method with micellar electrokinetic chromatography coupled to mass spectrometry for the determination of estrogenic compounds in milk and yogurt. Electrophoresis, 36, 615–625. European Commission (2004). Commission Decision of 21 January 2004 on emergency measures regarding hot chilli and hot chilli product. Official Journal of the European Union, L27, 52–54. European Commission (2006). SANCO – D.1(06)D/411990. Summary record of the standing committee on the food chain and animal health held in Brussels on 23 June 2006. https://ec.europa.eu/food/sites/food/files/safety/docs/reg-com_toxic_summary21_en.pdf Accessed 7 April 2019. European Commission (2015). 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Fig. 3. Extracted ion electropherograms of positive and negative samples. The SRM transitions of Q and I are available in Table SM1.

4. Concluding remarks A sensitive and selective MEKC-MS/MS method using APFO as volatile surfactant was developed for the determination of Sudan dyes in chili products. To the best of our knowledge, this is the first report of coupling MEKC to AJS-ESI-MS/MS for the determination of neutral compounds. Thus, our concept can be extended to the determination of non-polar neutral compounds in other matrices. The combination of UAE and freeze-out clean-up removed major lipid classes from the extracts without a significant effect on the recoveries. Moreover, this sample treatment diminished the matrix effect. Full validation was carried out for a wide range of chili products, obtaining LOQs lower than 22 µg kg−1. To demonstrate the method’s potential applicability for food quality/safety control, 20 chili products were analyzed using the developed method, and Sudan dye residues were found in two of them. However, since APFO is a POP, the direct coupling of MEKC with AJS-ESI-MS was achieved here at the expense of GAC principle No. 11. Thus, additional research would be needed to develop MEKC-MS approaches that are in more line with this green chemistry principle. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper

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