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Simultaneous Determination of Water-Soluble and Fat-Soluble http://www.paper.edu.cn Journal of Chromatography A, 1103 (2006) 170–176 Short communication Simultaneous determination of water-soluble and fat-soluble synthetic colorants in foodstuff by high-performance liquid chromatography–diode array detection–electrospray mass spectrometry Ming Ma a,b, Xubiao Luo a,b, Bo Chen b,∗, Shengpei Su b, Shouzhuo Yao a,b,∗ a State Key Laboratory of Chemo/Biosensing and Chemometrics, Hunan University, Changsha 410082, PR China b Key Laboratory of Chemical Biology & Traditional Chinese Medicine Research, Ministry of Education, Hunan Normal University, Changsha 410081, PR China Received 18 June 2005; received in revised form 12 November 2005; accepted 18 November 2005 Available online 7 December 2005 Abstract An accurate method was developed for the simultaneous determination of water-Tartrazine, Amaranth, Ponceau 4R, Sunset Yellow FCF, and fat-Sudan (I–IV), synthetic soluble colorants in foodstuff. This method uses dimethylsulfoxide (DMSO) as the extraction solvent in the sample preparation process and high performance liquid chromatography (HPLC)–diode array detector (DAD)–electrospray mass spectrometry (ESI-MS), applying selected ion recording in positive/negative alternate mode to acquire mass spectral data, as the analytical technique. Linearity of around three orders in the magnitude of concentration was generally obtained. Detection and quantification limits of the investigated dyes, which were evaluated at signal to noise ratio of 3 for detection limit and 10 for quantification limit, were in the ranges of 0.01–4 and 0.03–11.2 ng, respectively. The recoveries of the eight synthetic colorants in four matrices ranged from 93.2 to 108.3%. Relative standard deviations of less than 8.2% were also achieved. This method has been applied successfully in the determination of water-soluble colorants in the soft drink and the delicious ginger, and fat-soluble dyes in chilli powders and chilli spices. © 2005 Elsevier B.V. All rights reserved. Keywords: Soft drinks; Delicious ginger; Chilli powder; Chilli spice; Synthetic colorants; HPLC–DAD–ESI-MS; Dimethylsulfoxide 1. Introduction azo-dyes has been confirmed [5,6] and structure–activity rela- tionships have been assessed [7,8]. It is well known that Sudans Colorants are often added in food to enhance its visual aes- (I–IV) have been classified as category 3 carcinogen to humans thetics and to promote sales [1]. Although the allowable amount by International Agency for Research on Cancer [9], and the of synthetic colorants is reduced for consumer healthiness rea- use of Sudan I in foodstuff is forbidden in global food regu- sons in recent years, many kinds of synthetic food dyes are still lation act [10]. However, Sudan dyes were still found in food widely used all over the world due to their low price, high products exported in European countries [6]. For this reason, effectiveness and excellent stability [2]. Generally, synthetic accurate and reliable methods for the determination of synthetic colorants can be classified into water-soluble and fat-soluble col- dyes in foodstuff are required for the assurance of consumer orants based on their solubility. In China, the commonly used healthiness. water-soluble synthetic dyes that are permitted in foodstuff, are Thin-layer chromatography [11,12], spectrophotometry Amaranth, Brilliant Blue FCF, Ponceau 4R, Sunset Yellow FCF, [13–16], and adsorptive voltammetry [17] have been used for and Tartrazine (Fig. 1) [3,4]. Most fat-soluble synthetic colorants the determination of various water-soluble synthetic dyes. How- present in the market are azocompounds, such as Sudan I, Sudan ever, all methods mentioned above are either time-consuming or II, Sudan III, and Sudan IV (Fig. 1). The genetic toxicity of some not suitable for complicated colorant mixtures. To improve the efficiency and reliability of the determination, capillary elec- trophoresis [1,18–22], reversed-phase liquid chromatography ∗ Corresponding authors. Tel.: +86 731 8865515; fax: +86 731 8865515. (RPLC) [4,23–24], ion-pair RPLC [25–28], and anion-exchange E-mail address: [email protected] (S. Yao). chromatography [29–32] are successfully applied for water- 0021-9673/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2005.11.061 转载 中国科技论文在线 http://www.paper.edu.cn M. Ma et al. / J. Chromatogr. A 1103 (2006) 170–176 171 Fig. 1. Chemical structures of synthetic colorants investigated and the internal standard, Brilliant Blue FCF. soluble synthetic dyes. Detection limits of these methods are dilution combined with APCI tandem mass spectrometry for in the range of 0.1–10 ␮g/mL. Fat-soluble Sudan azo-dyes are trace determination of Sudan I contamination in foodstuff. In commonly determined by LC [33,34] based on the absorbance these works, acetone, ethanol, and acetonitrile were verified to properties of Sudan azo-dyes in the visible region of the be the suitable extractants for Sudan extracting. Water-soluble spectrum, while two dyes owned highly similar structure may colorants, Amaranth, Brilliant Blue FCF, Ponceau 4R, Sunset have the same color appearance, indistinguishable UV–vis Yellow FCF, and Tartrazine, are dissolvable in water. However, adsorption spectra, and close retention times that are not easily it is difficult for them to dissolve in ethanol and plant oil identified under HPLC analysis. Recently, Calbiani et al. [6] and [35]. In our preparation experiment, acetone, ethanol, and Tateo et al. [9] reported a successful determination of Sudan in acetonitrile were explored as solvents to dissolve dyes studied. hot chilli powder using LC–ESI-MS/MS and HPLC–APCI-MS Unfortunately, solubilities of water-soluble dyes mentioned methods. Di Donna et al. [10] developed the method of isotope above were unsatisfied. Up to now, simultaneous extraction 中国科技论文在线 http://www.paper.edu.cn 172 M. Ma et al. / J. Chromatogr. A 1103 (2006) 170–176 and determination of water-soluble and fat-soluble synthetic 2.4. Standard solutions colorants in foodstuff have not been found in the literatures. In this work, we developed an HPLC–DAD–ESI-MS method A standard stock solution containing Tartrazine (80 ␮g/mL), for the simultaneous identification and determination of water- Amaranth (80 ␮g/mL), Ponceau 4R (80 ␮g/mL), Sunset Yellow soluble and fat-soluble synthetic dyes. In this novel method, the FCF (80 ␮g/mL), Sudan I (20 ␮g/mL), Sudan II (32 ␮g/mL), simultaneous ionization in ESI-MS and simultaneous extraction Sudan III (56 ␮g/mL), and Sudan IV (72 ␮g/mL) was prepared using dimethylsulfoxide (DMSO) as the extraction solvent in the in DMSO. Internal standard solution (50 ␮g/mL) of Brilliant sample preparation have been successfully applied. Blue FCF was also prepared in DMSO. Four sets of stan- dard solutions were prepared in DMSO (matrix A), in extracts 2. Experimental of spiked delicious gingers (matrix B), spiked chilli powders (matrix C), and spiked chilli spices (matrix D), respectively. 2.1. Chemicals Each set of standard solutions contained six different concen- trations of dyes. The concentration range for each standard HPLC-grade methanol was obtained from Tedia Com- solution was: 0.40–40 ␮g/mL of Tartrazine, 0.40–40 ␮g/mL of pany Inc. (Fairfield, Ohio, USA). Analytical grade ammonium Amaranth, 0.40–40 ␮g/mL of Ponceau 4R, 0.40–40 ␮g/mL of acetate, acetate acid, and DMSO were purchased from Shang- Sunset Yellow FCF, 0.10–10 ␮g/mL of Sudan I, 0.16–16 ␮g/mL hai Chemical Reagent Co. Ltd. (Shanghai, China). Water was of Sudan II, 0.28–28 ␮g/mL of Sudan III, and 0.36–36 ␮g/mL purified using a Milli-Q Ultrapure water purification system of Sudan IV. The concentration of the internal standard was (Millipore, Bedford, MA, USA). Tartrazine, Amaranth, Ponceau 0.2 ␮g/mL. All solutions were stored at 4 ◦C, and the storage 4R, Sunset Yellow FCF, Sudan I, Sudan II, Sudan III, Sudan IV, life of each solution was at least half a year. and Brilliant Blue FCF were purchased from National Research Center for CRM’S (Beijing, China). 2.5. Sample preparation and HPLC–DAD–ESI-MS analysis 2.2. Samples Soft drink samples were filtered through a 0.45-␮m nylon membrane (Whatman, UK) and degassed, then 20 ␮Lof The following commercial samples: 1 kind of soft drink, 2 50 ␮g/mL internal standard solution of Brilliant Blue FCF was kinds of delicious ginger, 12 kinds of chilli powders and 28 added to 5 mL of sample solution, after being thoroughly mixed kinds of chilli spices, were purchased in big trades. The samples it was injected directly into the LC–MS system. of delicious ginger, chilli powder, and chilli spice that did not About 1 kg of fortified solid samples, delicious ginger, chilli show any detectable residues of target dyes were taken as the powder and chilli spice samples, were collected. Samples were blank, respectively. homogenized for 2 min and 20 g of aliquots was taken for extrac- tion purposes. Two hundred microliters of 50 ␮g/mL internal 2.3. Instrumentation standard solution of Brilliant Blue FCF followed by 50 mL of DMSO was added. After being sonicated for 15 min, the extract A Waters-2695 Alliance HPLC system (Waters Corpora- was centrifuged at 4000 rpm for 5 min. The supernatant was fil- tion, Milford, MA, USA) equipped with DAD detector at tered through a 0.45 ␮m nylon membrane filter, then transferred 254 nm was coupled to a Micromass ZQ 2000 electrospray into a 50-mL volumetric flask and diluted to the volume with mass spectrometer (Manchester, UK). The Masslynx v. 3.4 soft- DMSO before injection in the LC–MS system. Spiked delicious ware (Micromass) was used for data acquisition and process- ginger, chilli powder, and chilli spice samples were prepared by ing. An spherigel C18 analytical column (4.6 mm × 250 mm, adding calculated amount of synthetic colorants in 20 g of rel- 5 ␮m, Johnsson Dalian, China) was used.
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