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Dingguo Jiang1 ∗ Lisong Chen2 Wusheng Fu2 Hanquan Qiu2 1 Key
Laboratory of Food Safety Risk Assessment of Ministry of Health, China National Center for Food Safety Risk Assessment, Beijing, P. R. China 2 Fujian Provincial Key Laboratory of Zoonosis Research, Fujian Provincial Center for Disease Control and Prevention, Fuzhou, P. R. China Received October 7, 2014 Revised November 28, 2014 Accepted November 30, 2014
Research Article
Simultaneous determination of 11 fluorescent whitening agents in food-contact paper and board by ion-pairing high-performance liquid chromatography with fluorescence detection 4,4 -Diaminostilbene-2,2 -disulfonic acid based fluorescent whitening agents (DSD-FWAs) are prohibited in food-contact paper and board in many countries. In this work, a reliable high-performance liquid chromatography method was developed for the simultaneous determination of 11 common DSD-FWAs in paper material. Sample preparation and extraction as well as chromatographic separation of multicomponent DSD-FWAs were successfully optimized. DSD-FWAs in prepared samples were ultrasonically extracted with acetonitrile/water/triethylamine (40:60:1, v/v/v), separated on the C18 column with the mobile phase containing tetrabutylammonium bromide, and then detected by a fluorescence detector. The limits of detection were 0.12–0.24 mg/kg, and the calibration curves showed the linear correlation (R2 ࣙ 0.9994) within the range of 8.0–100 ng/mL, which was equivalent to the range of 0.80–10 mg/kg in the sample. The average recoveries and the RSDs were 81–106% and 2–9% at two fortification levels (1.0 and 5.0 mg/kg) in paper bowls, respectively. The successful determination of 11 DSD-FWAs in food-contact paper and board obtained from local markets indicated that the newly developed method was rapid, accurate, and highly selective. Keywords: Fluorescent whitening agents / Food-contact paper and board / Highperformance liquid chromatography DOI 10.1002/jssc.201401110
Additional supporting information may be found in the online version of this article at the publisher’s web-site
1 Introduction 4, 4 -Diaminostilbene-2, 2 -disulfonic acid based fluorescent whitening agents (DSD-FWAs) have strong fluorescent properties and can directly absorb UV light (290–400 nm) and then reradiate the blue-violet light (400–500 nm). Therefore, they have been widely used in the modern paper industry in the world [1]. The molecular structures of commonly used DSD-FWAs are shown in Table 1. According to the number of sulfonic acid groups, DSD-FWAs can be classified into three types: disulfonic acid type (2S, e.g. FWA Correspondence: Dr. Wusheng Fu, Fujian Provincial Key Laboratory of Zoonosis Research, Fujian Provincial Center for Disease Control and Prevention, 76 Jintai Road, Gulou District, Fuzhou 350001, P. R. China E-mail: [email protected] Fax: +86-591-87670235
Abbreviations: ACN, acetonitrile; DSD-FWA, 4,4’diaminostilbene-2,2’-disulfonic acid-based fluorescent whitening agent; FWA, fluorescent whitening agent; HCl, hydrochloric acid; MeOH, methanol; 2S, disulfonic acid; 4S, tetrasulfonic acid; 6S, hexasulfonic acid; TBA, tetrabutylammonium bromide C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
5BM, C.I.71, C.I.85, C.I.90, and C.I.113), tetrasulfonic acid type (4S, e.g. C.I.24, C.I.210, and C.I.220), and hexasulfonic acid type (6S, e.g. C.I.264, C.I.353, and C.I.357). In some toxicological experiments, the toxicity of some DSD-FWAs was relatively low [2–4]. However, in other studies, the 2S DSD-FWA (C.I.85) could improve the peroral infection of the recombinant virus [5], and some FWAs affected the wound healing process [6]. Moreover, the toxicity of many newly developed DSD-FWAs and their photo-isomers is still unknown. Therefore, in many countries, DSD-FWAs have not been approved for food-contact paper and board. In the European Union, according to good manufacturing practices, FWAs cannot be detected in food-contact paper and board [7]. In China, FWAs were forbidden in the production of food-contact paper in 1989 [8]. However, according to the regulations of U.S. Food and Drug Administration, only C.I.220 can be added to food-contact paper and board (http:// www.accessdata.fda.gov/scripts/fcn/fcndetailnavigation.cfm ?rpt=fcsListing&id=889). Recently, high levels of FWAs had been found in some paper bowls for popcorn or instant ∗ Additional corresponding: Dr. Dingguo Jiang, E-mail: [email protected]
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Table 1. Structures and information of 11 DSD-FWAs
Analytes
CAS No.
R1
FWA 5BM C.I.71
13863–31–5 16090–02–1
−N(CH3 )C2 H4 OH
C.I.85 C.I.90 C.I.113
12224–06–5 3426–43–5 12768–92–2
−NHC2 H4 OH −OCH3 −N(OCH2 CH3 )2
C.I.24
12224–02–1
−N(C2 H4 OH)2
C.I.210
28950–61–0
C.I.220
16470–24–9
−N(C2 H4 OH)2
C.I.264 C.I.353
76482–78–5 55585–28–9
−N(C2 H4 OH)2
C.I.357
41098–56–0
−N(CH2 CH3 )2
R2
Basic structure
HPLC methods can only detect a few DSD-FWAs (less than seven kinds), while some common DSD-FWAs in paper cannot be detected. The LC–MS method is relatively rapid and sensitive [22, 23], but the nonvolatile ion-pair reagents successfully used in the HPLC separation often suppress the response signal and easily lead to the contamination of ion source [24]. Additionally, the multisulfonic groups of DSDFWAs may hinder the progress toward multicomponent analysis because the properties of multiply charged ions of DSDFWAs make it difficult to tune the precursor ions. Although some LC–MS methods for 2S DSD-FWAs were reported, the studies on 4S or 6S DSD-FWAs have been seldom reported until now. In conclusion, the ion-pairing HPLC method may be the most suitable method for quantifying DSD-FWAs. In addition, different sample preparation methods may greatly affect the detection results because the distribution of DSD-FWAs in paper samples is often inhomogeneous. However, the representativeness or the homogeneity of the prepared paper samples has been not reported. Moreover, 2S, 4S, and 6S DSD-FWAs are different in the number of sulfonic groups, the solubility, and the adsorption capacity to paper fiber. The method for simultaneously and effectively extracting three types of DSD-FWAs from paper materials has been not reported. In this study, a sensitive, accurate, and rapid analytical method was successfully developed for the simultaneous determination of 11 common DSD-FWAs in food-contact paper and board by ion-pairing HPLC with fluorescence detection.
2 Materials and methods 2.1 Chemicals and materials noodles and some paper cups for milk in China (http://food. china.com/rdjj/11101747/20120809/17364485.html) [9]. The situation was probably caused by the illegal addition in papermaking or the recycling of waste paper containing FWAs. Therefore, it is very important to develop a rapid and accurate analytical method for the investigation of the contamination levels of DSD-FWAs in food-contact paper. UV irradiation [10], fluorospectrophotometry [11], UV spectrophotometry [12], TLC [13], CZE [14,15], HPLC [16–20], and LC–MS [21–23] have been applied in the analysis of DSDFWAs in food-contact paper. The UV irradiation method by visual inspection is still an official method in some countries, but it cannot be used to accurately detect the total FWAs because the detection results are easily influenced by artificial factors. HPLC is a relatively reliable method for the qualitative and quantitative determinations of DSD-FWAs. However, DSD-FWAs with some sulfonic acid groups have the strong polarity in aqueous solutions and are difficult to be retained on a C18 RP column. To increase the retention of DSD-FWAs on C18 column, some ion-pair reagents are usually added into the mobile phases to transform DSD-FWAs into ion-paired compounds. To date, because some DSD-FWAs have very similar structure and are difficult to separate, the reported C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
C.I.24, 85, 113, 210, 264, and 357 were purchased from American International Laboratory. C.I.71 and 210 were obtained from Zhejiang Hongda. C.I.353, 90 and FWA 5BM were kindly supplied by China National Center of Quality Supervision and Inspection for Dyestuff. All DSD-FWAs were of reagent grade. HPLC-grade methanol (MeOH) and acetonitrile (ACN) were purchased from Fisher Scientific. Chromatographically pure hexane was obtained from Merck. Analytically pure tetrabutylammonium bromide (TBA), triethylamine, ethanol, acetone, and hydrochloric acid (HCl) were purchased from Sinopharm Chemical Reagent. Ultrapure water was obtained from a Mill-Q system (Millipore, Bedford, MA, USA). Disposable paper cups, paper bowls, and paper bags were purchased from local markets in Fuzhou, China.
2.2 Preparation of standard solutions Single standard stock solutions (500 g/mL) of the analytes were prepared by dissolution in acetonitrile/water (2:3, v/v) containing 1% triethylamine and then stored in a freezer (–18⬚C). The mixed standard intermediate solution www.jss-journal.com
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(10.0 g/mL) was prepared by further dilution of single standard stock solutions with acetonitrile/water (2:3, v/v) and stored in a refrigerator (4⬚C). The mixed standard working solutions (from 1.0 to 100 ng/mL) were prepared by further dilution of the mixed standard intermediate solution with acetonitrile/water (2:3, v/v). All the preparation operations should be carried out in the lab environment where the light intensity was below 20 Lux and all volumetric flasks for standard solutions should be brown. The prepared solutions should be tightly wrapped with aluminium foil and stored in the dark.
2.3 Instrumentation All the analyses were performed with an LC-20AT HPLC system (Shimadzu, Japan). DSD-FWAs were separated on a Symmetry C18 column (250 × 4.6 mm, 5 m, Waters). The column temperature was maintained at 35⬚C. The mobile phase was composed of Eluent A (acetonitrile/methanol, 2:3, v/v) and Eluent B (methanol/water, 5:95, v/v, containing 25 mmol/L TBA, adjusted to pH 8.0 with triethylamine). The gradient elution at a flow rate of 1.0 mL/min was programmed as: 0–2 min, 40% B; 2–12 min, 40%B→30% B; 12–17 min, 30%B→20% B; 17–19 min, 20% B→0% B; 19–21 min, 0% B→40% B; 21–25 min, 40% B. The analytes were detected by an Rf-10AXL fluorescence detector (Shimadzu, Japan). The excitation wavelength and the emission wavelength for all DSD-FWAs were 350 and 430 nm, respectively.
2.4 Sample preparation The relatively hard paper samples, such as the disposable paper cups and paper bowls, were sheared into flat paperboard with surgical scissors. Then about 30 g flat paperboard was cut into pieces with a size of 2 × 6 mm with an X2 shredder (Anbixin, China). The relatively soft paper samples, about 30 g, were overlapped and cut into the pieces with the size of 10 × 10 mm with surgical scissors. The paper pieces were transferred into the DFY-80 high-speed grinder (Wenling Linda Machinery, China), and ground at 10 000 r/min for 15 s. The grinding process was repeated six times with the interval of 5 s. The ground fine paper fiber was then packaged in a clean polyethylene bag and stored in the dark.
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tracted at 50⬚C for 35 min. After cooling to room temperature, the extract was centrifuged at 3750 r/min for 5 min, and the supernatant was carefully transferred into a 50 mL volumetric flask. Then 12 and 10 mL of the same extract solvent were added into the sample residue, respectively. The above procedure of extraction and centrifugation was repeated except that the extraction time was reduced to 10 min. The extracts were merged into the same volumetric flask, adjusted to pH 8.0 with 0.5 mol/L HCl solution, diluted to the volume of 50 mL with acetonitrile/water (2:3, v/v), and thoroughly mixed. Then 2 mL of extracts and 0.5 mL of hexane were added into a 5 mL test tube, which was vibrated for 30 s. The solution was allowed to stand for 2 min for stratification and then the supernatant was removed. The lower liquid was transferred into a 2 mL plastic centrifuge tube and centrifuged at 10 000 r/min for 5 min. The supernatant was transferred into a 2 mL brown sample bottle for HPLC analysis with the injection volume of 20 L. The amount of sample equivalent in the final extract was about 0.01 g/mL.
3 Results and discussion 3.1 Optimization of HPLC separation 3.1.1 Gradient elution To quickly achieve the equilibrium between the mobile phase and the C18 stationary phase, the isocratic elution mode was often recommended for the separation of DSD-FWAs in the ion-pairing HPLC [17, 21]. In this study, the similar chromatographic conditions were tested. However, the results showed that some 2S DSD-FWAs had a relatively long retention time (about 35 min) and some 4S DSDFWAs had a very short retention time (about 3 min). Moreover, the chromatographic resolutions of C.I.24/C.I.210 and C.I.85/C.I.113 were 0.40 and 0.60, respectively. Therefore, optimization tests for gradient elution program were carried out. In the gradient elution mode, the rapid separation of 11 DSD-FWAs was achieved and the RSD of the peak areas was less than 8% in six intraday measurements of each DSD-FWA. Therefore, the gradient elution mode was selected. 3.1.2 Organic mobile phase (Eluent A)
2.5 Sample extraction To reduce the possible isomerization of DSD-FWAs, all of the following experiments were performed in the lab environment where the light intensity was below 20 Lux. About 0.5 g of the ground paper sample was weighed and transferred into a 50 mL plastic centrifuge tube. Then 25 mL of acetonitrile/water/triethylamine (40:60:1, v/v/v) was then added into the centrifuge tube, which was then placed in an ultrasonic device. Then the test sample was ultrasonically ex C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Acetonitrile and methanol are common elution solvents used in HPLC analysis of DSD-FWAs [14, 17]. In this work, the elution capabilities of acetonitrile and methanol to DSDFWAs were tested under the condition of the same gradient elution program and TBA concentration. The chromatographic results were shown in Fig. 1A and 1B. For acetonitrile, the retention time of 11 DSD-FWAs was between 10.0 and 12.5 min, which indicated that the chromatographic separation was poor. For methanol, the retention time of 11 DSD-FWAs was between 11 and 21 min, so their retention www.jss-journal.com
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Figure 1. Effects of the organic mobile phase on the separation of 11 DSD-FWAs. Eluent A was (A) ACN, (B) MeOH, (C) ACN/MeOH = 4:1, (D) ACN/MeOH = 3:2, (E) ACN/MeOH = 1:4, and (F) ACN/MeOH = 2:3. The concentration of TBA, Eluent B and the gradient elution program were the same as that in Section 2.3. The concentration of each DSD-FWA was 100 ng/mL. The peaks: 1. C.I.220; 2. C.I.24; 3. C.I.210; 4. C.I.85; 5. C.I.113; 6. C.I.264; 7. C.I.353; 8. C.I.357; 9. FWA 5BM; 10. C.I.90; 11. C.I.71.
behaviors were significantly different and their separations were greatly improved. Based on the above results, the separation effects of different proportions of acetonitrile to methanol on the 11 DSD-FWAs were tested in the further experiments. The results shown in Fig. 1C, 1D, 1E, and 1F were different from those in Fig. 1B in three aspects. Firstly, the elution order of some DSD-FWAs was changed significantly. Secondly, the retention capability of some 2S DSD-FWAs (C.I.85, 113) was reduced. Thirdly, the retention capability of the 6S DSD-FWAs was enhanced. Among the four proportions of acetonitrile to methanol, the separation effect of acetonitrile/methanol (2:3, v/v) on the 11 DSD-FWAs was the best, so the 11 DSD-FWAs could be better separated. Therefore, acetonitrile/methanol (2:3, v/v) was identified as the optimal organic elution solvent. 3.1.3 TBA concentrations in Eluent B Some studies showed that the higher the TBA concentration in water, the longer the retention time of DSD-FWAs on C18 stationery phase [17]. Meanwhile, the effect of TBA on the retention capacity of three types of DSD-FWAs was decreased in the sequence: 6S, 4S, and 2S [25]. Therefore, the separation effects of different TBA concentrations on DSD-FWAs were tested under the conditions of the same organic mobile phase and gradient elution program (Supporting Information Fig. S1). With the increase of TBA concentration in Eluent B (methanol/water, 5:95, v/v), the retention time of all DSDFWAs was increased, and the resolutions between C.I.24 and 210 and between C.I.264 and 353 were also improved, while the resolution between C.I.357 and FWA 5BM was decreased. The changes between C.I.357 and FWA 5BM could be interpreted below. The number of sulfonic acid groups of C.I.357 was three times that of FWA 5BM. Therefore, compared with FWA 5BM, C.I.357 was more significantly influenced by the change of TBA concentration. With the increase of the TBA concentration, the retention time of C.I.357 became gradually closer to that of FWA 5BM, leading to the decreased C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
resolution between C.I.357 and FWA 5BM. When the TBA concentration in methanol/water (5:95, v/v) was 25 mmol/L, all target compounds could be optimally separated. 3.1.4 pH of Eluent B In this work, the chromatographic separation of 11 DSDFWAs was hardly affected by the pH of Eluent B (methanol/water, 5:95, v/v, containing 25 mmol/L TBA). However, when the pH of Eluent B was between 5 and 6, about 3% of each 6S DSD-FWAs in the injected sample solution would be retained in the HPLC system and appeared in the chromatogram of the next injection. When the pH was adjusted to 8.0 with triethylamine, the residual phenomenon disappeared and the response of all 6S DSD-FWAs also increased. 6S DSD-FWAs were probably easily aggregated and precipitated in the weak acidic Eluent B, thus leading to the crossing contamination problem in HPLC analysis. Therefore, Eluent B should be adjusted to the weak alkaline condition (pH 8.0) with triethylamine. Under the optimized chromatographic conditions described above, the 11 DSD-FWAs could be well separated in the retention time range of 11.5–19.5 min. According to the structures of DSD-FWAs, the separation of DSD-FWAs on the C18 column was influenced by the type of R1 group and the number of the sulfonic acids in R2 group. When the number of sulfonic acids in R2 group was the same, the stronger polarity of the R1 group indicated the weaker retention capability of DSD-FWAs on the C18 column except that the retention time of C.I.24 and 210 was affected by the position of sulfonic acid group on phenyl ring (namely the steric effect). When the position of R1 group was the same, according to the polarity, the theoretical elution order of DSD-FWAs should be 6S, 4S, and 2S. However, the actual elution order was 4S, 6S, and 2S (e.g. C.I.210, C.I.353, and C.I.71), indicating that the retention of 6S DSD-FWAs was enhanced. Since 6S DSD-FWAs had more sulfonic acid groups than 4S DSDFWAs, the retention capacity of 6S DSD-FWAs was more www.jss-journal.com
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easily influenced by TBA. Moreover, 6S DSD-FWAs had the larger molecular weight, so they had the stronger adsorption with a C18 column than 4S DSD-FWAs.
3.2 Sample preparation In the reported methods [17, 22], the paper samples were often cut into pieces with the size of 1–2 cm2 . In this work, the real paper bags containing C.I.220 from local market were also prepared by simply cutting, and then C.I.220 was detected in the prepared samples. The results showed that RSD of the concentrations of C.I.220 in six replicates was 54%. Therefore, the distribution of DSD-FWAs in food-contact paper might be inhomogeneous and the samples prepared by simply cutting might not be representative. In this study, the sample preparation method described in Section 2.4 was thoroughly validated. Briefly, about 15 g paper bowl without DSD-FWA and about 15 g paper bag containing C.I.220, 85, 264, and 353 were cut into pieces with the size of 2 × 6 mm with shredders, respectively, and the pieces were mixed together and ground into fine fibers at 10 000 r/min. Then, C.I.220, 85, 264, and 353 in the prepared samples were analyzed according to Sections 2.3 and 2.5 (Supporting Information Table S1). For 0.5 g sample, the RSDs of four DSD-FWAs in six replicates were 1–12%. Moreover, the detection results of four sampling amounts (0.5, 1.0, 2.0, and 3.0 g) in three replicates were basically the same and showed no significant difference (p > 0.05). Therefore, the samples prepared according to Section 2.4 were homogeneous and representative.
3.3 Optimization of extraction conditions In the optimization experiments, all the spiked samples were prepared as follows: 500 L of the mixed standard solution of 11 DSD-FWAs (10 g/mL) was carefully added into 0.5 g of the ground blank paper fiber in a 50 mL centrifuge tube and then the centrifuge tube cap was tightened. The centrifuge tube was tightly wrapped in aluminum foil, and placed in the dark for 24 h to allow DSD-FWAs to be fully combined with paper fiber. The spiked samples were then used for a series of optimization experiments of the extraction conditions, including the extraction method, solvent, temperature, duration, and times. All the optimization experiments for each parameter were repeatedly carried out for three times to minimize the experimental deviations. 3.3.1 Extraction method and solvent In this work, water was used as the extraction solvent and the paper bag sample was the matrix. Hot-water extraction, ultrasonic extraction, shaking extraction, and Soxhlet extraction were tested. The extraction rates of ultrasonic extraction and Soxhlet extraction were about the same, which were about two times those of hot-water extraction and shaking extrac C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 2. The extraction efficiency of different acetonitrile concentrations in water (n = 3). The spiked level of 11 DSD-FWAs in paper bag was 20 mg/kg. The trend charts of other 2S, 4S, and 6S DSD-FWAs, which were omitted, were similar to those of FWA 5BM, C.I.220, and C.I.264.
tion. Therefore, the fast and simple ultrasonic extraction was selected as the optimal extraction method. In some studies, hot water was used to extract DSD-FWAs in paper samples [21–23]. However, it was reported that the extraction efficiency of hot water was relatively low, especially for 2S DSD-FWAs [17]. Therefore, the DSD-FWAs in spiked paper cups were extracted with 40% (volumetric concentration) ethanol, acetonitrile, methanol, and acetone in water, respectively. The results indicated that 40% acetonitrile in water was the most effective, but the extraction recoveries of DSD-FWAs by 40% methanol in water were merely 38– 83% (Supporting Information Fig. S2). Furthermore, the extraction efficiencies of different acetonitrile concentrations in water were investigated (Fig. 2). The recoveries of 2S DSDFWAs were 38–47% when the acetonitrile concentration was less than 30%, indicating that 2S DSD-FWAs with the relatively low polarity were difficult to be extracted by the solvent containing the high content of water. On the contrary, the extraction rates of 6S DSD-FWAs with the relatively high polarity decreased when the acetonitrile concentration was more than 50%. At the same time, the change of the acetonitrile concentration had little effect on the extraction rates of 4S DSD-FWAs. To simultaneously extract three types of DSDFWAs, 40% acetonitrile in water was selected as the optimal extraction solvent. 3.3.2 Extraction temperature, duration, and times The extraction temperature, duration, and time are key factors in the extraction of DSD-FWAs in samples. The optimization experiments showed that the extraction rates of 11 DSDFWAs were the highest after extracting for 35 min at 30⬚C, but the recoveries of some 2S DSD-FWAs (C.I.113, FWA 5BM) were only 51–60%. With the increase of the extraction temperature, the extraction rates of these 2S DSD-FWAs were also improved. The optimal extraction temperature was 50⬚C. C.I.220, 210, 85, 113, and 264 in the real paper bag sample were extracted at 50⬚C for 35 min every time and the total contents of five DSD-FWAs extracted four times were determined as the true values (Supporting Information www.jss-journal.com
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Table 2. Validation parameters of the optimized method
Analyte
FWA 5BM C.I.71 C.I.85 C.I.90 C.I.113 C.I.24 C.I.210 C.I.220 C.I.264 C.I.353 C.I.357
Linear range (ng/mL)
6.0–100 4.3–100 4.0–100 6.7–100 7.0–100 5.3–100 6.0–100 4.7–100 7.7–100 8.0–100 6.7–100
Sample equivalent in linear range (mg/kg)
0.60–10 0.43–10 0.40–10 0.67–10 0.70–10 0.53–10 0.60–10 0.47–10 0.77–10 0.80–10 0.67–10
LOD (mg/kg)
0.18 0.13 0.12 0.20 0.21 0.16 0.18 0.14 0.23 0.24 0.20
Recoverya) (%)
RSD (%)
1 mg/kg
5 mg/kg
1 mg/kg
5 mg/kg
90 98 92 95 92 97 97 106 81 86 85
89 90 94 95 87 92 88 100 99 90 95
3 9 3 3 4 2 2 4 5 6 9
5 5 6 4 6 6 6 5 7 5 5
a) Recovery is the mean of six measurements, and 1 and 5 mg/kg are the spiked levels in paper bowls (n = 6).
Fig. S3). In the first extraction, the extraction rates of five DSD-FWAs were only 60–80%. However, the extraction rates of five DSD-FWAs extracted for three times could reach at least 95%. Therefore, three times was selected as the extraction times. Because the contribution ratios of the second and third extractions were relatively low (14–29%), the second and third extractions were shortened to 10 min. In the further experiment, the total extraction rates of the shortened extraction program (35, 10, and 10 min) showed no significant difference with that of the consistent extraction program (35, 35, and 35 min; p > 0.05). 3.3.3 pH of extraction solvent Under the above optimal extraction conditions, the recoveries of all DSD-FWAs in the spiked paper cups and bags were relatively high (68–106%), but the recoveries of 6S DSDFWAs in the spiked paper bowls were low. The recoveries of 6S DSD-FWAs at three fortification levels (15, 5.0, and 1.0 mg/kg) in paper bowls were 76–83, 55–66, and 4–12%, respectively. It was reported that the appropriate pH range of extraction solvent for DSD-FWAs was between 7.5 and 9.5 [13, 19]. Therefore, different pH values of extraction solvent were adjusted with triethylamine and the extraction effects of different pH values on DSD-FWAs at the level of 1 mg/kg in paper bowls were tested (Supporting Information Fig. S4). Within the pH range of 7.5–9.5, the recoveries of 2S DSD-FWAs were 64–82%, but the recoveries of 4S and 6S DSD-FWAs were only 42–65 and 4–14%, respectively. With the increase of the pH value, the recoveries of all DSDFWAs were improved. When the pH was increased to 11.5 (acetonitrile/water/triethylamine 40:60:1, v/v/v), the recoveries of 6S DSD-FWAs were higher than 70%. It was inferred that 6S DSD-FWAs with more sulfonic acid groups had the stronger adsorption effect on paper fiber than 2S and 4S DSDFWAs when the pH values of extraction solvents were low. Therefore, the extraction solvents with the stronger alkalinity had the better extraction effect. pH 11.5 (adjusted with C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
triethylamine) was selected as the optimal pH of extraction solvent because the recoveries of all DSD-FWAs extracted by pH 11.5 extraction solvent were satisfactory.
3.4 Method validation The key parameters, including the linear range, LOD, accuracy, and precision, were fully validated in a single laboratory. To calculate LODs, low concentrations of the target compounds were injected (chromatographic concentrations from 1.0 to 5.0 ng/mL), and LODs were determined as the lowest concentration of analyte for which S/Ns were 3. LODs ranged from 0.12 to 0.24 mg/kg (Table 2). To evaluate linearity, the serial standard solutions were prepared as the method described in Section 2.2 and then their peak areas were obtained by HPLC. All target DSDFWAs exhibited good linearity with correlation coefficients (R2 ) ࣙ0.9994 in the concentration range of 8.0–100 ng/mL, which corresponded to the sample equivalent of 0.80– 0.10 mg/kg (Table 2). Accuracy and precision were evaluated by recovery studies at two concentration levels (1.0 and 5.0 mg/kg) in paper bowls. Six blank samples were spiked at each level. The results were provided in Table 2. The recoveries and RSDs of 11 DSD-FWAs at two concentration levels in paper bowls were 81–106 and 2–9%, respectively. Therefore, the established method was accurate and repeatable.
3.5 Application of the method Disposable paper cups, bags, and bowls were collected from local markets in China and tested by the proposed method. No obvious interfering peak was found in the chromatograms of all samples. Some DSD-FWAs were detected from 41% of samples (n = 87) and their contents were between 0.16 and 1869 mg/kg. Among all 11 DSD-FWAs, both the detectable www.jss-journal.com
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rate (40%) and the average content (75.3 mg/kg) of C.I.220 in samples were the highest. Therefore, the proposed method is very practical and reliable.
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[4] Keplinger, M., Fancher, O. E., Lyman, F. L., Calandra, J., Toxicol. Appl. Pharm. 1974, 27, 494–506. [5] Wang, B., Shang, J., Liu, X., Cui, W., Wu, X., Zhao, N., Curr. Microbiol. 2007, 54, 5–8.
4 Concluding remarks
[6] Gloxhuber, C., Bloching, H., Clin. Toxicol. 1978, 13, 171– 203.
A rapid, sensitive, and accurate method was developed for the simultaneous determination of 11 common DSD-FWAs in food-contact paper and board. Some problems in sample preparation, sample extraction, and chromatographic separation were successfully solved. Firstly, the distribution of DSD-FWAs in paper was often inhomogeneous. Therefore, a new sample preparation procedure was developed to guarantee the representativeness and homogeneity of the prepared paper sample. Secondly, 2S, 4S, and 6S DSD-FWAs were significantly different in the chemical properties, such as acidity, solubility, and adsorption capacity to the paper fiber. Some key factors such as the composition and pH of extraction solvent were optimized to effectively extract three types of DSDFWAs in paper fiber. Thirdly, DSD-FWAs have similar structures and are difficult to retain on a C18 column. Therefore, it was very difficult to separate some DSD-FWAs. The elution mode, the composition and the pH of the mobile phase, and the TBA concentration were successfully optimized to obtain the desirable separation of the 11 common DSD-FWAs.
[7] Escabasse, J. Y., Ottenio, D., Food Addit. Contam. 2002, 19(4), 79–92.
This work was supported by the National Natural Science Foundation of China (Grant No. 81072306) and Technology Foundation for Selected Overseas Chinese Scholar, Ministry of Human Resources and Social Security of China (2013).
[8] Ministry of Health of China. Hygienic standard of paper used for food packaging, 1989, GB 11680–1989. [9] Ren, Y. H., Commodity Quality 2011, 4, 12–14. [10] Ministry of Health of China. Method for analysis of hygienic standard of papers for food packaging, 2003, GB 5009.78–2003. [11] Sun, J. Y., Di, Q. M., Xu, Q. G., Appl. Mech. Mater. 2013, 401, 1135–1138. [12] Cao, Y., Griffith, J. F., Weisberg, S. B., Water Res. 2009, 43, 2273–2279. [13] Jasperse, J., Steiger, P. J., Am. Oil. Chem. Soc. 1992, 69, 621–625. [14] Wang, S. P., Huang, S. P., Electrophoresis 2001, 22, 2222– 2230. [15] Damant, A. P., Castle, L. J., Microcolumn. Sep. 1999, 11, 259–262. [16] Jeong, S. K., Do, H. K., Keon, K., Bull. Korean Chem. Soc. 2012, 33, 3971–3976. [17] Xian, Y. P., Guo, X. D., Luo, H. Y., Wu, Y. L., Chin. J. Chromatogr. 2013, 31(2), 162–169. [18] Shu, W. C., Ding, W. H., J. Chromatogr. A. 2005, 1088, 218–223. [19] Stoll, A., Giger W., Anal. Chem. 1997, 69, 2594–2599.
The authors have declared no conflict of interest.
5 References [1] Bilitewski, B., Darbra, R. M., Barcelo, D., Global RiskBased Management of Chemical Additives I, SpringerVerlag, Berlin and Heidelberg 2012, pp. 11–34. [2] Sturm, R., Williams, K., Macek, K., Water Res. 1975, 9, 211–219. [3] Lyman, F., Schulze, J., Ganz, C., Fd. Cosmet. Toxicol. 1975, 13, 521–527.
C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
[20] Shu, W. C., Ding, W. H., J. Chin. Chem. Soc. 2009, 56, 797–803. [21] Santos, M., Nerin, C., Domeno, C., Batlle, R., LC GC Eur. 2004, 17(11a), 6–13. [22] Chen, H. C., Ding, W. H., J. Chromatogr. A 2006, 1108, 202–207. [23] Shadkami, F., Helleur, R., Sithole, B. B., J. Wood. Chem. Technol. 2011, 31, 42–57. ˇ ˇ [24] Holcapek, M., Jandera, P., Zderadicka, P., J. Chromatogr. A. 2001, 926, 175–186. [25] Cecchi, T., Crit. Rev. Anal. Chem. 2008, 38, 161–213.
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J. Sep. Sci. 2015, 38, 605–611
Dingguo Jiang1 ∗ Lisong Chen2 Wusheng Fu2 Hanquan Qiu2 1 Key
Laboratory of Food Safety Risk Assessment of Ministry of Health, China National Center for Food Safety Risk Assessment, Beijing, P. R. China 2 Fujian Provincial Key Laboratory of Zoonosis Research, Fujian Provincial Center for Disease Control and Prevention, Fuzhou, P. R. China Received October 7, 2014 Revised November 28, 2014 Accepted November 30, 2014
Research Article
Simultaneous determination of 11 fluorescent whitening agents in food-contact paper and board by ion-pairing high-performance liquid chromatography with fluorescence detection 4,4 -Diaminostilbene-2,2 -disulfonic acid based fluorescent whitening agents (DSD-FWAs) are prohibited in food-contact paper and board in many countries. In this work, a reliable high-performance liquid chromatography method was developed for the simultaneous determination of 11 common DSD-FWAs in paper material. Sample preparation and extraction as well as chromatographic separation of multicomponent DSD-FWAs were successfully optimized. DSD-FWAs in prepared samples were ultrasonically extracted with acetonitrile/water/triethylamine (40:60:1, v/v/v), separated on the C18 column with the mobile phase containing tetrabutylammonium bromide, and then detected by a fluorescence detector. The limits of detection were 0.12–0.24 mg/kg, and the calibration curves showed the linear correlation (R2 ࣙ 0.9994) within the range of 8.0–100 ng/mL, which was equivalent to the range of 0.80–10 mg/kg in the sample. The average recoveries and the RSDs were 81–106% and 2–9% at two fortification levels (1.0 and 5.0 mg/kg) in paper bowls, respectively. The successful determination of 11 DSD-FWAs in food-contact paper and board obtained from local markets indicated that the newly developed method was rapid, accurate, and highly selective. Keywords: Fluorescent whitening agents / Food-contact paper and board / Highperformance liquid chromatography DOI 10.1002/jssc.201401110
Additional supporting information may be found in the online version of this article at the publisher’s web-site
1 Introduction 4, 4 -Diaminostilbene-2, 2 -disulfonic acid based fluorescent whitening agents (DSD-FWAs) have strong fluorescent properties and can directly absorb UV light (290–400 nm) and then reradiate the blue-violet light (400–500 nm). Therefore, they have been widely used in the modern paper industry in the world [1]. The molecular structures of commonly used DSD-FWAs are shown in Table 1. According to the number of sulfonic acid groups, DSD-FWAs can be classified into three types: disulfonic acid type (2S, e.g. FWA Correspondence: Dr. Wusheng Fu, Fujian Provincial Key Laboratory of Zoonosis Research, Fujian Provincial Center for Disease Control and Prevention, 76 Jintai Road, Gulou District, Fuzhou 350001, P. R. China E-mail: [email protected] Fax: +86-591-87670235
Abbreviations: ACN, acetonitrile; DSD-FWA, 4,4’diaminostilbene-2,2’-disulfonic acid-based fluorescent whitening agent; FWA, fluorescent whitening agent; HCl, hydrochloric acid; MeOH, methanol; 2S, disulfonic acid; 4S, tetrasulfonic acid; 6S, hexasulfonic acid; TBA, tetrabutylammonium bromide C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
5BM, C.I.71, C.I.85, C.I.90, and C.I.113), tetrasulfonic acid type (4S, e.g. C.I.24, C.I.210, and C.I.220), and hexasulfonic acid type (6S, e.g. C.I.264, C.I.353, and C.I.357). In some toxicological experiments, the toxicity of some DSD-FWAs was relatively low [2–4]. However, in other studies, the 2S DSD-FWA (C.I.85) could improve the peroral infection of the recombinant virus [5], and some FWAs affected the wound healing process [6]. Moreover, the toxicity of many newly developed DSD-FWAs and their photo-isomers is still unknown. Therefore, in many countries, DSD-FWAs have not been approved for food-contact paper and board. In the European Union, according to good manufacturing practices, FWAs cannot be detected in food-contact paper and board [7]. In China, FWAs were forbidden in the production of food-contact paper in 1989 [8]. However, according to the regulations of U.S. Food and Drug Administration, only C.I.220 can be added to food-contact paper and board (http:// www.accessdata.fda.gov/scripts/fcn/fcndetailnavigation.cfm ?rpt=fcsListing&id=889). Recently, high levels of FWAs had been found in some paper bowls for popcorn or instant ∗ Additional corresponding: Dr. Dingguo Jiang, E-mail: [email protected]
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Table 1. Structures and information of 11 DSD-FWAs
Analytes
CAS No.
R1
FWA 5BM C.I.71
13863–31–5 16090–02–1
−N(CH3 )C2 H4 OH
C.I.85 C.I.90 C.I.113
12224–06–5 3426–43–5 12768–92–2
−NHC2 H4 OH −OCH3 −N(OCH2 CH3 )2
C.I.24
12224–02–1
−N(C2 H4 OH)2
C.I.210
28950–61–0
C.I.220
16470–24–9
−N(C2 H4 OH)2
C.I.264 C.I.353
76482–78–5 55585–28–9
−N(C2 H4 OH)2
C.I.357
41098–56–0
−N(CH2 CH3 )2
R2
Basic structure
HPLC methods can only detect a few DSD-FWAs (less than seven kinds), while some common DSD-FWAs in paper cannot be detected. The LC–MS method is relatively rapid and sensitive [22, 23], but the nonvolatile ion-pair reagents successfully used in the HPLC separation often suppress the response signal and easily lead to the contamination of ion source [24]. Additionally, the multisulfonic groups of DSDFWAs may hinder the progress toward multicomponent analysis because the properties of multiply charged ions of DSDFWAs make it difficult to tune the precursor ions. Although some LC–MS methods for 2S DSD-FWAs were reported, the studies on 4S or 6S DSD-FWAs have been seldom reported until now. In conclusion, the ion-pairing HPLC method may be the most suitable method for quantifying DSD-FWAs. In addition, different sample preparation methods may greatly affect the detection results because the distribution of DSD-FWAs in paper samples is often inhomogeneous. However, the representativeness or the homogeneity of the prepared paper samples has been not reported. Moreover, 2S, 4S, and 6S DSD-FWAs are different in the number of sulfonic groups, the solubility, and the adsorption capacity to paper fiber. The method for simultaneously and effectively extracting three types of DSD-FWAs from paper materials has been not reported. In this study, a sensitive, accurate, and rapid analytical method was successfully developed for the simultaneous determination of 11 common DSD-FWAs in food-contact paper and board by ion-pairing HPLC with fluorescence detection.
2 Materials and methods 2.1 Chemicals and materials noodles and some paper cups for milk in China (http://food. china.com/rdjj/11101747/20120809/17364485.html) [9]. The situation was probably caused by the illegal addition in papermaking or the recycling of waste paper containing FWAs. Therefore, it is very important to develop a rapid and accurate analytical method for the investigation of the contamination levels of DSD-FWAs in food-contact paper. UV irradiation [10], fluorospectrophotometry [11], UV spectrophotometry [12], TLC [13], CZE [14,15], HPLC [16–20], and LC–MS [21–23] have been applied in the analysis of DSDFWAs in food-contact paper. The UV irradiation method by visual inspection is still an official method in some countries, but it cannot be used to accurately detect the total FWAs because the detection results are easily influenced by artificial factors. HPLC is a relatively reliable method for the qualitative and quantitative determinations of DSD-FWAs. However, DSD-FWAs with some sulfonic acid groups have the strong polarity in aqueous solutions and are difficult to be retained on a C18 RP column. To increase the retention of DSD-FWAs on C18 column, some ion-pair reagents are usually added into the mobile phases to transform DSD-FWAs into ion-paired compounds. To date, because some DSD-FWAs have very similar structure and are difficult to separate, the reported C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
C.I.24, 85, 113, 210, 264, and 357 were purchased from American International Laboratory. C.I.71 and 210 were obtained from Zhejiang Hongda. C.I.353, 90 and FWA 5BM were kindly supplied by China National Center of Quality Supervision and Inspection for Dyestuff. All DSD-FWAs were of reagent grade. HPLC-grade methanol (MeOH) and acetonitrile (ACN) were purchased from Fisher Scientific. Chromatographically pure hexane was obtained from Merck. Analytically pure tetrabutylammonium bromide (TBA), triethylamine, ethanol, acetone, and hydrochloric acid (HCl) were purchased from Sinopharm Chemical Reagent. Ultrapure water was obtained from a Mill-Q system (Millipore, Bedford, MA, USA). Disposable paper cups, paper bowls, and paper bags were purchased from local markets in Fuzhou, China.
2.2 Preparation of standard solutions Single standard stock solutions (500 g/mL) of the analytes were prepared by dissolution in acetonitrile/water (2:3, v/v) containing 1% triethylamine and then stored in a freezer (–18⬚C). The mixed standard intermediate solution www.jss-journal.com
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(10.0 g/mL) was prepared by further dilution of single standard stock solutions with acetonitrile/water (2:3, v/v) and stored in a refrigerator (4⬚C). The mixed standard working solutions (from 1.0 to 100 ng/mL) were prepared by further dilution of the mixed standard intermediate solution with acetonitrile/water (2:3, v/v). All the preparation operations should be carried out in the lab environment where the light intensity was below 20 Lux and all volumetric flasks for standard solutions should be brown. The prepared solutions should be tightly wrapped with aluminium foil and stored in the dark.
2.3 Instrumentation All the analyses were performed with an LC-20AT HPLC system (Shimadzu, Japan). DSD-FWAs were separated on a Symmetry C18 column (250 × 4.6 mm, 5 m, Waters). The column temperature was maintained at 35⬚C. The mobile phase was composed of Eluent A (acetonitrile/methanol, 2:3, v/v) and Eluent B (methanol/water, 5:95, v/v, containing 25 mmol/L TBA, adjusted to pH 8.0 with triethylamine). The gradient elution at a flow rate of 1.0 mL/min was programmed as: 0–2 min, 40% B; 2–12 min, 40%B→30% B; 12–17 min, 30%B→20% B; 17–19 min, 20% B→0% B; 19–21 min, 0% B→40% B; 21–25 min, 40% B. The analytes were detected by an Rf-10AXL fluorescence detector (Shimadzu, Japan). The excitation wavelength and the emission wavelength for all DSD-FWAs were 350 and 430 nm, respectively.
2.4 Sample preparation The relatively hard paper samples, such as the disposable paper cups and paper bowls, were sheared into flat paperboard with surgical scissors. Then about 30 g flat paperboard was cut into pieces with a size of 2 × 6 mm with an X2 shredder (Anbixin, China). The relatively soft paper samples, about 30 g, were overlapped and cut into the pieces with the size of 10 × 10 mm with surgical scissors. The paper pieces were transferred into the DFY-80 high-speed grinder (Wenling Linda Machinery, China), and ground at 10 000 r/min for 15 s. The grinding process was repeated six times with the interval of 5 s. The ground fine paper fiber was then packaged in a clean polyethylene bag and stored in the dark.
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tracted at 50⬚C for 35 min. After cooling to room temperature, the extract was centrifuged at 3750 r/min for 5 min, and the supernatant was carefully transferred into a 50 mL volumetric flask. Then 12 and 10 mL of the same extract solvent were added into the sample residue, respectively. The above procedure of extraction and centrifugation was repeated except that the extraction time was reduced to 10 min. The extracts were merged into the same volumetric flask, adjusted to pH 8.0 with 0.5 mol/L HCl solution, diluted to the volume of 50 mL with acetonitrile/water (2:3, v/v), and thoroughly mixed. Then 2 mL of extracts and 0.5 mL of hexane were added into a 5 mL test tube, which was vibrated for 30 s. The solution was allowed to stand for 2 min for stratification and then the supernatant was removed. The lower liquid was transferred into a 2 mL plastic centrifuge tube and centrifuged at 10 000 r/min for 5 min. The supernatant was transferred into a 2 mL brown sample bottle for HPLC analysis with the injection volume of 20 L. The amount of sample equivalent in the final extract was about 0.01 g/mL.
3 Results and discussion 3.1 Optimization of HPLC separation 3.1.1 Gradient elution To quickly achieve the equilibrium between the mobile phase and the C18 stationary phase, the isocratic elution mode was often recommended for the separation of DSD-FWAs in the ion-pairing HPLC [17, 21]. In this study, the similar chromatographic conditions were tested. However, the results showed that some 2S DSD-FWAs had a relatively long retention time (about 35 min) and some 4S DSDFWAs had a very short retention time (about 3 min). Moreover, the chromatographic resolutions of C.I.24/C.I.210 and C.I.85/C.I.113 were 0.40 and 0.60, respectively. Therefore, optimization tests for gradient elution program were carried out. In the gradient elution mode, the rapid separation of 11 DSD-FWAs was achieved and the RSD of the peak areas was less than 8% in six intraday measurements of each DSD-FWA. Therefore, the gradient elution mode was selected. 3.1.2 Organic mobile phase (Eluent A)
2.5 Sample extraction To reduce the possible isomerization of DSD-FWAs, all of the following experiments were performed in the lab environment where the light intensity was below 20 Lux. About 0.5 g of the ground paper sample was weighed and transferred into a 50 mL plastic centrifuge tube. Then 25 mL of acetonitrile/water/triethylamine (40:60:1, v/v/v) was then added into the centrifuge tube, which was then placed in an ultrasonic device. Then the test sample was ultrasonically ex C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Acetonitrile and methanol are common elution solvents used in HPLC analysis of DSD-FWAs [14, 17]. In this work, the elution capabilities of acetonitrile and methanol to DSDFWAs were tested under the condition of the same gradient elution program and TBA concentration. The chromatographic results were shown in Fig. 1A and 1B. For acetonitrile, the retention time of 11 DSD-FWAs was between 10.0 and 12.5 min, which indicated that the chromatographic separation was poor. For methanol, the retention time of 11 DSD-FWAs was between 11 and 21 min, so their retention www.jss-journal.com
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Figure 1. Effects of the organic mobile phase on the separation of 11 DSD-FWAs. Eluent A was (A) ACN, (B) MeOH, (C) ACN/MeOH = 4:1, (D) ACN/MeOH = 3:2, (E) ACN/MeOH = 1:4, and (F) ACN/MeOH = 2:3. The concentration of TBA, Eluent B and the gradient elution program were the same as that in Section 2.3. The concentration of each DSD-FWA was 100 ng/mL. The peaks: 1. C.I.220; 2. C.I.24; 3. C.I.210; 4. C.I.85; 5. C.I.113; 6. C.I.264; 7. C.I.353; 8. C.I.357; 9. FWA 5BM; 10. C.I.90; 11. C.I.71.
behaviors were significantly different and their separations were greatly improved. Based on the above results, the separation effects of different proportions of acetonitrile to methanol on the 11 DSD-FWAs were tested in the further experiments. The results shown in Fig. 1C, 1D, 1E, and 1F were different from those in Fig. 1B in three aspects. Firstly, the elution order of some DSD-FWAs was changed significantly. Secondly, the retention capability of some 2S DSD-FWAs (C.I.85, 113) was reduced. Thirdly, the retention capability of the 6S DSD-FWAs was enhanced. Among the four proportions of acetonitrile to methanol, the separation effect of acetonitrile/methanol (2:3, v/v) on the 11 DSD-FWAs was the best, so the 11 DSD-FWAs could be better separated. Therefore, acetonitrile/methanol (2:3, v/v) was identified as the optimal organic elution solvent. 3.1.3 TBA concentrations in Eluent B Some studies showed that the higher the TBA concentration in water, the longer the retention time of DSD-FWAs on C18 stationery phase [17]. Meanwhile, the effect of TBA on the retention capacity of three types of DSD-FWAs was decreased in the sequence: 6S, 4S, and 2S [25]. Therefore, the separation effects of different TBA concentrations on DSD-FWAs were tested under the conditions of the same organic mobile phase and gradient elution program (Supporting Information Fig. S1). With the increase of TBA concentration in Eluent B (methanol/water, 5:95, v/v), the retention time of all DSDFWAs was increased, and the resolutions between C.I.24 and 210 and between C.I.264 and 353 were also improved, while the resolution between C.I.357 and FWA 5BM was decreased. The changes between C.I.357 and FWA 5BM could be interpreted below. The number of sulfonic acid groups of C.I.357 was three times that of FWA 5BM. Therefore, compared with FWA 5BM, C.I.357 was more significantly influenced by the change of TBA concentration. With the increase of the TBA concentration, the retention time of C.I.357 became gradually closer to that of FWA 5BM, leading to the decreased C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
resolution between C.I.357 and FWA 5BM. When the TBA concentration in methanol/water (5:95, v/v) was 25 mmol/L, all target compounds could be optimally separated. 3.1.4 pH of Eluent B In this work, the chromatographic separation of 11 DSDFWAs was hardly affected by the pH of Eluent B (methanol/water, 5:95, v/v, containing 25 mmol/L TBA). However, when the pH of Eluent B was between 5 and 6, about 3% of each 6S DSD-FWAs in the injected sample solution would be retained in the HPLC system and appeared in the chromatogram of the next injection. When the pH was adjusted to 8.0 with triethylamine, the residual phenomenon disappeared and the response of all 6S DSD-FWAs also increased. 6S DSD-FWAs were probably easily aggregated and precipitated in the weak acidic Eluent B, thus leading to the crossing contamination problem in HPLC analysis. Therefore, Eluent B should be adjusted to the weak alkaline condition (pH 8.0) with triethylamine. Under the optimized chromatographic conditions described above, the 11 DSD-FWAs could be well separated in the retention time range of 11.5–19.5 min. According to the structures of DSD-FWAs, the separation of DSD-FWAs on the C18 column was influenced by the type of R1 group and the number of the sulfonic acids in R2 group. When the number of sulfonic acids in R2 group was the same, the stronger polarity of the R1 group indicated the weaker retention capability of DSD-FWAs on the C18 column except that the retention time of C.I.24 and 210 was affected by the position of sulfonic acid group on phenyl ring (namely the steric effect). When the position of R1 group was the same, according to the polarity, the theoretical elution order of DSD-FWAs should be 6S, 4S, and 2S. However, the actual elution order was 4S, 6S, and 2S (e.g. C.I.210, C.I.353, and C.I.71), indicating that the retention of 6S DSD-FWAs was enhanced. Since 6S DSD-FWAs had more sulfonic acid groups than 4S DSDFWAs, the retention capacity of 6S DSD-FWAs was more www.jss-journal.com
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easily influenced by TBA. Moreover, 6S DSD-FWAs had the larger molecular weight, so they had the stronger adsorption with a C18 column than 4S DSD-FWAs.
3.2 Sample preparation In the reported methods [17, 22], the paper samples were often cut into pieces with the size of 1–2 cm2 . In this work, the real paper bags containing C.I.220 from local market were also prepared by simply cutting, and then C.I.220 was detected in the prepared samples. The results showed that RSD of the concentrations of C.I.220 in six replicates was 54%. Therefore, the distribution of DSD-FWAs in food-contact paper might be inhomogeneous and the samples prepared by simply cutting might not be representative. In this study, the sample preparation method described in Section 2.4 was thoroughly validated. Briefly, about 15 g paper bowl without DSD-FWA and about 15 g paper bag containing C.I.220, 85, 264, and 353 were cut into pieces with the size of 2 × 6 mm with shredders, respectively, and the pieces were mixed together and ground into fine fibers at 10 000 r/min. Then, C.I.220, 85, 264, and 353 in the prepared samples were analyzed according to Sections 2.3 and 2.5 (Supporting Information Table S1). For 0.5 g sample, the RSDs of four DSD-FWAs in six replicates were 1–12%. Moreover, the detection results of four sampling amounts (0.5, 1.0, 2.0, and 3.0 g) in three replicates were basically the same and showed no significant difference (p > 0.05). Therefore, the samples prepared according to Section 2.4 were homogeneous and representative.
3.3 Optimization of extraction conditions In the optimization experiments, all the spiked samples were prepared as follows: 500 L of the mixed standard solution of 11 DSD-FWAs (10 g/mL) was carefully added into 0.5 g of the ground blank paper fiber in a 50 mL centrifuge tube and then the centrifuge tube cap was tightened. The centrifuge tube was tightly wrapped in aluminum foil, and placed in the dark for 24 h to allow DSD-FWAs to be fully combined with paper fiber. The spiked samples were then used for a series of optimization experiments of the extraction conditions, including the extraction method, solvent, temperature, duration, and times. All the optimization experiments for each parameter were repeatedly carried out for three times to minimize the experimental deviations. 3.3.1 Extraction method and solvent In this work, water was used as the extraction solvent and the paper bag sample was the matrix. Hot-water extraction, ultrasonic extraction, shaking extraction, and Soxhlet extraction were tested. The extraction rates of ultrasonic extraction and Soxhlet extraction were about the same, which were about two times those of hot-water extraction and shaking extrac C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 2. The extraction efficiency of different acetonitrile concentrations in water (n = 3). The spiked level of 11 DSD-FWAs in paper bag was 20 mg/kg. The trend charts of other 2S, 4S, and 6S DSD-FWAs, which were omitted, were similar to those of FWA 5BM, C.I.220, and C.I.264.
tion. Therefore, the fast and simple ultrasonic extraction was selected as the optimal extraction method. In some studies, hot water was used to extract DSD-FWAs in paper samples [21–23]. However, it was reported that the extraction efficiency of hot water was relatively low, especially for 2S DSD-FWAs [17]. Therefore, the DSD-FWAs in spiked paper cups were extracted with 40% (volumetric concentration) ethanol, acetonitrile, methanol, and acetone in water, respectively. The results indicated that 40% acetonitrile in water was the most effective, but the extraction recoveries of DSD-FWAs by 40% methanol in water were merely 38– 83% (Supporting Information Fig. S2). Furthermore, the extraction efficiencies of different acetonitrile concentrations in water were investigated (Fig. 2). The recoveries of 2S DSDFWAs were 38–47% when the acetonitrile concentration was less than 30%, indicating that 2S DSD-FWAs with the relatively low polarity were difficult to be extracted by the solvent containing the high content of water. On the contrary, the extraction rates of 6S DSD-FWAs with the relatively high polarity decreased when the acetonitrile concentration was more than 50%. At the same time, the change of the acetonitrile concentration had little effect on the extraction rates of 4S DSD-FWAs. To simultaneously extract three types of DSDFWAs, 40% acetonitrile in water was selected as the optimal extraction solvent. 3.3.2 Extraction temperature, duration, and times The extraction temperature, duration, and time are key factors in the extraction of DSD-FWAs in samples. The optimization experiments showed that the extraction rates of 11 DSDFWAs were the highest after extracting for 35 min at 30⬚C, but the recoveries of some 2S DSD-FWAs (C.I.113, FWA 5BM) were only 51–60%. With the increase of the extraction temperature, the extraction rates of these 2S DSD-FWAs were also improved. The optimal extraction temperature was 50⬚C. C.I.220, 210, 85, 113, and 264 in the real paper bag sample were extracted at 50⬚C for 35 min every time and the total contents of five DSD-FWAs extracted four times were determined as the true values (Supporting Information www.jss-journal.com
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Table 2. Validation parameters of the optimized method
Analyte
FWA 5BM C.I.71 C.I.85 C.I.90 C.I.113 C.I.24 C.I.210 C.I.220 C.I.264 C.I.353 C.I.357
Linear range (ng/mL)
6.0–100 4.3–100 4.0–100 6.7–100 7.0–100 5.3–100 6.0–100 4.7–100 7.7–100 8.0–100 6.7–100
Sample equivalent in linear range (mg/kg)
0.60–10 0.43–10 0.40–10 0.67–10 0.70–10 0.53–10 0.60–10 0.47–10 0.77–10 0.80–10 0.67–10
LOD (mg/kg)
0.18 0.13 0.12 0.20 0.21 0.16 0.18 0.14 0.23 0.24 0.20
Recoverya) (%)
RSD (%)
1 mg/kg
5 mg/kg
1 mg/kg
5 mg/kg
90 98 92 95 92 97 97 106 81 86 85
89 90 94 95 87 92 88 100 99 90 95
3 9 3 3 4 2 2 4 5 6 9
5 5 6 4 6 6 6 5 7 5 5
a) Recovery is the mean of six measurements, and 1 and 5 mg/kg are the spiked levels in paper bowls (n = 6).
Fig. S3). In the first extraction, the extraction rates of five DSD-FWAs were only 60–80%. However, the extraction rates of five DSD-FWAs extracted for three times could reach at least 95%. Therefore, three times was selected as the extraction times. Because the contribution ratios of the second and third extractions were relatively low (14–29%), the second and third extractions were shortened to 10 min. In the further experiment, the total extraction rates of the shortened extraction program (35, 10, and 10 min) showed no significant difference with that of the consistent extraction program (35, 35, and 35 min; p > 0.05). 3.3.3 pH of extraction solvent Under the above optimal extraction conditions, the recoveries of all DSD-FWAs in the spiked paper cups and bags were relatively high (68–106%), but the recoveries of 6S DSDFWAs in the spiked paper bowls were low. The recoveries of 6S DSD-FWAs at three fortification levels (15, 5.0, and 1.0 mg/kg) in paper bowls were 76–83, 55–66, and 4–12%, respectively. It was reported that the appropriate pH range of extraction solvent for DSD-FWAs was between 7.5 and 9.5 [13, 19]. Therefore, different pH values of extraction solvent were adjusted with triethylamine and the extraction effects of different pH values on DSD-FWAs at the level of 1 mg/kg in paper bowls were tested (Supporting Information Fig. S4). Within the pH range of 7.5–9.5, the recoveries of 2S DSD-FWAs were 64–82%, but the recoveries of 4S and 6S DSD-FWAs were only 42–65 and 4–14%, respectively. With the increase of the pH value, the recoveries of all DSDFWAs were improved. When the pH was increased to 11.5 (acetonitrile/water/triethylamine 40:60:1, v/v/v), the recoveries of 6S DSD-FWAs were higher than 70%. It was inferred that 6S DSD-FWAs with more sulfonic acid groups had the stronger adsorption effect on paper fiber than 2S and 4S DSDFWAs when the pH values of extraction solvents were low. Therefore, the extraction solvents with the stronger alkalinity had the better extraction effect. pH 11.5 (adjusted with C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
triethylamine) was selected as the optimal pH of extraction solvent because the recoveries of all DSD-FWAs extracted by pH 11.5 extraction solvent were satisfactory.
3.4 Method validation The key parameters, including the linear range, LOD, accuracy, and precision, were fully validated in a single laboratory. To calculate LODs, low concentrations of the target compounds were injected (chromatographic concentrations from 1.0 to 5.0 ng/mL), and LODs were determined as the lowest concentration of analyte for which S/Ns were 3. LODs ranged from 0.12 to 0.24 mg/kg (Table 2). To evaluate linearity, the serial standard solutions were prepared as the method described in Section 2.2 and then their peak areas were obtained by HPLC. All target DSDFWAs exhibited good linearity with correlation coefficients (R2 ) ࣙ0.9994 in the concentration range of 8.0–100 ng/mL, which corresponded to the sample equivalent of 0.80– 0.10 mg/kg (Table 2). Accuracy and precision were evaluated by recovery studies at two concentration levels (1.0 and 5.0 mg/kg) in paper bowls. Six blank samples were spiked at each level. The results were provided in Table 2. The recoveries and RSDs of 11 DSD-FWAs at two concentration levels in paper bowls were 81–106 and 2–9%, respectively. Therefore, the established method was accurate and repeatable.
3.5 Application of the method Disposable paper cups, bags, and bowls were collected from local markets in China and tested by the proposed method. No obvious interfering peak was found in the chromatograms of all samples. Some DSD-FWAs were detected from 41% of samples (n = 87) and their contents were between 0.16 and 1869 mg/kg. Among all 11 DSD-FWAs, both the detectable www.jss-journal.com
J. Sep. Sci. 2015, 38, 605–611
rate (40%) and the average content (75.3 mg/kg) of C.I.220 in samples were the highest. Therefore, the proposed method is very practical and reliable.
Liquid Chromatography
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[4] Keplinger, M., Fancher, O. E., Lyman, F. L., Calandra, J., Toxicol. Appl. Pharm. 1974, 27, 494–506. [5] Wang, B., Shang, J., Liu, X., Cui, W., Wu, X., Zhao, N., Curr. Microbiol. 2007, 54, 5–8.
4 Concluding remarks
[6] Gloxhuber, C., Bloching, H., Clin. Toxicol. 1978, 13, 171– 203.
A rapid, sensitive, and accurate method was developed for the simultaneous determination of 11 common DSD-FWAs in food-contact paper and board. Some problems in sample preparation, sample extraction, and chromatographic separation were successfully solved. Firstly, the distribution of DSD-FWAs in paper was often inhomogeneous. Therefore, a new sample preparation procedure was developed to guarantee the representativeness and homogeneity of the prepared paper sample. Secondly, 2S, 4S, and 6S DSD-FWAs were significantly different in the chemical properties, such as acidity, solubility, and adsorption capacity to the paper fiber. Some key factors such as the composition and pH of extraction solvent were optimized to effectively extract three types of DSDFWAs in paper fiber. Thirdly, DSD-FWAs have similar structures and are difficult to retain on a C18 column. Therefore, it was very difficult to separate some DSD-FWAs. The elution mode, the composition and the pH of the mobile phase, and the TBA concentration were successfully optimized to obtain the desirable separation of the 11 common DSD-FWAs.
[7] Escabasse, J. Y., Ottenio, D., Food Addit. Contam. 2002, 19(4), 79–92.
This work was supported by the National Natural Science Foundation of China (Grant No. 81072306) and Technology Foundation for Selected Overseas Chinese Scholar, Ministry of Human Resources and Social Security of China (2013).
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The authors have declared no conflict of interest.
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