Journal of Chromatography A, 1336 (2014) 67–75

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Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma

Col Liquid Chromatography

Analysis of additives in dairy products by liquid chromatography coupled to quadrupole-orbitrap mass spectrometry Wei Jia a,b , Yun Ling a , Yuanhui Lin c , James Chang d , Xiaogang Chu a,∗ a

Institute of Food Safety, Chinese Academy of Inspection and Quarantine, Beijing 100123, China College of chemistry and chemical Engineering, Shaanxi University of Science and Technology, Xi’an 710021, China Beijing Entry-Exit Inspection and Quarantine Bureau, Beijing 100026, China d ThermoFisher Scientific, 355 River Oaks Parkway, San Jose, California 95134, USA b c

a r t i c l e

i n f o

Article history: Received 23 December 2013 Received in revised form 8 February 2014 Accepted 10 February 2014 Available online 18 February 2014 Keywords: antioxidants preservatives synthetic sweeteners QuEChERS Q-Orbitrap dairy products.

a b s t r a c t A new method combining QuEChERS with ultrahigh-performance liquid chromatography and electrospray ionization quadrupole Orbitrap high-resolution mass spectrometry (UHPLC/ESI Q-Orbitrap) was developed for the highly accurate and sensitive screening of 43 antioxidants, preservatives and synthetic sweeteners in dairy products. Response surface methodology was employed to optimize a quick, easy, cheap, effective, rugged, and safe (QuEChERS) sample preparation method for the determination of 42 different analytes in dairy products for the first time. After optimization, the maximum predicted recovery was 99.33% rate for aspartame under the optimized conditions of 10 mL acetionitrile, 1.52 g sodium acetate, 410 mg PSA and 404 mg C18 . For the matrices studied, the recovery rates of the other 42 compounds ranged from 89.4% to 108.2%, with coefficient of variation 0.999. The limits of detection for the analytes are in the range 0.0001–3.6 ␮g kg−1 . This method has been successfully applied on screening of antioxidants, preservatives and synthetic sweeteners in commercial dairy product samples, and it is very useful for fast screening of different food additives. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Antioxidants are widely used as food additives to compensate for the loss of natural antioxidants, which are destroyed during storage and processing, and to help guard against food deterioration and rancidity [1–3]. Therefore, many antioxidants including natural antioxidants such as caffeic acid, glycyrrhizic acid and ursolic acid, as well as synthetic antioxidants such as propyl gallate, tertiary butylhydroquinone, butylated hydroxyanisole and butylated hydroxytoluene are used in foods [4]. Preservatives such as (2,4-dichlorophenoxy)-acetic acid, dehydro acetic acid and 4-Hexyl-resorcinol, are often added to food to prevent their spoilage, or to retain their nutritional value and flavor for a longer period [5,6]. The US Food and Drug Administration (FDA) regulates synthetic sweeteners such as aspartame, acesulfame potassium,

∗ Corresponding author. Tel.: +86 010 85778904. E-mail addresses: [email protected], [email protected] (X. Chu). http://dx.doi.org/10.1016/j.chroma.2014.02.028 0021-9673/© 2014 Elsevier B.V. All rights reserved.

sodium cyclamate, neotame and sucralose as food additives that duplicate the effect of sugar in taste, usually with less food energy. According to market analysts Freedonia, the United States artificial sweetener market is set to grow at around 8% per year to $189 million in 2012 [7,8]. Although more and more evidences in recent years indicate that the abuse of antioxidants, preservatives and synthetic sweeteners may produce toxicities or mutagenicites, and thus endanger the health of people [9], many kinds of antioxidants, preservatives and synthetic sweeteners are still widely used because of their high performance, low cost and excellent stability [10,11]. To protect public health, many countries have established strict regulations for the allowable kinds and concentration of antioxidants, preservatives and synthetic sweeteners [12]. However, some food producers may still add banned compounds to their products putting sensitive population in health risk. Therefore, there are still genuine needs to develop an accurate, convenient, sensitive, qualitative and quantitative method for analytically monitoring the proper use of illegal antioxidants, preservatives and synthetic

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sweeteners and the super-scale use of permitted antioxidants, preservatives and synthetic sweeteners in foods to ensure food safety. Various methods for the determination of antioxidants, preservatives and synthetic sweeteners in food have been reported, including high performance liquid chromatography (HPLC) with ultraviolet or diode-array detector (DAD) detection [13–15], capillary electrophoresis (CE) [16–18], liquid chromatography-mass spectrometry (LC-MS) [19–22], and gas chromatography-mass spectrometry (GC-MS) [23–25]. However, these methods are not suitable for simultaneous screening large number of antioxidants, preservatives and synthetic sweeteners because the multiple isomers and structural analogs of analytes are difficult to separate. In many instances blended food additives are added to foodstuff which often contains many kinds of food additives including antioxidants, preservatives and synthetic sweeteners. Therefore, analytical methods that can simultaneously determine antioxidants, preservatives and synthetic sweeteners are advantageous. But most of the methods only allow antioxidants, preservatives or synthetic sweeteners to be determined, respectively. Besides, false positives caused by complex food matrices are frequently encountered [26,27]. From last year the role of UHPLC/ESI Q-Orbitrap and related techniques is increasingly built up as enabling tool in food safety analysis for it can provide detailed structural information. In spite of the potential value of the application, to the best of our knowledge, so far no one has reported the application of Q-Orbitrap mass spectrometry combined with high performance liquid chromatography for simultaneous determination for a group of antioxidants, preservatives and synthetic sweeteners in foods [28–30]. Current procedures used to prepare samples for the determination of antioxidants, preservatives and synthetic sweeteners involve extraction by liquid–liquid extraction (LLE) and solid-phase extraction (SPE), LLE being gradually replaced by SPE [16,18,22]. Some of these methods present disadvantages, such as lengthy analysis time, high cost, and high solvent consumption, which make them inadequate for routine analyses. Hence, new straightforward approaches involving simpler and fewer steps would be welcome for a more effective clean-up of complex matrices such as dairy products samples. In this way, QuEChERS has been checked elsewhere for the extraction of pesticide residues such as vinclozolin, penconazole, dieldrin and fenbuconazole in milk, but to date, no work focused on the determination of antioxidants, preservatives or synthetic sweeteners in dairy products using QuEChERS has been published [31]. Difficulties may arise when the official methods are applied to emulsified fatty foods such as dairy products. Some compounds have been shown to be poorly recovered from milk samples, and the recovery rate tended to decrease as the fat content increased [32]. The FDA defined fatty foods as those with a fat content >2% of total composition (United States Food and Drug Administration, 1994). On average, dairy products contain 3.3–4.7% emulsified fat components [33]. The optimization of procedures for the extraction of antioxidants, preservatives and synthetic sweeteners in dairy products is time-consuming due to the number of target analytes to be extracted as well as number of variables that may affect the process. Hence, experimental design can be used to reduce series of experiments and find the relevant factors influencing the process. One of the goals of this study was to develop an analytical QuEChERS method for analysis of antioxidants, preservatives and synthetic sweeteners in dairy products by using response surface methodology to easily optimize the conditions of various sorbents and the volume of acetonitrile (MeCN) used in sample procedure. So far, no one has reported the application of response surface methodology to improve the pretreatment steps for food additives analysis methods. In this work, we developed a highly sensitive and accurate UHPLC/ESI Q-Orbitrap method to simultaneously screen 43

antioxidants, preservatives and synthetic sweeteners in dairy products. The modified QuEChERS sample preparation, composition of mobile phases, and the Q-Orbitrap mass spectrometric parameters for each target compound were optimized in detail. Several analytical parameters, such as sensitivity, precision, mass accuracy, selectivity linearity and limits of detection are evaluated. This method has been successfully applied on screening of antioxidants, preservatives and synthetic sweeteners in dairy products samples from local market. 2. Experimental 2.1. Chemicals and reagents Standards of 2,4,4 -trichloro-2 -hydroxydiphenyl ether (THE), benzyl 4-hydroxybenzoate (BPB), propyl benzoate (PB), n-butyl benzoate (BB), n-octyl 4-hydroxybenzoate (OHB), cinnamaldehyde (CH), benzyl benzoate (BZB), dimethyl fumarate (DF), isopropyl paraben (IPP), heptyl 4-hydroxybenzoate (HP), ethyl phydroxybenzoate (EP), methyl p-hydroxybenzoate (MP) and 2(1,3-Thiazol-4-yl)-1H-benzimidazole (TBZ) were obtained from Fluka (Buchs, Switzerland). Reference srtandards of dehydro acetic acid, (DHAA) isobutyl ester (IBP), n-butyl p-hydroxybenzoate (BP), methenamine (ME), (2,4-dichlorophenoxy)-acetic acid (2,4D), propyl-p-hydroxybenzoate (PP), propyl gallate (PG), octyl gallate (OG), dodecyl gallate (DG), 4-hexyl-resorcinol (HR), nordihydroguaiaretic acid (NDGA), 2 ,4 ,5 -trihydroxy-butyrophenon (THBP), 3,5-di-tert-butyl-4-hydroxybenzyl alcohol (IONOX), dilauryl thiodipropionate (DLTP), ethoxyquin (EQ), dl-thioctic acid (DLTA), ursolic acid (UA), epigallocatechin gallate (EGCG), glycyrrhizic acid (GA), caffeic acid (CA), 2,5-dihydroxy-1,4-di-tertbutylbenzene (BHQ), butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and tertiary butylhydroquinone (TBHQ) were supplied by Sigma–Aldrich (Steinheim, Germany) and aspartame (AP), sodium cyclamate (AC), acesulfame potassium (AK), saccharin sodium (SS), sucralose (TGS) and neotame (NT) were purchased from Dr. Ehrenstorfer (Augsburg, Germany). All reference compounds have a purified of >98.0%. MeCN, ascorbic acid, acetic acid, sodium acetate, ammonium formate, sodium chloride and anhydrous magnesium sulfate were purchased from Sigma–Aldrich (Steinheim, Germany). All reagents were of analytical grade. Anhydrous magnesium sulfate of reagent grade was dried at 110 ◦ C for 16 h before using. Ultrapure solvents (ethanol, methanol, formic acid) for liquid chromatography were sourced from Merck (Darmstadt, Germany). Bondesil primary secondary amine (PSA), BAKERBOND® octadecyl (C18 ) and Ceramic homogenizers obtained from Agilent Technologies (Harbor City, USA). Ultrapure water (resistivity, 18.2 M) was generated inhouse using a Millipore water purification system (Bedford, USA). Locally purchased milk, milk beverages and yogurt samples found to contain no response at the retention times of reference compounds or metabolite were selected for use as negative controls and stored at 4 ◦ C prior to analysis. 2.2. Standard solutions Stock standards solutions of individual compounds (with concentrations between 3000 and 5000 mg L−1 ) were prepared by exact weighing of the powder and dissolved in 100 mL of ethanol, which were then stored at −20 ◦ C in the dark. A multicompound working standard solution at a concentration of 1000 mg L−1 of each compound was prepared by appropriate dilutions of the stock solutions with water/methanol (1:1, v/v) and stored in screwcapped glass tubes at −20 ◦ C in the dark. An ascorbic acid mixture was also prepared daily by adding 1 g of ascorbic acid to 1000 mL of

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Table 1 Variables and levels evaluated in the central composite design to optimize the extraction conditions. Independent variable

Unit

Symbol

Extraction solvent volume Na Acetate quantity PSA quantity C18 quantity

mL G Mg Mg

X1 X2 X3 X4

the mixed solvent. All solutions were used within one month. The standard stock solutions and ascorbic acid mixture were employed to prepare standard working solutions that were used within one week. Corresponding standard solutions for the calibration curve were prepared using the standard working solutions and ascorbic acid mixture, and were used immediately after preparation. 2.3. Instrumentation The UHPLC/ESI Q-Orbitrap system consisted of an Accela 1250 LC pump and a CTC Analytics PAL open autosampler coupled with a Q Exactive mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). The system was controlled by Exactive Tune 1.1 and Xcalibur 2.2 software (Thermo Fisher Scientific, San Jose, USA). ToxID software was also used for data processing (Thermo Fisher Scientific). 2.4. Analytical procedure 2.4.1. Sample preparation Milk, milk beverages and yogurt samples (15 g) were weighed into 50-mL screw-cap centrifuge tubes (Waters, Milford, USA), fortified with the forty-three different antioxidants, preservatives and synthetic sweeteners and let to stand for 15 min. A 10-mL volume of MeCN in 1% acetic acid was added as an extraction solvent and the tube was tightly capped and vigorously mixed for 1 min using a vortex (Scientific Industries, New York, USA) mixer at maximum speed. Anhydrous MgSO4 (6 g), sodium acetate (1.52 g) and ceramic homogenizers were added to the tube, to induce phase separation. Samples were immediately shaken for 1 min, and then centrifuged for 5 min at 1500 rcf at 4 ◦ C (Beckman Couler, Brea, USA). Dispersive-SPE (dSPE) of the samples was carried out by pouring the supernatant (8 mL) into a centrifuge tube (50 mL) containing MgSO4 (1.2 g), PSA (410 mg) and C18 (404 mg). The sample was vortexed for 1 min and centrifuged for 5 min at 1500 rcf at 4 ◦ C. An aliquot of the final upper layer (200 ␮L) was transferred into a Mini-UniPrep vial, 300 ␮L methanol and 500 ␮L 8 mM ammonium formate buffer were added. The vials were capped, vortexed for 30 s, and filtered through a 0.22 ␮m nylon filter (Pall Corporation, Harbor, USA) into an autosampler vial. 5 ␮L of the filtrate was injected to the UHPLC/ESI Q-Orbitrap system. 2.4.2. Experimental design for response surface methodology (RSM) Response surface methodology (RSM) was employed to investigate the variations in recovery rates with respect to the preparation of conditions including extraction solvent volume, the amounts of sodium acetate, PSA, and C18 . The optimal composition of the 4 variables was determined by using a central composite design (CCD) approach. Table 1 indicates the coded and CCD-processed variables for the optimization of the QuEChERS method for samples. The resulting 30 experiments were carried out randomly. The goodness of fit of the regression model and the significance of parameter estimates were determined through appropriate statistical methods. Design Expert trial version 8.0.6.0 was used (Stat-Ease Inc., Minneapolis, MN).

Coded levels −␣

−1

0

+1

+␣

2 0.5 0 0

6 1.0 200 200

10 1.5 400 400

14 2.0 600 600

18 2.5 800 800

2.4.3. UHPLC-high resolution mass spectrometry analysis The chromatography was performed on the Thermo Accucore C18 aQ analytical column, 100 mm × 2.1 mm, 2.6 ␮m (Thermo Fisher Scientific, San Jose, USA) with an Accucore C-18 aQ guard column 10 mm × 2.1 mm (Thermo Fisher Scientific). The column was maintained at 30 ◦ C and the tray of the autosampler at 5 ◦ C. A gradient containing 0.1% formic acid and 4 mM ammonium formate in water (A), methanol with 0.1% formic acid and 4 mM ammonium formate (B) was applied. The following gradient was used: 0.0–1.0 min 100% A, 1.0–7.0 min 100–0% A, 7.0–12.0 min 0% A, 12.0–13.0 min 0–100% A. From 13.0 to 15.0 min the system was equilibrated with initial conditions 100% A. The run time for each injection was 15.0 min, and the flow rate was 0.3 mL min−1 . The retention times for the compounds are shown in Table 2. Q-Orbitrap ion source was equipped with a Heated Electrospray Ionization II (HESI II) probe. The optimized HESI II temperature was set at 350 ◦ C, the capillary temperature at 320 ◦ C, the electrospray voltage at 3.5 kV and 3.0 kV for positive and negative modes, respectively. Sheath and auxiliary gas were 18 and 3 L min−1 , respectively. The mass calibration of Q-Orbitrap was performed every five days to ensure a working mass accuracy of lower than 5 ppm. 3. Results and discussion Dairy product is one of the most complex food matrices due to the presence of numerous interferences. Forty-three target ions are searched daily in dairy products that are screened with a continuous and unlimited addition of new analytes to the method. The goal of this study was to develop a simple and accurate UHPLC/ESI Q-Orbitrap method to simultaneously screen forty-three antioxidants, preservatives and synthetic sweeteners. 3.1. Sample treatment MeCN, ethyl acetate and acetone, commonly used in the QuEChERS technique, were compared for solvent extraction of a blank sample of commercial milk spiked with the forty-three target analytes at a concentration 100 ␮g kg−1 . The experiments were performed as mentioned in Section 2.4.1, except that the type of solvent was varied. In comparison with that obtained using ethyl acetate and acetone, MeCN provided better extraction efficiency for all forty-three target analytes with recoveries in the range of 89–108% (Fig. 1), while acetone was the worst. MeCN was chosen as the extraction solvent because it good miscibility with water, and its ability to recover the analytes without extracting high quantities of lipophilic material (Fig. 2). To optimize the sample preparation conditions, the effect of each sorbent on the recovery of forty-three target analytes was considered. The most common sorbents to be used in QuEChERS for the treatment of dairy products are sodium acetate, PSA (primary and secondary amine) and C18 [34]. Compared with those from a non-buffered QuEChERS method, the acetate buffered method improved stabilities and recoveries of certain pH-dependent compounds (e.g. Dehydro acetic acid, Isopropyl p-hydroxybenzoate, Propyl-p-hydroxybenzoate, 2,4,4 Trichloro-2 -hydroxydiphenyl Ether and Aspartame), while the

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Table 2 The retention time, elemental composition, ionization mode, accurate mass, measured mass and mass deviations for all compounds. Peak

Compound

RT (min)

Elemental composition

Ionization mode

Theoretical (m/z)

Measured (m/z)

Accuracy (ppm)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43

Methenamine Acesulfame potassium Saccharin sodium Sodium cyclamate Epigallocatechin gallate Caffeic acid Sucralose 2-(1,3-Thiazol-4-yl)-1H-benzimidazole Dimethyl fumarate Aspartame Propyl gallate Methyl p-hydroxybenzoate Dehydro acetic acid Butylated hydroxyanisole Tertiary butylhydroquinone 2 ,4 ,5 -trihydroxy-butyrophenon Ethyl phydroxybenzoate Cinnamaldehyde Isopropyl paraben (2,4-dichlorophenoxy)-Acetic acid DL-thioctic acid Propyl-p-hydroxybenzoate Ethoxyquin Neotame Nordihydroguaiaretic acid Isobutyl ester 2,5-Dihydroxy-1,4-di-tert-butylbenzene n-Butyl p-hydroxybenzoate Benzyl 4-hydroxybenzoate Glycyrrhizic acid 4-Hexyl-resorcinol 3,5-di-tert-Butyl-4-hydroxybenzyl alcohol Propyl benzoate Octyl gallate Benzyl benzoate n-Butyl benzoate Heptyl 4-hydroxybenzoate 2,4,4 -Trichloro-2 -hydroxydiphenyl Ether n-Octyl 4-hydroxybenzoate Dodecyl gallate Butylated hydroxytoluene Ursolic acid Dilauryl thiodipropionate

0.74 1.50 3.58 3.71 4.80 4.90 4.95 5.19 5.40 5.52 5.75 5.81 5.85 6.03 6.18 6.20 6.33 6.37 6.68 6.69 6.80 6.81 6.83 6.87 7.02 7.13 7.17 7.19 7.20 7.34 7.46 7.50 7.52 7.64 7.82 7.87 8.10 8.11 8.33 8.51 8.74 9.08 12.91

C6 H12 N4 C4 H4 KNO4 S C7 H4 NO3 SNa C6 H12 NNaO3 S C22 H18 O11 C9 H8 O4 C12 H19 Cl3 O8 C10 H7 N3 S C6 H8 O4 C14 H18 N2 O5 C10 H12 O5 C8 H8 O3 C8 H8 O4 C11 H16 O2 C10 H14 O2 C10 H12 O4 C9 H10 O3 C9 H8 O C10 H12 O3 C8 H6 Cl2 O3 C8 H14 O2 S2 C10 H12 O3 C14 H19 NO C20 H30 N2 O5 C18 H22 O4 C11 H14 O3 C14 H22 O2 C11 H14 O3 C14 H12 O3 C42 H62 O16 C12 H18 O2 C15 H24 O2 C10 H12 O2 C15 H22 O5 C14 H12 O2 C11 H14 O2 C14 H20 O3 C12 H7 Cl3 O2 C15 H22 O3 C19 H30 O5 C15 H24 O C30 H48 O3 C30 H58 O4 S

[M + H]+ [M − K]− [M − Na]− [M − Na]− [M − H][M − H]− [M + Na]+ [M + H]+ [M + H]+ [M + H]+ [M − H]− [M − H]− [M + H]+ [M + NH4 ]+ [M − H]− [M − H]− [M − H]− [M + H]+ [M − H]− [M − H]− [M + Na]+ [M − H]− [M + H]+ [M + H]+ [M − H]− [M − H]− [M + NH4 ]+ [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M + H]+ [M − H]− [M + NH4 ]+ [M + H]+ [M − H]− [M − H]− [M − H]− [M − H]− [M − H]− [M + H]+ [M + H]+

141.11347 161.98665 181.99173 178.05433 457.07763 179.03498 419.00377 202.04334 145.04954 295.12885 211.06120 151.04007 169.04954 198.14886 165.09210 195.06628 165.05572 133.06479 179.07137 218.96212 229.03274 179.07137 218.15394 379.22275 301.14453 193.08702 240.19581 193.08702 227.07137 821.39651 193.12340 235.17035 165.09101 281.13945 230.11756 179.10666 235.13397 286.94389 249.14962 337.20205 219.17543 457.36762 515.41286

141.11357 161.98674 181.99184 178.05435 457.07800 179.03503 419.00394 202.04343 145.04970 295.12897 211.06133 151.04015 169.04965 198.14896 165.09222 195.06635 165.05573 133.06496 179.07146 218.96227 229.03290 179.07146 218.15382 379.22290 301.14478 193.08707 240.19589 193.08707 227.07158 821.39752 193.12341 235.17053 165.09103 281.13962 230.11774 179.10663 235.13402 286.94427 249.14964 337.20245 219.17551 457.36719 515.41290

0.71 0.54 0.57 0.07 0.81 0.28 0.41 0.45 1.10 0.41 0.62 0.55 0.65 0.50 0.73 0.36 0.08 1.28 0.50 0.69 0.70 0.50 0.55 0.40 0.83 0.26 0.33 0.26 0.92 1.23 0.04 0.77 0.12 0.60 0.78 0.17 0.21 1.32 0.08 1.19 0.32 0.94 0.08

recoveries of other target compounds were found of no apparent difference. Sodium acetate also has a dissolving effect on milk fat globules, which could affect recovery rates. PSA could efficiently remove interferences from polar organic acids, polar pigments, sugars and fatty acids. Strong interaction between PSA and acid functional groups of matrices removed most fatty acids and other organic acids in extracts of dairy products. The use of C18 sorbent retains trace amounts of lipid matrix components and is commonly used to remove apolar interferences, in combination with PSA. The combination of sodium acetate, PSA and C18 gave improved cleanup of matrix components from dairy products, but their amounts must be carefully selected to prevent analytes being removed. In general, higher amount of extraction solvent leads to a higher analytical signal in almost all analytes, but there was no preliminary study performed testing different ratios of mass (g) of sample per volume (mL) of extraction solvent. The best ratio needs to be found. Therefore, the amounts of MeCN, sodium acetate, PSA and C18 used in this study were determined by the RSM variables. Antioxidants, preservatives and synthetic sweeteners were selected according to the FDA food safety data program (FDA, 2006), Commission Regulation No 1129/2011 (EU, 2011), Canada Food and Drug Regulations (C.R.C., 2008), Codex General Standard for Food Additives (CAC, 2011), Japan Standards for Use (MHLW, 2011) and

the Ministry of Health, P.R. China official regulations for dairy products (MOHC, 2011). Taking in account the results obtained in preliminary studies described above, a full factorial central composite design was built with 30 experimental points for appropriate optimization of most important factors affecting QuEChERS clean up. The individual recoveries of all analytes were introduced separately as the response in the statistical program. The results from individual analysis of each compound were studied. Aspartame was selected to demonstrate the regression coefficient values calculated for the recovery of a compound. From the analysis of the effects (Table 3), it can be seen that the most significant variable was the volume of MeCN used in the extraction. An increase in the volume of MeCN from 6 to 14 mL provided a 15% increase in the extraction efficiency for aspartame. The best ratio of mass (g) of sample per volume (mL) of extraction solvent was found to be 1.50 because of the most suitable dispersion and the best homogenization between the dairy products and the extraction solvent were obtained. The interaction between the amount of sodium acetate and PSA was relevant well. This finding indicated that the optimization of these two variables should be carried out simultaneously as the adsorption of PSA depended on the pH. In addition, the amount of sodium acetate was the most important factor affecting the recoveries of the pH-dependent

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Fig. 1. Average recovery (n = 7 batches) of forty-three target analytes obtained from the QuEChERS sample preparation using MeCN, EtOAc and acetone for the solvent extraction of a blank sample spiked with forty-three target analytes at 100 ␮g kg−1 each. Other QuEChERS conditions are given in Section 2.4.1.

Table 3 ANOVA used to fit the quadratic model to the aspartame data. Variable

Sum of squares

Degrees of freedom

Mean square

F value

P-value prob > F

Model Extr. volume(X1 ) Na acetate(X2 ) PSA(X3 ) C18(X4 ) X1 × X2 X1 × X3 X1 × X4 X2 × X3 X2 × X4 X3 × X4 X1 2 X2 2 X3 2 X4 2 Residual Lack of fit pure error Cor total R2

17857.97 1466.72 6.39 983.30 1435.62 0.35 0.35 0.01 11.59 1.20 0.82 2434.24 8323.97 5220.19 3271.13 6.59 5.26 1.33 17864.56 0.9996

14 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 10 5 29

1275.57 1466.72 6.39 983.30 1435.62 0.35 0.35 0.01 11.59 1.20 0.82 2434.24 8323.97 5220.19 3271.13 0.44 0.53 0.27

2903.35 3338.43 14.54 2238.10 3267.63 0.81 0.81 0.021 26.39 2.73 1.86 5540.61 18946.35 11881.76 7445.49

< 0.0001 < 0.0001 0.0017 < 0.0001 < 0.0001 0.3835 0.3835 0.8879 0.0001 0.1193 0.1923 < 0.0001 < 0.0001 < 0.0001 < 0.0001

compounds. Under strongly acidic or alkaline conditions, aspartame may generate methanol by hydrolysis. At room temperature, it is most stable at pH 4.3, where its half-life is nearly 300 days. At pH 7, however, its half-life is only a few days. Therefore, the response surfaces generated suggest that the best extraction conditions for aspartame were a volume of 10.12 mL of MeCN and add 1.52 g sodium acetate in the centrifuge tubes. The amount of PSA and C18 reduces the peak area of interfering compounds but also reduces the chromatographic peak area of the analytes that are significant variables in the statistical program. The following optimum experimental conditions were obtained: 410 mg PSA, 404 mg C18 . While the optimized regression equations had been obtained and the predicted maximum recoveries of forty-three target

1.97

0.2350

analytes calculated, the simultaneous extraction of forty-three different antioxidants, preservatives and synthetic sweeteners with different properties from a complex matrix such as milk requires a compromise between each individual extraction optimum condition to perform simultaneous analysis of multiple compounds with a single pretreatment. For this reason, multiresidue methods for recovery were tested under the conditions optimized for aspartame, which gave the highest observed recovery in the single recovery tests and were therefore used to test the multiresidual compound quantification. The optimized conditions for aspartame were 10.00 mL MeCN, 1.52 g Na Acetate, 410 mg PSA, 404 mg C18 . The results of the multiresidue methods recovery test and validation information for each analyte are shown in Table 2. In

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Fig. 2. Response surfaces and contour curves of the extraction efficiency of aspartame showing the effects of varying the amounts of MeCN and sodium acetate.

summary, the resulting conditions allowed reliable simultaneous analysis of forty-three of the target analytes with recoveries in the range of 89.4–108.2%. 3.2. Chromatographic separation Traditional methods use HPLC coupled with triple Quadrupole Mass Spectrometer or DAD detection for determining antioxidants, preservatives or synthetic sweeteners in dairy products. However, structural analogs and multiple isomers of the antioxidants, preservatives and synthetic sweeteners are difficult to separate and determine. For instance, Li et al. used a Acquity BEH C18 column (50 mm × 2.1 mm, 1.7 ␮m) to separate ten synthetic preservatives in foodstuff, but many preservatives were overlapped [35]. Some overlapped peaks cannot be quantified by triple Quadrupole Mass Spectrometer or DAD detection, such as Isobutyl ester (m/z at 193.08702) retention time is 7.13 min, 4-Hexyl-resorcinol (m/z at 193.12340) retention time is 7.46. Three columns were tested to obtain the best resolution for these analytes, including Thermo Scientific Accucore aQ C18 (100 mm × 2.1 mm, 2.6 ␮m), Thermo Scientific Accucore RP-MS C18 (100 mm × 2.1 mm, 2.6 ␮m) and Kinetex XB C18 (100 mm × 2.1 mm, 1.7 ␮m). After optimizing the mobile phase conditions, the results showed that the Accucore aQ C18 column achieved the best resolution when formic acid–ammonium formate in water and methanol was used as the mobile phase. Several gradient profiles were studied, obtaining good response with the gradient described in Section 2. Under these conditions retention times of the analytes were constant, ranging from 0.76 (Methenamine) to 12.45 (Dilauryl thiodipropionate). 3.3. MS/MS conditions It is important to note that the results presented in this paper were obtained without any lock mass and with only external calibration. In addition, in this study, it has used the Customer Calibration to improve mass accuracy for low m/z measurement. For instance, the lowest m/z (SDS, sodium dodecyl sulfate) in the standard solution for ESI-calibration is not enough to guarantee the mass accuracy of chemicals with low molecular weight (Methyl p-hydroxybenzoate [M − H]− m/z 151.04007, Standard Calibration Mass Deviation 10.38 ppm.). This phenomenon is probably due to

Fig. 3. Effect of calibration mode on mass deviation: Customer ESI-Calibration (A) Standard ESI-Calibration (B). It can be seen that Customer Calibration improved mass accuracy for low m/z measurement.

unstable electrospray in the negative ion mode in combination with UHPLC separation technique on Q-Orbitrap. Citric acid was inserted into the standard ESI-calibration solution with final concentration 10 mg kg−1 for customer calibration, 20 eV in-source CID energy was used to induce citric acid (m/z 191.01973) fragmentation to get lower m/z product ions (111.00877, 87.00877), considering that citric acid not small enough. Finally, Customer Calibration improved mass accuracy for low m/z measurement (Fig. 3, Methyl p-hydroxybenzoate, Customer Calibration Mass Deviation 0.55 ppm). After full scan analysis, specific mass windows were extracted to screen the data for the presence of analytes. In principle, the selectivity obtained during data evaluation increase with narrowing the mass extraction window, when the mass extraction window is increased, the selectivity of the method is decreased, resulting in a higher background and reduction in sensitivity. But if the selected extraction window is too narrow and the mass accuracy is too high, thereby resulting in false negative results. The effect of the mass extraction window on selectivity for analytes in dairy products at a concentration 100 ␮g kg−1 (2, 3, 5, 8 and 10 ppm mass deviations) was tested. The best results were obtained when mass extraction windows of 3 ppm were employed. 3.4. Evaluation of matrix effect Matrix components can have an influence on the Q-Orbitrap signal intensity of the target compounds. Matrix effects may occur by ion enhancement (increasing) or ion suppression (decreasing) the MS signal and can have profound effects on assay precision and trueness in quantitative analysis, and its effects vary from sample to sample. It is necessary to evaluate the degree of matrix effects so that proper procedures can be taken to compensate or reduce for the effects. The matrix effect was tested by comparing the slopes of the matrix-free calibration curves to the matrix-matched calibration curves. Matrix effect was investigated by calculating the percentage (C%) of signal enhancement or suppression, according to Eq. (1). C% =



1−

ss sm



× 100

(1)

W. Jia et al. / J. Chromatogr. A 1336 (2014) 67–75

73

Table 4 Validation parameters of the developed method for milk sample. Compound

Extraction Recoveries %a

Calibration equation

Dynamic range (␮g kg−1 )

CC␣

CC␤

r

(y=)

Levels(n = 9 days) −1

Methenamine Acesulfame potassium Saccharin sodium Sodium cyclamate Epigallocatechin gallate Caffeic acid Sucralose 2-(1,3-Thiazol-4-yl)-1Hbenzimidazole Dimethyl fumarate Aspartame Propyl gallate Methyl p-hydroxybenzoate Dehydro acetic acid Butylated hydroxyanisole Tertiary butylhydroquinone 2 ,4 ,5 -trihydroxy-butyrophenon Ethyl phydroxybenzoate Cinnamaldehyde Isopropyl paraben (2,4-dichlorophenoxy)-acetic acid Propyl-p-hydroxybenzoate DL-thioctic acid Neotame Ethoxyquin Nordihydroguaiaretic acid Isobutyl ester n-Butyl p-hydroxybenzoate Benzyl 4-hydroxybenzoate 2,5-Dihydroxy-1,4-di-tertbutylbenzene Glycyrrhizic acid 4-Hexyl-resorcinol 3,5-di-tert-Butyl-4hydroxybenzyl alcohol Propyl benzoate Octyl gallate Benzyl benzoate n-Butyl benzoate Heptyl 4-hydroxybenzoate 2,4,4 -Trichloro-2 hydroxydiphenyl Ether n-Octyl 4-hydroxybenzoate Dodecyl gallate Butylated hydroxytoluene Ursolic acid Dilauryl thiodipropionate a

RSD % at three

89.4 98.2 99.9 89.8 104.7 95.8 99.4 101.5

−2910 + 6971x −3413 + 5633x −6851 + 9133x −8951 + 7869x −4259 + 3738x −5238 + 1185x −5379 + 1515x −185 + 397x

0.2–150 0.2–150 0.5–500 0.5–500 0.1–100 0.1–100 0.05–50 0.01–10

(␮g kg 0.13 0.12 0. 4 0.5 0.10 0.04 0.03 0.009

98.4 100.1 95.7 99.0 96.8 89.7 100.9 98.8 103.7 101 103.7 102.7 98. 98.5 96.7 93.5 94.3 98.6 97.8 89.9 96.8

−45790 + 1610x −3141 + 9509x −511 + 1605x 3714 + 1999x −3715 + 1469x −6307 + 4280x −21135 + 2200x −128 + 1034x −651 + 2629x −741 + 1919x −875 + 3824x −313 + 2730x 571 + 1857x −8265 + 2631x −7943 + 2656x −238 + 1392x −271 + 2431x −545 + 4562x −103 + 7923x −215 + 3138x −3178 + 1519x

5–1000 0.5–500 0.005–50 0.05–10 0.005–5 0.1–50 2–1000 0.001–1 0.01–10 0.01–10 0.01–10 0.01–10 0.01–10 0.1–100 0.1–100 0.005–5 0.01–10 0.01–10 0.01–10 0.01–10 0.2–100

3.6 0.3 0.003 0.03 0.003 0.15 1.8 0.0007 0.009 0.011 0.022 0.009 0.011 0.19 0.06 0.004 0.006 0.011 0.009 0.006 0.22

99.7 108.2 108.1

−1514 + 2599x 2943 + 4548x −1653 + 5747x

0.1–50 0.2–100 0.2–100

98.7 90.1 98.4 102.8 100.6 107.3

−211 + 821x −376 + 1670x −3665 + 9878x −5142 + 10012x −9363 + 7236x −6043 + 5423x

106.7 103.8 100.2 98.6 99.7

−6310 + 9778x −247 + 6319x −31270 + 2724x −6235 + 9973x –94456 + 3035x

)

−1

(␮g kg 0.22 0.21 0.8 0.9 0.18 0.07 0.06 0.015

) 0.9998 0.9997 0.9991 0.9996 0.9999 0.9996 0.9995 0.9991

Level 1 4.2 5.1 4.1 5.7 5.0 4.1 4.2 3.6

Level 2 2.2 1.0 2.8 1.7 3.1 1.3 3.0 4.6

Level 3 1.6 3.2 2.2 4.4 2.9 0.4 3.4 0.8

6 0.5 0.006 0.06 0.006 0.26 3 0.0012 0.015 0.019 0.038 0.016 0.019 0.32 0.1 0.007 0.011 0.019 0.015 0.01 0.37

0.9992 0.9997 0.9998 0.9994 0.9992 0.9993 0.9998 0.9996 0.999 0.9997 0.9996 0.9998 0.9996 0.9996 0.9992 0.9991 0.9994 0.9992 0.9999 0.9993 0.9994

2.6 3.6 2.7 3.4 2.1 3.9 0.2 4.6 1.9 2.7 4.1 3.2 5.2 5.7 4.3 5.7 3.0 3.5 4.4 0.3 3.4

3.4 3.9 5.0 3.9 1.8 3.4 1.5 3.9 2.8 4.2 2.3 3.8 3.8 2.1 3.7 3.4 3.8 6.4 1.8 3.9 3.2

3.2 2.5 2.5 2.9 2.9 2.7 3.0 0.4 3.9 2.6 2.7 1.2 2.2 3.2 1.3 3.2 3.9 0.6 4.5 4.3 1.5

0.096 0.29 0.24

0.16 0.49 0.41

0.9994 0.9993 0.9995

3.9 3.8 2.1

4.4 2.6 1.8

2.5 1.6 1.0

0.01–10 0.001–5 0.2–100 0.5–200 0.1–50 0.1–50

0.04 0.0001 0.22 0.5 0.07 0.10

0.08 0.0012 0.38 0.9 0.13 0.18

0.9992 0.9998 0.9991 0.9998 0.9991 0.9997

1.0 4.9 5.2 0.3 4.1 3.4

4.0 2.1 3.3 3.0 4.8 2.2

1.3 3.4 0.9 3.9 3.9 2.5

0.1–50 0.001–5 1–500 0.1–50 1–500

0.10 0.0012 1.4 0.09 1.3

0.17 0.0017 2.4 0.16 2.2

0.9997 0.9991 0.9992 0.9996 0.9993

4.8 1.0 2.6 4.4 2.3

4.5 0.9 4.5 3.1 3.3

1.6 2.0 1.8 2.7 2.0

Average of three concentration levels – CC␤, 2 CC␤, 4 CC␤

Where ss is the slope of calibration plot with matrix-matched calibration solutions and sm is the slope of calibration plot with calibration solutions in solvent. In the 43 analytes, 39 did not display significant changes in the slopes between standard calibration curves and matrix-matched calibration curves (−50% < C% < +50%). The remaining 4 compounds that had significant matrix effect were namely Sodium cyclamate, Butylated hydroxyanisole, Tertiary butylhydroquinone and n-Octyl 4-hydroxybenzoate (C% < −50% or C% > +50%). Apparently, QuEChERS was as effective for antioxidants, preservatives and synthetic sweeteners extraction and sample cleanup for dairy products. It is interesting to notice that the matrix effects from different types of dairy products showed a similar ion enhancement or suppression profile (Fig. 4). Therefore, any dairy product may be chosen as a standard matrix to construct matrix-matched calibration curves for quantification in routine practice.

3.5. Method validation The optimized method with Q-Orbitrap as acquisition mode was validated according to Commission Decision 2002/657/EC. The parameters evaluated were extraction recovery, specificity, linearity, precision, trueness, CC␣ and CC␤. The results of this validation are summarized in Table 4. Extraction recoveries were assessed by spiking blank dairy samples before and after extraction at three concentration levels(CC␤, 2 CC␤, 4 CC␤) with five replicates at each level. Linearity was assessed with spiked blank matrix at four concentration levels ranging between 0.001 and 1000 ␮g kg−1 . For all analytes, r2 was greater than 0.99 and the deviation of each point from the calibration line was lower than 15%. Specificity was assessed by verifying the presence of interference at the retention time of analytes greater than a signal-to-noise ration of three. Trueness was calculated as the percentage of error between spiked

74

W. Jia et al. / J. Chromatogr. A 1336 (2014) 67–75

Fig. 4. UHPLC/ESI Q-Orbitrap matrix effects of 3 dairy products. The evaluation was done at 100 ␮g kg−1 equivalent in samples.

and found concentrations. Three consecutive injections on-column at 100 ␮g kg−1 were carried out to detect any decrease in mass accuracy. No significant decrease was observed and the maximum mass deviation ranged from 0.05 to 1.52 ppm. This minor deviation in mass accuracy demonstrated the wide dynamic range of Q-Orbitrap for qualitative analysis at a resolving power of 70,000 FWHM. The CC␣ values of the proposed Q-Orbitrap mass spectrometer method are comparable to the limits of the previously reported triple quadrupole mass spectrometer methods. Comparing with the detection limits reported in literatures [36–38], the detection sensitivity and accuracy was improved more than 5 times. 3.6. Sample analysis In order to detect illegal antioxidants, preservatives and synthetic sweeteners, this UHPLC/ESI Q-Orbitrap method was applied to 30 samples from the local market. No illegal antioxidants, preservatives and synthetic sweeteners were detected. Aspartame, Acesulfame potassium, Sodium cyclamate, Caffeic acid, Dehydro acetic acid and Neotame were identified at levels lower than their legal limits (MOHC, 2011) [39]. Table 5 summarizes the screening results of the positive samples. Fig. 5 shows the typical chromatograms and spectra from a full MS/dd-MS2 experiment of analytes detected in positive samples.

Fig. 5. Examples of typical UHPLC/ESI Q-Orbitrap MS chromatograms and spectra from a full MS/dd-MS2 experiment: (A1) extracted ion chromatogram (displayed as a stick per scan) of Sodium cyclamate [M − Na]− m/z 178.05433 in sample No. 2.; (A2) dd-MS2 total ion chromatogram of Sodium cyclamate [M − Na]− m/z 178.05433 in sample No. 2.

4. Conclusions A new analytical method has been developed and applied in routine for screening and quantitation of antioxidants, preservatives and synthetic sweeteners in dairy products. In summary, by combining QuEChERS extraction procedure and UHPLC/ESI Q-Orbitrap, an accurate and highly sensitive method was developed to screen forty-three antioxidants, preservatives and synthetic sweeteners in foods. Compared with traditional methods, the sensitivity was enhanced, and the accuracy was improved by more than 5 times, leading to a powerful method for screening illegal antioxidants, preservatives and synthetic sweeteners in foods. Acknowledgments We wish to thank ThermoFisher Scientific (San Jose, USA) for providing us with UHPLC/ESI Q-Orbitrap. We would also like to thank James Chang for the technical support. The present research was financially supported by the grants from the project of General Administration of Quality Supervision, Inspection and Quarantine of the People’s Republic of China (Project number: 2012104002; 2013IK198). References

Table 5 Quantification results for target analytes in positive soft drinks samples analyzed by UHPLC/ESI Q-Orbitrap. Sample

Compound

Concentration (␮g kg−1 )

RSD (%)

No. 2

Saccharin sodium Sodium cyclamate Acesulfame potassium Sodium cyclamate Aspartame Sodium cyclamate Caffeic acid Acesulfame potassium Sodium cyclamate Aspartame Dehydro acetic acid Aspartame Sodium cyclamate Acesulfame potassium Neotame

30.1 321.5 101.2 298.3 73.1 46.4 89.7 102.3 489.5 246.8 0.12 103.4 36.7 99.7 64.1

0.7 2.9 2.3 1.7 1.6 0.7 1.2 1.4 2.9 1.8 1.6 0.6 2.8 2.3 2.6

No. 6 No. 9

No. 18 No. 27 No. 29

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