Journal of Chromatography B, 989 (2015) 21–26
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Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Simultaneous determination of 18 preservative residues in vegetables by ultra high performance liquid chromatography coupled with triple quadrupole/linear ion trap mass spectrometry using a dispersive-SPE procedure Xue Zhou a , Shurui Cao b , Xianliang Li b , Bobin Tang b , Xiaowen Ding c , Cunxian Xi b , Jiangtao Hu d , Zhiqiong Chen a,∗ a
College of Pharmacy, Chongqing Medical University, Chongqing 400016, China Chongqing Entry-Exit Inspection and Quarantine Bureau, Chongqing Engineering Technology Research Center of Import and Export Food Safety, Chongqing 400020, China c College of Food Science and Engineering, Southwest University, Chongqing 400715, China d Sichuan Entry-Exit Inspection and Quarantine Bureau, Sichuan Engineering Technology Research Center of Import and Export Food Safety, Chengdu 610041, China b
a r t i c l e
i n f o
Article history: Received 2 December 2014 Accepted 21 February 2015 Available online 10 March 2015 Keywords: UHPLC–QTRAP Vegetables Preservatives Dispersive-SPE
a b s t r a c t A new method combining dispersive-solid phase purification procedure with ultra high performance liquid chromatography–triple quadrupole/linear ion trap mass spectrometry was developed for simultaneous determination of 18 preservative residues in vegetables. The new method not only had the advantages of dispersive-solid phase purification procedure such as high recoveries, easy operation, rapid analysis, little solvent usage and wide analysis range of preservatives, but also had the advantages of ultra high performance liquid chromatography–triple quadrupole/linear ion trap mass spectrometry to be operated in positive mode and negative mode simultaneously. The method was validated for the following representative matrices: radish (tuber), tomato (eggplant fruit), cabbage (leafy), cowpea (bean), cucumber (melon) and so on. Samples were extracted with hexane–ethyl acetate (1:2, v/v), and then detected by ultra high performance liquid chromatography–triple quadrupole/linear ion trap mass spectrometry after being cleaned up with dispersive-solid phase purification procedure. Significant matrix effects were compensated by using the matrix-matched calibration curves. 18 preservatives showed good linearity over the range of 5.0–100.0 g/L with correlation coefficients of 0.9904–1.000. The limits of detections were in the range of 0.04–4.16 g/kg and the limits of quantity were in the range of 0.13–13.85 g/kg. The recoveries of 18 preservatives ranged from 76.0% to 120.0% with the spiked levels of 2, 4 and 10 g/kg into homogenized vegetables, and the relative standard deviations (RSDs) ranged from 0.3% to 14.8%. Compared with the reported literatures, the method is more rapid, simple, highly sensitive, reliable and can meet testing requirements of 18 preservative residues in vegetables. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Vegetables are essential in people’s daily diet which can provide a variety of vitamins and minerals. Eating plenty of vegetables will be beneficial for the health such as enhancing nutrition, improving digestion, softening blood vessels and so on. But vegetables are often infected by microorganisms during their growth in the field, which is resulting in alteration and degradation soon after picking
∗ Corresponding author. Tel.: +86 13032398667; fax: +86 23 68485161. http://dx.doi.org/10.1016/j.jchromb.2015.02.030 1570-0232/© 2015 Elsevier B.V. All rights reserved.
[1–4]. Preservatives are usually used during the growth and storage of the vegetables to keep the freshness of the vegetables. The promotion and use of efficient and lowly toxic preservatives have a favor of vegetables year-round sales, corruption reducing, longdistance transport, etc. [5]. However, people are no longer evaluating preservative which has a preservative effect and can improve the economic efficiency of vegetables. Social benefits without the environmental and healthy damage are more focused on. The extensive use of preservatives has been a concern because of their potential harm to the environment and known or suspected toxic effects on humans, such as neurodevelopmental impairment, acute
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neurological toxicity, reproductive and endocrine systems, possible dysfunction of the immune, chronic kidney diseases, intolerance, cancer and other potential diseases [6,7]. Due to these concerns, the determination of preservative residues in vegetables has a very important significance, especially the development of the method for the simultaneous determination of many kinds of preservative residues is imperative. Simultaneous determination of a wider range of preservative residues in vegetables will not only protect healthy eating of vegetables but also can reduce human labor. Different methods have been developed for the determination of preservative residues, including enzyme-linked immunosorbent assay (ELISA) [8,9], biosensor [10,11], capillary electrophoresis (CE) [12,13], high-performance liquid chromatography (HPLC) [14,15], gas chromatography (GC) [16,17], liquid chromatography (LC) and gas chromatography coupled to mass spectrometric (MS) detectors [18,19]. Biosensor is a newly developed and promising analytical technique for preservatives, but this technique can only detect organophosphorous preservatives [10]. GC and GC–MS suit for volatile preservatives [20,21]. ELISA, CE and other detection instruments can not be compared with ultra high performance liquid chromatography-tandem mass spectrometry (UPLC–MS/MS) in many fields such as sensitivity, application range and so on. UPLC–MS/MS is already a proven technique with a wide range of applications [22]. Paper has been published in which the detection limit was low to 0.12 g/kg [23]. But almost all the UPLC–MS/MS have to select an electrospray ionization source (ESI) in positive mode or in negative mode separately [22,24]. Ultra high performance liquid chromatography–triple quadrupole/linear ion trap mass spectrometry (UHPLC–QTRAP) that we used could detect positive and negative ions simultaneously, without the need for separate detection of positive and negative ions. The determination of preservative residues in vegetables using UHPLC–QTRAP is rarely reported. Many experimental conditions of 18 preservatives in vegetables were investigated such as extraction and purification. This method made full use of the advantages of ultra-fast scanning speed, strong specificity, high efficiency and sensitivity of UHPLC–QTRAP. The UHPLC–QTRAP method coupled with dispersive-SPE procedure for the simultaneous determination of 18 preservative residues in vegetables was issued. The method was fully validated for linearity, limits of detection, matrix effect, accuracy, and precision, which can provide technical support for the rapid determination of preservative residues in vegetables.
2. Materials and methods 2.1. Chemicals and reagents All compounds (listed in Table 1; analytic grade ≥99.0% purity) were purchased from AccuStandard (New Haven, CT, USA). Acetonitrile, hexane, ethyl acetate, acetone, toluene, and methanol (HPLC grade) were obtained from Tedia (Fairfield, OH, USA). Ethylenediamine-N-propyl silane’s (PSA’s) size was 60 m, which was purchased from Agilent Technologies (Santa Clara, CA, USA). Sodium chloride, ammonium acetate and magnesium sulfate were analytical grade. Ultra-pure water was prepared by Milli-Q-plus ultra-pure water system (Milford, MA, USA) throughout the study. All of the vegetables were purchased from various local supermarkets (Chongqing, China). 2.2. Instrumentation LC-30 ultra performance liquid chromatography (Shimadzu, Japan)-API5500Q triple quadrupole/linear ion trap mass spectrometer (AB Sciex, USA); XH-B vortex mixer (Jiangsu healthcare medical supplies company, LTD); 3–30 K refrigerated centrifuge (German, Sigma company); Rotary vacuum evaporators (4011 Digital) (German, Heidolph Company); N-EVAP116 nitrogen dry instrument (Organomation Associates, USA); Pipettes (adjustable range: 10–100 L, 20–200 L, 10–1000 L, 1000–5000 L, 1–10 mL) (German, Eppendorf Company); SR-2DS powerful oscillator (TAITEC, Japan). 2.3. Standard solutions Each stock solution of the studied standards was individually prepared by dissolving the weighed compound in acetonitrile at final concentration of 1 mg/mL in glass vials, which was stored at −18 ◦ C. An aliquot of each stock solution was added to appropriate acetonitrile–2 mmol/L ammonium acetate (1:1, v/v) to prepare a mixture solution of the 18 preservatives, with 10 mg/L in 50 mL and stored at −18 ◦ C prior to use. This stock solution was further diluted by acetonitrile–2 mmol/L ammonium acetate (1:1, v/v) to make standard solutions of all preservatives, which was prepared daily. The concentrations of standard solutions were 5, 10, 20, 50 and 100 g/L.
Table 1 LC–MS/MS acquisition parameters for 18 preservatives. No.
Compound
Retention time (min)
Ion pair Q1 /Q3 (m/z)
Declustering potential DP/V
Collision voltage CE/V
Collision chamber outlet voltage CXP/V
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Enilconazole Chlorpyrifos Carbendazim 2-(4-thiazolyl)benzimidazole Prochloraz Pyrimethanil Myclobutanil Flusilazole Propiconazole Triadimefon Iprodione Fenethanil Butyl paraben Propyl paraben Ethyl paraben Methyl paraben 2-Naphthol 4-Phenylphenol
5.85 7.21 3.65 3.86 6.16 5.45 5.70 5.90 6.12 5.75 5.99 5.93 5.39 4.95 4.46 3.93 4.84 5.31
297.1/159.1a , 297.1/255.1 350/97a , 350/198 192.2/131.1a , 192.2/160.1 202.2/131.1a , 202.2/175.1 376/266.2, 376/308.1a 200.2/107.1a , 200.2/168.1 289.1/70.1a , 289.1/125.1 316.1/165.1a , 316.1/247.1 342.1/159a , 342.1/205 294.1/197.1a , 294.1/225.1 330.1/245.0a , 330.1/288.1 337.1/125.1a , 337.1/70.0 192.9/91.8a , 192.9/136.1 178.9/92.0a , 178.9/136.0 165.0/92.0a , 165.0/149.0 151.0/92.0a , 151.0/135.9 142.8/114.9a , 142.8/124.8 160.9/93.0a , 160.9/140.9, 160.9/114.9
50 50 58 106 26 96 55 37 63 41 84 91 −110 −83 −70 −57 −130 −114
29, 25 47, 32 43, 24 44, 34 22, 16 34, 41 21, 47 36, 26 42, 24 21, 17 19, 18 23, 44 −25, −24 −30, −22 −29, −19 −29, −18 −34, −12 −40, −37, −41
9.8, 5 4.9, 6 7.2, 10 8.9, 11.6 8.4, 6.8 14, 9 8.5, 7.4 8.1, 15.8 8.8, 6.0 10.2, 14 11, 11 7.3, 8.1 −10, −10 −5.6, −8 −8.8, −7.3 −4.8, −6.5 −15, −16 −8.4, −9.9, −5.3
a
Quantitative ion.
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23
a
b
c
Fig. 1. (a) Separation effect of different gradient elution programs. (b) Separation effect of different gradient elution programs. (c) Separation effect of different gradient elution programs.
2.4. UHPLC–QTRAP analysis Analyses were done in a Shimadzu UHPLC system coupled with an API 5500Q triple quadrupole/linear ion trap mass spectrometer. For the UHPLC system, chromatographic separation was achieved in an (Waters) ACQUITY UPLC BEH C18 (2.1 mm × 50 mm, 1.7 m). The mobile phase and a gradient elution program are shown in the picture III of Fig. 1. The flow rate was kept at 0.3 mL/min. The injection volume was 1 L, and the column temperature was maintained at 40 ◦ C. For the mass spectrometric analysis, a mass spectrometer equipped with the manufacturer’s electrospray (ESI) source, operated in positive ion mode and negative ion mode simultaneously, was applied in multiple reaction monitoring (MRM) mode. The total online UHPLC–QTRAP analysis time per sample was 10 min. Electrospray voltage (IS) was 5500 V and collision chamber inlet voltage (EP) was 10.0 V when ionization mode was in ESI+ mode. While the ionization mode was in ESI− mode, IS was −4500 V and EP was −10.0 V. Atomization gas pressure (GS1) was 50.0 psi, air curtain gas pressure (CUR) was 30.0 psi, auxiliary air pressure (GS2) was 50.0 psi and the temperature was 550 ◦ C, no matter the ionization mode was in ESI+ mode or in ESI− mode. Mass spectrum parameters of all compounds are shown in Table 1. 2.5. Preparation and treatment of the samples 2.5.1. Samples preparation Extraction procedure was as follows: accurately weighed 10.0 g of the homogenized vegetable (accurate to 0.01 g) into a 50 mL teflon centrifuge tube, 20 mL hexane–ethyl acetate (1:2, v/v) and 4 mL 2 mol/L ammonium acetate solution were added. The sample was extracted for 8 min by powerful oscillator. After the addition of 5.0 g (accurate to 0.01 g) NaCl, the tube was vortexed for 1 min, followed by centrifugation at 5000 rpm for 3 min. Then, 10 mL of the supernatant were transferred to a 15 mL teflon centrifuge tube with bottom containing 100 mg of PSA and 500 mg of anhydrous MgSO4 . The tube was vortexed for 2 min, followed by centrifugation at 5000 rpm for 3 min. The supernatant was placed into
a 25 mL round-bottom flask. The supernatant was concentrated to almost dryness on rotary vacuum evaporator below 40 ◦ C, and residues were dissolved with 1 mL acetonitrile–2 mmol/L ammonium acetate (1:1, v/v). The content was filtered through a 0.22 m nylon membrane filter and analyzed by UHPLC–QTRAP. 2.5.2. Preparation of calibration curve 10.0 g of the homogenized vegetable were treated as described in Section 2.5.1 without adding standard solution in. When the extract was concentrated to almost dryness on rotary vacuum evaporator below 40 ◦ C, 1 mL of each standard solution of 5, 10, 20, 50 and 100 g/L was spiked to blank residue to obtain a series of matrix-matched calibration solutions (5, 10, 20, 50 and 100 g/L), which were analyzed by UHPLC–QTRAP. Matrix-matched calibration curve was used for quantification and matrix effect assessment. 3. Results and discussion 3.1. Optimization of UHPLC–QTRAP The UHPLC–QTRAP with ESI source and MRM mode provided a highly selective and sensitive method for the determination of 18 preservatives. According to the chemical structure characteristics of the targets, most of the compounds fitted in ESI+ mode form [M+H]+ ions; the few compounds were suitable for ESI− mode form [M−H]− ions. Therefore, standard solution (1 mg/L) was injected in and [M+H]+ or [M−H]− molecular ion was determined by the primary mass spectrometry, then fragment ions information was obtained by the secondary mass spectrometry. Two characteristic ions of maximum sensitivity were selected as the quantification and identification ions to optimize the MS/MS parameters (Table 1). Positive and negative ions can be detected simultaneously, without the need for separate detection of positive and negative ions. The commonly used mobile-phase compositions such as acetonitrile–water and methanol–water were optimized. The results showed that acetonitrile–2 mmol/L ammonium acetate was the best mobile-phase compositions. Several gradient elution programs were optimized (Fig. 1). As shown in Fig. 1, separation of 18
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acetonitrile’s proportion was high. So proportion of acetonitrile and 2 mmol/L ammonium acetate was considered to be 50:50, which was 1:1. 3.4. Method validation
Fig. 2. Effect of different extraction solvents on recovery.
preservatives in picture c was the worst. The change rate of gradient elution 1 in picture c was bigger than picture b, which resulted in that the peak times of most preservatives were in gradient elution 1 of picture c, so that separation of 18 preservatives in picture b was better than picture c. In picture a, because the proportion of the organic phase was reduced and the change rate of gradient elution of picture a was appropriate to be decreased, the separation of 18 preservatives was the best. The gradient elution program in the picture c of Fig. 1 was developed to provide baseline separation for 18 preservatives. The columns were investigated including ACQUITY UPLC BEH C18 , Shim-pack VP-ODS, Inertsil ODS-SP and Kinetex C18 . It turned out that using ACQUITY UPLC BEH C18 column to separate and using acetonitrile–2 mmol/L aqueous ammonium acetate as mobile phase could improve the ionization efficiency of the target compounds. Under the optimal operating conditions of UHPLC–QTRAP, all compounds were eluted within the 10 min analysis time. 3.2. Optimization of sample extraction solvent Acetonitrile, hexane–ethyl acetate (1:1, v/v), hexane–ethyl acetate (1:2, v/v) and acetone–ethyl acetate (1:1, v/v), commonly used in the extraction of preservatives, were compared for solvent extraction of blank samples of radish spiked with 18 preservatives at a concentration 2 g/kg. All the sample extraction solvent were investigated three times. The average of two extraction recoveries of each sample extraction solvent was accepted. The experiments were performed as mentioned in Section 2.5.1, except that the type of solvent was varied. In comparison with that obtained using acetonitrile, hexane–ethyl acetate (1:1, v/v) and acetone–ethyl acetate (1:1, v/v), hexane–ethyl acetate (1:2, v/v) provided better extraction efficiency for all 18 preservatives with recoveries in the range of 79.6–106.5% (Fig. 2). In comparison with acetonitrile and acetone–ethyl acetate (1:1, v/v), there were less impurities when extracted by hexane–ethyl acetate (1:2, v/v). 3.3. Optimization of dissolved solution Different proportions of acetonitrile and 2 mmol/L ammonium acetate (100:0, 90:10, 80:20, 50:50, 40:60, 30:70, 20:80, 10:90, 5:95, v/v) were investigated, which were used to dissolve residues. All compounds showed good peak shapes in different proportions of acetonitrile and 2 mmol/L ammonium, but not all compounds could be dissolved when 2 mmol/L ammonium acetate’s proportion was high and non-target residues would be dissolved when
3.4.1. Linearity, detection and quantitation limits Linearity was studied in the range of 5–100 g/L for 18 preservatives with different calibration levels by matrix-matched standard calibration in blank extracts or by standard calibration in solvent. Linearity values, calculated as determination coefficients (R2 ) for 18 preservatives from the matrix-matched calibration plots or just from the calibration plots, are shown in Table S1. Good linearity was found for 18 preservatives within R2 values of 0.9904–1.000 from the matrix-matched calibration plots and 0.9927–1.000 just from the calibration plots. LOD and LOQ are two fundamental elements of method validation that define the limitation of an analytical method. The LOD and LOQ for 18 preservatives ranged from 0.04 to 4.16 g/kg and from 0.13 to 13.85 g/kg at the signal-to-noise ratios (S/N) of 3 and 10, respectively, which were calculated from standard solution at a concentration of 10 g/L. 3.4.2. Evaluation of matrix effect In mass spectrometry, co-elution of residual components from the sample matrix may occur by ion enhancement (increasing) or ion suppression (decreasing) the MS/MS signal and have profound effects on assay precision and trueness in quantitative analysis. Matrix effects vary from sample to sample. Therefore, the evaluation of matrix effect on the quantitative analysis was an important assay validation. The matrix effect (ME) was defined by the ratio between the slope of matrix-matched calibration curve and the slope of standard solution curve subtracted 1, and then multiplied by 100% [28–30]. Formula was showed in Eq. (1). A negative ME value indicates that the MS/MS signal of target compound was suppressed. Contrarily, the positive ME value means an ionization enhancement effect. Absolute value varies from 0 to 20%, indicating the presence of a weak matrix effect; Absolute value varies from 20% to 50%, indicating the presence of moderate matrix effect; Absolute value is above 50%, indicating that there is a strong matrix effect. ME% =
S
s
Sm
− 1 × 100%
(1)
where Ss is the slope of calibration plot with matrix-matched calibration solutions, Sm is the slope of calibration plot with calibration solutions in solvent and ME% is the calculating percentage of signal enhancement or suppression. In this research, matrix-matched calibration curves were performed at five concentration levels (5, 10, 20, 50 and 100 g/L) by spiking the extracts obtained from blank sample. As shown in Table S1, matrix effects of 18 preservatives were different in five kinds of matrix. The absolute values range from 4.01% to 78.1%. Most of the matrix effects were suppressed and moderate. To eliminate matrix influence, this method was performed by matrix-matched calibration curves. 3.4.3. Recovery and repeatability Recovery and repeatability of the method were established to evaluate the performance of method. The repeatability and the recovery of the method were studied by carrying out six consecutive extractions (n = 6) of spiked matrices at three concentration levels (2, 4 and 10 g/kg). Table S2 provides the recoveries and RSDs. The recoveries ranged from 83.6% to 119.2%, from 79.7% to 119.8%, from 76.0% to 108.6%, from 81.6% to 119.5% and from 78.1% to 110.0% when the matrices were radish, tomato, cabbage, cowpea and cucumber respectively. RSDs were below 15% in all cases.
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Table 2 Comparison of different methods. Category
GB/T 20769-2008 [25]
Literature 1 [26]
Literature 2 [27]
The method of this research
Species (n) Purification Experimental time (min) The detection time (min) Recovery (%) LOD (g/kg)
11 NH2 column 266 30.40 / 0.12–13.45
5 QuEChERS: PSA, MgSO4 19 17.73 90–120.0% /
4 / 35 25 91.0–115% 0.1–0.5
18 QuEChERS: PSA, MgSO4 37 10.00 78.1–120.0% 0.04–4.16
Table 3 The amount of preservative residues in vegetables (g/kg). No.
Radish
Tomato
Cabbage
Cowpea
Cucumber
No.
Radish
Tomato
Cabbage
Cowpea
Cucumber
1 2 3 4 5 6 7 8 9
0.2 7.6 0.5 0.5 0.3 ND ND 0.1 ND
ND 5.7 0.7 ND ND ND ND ND ND
ND 4.4 0.2 ND ND ND ND ND ND
ND 7.0 6.5 ND 7.5 0.4 0.4 0.1 0.1
ND 5.1 0.6 ND ND ND ND ND ND
10 11 12 13 14 15 16 17 18
ND ND ND ND 0.2 2.0 5.0 0.3 0.1
ND ND ND ND 0.2 0.5 1.5 0.2 ND
ND 0.3 ND ND 1.2 1.6 1.3 ND ND
ND 2.1 ND 0.1 0.5 ND 0.9 0.2 0.3
0.2 0.7 ND ND 0.8 0.1 1.0 0.1 ND
Note: ND indicates that the content of the sample is less than the detection limit of the method.
3.4.4. Comparison with other methods This study, GB/T 20769-2008 [25], the literature 1 [26] and 2 [27] were compared. NH2 columns were used for cleanup in GB/T 20769-2008, but there was no purification in literature 2. The results showed that the time of GB/T 20769-2008 was too long and the spiked levels of literature 2 were too high. This study used dispersive-SPE procedure for cleanup, which is same to literature 1, but the detected species of literature 1 were so few. On the one hand, the time consuming of the issued method in the pretreatment process is short, on the other hand, UHPLC–QTRAP can be operated in positive mode and negative mode simultaneously. What is more important, the detected species of preservatives were the most. Compared with other methods, the detected speed of this method was increased by 35%. The detailed results were shown in Table 2.
Acknowledgments This work was supported by “Food and Agricultural Research fund project (No. cstc2013yykfB0165)” and “Youth Science and Technology talents fund project (No. cstc2014kjrc-qnrc00002)” of Chongqing Municipal Science and Technology Commission of the People’s Republic of China. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jchromb. 2015.02.030. References
3.4.5. Analysis of real samples A real sample survey was conducted to check the effectiveness of the validated method and its fitness in routine analysis. This method had been successfully applied to detect preservatives in 5 kinds of vegetables, which were purchased randomly from local supermarkets. The results were shown in Table 3. Although a few preservatives in some vegetables were determined, their content did not exceed the maximum residue of national requirements in vegetables. 4. Conclusion In conclusion, a method for simultaneous determination of 18 preservatives in vegetables was developed and validated. The samples were extracted with the dispersive-SPE method and preservatives were analyzed by UHPLC–QTRAP. All compounds showed satisfying recoveries and RSDs while method was validated. Simple pre-treatment, rapid and efficient cleanup, low consumption of organic solvents and low cost are the advantages of the developed method. The developed method has been proven to exhibit sufficient sensitivity for simultaneous determination of 18 preservatives in actual vegetable samples.
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Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Simultaneous determination of 18 preservative residues in vegetables by ultra high performance liquid chromatography coupled with triple quadrupole/linear ion trap mass spectrometry using a dispersive-SPE procedure Xue Zhou a , Shurui Cao b , Xianliang Li b , Bobin Tang b , Xiaowen Ding c , Cunxian Xi b , Jiangtao Hu d , Zhiqiong Chen a,∗ a
College of Pharmacy, Chongqing Medical University, Chongqing 400016, China Chongqing Entry-Exit Inspection and Quarantine Bureau, Chongqing Engineering Technology Research Center of Import and Export Food Safety, Chongqing 400020, China c College of Food Science and Engineering, Southwest University, Chongqing 400715, China d Sichuan Entry-Exit Inspection and Quarantine Bureau, Sichuan Engineering Technology Research Center of Import and Export Food Safety, Chengdu 610041, China b
a r t i c l e
i n f o
Article history: Received 2 December 2014 Accepted 21 February 2015 Available online 10 March 2015 Keywords: UHPLC–QTRAP Vegetables Preservatives Dispersive-SPE
a b s t r a c t A new method combining dispersive-solid phase purification procedure with ultra high performance liquid chromatography–triple quadrupole/linear ion trap mass spectrometry was developed for simultaneous determination of 18 preservative residues in vegetables. The new method not only had the advantages of dispersive-solid phase purification procedure such as high recoveries, easy operation, rapid analysis, little solvent usage and wide analysis range of preservatives, but also had the advantages of ultra high performance liquid chromatography–triple quadrupole/linear ion trap mass spectrometry to be operated in positive mode and negative mode simultaneously. The method was validated for the following representative matrices: radish (tuber), tomato (eggplant fruit), cabbage (leafy), cowpea (bean), cucumber (melon) and so on. Samples were extracted with hexane–ethyl acetate (1:2, v/v), and then detected by ultra high performance liquid chromatography–triple quadrupole/linear ion trap mass spectrometry after being cleaned up with dispersive-solid phase purification procedure. Significant matrix effects were compensated by using the matrix-matched calibration curves. 18 preservatives showed good linearity over the range of 5.0–100.0 g/L with correlation coefficients of 0.9904–1.000. The limits of detections were in the range of 0.04–4.16 g/kg and the limits of quantity were in the range of 0.13–13.85 g/kg. The recoveries of 18 preservatives ranged from 76.0% to 120.0% with the spiked levels of 2, 4 and 10 g/kg into homogenized vegetables, and the relative standard deviations (RSDs) ranged from 0.3% to 14.8%. Compared with the reported literatures, the method is more rapid, simple, highly sensitive, reliable and can meet testing requirements of 18 preservative residues in vegetables. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Vegetables are essential in people’s daily diet which can provide a variety of vitamins and minerals. Eating plenty of vegetables will be beneficial for the health such as enhancing nutrition, improving digestion, softening blood vessels and so on. But vegetables are often infected by microorganisms during their growth in the field, which is resulting in alteration and degradation soon after picking
∗ Corresponding author. Tel.: +86 13032398667; fax: +86 23 68485161. http://dx.doi.org/10.1016/j.jchromb.2015.02.030 1570-0232/© 2015 Elsevier B.V. All rights reserved.
[1–4]. Preservatives are usually used during the growth and storage of the vegetables to keep the freshness of the vegetables. The promotion and use of efficient and lowly toxic preservatives have a favor of vegetables year-round sales, corruption reducing, longdistance transport, etc. [5]. However, people are no longer evaluating preservative which has a preservative effect and can improve the economic efficiency of vegetables. Social benefits without the environmental and healthy damage are more focused on. The extensive use of preservatives has been a concern because of their potential harm to the environment and known or suspected toxic effects on humans, such as neurodevelopmental impairment, acute
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neurological toxicity, reproductive and endocrine systems, possible dysfunction of the immune, chronic kidney diseases, intolerance, cancer and other potential diseases [6,7]. Due to these concerns, the determination of preservative residues in vegetables has a very important significance, especially the development of the method for the simultaneous determination of many kinds of preservative residues is imperative. Simultaneous determination of a wider range of preservative residues in vegetables will not only protect healthy eating of vegetables but also can reduce human labor. Different methods have been developed for the determination of preservative residues, including enzyme-linked immunosorbent assay (ELISA) [8,9], biosensor [10,11], capillary electrophoresis (CE) [12,13], high-performance liquid chromatography (HPLC) [14,15], gas chromatography (GC) [16,17], liquid chromatography (LC) and gas chromatography coupled to mass spectrometric (MS) detectors [18,19]. Biosensor is a newly developed and promising analytical technique for preservatives, but this technique can only detect organophosphorous preservatives [10]. GC and GC–MS suit for volatile preservatives [20,21]. ELISA, CE and other detection instruments can not be compared with ultra high performance liquid chromatography-tandem mass spectrometry (UPLC–MS/MS) in many fields such as sensitivity, application range and so on. UPLC–MS/MS is already a proven technique with a wide range of applications [22]. Paper has been published in which the detection limit was low to 0.12 g/kg [23]. But almost all the UPLC–MS/MS have to select an electrospray ionization source (ESI) in positive mode or in negative mode separately [22,24]. Ultra high performance liquid chromatography–triple quadrupole/linear ion trap mass spectrometry (UHPLC–QTRAP) that we used could detect positive and negative ions simultaneously, without the need for separate detection of positive and negative ions. The determination of preservative residues in vegetables using UHPLC–QTRAP is rarely reported. Many experimental conditions of 18 preservatives in vegetables were investigated such as extraction and purification. This method made full use of the advantages of ultra-fast scanning speed, strong specificity, high efficiency and sensitivity of UHPLC–QTRAP. The UHPLC–QTRAP method coupled with dispersive-SPE procedure for the simultaneous determination of 18 preservative residues in vegetables was issued. The method was fully validated for linearity, limits of detection, matrix effect, accuracy, and precision, which can provide technical support for the rapid determination of preservative residues in vegetables.
2. Materials and methods 2.1. Chemicals and reagents All compounds (listed in Table 1; analytic grade ≥99.0% purity) were purchased from AccuStandard (New Haven, CT, USA). Acetonitrile, hexane, ethyl acetate, acetone, toluene, and methanol (HPLC grade) were obtained from Tedia (Fairfield, OH, USA). Ethylenediamine-N-propyl silane’s (PSA’s) size was 60 m, which was purchased from Agilent Technologies (Santa Clara, CA, USA). Sodium chloride, ammonium acetate and magnesium sulfate were analytical grade. Ultra-pure water was prepared by Milli-Q-plus ultra-pure water system (Milford, MA, USA) throughout the study. All of the vegetables were purchased from various local supermarkets (Chongqing, China). 2.2. Instrumentation LC-30 ultra performance liquid chromatography (Shimadzu, Japan)-API5500Q triple quadrupole/linear ion trap mass spectrometer (AB Sciex, USA); XH-B vortex mixer (Jiangsu healthcare medical supplies company, LTD); 3–30 K refrigerated centrifuge (German, Sigma company); Rotary vacuum evaporators (4011 Digital) (German, Heidolph Company); N-EVAP116 nitrogen dry instrument (Organomation Associates, USA); Pipettes (adjustable range: 10–100 L, 20–200 L, 10–1000 L, 1000–5000 L, 1–10 mL) (German, Eppendorf Company); SR-2DS powerful oscillator (TAITEC, Japan). 2.3. Standard solutions Each stock solution of the studied standards was individually prepared by dissolving the weighed compound in acetonitrile at final concentration of 1 mg/mL in glass vials, which was stored at −18 ◦ C. An aliquot of each stock solution was added to appropriate acetonitrile–2 mmol/L ammonium acetate (1:1, v/v) to prepare a mixture solution of the 18 preservatives, with 10 mg/L in 50 mL and stored at −18 ◦ C prior to use. This stock solution was further diluted by acetonitrile–2 mmol/L ammonium acetate (1:1, v/v) to make standard solutions of all preservatives, which was prepared daily. The concentrations of standard solutions were 5, 10, 20, 50 and 100 g/L.
Table 1 LC–MS/MS acquisition parameters for 18 preservatives. No.
Compound
Retention time (min)
Ion pair Q1 /Q3 (m/z)
Declustering potential DP/V
Collision voltage CE/V
Collision chamber outlet voltage CXP/V
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Enilconazole Chlorpyrifos Carbendazim 2-(4-thiazolyl)benzimidazole Prochloraz Pyrimethanil Myclobutanil Flusilazole Propiconazole Triadimefon Iprodione Fenethanil Butyl paraben Propyl paraben Ethyl paraben Methyl paraben 2-Naphthol 4-Phenylphenol
5.85 7.21 3.65 3.86 6.16 5.45 5.70 5.90 6.12 5.75 5.99 5.93 5.39 4.95 4.46 3.93 4.84 5.31
297.1/159.1a , 297.1/255.1 350/97a , 350/198 192.2/131.1a , 192.2/160.1 202.2/131.1a , 202.2/175.1 376/266.2, 376/308.1a 200.2/107.1a , 200.2/168.1 289.1/70.1a , 289.1/125.1 316.1/165.1a , 316.1/247.1 342.1/159a , 342.1/205 294.1/197.1a , 294.1/225.1 330.1/245.0a , 330.1/288.1 337.1/125.1a , 337.1/70.0 192.9/91.8a , 192.9/136.1 178.9/92.0a , 178.9/136.0 165.0/92.0a , 165.0/149.0 151.0/92.0a , 151.0/135.9 142.8/114.9a , 142.8/124.8 160.9/93.0a , 160.9/140.9, 160.9/114.9
50 50 58 106 26 96 55 37 63 41 84 91 −110 −83 −70 −57 −130 −114
29, 25 47, 32 43, 24 44, 34 22, 16 34, 41 21, 47 36, 26 42, 24 21, 17 19, 18 23, 44 −25, −24 −30, −22 −29, −19 −29, −18 −34, −12 −40, −37, −41
9.8, 5 4.9, 6 7.2, 10 8.9, 11.6 8.4, 6.8 14, 9 8.5, 7.4 8.1, 15.8 8.8, 6.0 10.2, 14 11, 11 7.3, 8.1 −10, −10 −5.6, −8 −8.8, −7.3 −4.8, −6.5 −15, −16 −8.4, −9.9, −5.3
a
Quantitative ion.
X. Zhou et al. / J. Chromatogr. B 989 (2015) 21–26
23
a
b
c
Fig. 1. (a) Separation effect of different gradient elution programs. (b) Separation effect of different gradient elution programs. (c) Separation effect of different gradient elution programs.
2.4. UHPLC–QTRAP analysis Analyses were done in a Shimadzu UHPLC system coupled with an API 5500Q triple quadrupole/linear ion trap mass spectrometer. For the UHPLC system, chromatographic separation was achieved in an (Waters) ACQUITY UPLC BEH C18 (2.1 mm × 50 mm, 1.7 m). The mobile phase and a gradient elution program are shown in the picture III of Fig. 1. The flow rate was kept at 0.3 mL/min. The injection volume was 1 L, and the column temperature was maintained at 40 ◦ C. For the mass spectrometric analysis, a mass spectrometer equipped with the manufacturer’s electrospray (ESI) source, operated in positive ion mode and negative ion mode simultaneously, was applied in multiple reaction monitoring (MRM) mode. The total online UHPLC–QTRAP analysis time per sample was 10 min. Electrospray voltage (IS) was 5500 V and collision chamber inlet voltage (EP) was 10.0 V when ionization mode was in ESI+ mode. While the ionization mode was in ESI− mode, IS was −4500 V and EP was −10.0 V. Atomization gas pressure (GS1) was 50.0 psi, air curtain gas pressure (CUR) was 30.0 psi, auxiliary air pressure (GS2) was 50.0 psi and the temperature was 550 ◦ C, no matter the ionization mode was in ESI+ mode or in ESI− mode. Mass spectrum parameters of all compounds are shown in Table 1. 2.5. Preparation and treatment of the samples 2.5.1. Samples preparation Extraction procedure was as follows: accurately weighed 10.0 g of the homogenized vegetable (accurate to 0.01 g) into a 50 mL teflon centrifuge tube, 20 mL hexane–ethyl acetate (1:2, v/v) and 4 mL 2 mol/L ammonium acetate solution were added. The sample was extracted for 8 min by powerful oscillator. After the addition of 5.0 g (accurate to 0.01 g) NaCl, the tube was vortexed for 1 min, followed by centrifugation at 5000 rpm for 3 min. Then, 10 mL of the supernatant were transferred to a 15 mL teflon centrifuge tube with bottom containing 100 mg of PSA and 500 mg of anhydrous MgSO4 . The tube was vortexed for 2 min, followed by centrifugation at 5000 rpm for 3 min. The supernatant was placed into
a 25 mL round-bottom flask. The supernatant was concentrated to almost dryness on rotary vacuum evaporator below 40 ◦ C, and residues were dissolved with 1 mL acetonitrile–2 mmol/L ammonium acetate (1:1, v/v). The content was filtered through a 0.22 m nylon membrane filter and analyzed by UHPLC–QTRAP. 2.5.2. Preparation of calibration curve 10.0 g of the homogenized vegetable were treated as described in Section 2.5.1 without adding standard solution in. When the extract was concentrated to almost dryness on rotary vacuum evaporator below 40 ◦ C, 1 mL of each standard solution of 5, 10, 20, 50 and 100 g/L was spiked to blank residue to obtain a series of matrix-matched calibration solutions (5, 10, 20, 50 and 100 g/L), which were analyzed by UHPLC–QTRAP. Matrix-matched calibration curve was used for quantification and matrix effect assessment. 3. Results and discussion 3.1. Optimization of UHPLC–QTRAP The UHPLC–QTRAP with ESI source and MRM mode provided a highly selective and sensitive method for the determination of 18 preservatives. According to the chemical structure characteristics of the targets, most of the compounds fitted in ESI+ mode form [M+H]+ ions; the few compounds were suitable for ESI− mode form [M−H]− ions. Therefore, standard solution (1 mg/L) was injected in and [M+H]+ or [M−H]− molecular ion was determined by the primary mass spectrometry, then fragment ions information was obtained by the secondary mass spectrometry. Two characteristic ions of maximum sensitivity were selected as the quantification and identification ions to optimize the MS/MS parameters (Table 1). Positive and negative ions can be detected simultaneously, without the need for separate detection of positive and negative ions. The commonly used mobile-phase compositions such as acetonitrile–water and methanol–water were optimized. The results showed that acetonitrile–2 mmol/L ammonium acetate was the best mobile-phase compositions. Several gradient elution programs were optimized (Fig. 1). As shown in Fig. 1, separation of 18
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acetonitrile’s proportion was high. So proportion of acetonitrile and 2 mmol/L ammonium acetate was considered to be 50:50, which was 1:1. 3.4. Method validation
Fig. 2. Effect of different extraction solvents on recovery.
preservatives in picture c was the worst. The change rate of gradient elution 1 in picture c was bigger than picture b, which resulted in that the peak times of most preservatives were in gradient elution 1 of picture c, so that separation of 18 preservatives in picture b was better than picture c. In picture a, because the proportion of the organic phase was reduced and the change rate of gradient elution of picture a was appropriate to be decreased, the separation of 18 preservatives was the best. The gradient elution program in the picture c of Fig. 1 was developed to provide baseline separation for 18 preservatives. The columns were investigated including ACQUITY UPLC BEH C18 , Shim-pack VP-ODS, Inertsil ODS-SP and Kinetex C18 . It turned out that using ACQUITY UPLC BEH C18 column to separate and using acetonitrile–2 mmol/L aqueous ammonium acetate as mobile phase could improve the ionization efficiency of the target compounds. Under the optimal operating conditions of UHPLC–QTRAP, all compounds were eluted within the 10 min analysis time. 3.2. Optimization of sample extraction solvent Acetonitrile, hexane–ethyl acetate (1:1, v/v), hexane–ethyl acetate (1:2, v/v) and acetone–ethyl acetate (1:1, v/v), commonly used in the extraction of preservatives, were compared for solvent extraction of blank samples of radish spiked with 18 preservatives at a concentration 2 g/kg. All the sample extraction solvent were investigated three times. The average of two extraction recoveries of each sample extraction solvent was accepted. The experiments were performed as mentioned in Section 2.5.1, except that the type of solvent was varied. In comparison with that obtained using acetonitrile, hexane–ethyl acetate (1:1, v/v) and acetone–ethyl acetate (1:1, v/v), hexane–ethyl acetate (1:2, v/v) provided better extraction efficiency for all 18 preservatives with recoveries in the range of 79.6–106.5% (Fig. 2). In comparison with acetonitrile and acetone–ethyl acetate (1:1, v/v), there were less impurities when extracted by hexane–ethyl acetate (1:2, v/v). 3.3. Optimization of dissolved solution Different proportions of acetonitrile and 2 mmol/L ammonium acetate (100:0, 90:10, 80:20, 50:50, 40:60, 30:70, 20:80, 10:90, 5:95, v/v) were investigated, which were used to dissolve residues. All compounds showed good peak shapes in different proportions of acetonitrile and 2 mmol/L ammonium, but not all compounds could be dissolved when 2 mmol/L ammonium acetate’s proportion was high and non-target residues would be dissolved when
3.4.1. Linearity, detection and quantitation limits Linearity was studied in the range of 5–100 g/L for 18 preservatives with different calibration levels by matrix-matched standard calibration in blank extracts or by standard calibration in solvent. Linearity values, calculated as determination coefficients (R2 ) for 18 preservatives from the matrix-matched calibration plots or just from the calibration plots, are shown in Table S1. Good linearity was found for 18 preservatives within R2 values of 0.9904–1.000 from the matrix-matched calibration plots and 0.9927–1.000 just from the calibration plots. LOD and LOQ are two fundamental elements of method validation that define the limitation of an analytical method. The LOD and LOQ for 18 preservatives ranged from 0.04 to 4.16 g/kg and from 0.13 to 13.85 g/kg at the signal-to-noise ratios (S/N) of 3 and 10, respectively, which were calculated from standard solution at a concentration of 10 g/L. 3.4.2. Evaluation of matrix effect In mass spectrometry, co-elution of residual components from the sample matrix may occur by ion enhancement (increasing) or ion suppression (decreasing) the MS/MS signal and have profound effects on assay precision and trueness in quantitative analysis. Matrix effects vary from sample to sample. Therefore, the evaluation of matrix effect on the quantitative analysis was an important assay validation. The matrix effect (ME) was defined by the ratio between the slope of matrix-matched calibration curve and the slope of standard solution curve subtracted 1, and then multiplied by 100% [28–30]. Formula was showed in Eq. (1). A negative ME value indicates that the MS/MS signal of target compound was suppressed. Contrarily, the positive ME value means an ionization enhancement effect. Absolute value varies from 0 to 20%, indicating the presence of a weak matrix effect; Absolute value varies from 20% to 50%, indicating the presence of moderate matrix effect; Absolute value is above 50%, indicating that there is a strong matrix effect. ME% =
S
s
Sm
− 1 × 100%
(1)
where Ss is the slope of calibration plot with matrix-matched calibration solutions, Sm is the slope of calibration plot with calibration solutions in solvent and ME% is the calculating percentage of signal enhancement or suppression. In this research, matrix-matched calibration curves were performed at five concentration levels (5, 10, 20, 50 and 100 g/L) by spiking the extracts obtained from blank sample. As shown in Table S1, matrix effects of 18 preservatives were different in five kinds of matrix. The absolute values range from 4.01% to 78.1%. Most of the matrix effects were suppressed and moderate. To eliminate matrix influence, this method was performed by matrix-matched calibration curves. 3.4.3. Recovery and repeatability Recovery and repeatability of the method were established to evaluate the performance of method. The repeatability and the recovery of the method were studied by carrying out six consecutive extractions (n = 6) of spiked matrices at three concentration levels (2, 4 and 10 g/kg). Table S2 provides the recoveries and RSDs. The recoveries ranged from 83.6% to 119.2%, from 79.7% to 119.8%, from 76.0% to 108.6%, from 81.6% to 119.5% and from 78.1% to 110.0% when the matrices were radish, tomato, cabbage, cowpea and cucumber respectively. RSDs were below 15% in all cases.
X. Zhou et al. / J. Chromatogr. B 989 (2015) 21–26
25
Table 2 Comparison of different methods. Category
GB/T 20769-2008 [25]
Literature 1 [26]
Literature 2 [27]
The method of this research
Species (n) Purification Experimental time (min) The detection time (min) Recovery (%) LOD (g/kg)
11 NH2 column 266 30.40 / 0.12–13.45
5 QuEChERS: PSA, MgSO4 19 17.73 90–120.0% /
4 / 35 25 91.0–115% 0.1–0.5
18 QuEChERS: PSA, MgSO4 37 10.00 78.1–120.0% 0.04–4.16
Table 3 The amount of preservative residues in vegetables (g/kg). No.
Radish
Tomato
Cabbage
Cowpea
Cucumber
No.
Radish
Tomato
Cabbage
Cowpea
Cucumber
1 2 3 4 5 6 7 8 9
0.2 7.6 0.5 0.5 0.3 ND ND 0.1 ND
ND 5.7 0.7 ND ND ND ND ND ND
ND 4.4 0.2 ND ND ND ND ND ND
ND 7.0 6.5 ND 7.5 0.4 0.4 0.1 0.1
ND 5.1 0.6 ND ND ND ND ND ND
10 11 12 13 14 15 16 17 18
ND ND ND ND 0.2 2.0 5.0 0.3 0.1
ND ND ND ND 0.2 0.5 1.5 0.2 ND
ND 0.3 ND ND 1.2 1.6 1.3 ND ND
ND 2.1 ND 0.1 0.5 ND 0.9 0.2 0.3
0.2 0.7 ND ND 0.8 0.1 1.0 0.1 ND
Note: ND indicates that the content of the sample is less than the detection limit of the method.
3.4.4. Comparison with other methods This study, GB/T 20769-2008 [25], the literature 1 [26] and 2 [27] were compared. NH2 columns were used for cleanup in GB/T 20769-2008, but there was no purification in literature 2. The results showed that the time of GB/T 20769-2008 was too long and the spiked levels of literature 2 were too high. This study used dispersive-SPE procedure for cleanup, which is same to literature 1, but the detected species of literature 1 were so few. On the one hand, the time consuming of the issued method in the pretreatment process is short, on the other hand, UHPLC–QTRAP can be operated in positive mode and negative mode simultaneously. What is more important, the detected species of preservatives were the most. Compared with other methods, the detected speed of this method was increased by 35%. The detailed results were shown in Table 2.
Acknowledgments This work was supported by “Food and Agricultural Research fund project (No. cstc2013yykfB0165)” and “Youth Science and Technology talents fund project (No. cstc2014kjrc-qnrc00002)” of Chongqing Municipal Science and Technology Commission of the People’s Republic of China. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jchromb. 2015.02.030. References
3.4.5. Analysis of real samples A real sample survey was conducted to check the effectiveness of the validated method and its fitness in routine analysis. This method had been successfully applied to detect preservatives in 5 kinds of vegetables, which were purchased randomly from local supermarkets. The results were shown in Table 3. Although a few preservatives in some vegetables were determined, their content did not exceed the maximum residue of national requirements in vegetables. 4. Conclusion In conclusion, a method for simultaneous determination of 18 preservatives in vegetables was developed and validated. The samples were extracted with the dispersive-SPE method and preservatives were analyzed by UHPLC–QTRAP. All compounds showed satisfying recoveries and RSDs while method was validated. Simple pre-treatment, rapid and efficient cleanup, low consumption of organic solvents and low cost are the advantages of the developed method. The developed method has been proven to exhibit sufficient sensitivity for simultaneous determination of 18 preservatives in actual vegetable samples.
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