Hollow Fiber Supported Ionic Liquids Liquid-Phase Micro-extraction Followed by High-Performance Liquid Chromatography for the Determination of Polycyclic Aromatic Hydrocarbons in Milk Samples

Hollow Fiber Supported Ionic Liquids Liquid-Phase Micro-extraction Followed by High-Performance... Abstract A rapid, simple, reliable and efficient hollow fiber supported ionic liquids liquid-phase micro-extraction method (IL-HF-LPME) followed by high-performance liquid chromatography was successfully applied to the determination of four kinds of polycyclic aromatic hydrocarbons (PAHs) in milk samples. In the IL-HF-LPME method, a mixture of [OMIM]PF6 and lauric acid, in a ratio of 3:1, was immobilized in the pores of a polypropylene hollow fiber used as extraction solvent. A series of essential parameters influencing the extraction efficiency were investigated and optimized. Under the optimal conditions, the extraction equilibrium is achieved within 3 min, the good linearity was >0.9990, the limits of detection varied from 0.14 to 0.71 ng/mL, the limit of quantification values were between 0.4 and 1.8 ng/mL, and the relative standard deviations were in the range of 1.24–3.27% (n = 5). The proposed method was applied to analyze four PAHs in milk samples and recoveries were between 93.6 and 102.8%. Introduction Polycyclic aromatic hydrocarbons (PAHs) are a huge and diverse group of carcinogenic and persistent organic compounds containing at least two fused benzene rings arranged in linear, angular or cluster structures. They are ubiquitously distributed as industrial–environmental pollutants, which are mainly produced by the natural processes of incomplete combustion or pyrolysis of organic matter at high temperature, but a few of them can also be of biological origin (1–3). Owing to their potential mutagenic, carcinogenic properties and significant toxicity, quite a few of PAHs, as well as their metabolites and degradation products, have been found widespread in several different kind of samples, e.g., environmental water (4–7), atmosphere (3), soil (8, 9) or dust particles (10). At the same time, most concerned still have been focused on their contamination in myriad foods, such as oils (11, 12), milk (2, 13), coffee (14, 15), fruits, vegetables (16) and roasted and smoked meat (1, 13, 17, 18). In particular, the highly carcinogenic benzo[a]pyrene has been detected in several foods at concentration levels varied from 0.1 to 100 mg/kg (14, 15). PAHs have appealed to a wide range of attention owing to their physical or chemical properties of lipophicity, poor solubility and aggregation to the precipitants (1, 19). Either the United States Environmental Protection Agency (US EPA) or the International Agency for Research on Cancer (IARC) has been reasonably enumerated 16 PAHs as the higher priority contaminants and genotoxic carcinogens (2, 11, 17, 19). Based on the Scientific Committee on Food, benzo[a]pyrene (BaP) has been applied as a significant indicator of the epidemiology, intake evaluation, system analysis methodology, occurrence and impact of total carcinogenic PAHs in food (1, 17, 20). In fact, four PAHs were chosen in this study: pyrene (Pyr), benzo[a]anthracene (BaA), 1-hydroxypyrene (1-OHP) and benzo[a]pyrene (BaP). They all have obvious carcinogenic and genotoxic properties (13). Since the pollution of PAHs in food has already become a serious threat to human health, it is particularly important and essential to establish a rapid, reliable and accurate analytical approach to accurately analyze levels of contamination from various PAHs in food (6, 14, 17). At present, various available sample pre-concentration and extraction methods with specialized analytical equipments have already been exhaustively researched for the determination of PAHs in various samples, including liquid–liquid extraction (LLE) (21), liquid-phase micro-extraction (LPME) (22), disperse liquid–liquid micro-extraction (DLLME) (6, 7), cloud-point extraction (CPE) (23), solid-phase extraction (SPE) (14), solid-phase micro-extraction (SPME) (4, 20), hollow fiber liquid-phase micro-extraction (HF-LPME) (24–26), magnetic solid-phase extraction (MSPE) (27), miniaturized homogeneous liquid–liquid extraction (MHLLE) (28) integrated with high-performance liquid chromatography and diode array detector (HPLC–DAD) (6, 12, 19) or fluorescence detection (HPLC–FLD) (1, 3, 13), and gas chromatography coupled to mass spectrometry (GC–MS) (7, 15, 21). LPME was a novel practically feasible, and suitable sample enrichment method due to their characteristics such as simplification, rapidness and high efficiency (29). In various LPME modes, HF-LPME, as the improvement of the conventional LLE technique, was on the basis of porous polypropylene hollow fiber which served as contact interfaces between micro-volumes of the extraction agent and the target analytes (26). According to the HF-LPME approach, the hollow fiber cannot only play a vital role in the purification of samples, but also absolutely remove the influence of cross contamination of samples and ensure repeatability (30). Moreover, the HF-LPME method can offer some advantages as follows: high enrichment factor, low cost, high repeatability, rapid sample processing, high sensitivity, etc. (30, 31). Overall, the extraction, enrichment and purification of the sample were carried out in one step. Hydrophobic ionic liquids (ILs) are incorporated with organic cations and various anions, and have several obvious superiorities over organic solvents as follows: low volatility, high heat stability and the capacity to interact with multifarious organic and inorganic compounds (32). In addition, they are regarded to be environmentally-friendly solvents, and thereby applied as great substitutes to traditional organic solvents. Our research groups have studied ionic liquid-HF-LPME (IL-HF-LPME) technology and applied for the analysis of phthalate esters (33). In the current work, the IL-HF-LPME method integrated with HPLC–DAD technique was established for the highly sensitive determination of four PAHs: pyrene (Pyr), benzo[a]anthracene (BaA), 1-hydroxypyrene (1-OHP) and benzo[a]pyrene (BaP). ILs were considered as the extractant and lauric acid was not only applied as the synergistic extraction solvent, but also as a solvent to reduce the viscosity of ionic liquids. The IL-HF-LPME provide very clean method that can be directly injected to the high-performance liquid chromatographic system, and has the excellent stable baseline, and enhance the practicability of the developed method (30). Various essential experimental parameters were screened and the optimized procedure was certified. The serviceability of the developed method was researched for the analysis of the trace levels of PAHs in milk samples. Experimental Materials Standards of 1-hydroxypyrene (1-OHP), benzo[a]anthracene (BaA), benzo[a]pyrene (BaP) and pyrene (Pyr) were supplied by Tokyo Chemical Industry Co., Ltd (Japan). The purity of each compound was above 98%. 1-butyl-3-methylimidazolium hexafluorophosphate ([BMIM]PF6, 99%), 1-hexyl-3-methylimidazolium hexafluorophosphate ([HMIM]PF6, 99%), 1-octyl-3-methylimidazolium hexafluorophosphate ([OMIM]PF6, 99%) and lauric acid (C12H24O2, 98%) were supplied by Aladdin (Shanghai, China). Acetonitrile (HPLC grade) was provided by Merck (Darmstadt, Germany). Acetone was obtained from Fengchuan Chemical Reagent Co., Ltd (Tianjin, China). The deionized water was provided by Milli-Q systems (USA). All other chemicals and solvents were at or above the analytical reagent-grade. Instruments An ultrasonic cleaner (Henao ultra-sonic instrument plant, Hu-10260T, Tianjin, China) was served to eliminate fomites in hollow fibers. The vortex mixer (Jintan experiment Instrument Co. Ltd, SK-1, Jiangsu, China) was applied to vortex-mixing. The centrifuge (Hunan experiment Instrument Co., Ltd., L420, Hunan, China) was applied in phase separation. The accurel Q3/2 polypropylene hollow fiber membrane was supplied by Membrana (Wuppertal, Germany), and the inner diameter was 600 μm, the wall thickness was 200 μm, and the pore size was 0.2 μm. A 1.0 mL sterile injection syringe (Shanghai medical Instrument Co., Ltd, Shanghai, China) was supplied to inject the extracting agent into the hollow fiber lumen. The experiments in liquid chromatography were actualized with Agilent 1260 series HPLC system (Agilent Technologies, CA, USA) combined with a photodiode-array detector (DAD). Chromatographic conditions The chromatographic separations and quantitative analysis were developed employing a reversed-phase Agilent TC-C18 column (250 mm × 4.6 mm i.d., 5 μm) with the column temperature of 30°C, and the detector wavelength was set at 254 nm. Acetonitrile–water (85:15, v/v) was employed as the mobile phase with the flow rate of 1 mL/min and the injection volume was 20 μL. Preparation of standard solutions and milk samples Stock standard solutions of Pyr, BaA, 1-OHP and BaP were dissolved in acetonitrile (1,000 μg mL−1). Working standard solutions were prepared by dilution of the stock solution. The stock solutions were stored in a fridge at 4°C (stable for at least 2 month) and then brought to room temperature prior to use. Milk samples were gained from the local supermarket in Kunming City (Yunnan Province, China), and stored at 4°C before analyzing. For protein precipitation, a 10 mL centrifuge vial was charged with a 5 mL milk sample containing NaAc (0.1 g), MgSO4 (0.4 g) and acetonitrile (2.0 mL), which was then completely blended by the vortex mixer within 2 min. After centrifugation at 4,000 rpm for 5 min, the supernatants were easily transferred into another new vial, and repeated once again based on the above method. Finally, supernatants were merged and diluted with deionized water to 25 mL. Preparation of hollow fiber and IL-HF-LPME extraction procedure Hollow fibers were cut into 6 cm segments, and washed with acetone for 5 min in an ultrasonic bath to eliminate any impurity pollution in the fiber, and then naturally drying in air. 1-octyl-3-methylimidazolium hexafluorophosphate [OMIM]PF6 (as the extraction agent) and lauric acid (as the synergistic extraction solvent) were dissolved by acetonitrile in a mixture ratio of 3:1, and then injected slowly into the hollow fiber through using the 1.0 mL sterile injection syringe. Each port of the fiber was sealed with a lighter and then the hollow fiber was bent into a U-shape and macerated in the 5 mL milk sample solution. The mixture was sufficiently vortex-mixed for 3 min at a temperature (ca. 30°C). Then, the fiber was removed, both of the ports were cut and 200 μL acetonitrile was injected into one of its port to dissolve the analytes, and then introduced into HPLC system for analyses. Each new hollow fiber was used only once for extraction. Results Optimization of the IL-HF-LPME procedure A series of disparate experimental parameters affecting the extraction efficiency for the IL-HF-LPME procedure have been investigated, such as the type and mixture ratio of extraction solvent, fiber length, the extraction time, sample pH value, salt addition and extraction temperature. The study and optimization of the above-mentioned variables were performed using the one variable at a time method. Finally, the optimized experimental conditions were established. Selection of extraction solvent The selection of an appropriate extraction solvent was very important for the IL-HF-LPME method. In general, the solvent selected should be compatible with the polypropylene fiber so as to be able to easily fill its wall pores. The immiscibility with the water sample and the acceptor phase should also be seriously considered as it acts as a suitable medium between the two phases. In the proposed method, according to the polarity of the analytes, several solvents were examined, including lauric acid dissolved by acetonitrile (concentration was 5%, w/v), [BMIM]PF6, [HMIM]PF6 and [OMIM]PF6. As shown in Figure 1, [OMIM]PF6 and a mixture of [OMIM]PF6 and lauric acid in a ratio of 3:1 provided similar satisfactory results. However, the viscosity of [BMIM][PF6], [HMIM][PF6] and [OMIM][PF6] were 450, 585 and 685 cP, respectively (34), and a high viscosity of extractant could reduce mass diffusion rates of PAHs into the ionic liquid phase during extraction. A mixture of [OMIM]PF6 and lauric acid in a ratio of 3:1 has suitable viscosity and less volatility, so it can impregnate the pores of HF and has form mixed extraction efficiency to PAHs. Consequently, a mixture of [OMIM]PF6 and lauric acid, in a ratio of 3:1, was adopted as the extraction agent in the following experiments. Figure 1. View largeDownload slide Effect of different kinds of extraction solvent on the extraction efficiency. Figure 1. View largeDownload slide Effect of different kinds of extraction solvent on the extraction efficiency. Effect of fiber length The length of hollow fiber determines the volume of extraction solvent, which was a critical parameter in the extraction. For the sake of examining the length of the hollow fiber, the mixed extraction solvent involving varied volumes were subjected to the identical IL-HF-LPME approach. Hence, the length of the hollow fiber from 2 to 10 cm was discussed. The results in Figure 2 illustrated that the maximum extraction efficiency was at 6 cm, and no discernible influence was changed when the fiber length increased from 8 to 10 cm. The short fiber length enhanced a high concentration of the trace analytes in the IL-HF-LPME method. Nevertheless, fiber membranes cannot be too short to offer adequate extraction solvent to facilitate transport of the analytes (35). Thus, it could be seen that the increase of fiber length was not beneficial to the extraction of the analytes. After comprehensive consideration, 6 cm the hollow fiber containing around 20 μL extraction solvent was utilized in the subsequent experiments. Figure 2. View largeDownload slide Effect of fiber length on the extraction efficiency. Figure 2. View largeDownload slide Effect of fiber length on the extraction efficiency. Effect of the extraction time The HF-LPME process mainly rested with the vortex-mixing of the target analytes between the extraction solvent and the sample solution (36), so it was important to find a minimum extraction time without losing sensitivity. To appraise the optimal extraction time, experiments were actualized by changing the time from 1 to 6 min, respectively. As shown in Figure 3, the results suggested that the system essentially reached equilibrium within 3 min, and there was no significant increase in the extraction efficiency at longer extraction time. Accordingly, in accordance with the above analysis results, 3 min was adopted as the optimized extraction time in the subsequent experiments. Figure 3. View largeDownload slide Effect of the extraction time on the extraction efficiency. Figure 3. View largeDownload slide Effect of the extraction time on the extraction efficiency. Effect of pH value Sample pH value was closely related to the solubility of the target analytes, which could change the partition coefficient of the analytes between the sample solution and the extraction solvent. The effect of sample pH value on the IL-HF-LPME method was studied within the range of 4.0–9.0 in present experiment. It was observed from Figure 4 that the maximum extraction efficiency could be obtained without changing the pH. PAHs were a group of organic compounds with strong inert and stable properties, and the pH value of donor phase did not produce an effect on the extraction efficiency (32). Consequently, there was no need to change the pH of the solution in following further experiments. Figure 4. View largeDownload slide Effect of pH on the extraction efficiency. Figure 4. View largeDownload slide Effect of pH on the extraction efficiency. Effect of salt addition As a result of the salting-out effect, addition of salt to the sample solution would increase the ionic strength of the solution and then reduce the solubility of the analytes in the IL-HF-LPME method. The impact of salting strength on the extraction of PAHs was carried out by increasing various dosages of NaCl within the range of 0.2–1.0 g. Figure 5 shows the extraction efficiency of PAHs in diverse amounts of NaCl. As expected (Figure 5), the results revealed that the extraction efficiency was almost unchanged. This phenomenon could be explained by the non-polarity of the PAHs compounds (6). Hence, no addition of salt to the sample solution was employed in the following whole experiments. Figure 5. View largeDownload slide Effect of the amount of salt on the extraction efficiency. Figure 5. View largeDownload slide Effect of the amount of salt on the extraction efficiency. Effect of extraction temperature Extraction temperature could also influence the extraction performance of the developed method, because it would affect both kinetics and thermodynamics of the analytes diffusion process in extraction procedure. The temperature of the sample solution was increased in the investigated range from 20 to 40°C, respectively. The results (Figure 6) revealed that the extraction efficiency increased with an increase temperature until the temperature was 30°C, and then decreased slightly when the temperature of the sample solution was further increased from 30 to 40°C. The mass transfer rates of analytes would increase with the increasing of temperature, thereby increasing the extraction efficiency. Nevertheless, high temperature might raise loss of organic phase and decrease precision. Therefore, the optimum sample temperature was chosen to be 30°C ± 1. Figure 6. View largeDownload slide Effect of extraction temperature on the extraction efficiency. Figure 6. View largeDownload slide Effect of extraction temperature on the extraction efficiency. Discussion Method validation To investigate the good recovery and excellent reproducibility of the developed method, PAHs were spiked into milk samples at three concentrations (5, 10 and 50 ng/mL), and analyzed five times per concentration level under the final optimized conditions. Linear calibration curves were acquired through plotting the peak area against the concentration of the respective compounds. All analytes showed a good linearity over the calibration range from 5 to 1,000 ng/mL with the correlation coefficients values (R2) higher than 0.9990. The limit of detections (LODs), on the basis of signal-to-noise ratio (S/N) of 3, were in the range of 0.14–0.71 ng/mL for the developed method. All the detailed analytical results were revealed in Table I. Table I. Analytical Parameters of the Proposed Method (n = 5) Analytes  Linearity range (ng/mL)  Calibration equations  R2  LODs (ng/mL)  1-OHP  5–1,000  Y = 1.468X − 0.720  0.999  0.71  Pyr  5–1,000  Y = 1.922X + 0.137  1.000  0.25  BaA  5–1,000  Y = 4.072X + 0.183  1.000  0.14  BaP  5–1,000  Y = 2.211X − 0.441  0.999  0.19  Analytes  Linearity range (ng/mL)  Calibration equations  R2  LODs (ng/mL)  1-OHP  5–1,000  Y = 1.468X − 0.720  0.999  0.71  Pyr  5–1,000  Y = 1.922X + 0.137  1.000  0.25  BaA  5–1,000  Y = 4.072X + 0.183  1.000  0.14  BaP  5–1,000  Y = 2.211X − 0.441  0.999  0.19  Analysis of milk samples To appraise the proposed IL-HF-LPME technique, milk samples purchased from a local supermarket in Kunming were selected to separate and determine their PAHs contents under the optimal experimental situations. The results were listed in Table II. Figure 7 showed the typical HPLC–DAD chromatograms of milk samples. As can be seen that 1-OHP was detected in milk samples. The results clarified that the IL-HF-LPME method was a rapid, effective and feasible technique for high selectivity extraction and high sensitivity determination of trace concentrations of four PAHs in milk samples. Table II. Analytical Results for Milk Samples Over 2 Days by the Proposed Method Analyte  Added (ng/mL)  Found (ng/mL) Day 1  Recovery (%)  RSD (%) (n = 5)  Found (ng/mL) Day 2  Recovery (%)  RSD (%) (n = 5)  1-OHP  0  7.23  –  –  6.89  –  –  5  12.17  98.8  3.05  12.03  102.8  3.14  10  17.05  98.2  2.47  16.84  99.5  2.57  50  58.09  101.7  1.98  56.35  98.9  1.24  Pyr  0  nd  –  –  nd  –  –  5  4.98  99.6  2.96  4.83  96.6  2.74  10  9.36  93.6  2.07  10.24  102.4  2.39  50  49.59  99.2  1.59  49.25  98.5  2.45  BaA  0  nd  –  –  nd  –  –  5  4.76  95.2  3.27  4.68  93.6  3.19  10  9.89  98.9  2.19  9.27  92.7  2.56  50  50.43  100.9  2.49  49.91  99.8  1.97  BaP  0  nd  –  –  nd  –  –  5  4.85  97.0  3.09  4.96  99.2  2.83  10  10.19  101.9  1.94  9.53  95.3  2.36  50  49.62  99.2  2.28  50.85  101.7  1.96  Analyte  Added (ng/mL)  Found (ng/mL) Day 1  Recovery (%)  RSD (%) (n = 5)  Found (ng/mL) Day 2  Recovery (%)  RSD (%) (n = 5)  1-OHP  0  7.23  –  –  6.89  –  –  5  12.17  98.8  3.05  12.03  102.8  3.14  10  17.05  98.2  2.47  16.84  99.5  2.57  50  58.09  101.7  1.98  56.35  98.9  1.24  Pyr  0  nd  –  –  nd  –  –  5  4.98  99.6  2.96  4.83  96.6  2.74  10  9.36  93.6  2.07  10.24  102.4  2.39  50  49.59  99.2  1.59  49.25  98.5  2.45  BaA  0  nd  –  –  nd  –  –  5  4.76  95.2  3.27  4.68  93.6  3.19  10  9.89  98.9  2.19  9.27  92.7  2.56  50  50.43  100.9  2.49  49.91  99.8  1.97  BaP  0  nd  –  –  nd  –  –  5  4.85  97.0  3.09  4.96  99.2  2.83  10  10.19  101.9  1.94  9.53  95.3  2.36  50  49.62  99.2  2.28  50.85  101.7  1.96  nd, not detected. Figure 7. View largeDownload slide HPLC–UV chromatograms: (a) milk sample which has not spiked with PAHs, (b) milk sample spiked with PAHs (10 ng/mL) without the IL-HF-LPME method and (c) milk sample spiked with PAHs (10 ng/mL) after using the IL-HF-LPME method. Figure 7. View largeDownload slide HPLC–UV chromatograms: (a) milk sample which has not spiked with PAHs, (b) milk sample spiked with PAHs (10 ng/mL) without the IL-HF-LPME method and (c) milk sample spiked with PAHs (10 ng/mL) after using the IL-HF-LPME method. Comparison of the developed method with other extraction methods According to the view point of sample preparation, extraction efficiency, extraction time and LODs, the proposed IL-HF-LPME method was in comparison with other extraction methods in extracting and determining PAHs. Judging from the analytical results in Table III, the developed method revealed that the LODs were equally matched with great majority of the previously mentioned methods, yet the relative standard deviation (RSD) for the developed technique were lower than other reported methods. The highlight of the proposed method was that the extraction time was shorter than those of previously published methods. The comparison results revealed that the proposed technique was a rapid, simple, sensitive and accurate method which would be developed for the extraction of PAHs in milk samples. Table III. Comparison of Different Methods for the Determination of PAHs Matrix  Method  LODs (ng/mL)  Time (min)  Recovery (%)  RSD (%)  Reference  Fresh milk samples  HF-LPME-GC–MS  0.07–1.4  30  85–110  3.1–6.0  (37)  Milk samples  DI-SPME-GC–MS  0.003–1.5  60  87.6–112  <20  (20)  Water samples  CPE-HPLC/FID  0.26–2.56  60  72.0–98.8  1.52–8.07  (23)  Aqueous samples  IL-LPME-HPLC/FID  –  30  –  2.8–12  (22)  Milk samples  IL-HF-LPME-HPLC/UV  0.14–0.71  5  91.8–101.6  1.19–3.56  This work  Matrix  Method  LODs (ng/mL)  Time (min)  Recovery (%)  RSD (%)  Reference  Fresh milk samples  HF-LPME-GC–MS  0.07–1.4  30  85–110  3.1–6.0  (37)  Milk samples  DI-SPME-GC–MS  0.003–1.5  60  87.6–112  <20  (20)  Water samples  CPE-HPLC/FID  0.26–2.56  60  72.0–98.8  1.52–8.07  (23)  Aqueous samples  IL-LPME-HPLC/FID  –  30  –  2.8–12  (22)  Milk samples  IL-HF-LPME-HPLC/UV  0.14–0.71  5  91.8–101.6  1.19–3.56  This work  Conclusion 1-OHP, Pyr, BaA and BaP in milk samples were concurrently separated and determined by the IL-HF-LPME method integrated with HPLC and DAD. It was demonstrated that a mixture of [OMIM]PF6 and lauric acid, in a ratio of 3:1, was applied as an excellent extraction solvent for the analysis of four PAHs. Compared to other previously published methods, the proposed method in this study not only reduced the extraction time, but also provided higher extraction efficiency. The method proposed in this paper has been proven to be a simple, rapid, low-cost, sensitive and accurate method for the quantitative and routine analysis of four PAHs assay in milk samples. On balance, the IL-HF-LPME method will have a broad potential in the analysis of other PAHs in sample preparation. Funding The work was strongly supported by the Analysis and Testing Foundation of Kunming University of Science and was supported by the National Natural Science Foundation of China (No. 21365026). Conflict of interest statement. The authors claim that there is no conflict of interest between them. 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Google Scholar CrossRef Search ADS PubMed  37 Sanagi, M.M., Loh, S.H., Ibrahim, W.A.W., Hasan, M.N., Enein, H.Y.A.; Determination of polycyclic aromatic hydrocarbons in fresh milk by hollow fiber liquid-phase microextraction–gas chromatography mass spectrometry; Journal of Chromatographic Science , ( 2013); 51( 2): 112– 116. Google Scholar CrossRef Search ADS PubMed  © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Chromatographic Science Oxford University Press

Hollow Fiber Supported Ionic Liquids Liquid-Phase Micro-extraction Followed by High-Performance Liquid Chromatography for the Determination of Polycyclic Aromatic Hydrocarbons in Milk Samples

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Oxford University Press
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© The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com
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0021-9665
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1945-239X
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10.1093/chromsci/bmx075
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Abstract

Abstract A rapid, simple, reliable and efficient hollow fiber supported ionic liquids liquid-phase micro-extraction method (IL-HF-LPME) followed by high-performance liquid chromatography was successfully applied to the determination of four kinds of polycyclic aromatic hydrocarbons (PAHs) in milk samples. In the IL-HF-LPME method, a mixture of [OMIM]PF6 and lauric acid, in a ratio of 3:1, was immobilized in the pores of a polypropylene hollow fiber used as extraction solvent. A series of essential parameters influencing the extraction efficiency were investigated and optimized. Under the optimal conditions, the extraction equilibrium is achieved within 3 min, the good linearity was >0.9990, the limits of detection varied from 0.14 to 0.71 ng/mL, the limit of quantification values were between 0.4 and 1.8 ng/mL, and the relative standard deviations were in the range of 1.24–3.27% (n = 5). The proposed method was applied to analyze four PAHs in milk samples and recoveries were between 93.6 and 102.8%. Introduction Polycyclic aromatic hydrocarbons (PAHs) are a huge and diverse group of carcinogenic and persistent organic compounds containing at least two fused benzene rings arranged in linear, angular or cluster structures. They are ubiquitously distributed as industrial–environmental pollutants, which are mainly produced by the natural processes of incomplete combustion or pyrolysis of organic matter at high temperature, but a few of them can also be of biological origin (1–3). Owing to their potential mutagenic, carcinogenic properties and significant toxicity, quite a few of PAHs, as well as their metabolites and degradation products, have been found widespread in several different kind of samples, e.g., environmental water (4–7), atmosphere (3), soil (8, 9) or dust particles (10). At the same time, most concerned still have been focused on their contamination in myriad foods, such as oils (11, 12), milk (2, 13), coffee (14, 15), fruits, vegetables (16) and roasted and smoked meat (1, 13, 17, 18). In particular, the highly carcinogenic benzo[a]pyrene has been detected in several foods at concentration levels varied from 0.1 to 100 mg/kg (14, 15). PAHs have appealed to a wide range of attention owing to their physical or chemical properties of lipophicity, poor solubility and aggregation to the precipitants (1, 19). Either the United States Environmental Protection Agency (US EPA) or the International Agency for Research on Cancer (IARC) has been reasonably enumerated 16 PAHs as the higher priority contaminants and genotoxic carcinogens (2, 11, 17, 19). Based on the Scientific Committee on Food, benzo[a]pyrene (BaP) has been applied as a significant indicator of the epidemiology, intake evaluation, system analysis methodology, occurrence and impact of total carcinogenic PAHs in food (1, 17, 20). In fact, four PAHs were chosen in this study: pyrene (Pyr), benzo[a]anthracene (BaA), 1-hydroxypyrene (1-OHP) and benzo[a]pyrene (BaP). They all have obvious carcinogenic and genotoxic properties (13). Since the pollution of PAHs in food has already become a serious threat to human health, it is particularly important and essential to establish a rapid, reliable and accurate analytical approach to accurately analyze levels of contamination from various PAHs in food (6, 14, 17). At present, various available sample pre-concentration and extraction methods with specialized analytical equipments have already been exhaustively researched for the determination of PAHs in various samples, including liquid–liquid extraction (LLE) (21), liquid-phase micro-extraction (LPME) (22), disperse liquid–liquid micro-extraction (DLLME) (6, 7), cloud-point extraction (CPE) (23), solid-phase extraction (SPE) (14), solid-phase micro-extraction (SPME) (4, 20), hollow fiber liquid-phase micro-extraction (HF-LPME) (24–26), magnetic solid-phase extraction (MSPE) (27), miniaturized homogeneous liquid–liquid extraction (MHLLE) (28) integrated with high-performance liquid chromatography and diode array detector (HPLC–DAD) (6, 12, 19) or fluorescence detection (HPLC–FLD) (1, 3, 13), and gas chromatography coupled to mass spectrometry (GC–MS) (7, 15, 21). LPME was a novel practically feasible, and suitable sample enrichment method due to their characteristics such as simplification, rapidness and high efficiency (29). In various LPME modes, HF-LPME, as the improvement of the conventional LLE technique, was on the basis of porous polypropylene hollow fiber which served as contact interfaces between micro-volumes of the extraction agent and the target analytes (26). According to the HF-LPME approach, the hollow fiber cannot only play a vital role in the purification of samples, but also absolutely remove the influence of cross contamination of samples and ensure repeatability (30). Moreover, the HF-LPME method can offer some advantages as follows: high enrichment factor, low cost, high repeatability, rapid sample processing, high sensitivity, etc. (30, 31). Overall, the extraction, enrichment and purification of the sample were carried out in one step. Hydrophobic ionic liquids (ILs) are incorporated with organic cations and various anions, and have several obvious superiorities over organic solvents as follows: low volatility, high heat stability and the capacity to interact with multifarious organic and inorganic compounds (32). In addition, they are regarded to be environmentally-friendly solvents, and thereby applied as great substitutes to traditional organic solvents. Our research groups have studied ionic liquid-HF-LPME (IL-HF-LPME) technology and applied for the analysis of phthalate esters (33). In the current work, the IL-HF-LPME method integrated with HPLC–DAD technique was established for the highly sensitive determination of four PAHs: pyrene (Pyr), benzo[a]anthracene (BaA), 1-hydroxypyrene (1-OHP) and benzo[a]pyrene (BaP). ILs were considered as the extractant and lauric acid was not only applied as the synergistic extraction solvent, but also as a solvent to reduce the viscosity of ionic liquids. The IL-HF-LPME provide very clean method that can be directly injected to the high-performance liquid chromatographic system, and has the excellent stable baseline, and enhance the practicability of the developed method (30). Various essential experimental parameters were screened and the optimized procedure was certified. The serviceability of the developed method was researched for the analysis of the trace levels of PAHs in milk samples. Experimental Materials Standards of 1-hydroxypyrene (1-OHP), benzo[a]anthracene (BaA), benzo[a]pyrene (BaP) and pyrene (Pyr) were supplied by Tokyo Chemical Industry Co., Ltd (Japan). The purity of each compound was above 98%. 1-butyl-3-methylimidazolium hexafluorophosphate ([BMIM]PF6, 99%), 1-hexyl-3-methylimidazolium hexafluorophosphate ([HMIM]PF6, 99%), 1-octyl-3-methylimidazolium hexafluorophosphate ([OMIM]PF6, 99%) and lauric acid (C12H24O2, 98%) were supplied by Aladdin (Shanghai, China). Acetonitrile (HPLC grade) was provided by Merck (Darmstadt, Germany). Acetone was obtained from Fengchuan Chemical Reagent Co., Ltd (Tianjin, China). The deionized water was provided by Milli-Q systems (USA). All other chemicals and solvents were at or above the analytical reagent-grade. Instruments An ultrasonic cleaner (Henao ultra-sonic instrument plant, Hu-10260T, Tianjin, China) was served to eliminate fomites in hollow fibers. The vortex mixer (Jintan experiment Instrument Co. Ltd, SK-1, Jiangsu, China) was applied to vortex-mixing. The centrifuge (Hunan experiment Instrument Co., Ltd., L420, Hunan, China) was applied in phase separation. The accurel Q3/2 polypropylene hollow fiber membrane was supplied by Membrana (Wuppertal, Germany), and the inner diameter was 600 μm, the wall thickness was 200 μm, and the pore size was 0.2 μm. A 1.0 mL sterile injection syringe (Shanghai medical Instrument Co., Ltd, Shanghai, China) was supplied to inject the extracting agent into the hollow fiber lumen. The experiments in liquid chromatography were actualized with Agilent 1260 series HPLC system (Agilent Technologies, CA, USA) combined with a photodiode-array detector (DAD). Chromatographic conditions The chromatographic separations and quantitative analysis were developed employing a reversed-phase Agilent TC-C18 column (250 mm × 4.6 mm i.d., 5 μm) with the column temperature of 30°C, and the detector wavelength was set at 254 nm. Acetonitrile–water (85:15, v/v) was employed as the mobile phase with the flow rate of 1 mL/min and the injection volume was 20 μL. Preparation of standard solutions and milk samples Stock standard solutions of Pyr, BaA, 1-OHP and BaP were dissolved in acetonitrile (1,000 μg mL−1). Working standard solutions were prepared by dilution of the stock solution. The stock solutions were stored in a fridge at 4°C (stable for at least 2 month) and then brought to room temperature prior to use. Milk samples were gained from the local supermarket in Kunming City (Yunnan Province, China), and stored at 4°C before analyzing. For protein precipitation, a 10 mL centrifuge vial was charged with a 5 mL milk sample containing NaAc (0.1 g), MgSO4 (0.4 g) and acetonitrile (2.0 mL), which was then completely blended by the vortex mixer within 2 min. After centrifugation at 4,000 rpm for 5 min, the supernatants were easily transferred into another new vial, and repeated once again based on the above method. Finally, supernatants were merged and diluted with deionized water to 25 mL. Preparation of hollow fiber and IL-HF-LPME extraction procedure Hollow fibers were cut into 6 cm segments, and washed with acetone for 5 min in an ultrasonic bath to eliminate any impurity pollution in the fiber, and then naturally drying in air. 1-octyl-3-methylimidazolium hexafluorophosphate [OMIM]PF6 (as the extraction agent) and lauric acid (as the synergistic extraction solvent) were dissolved by acetonitrile in a mixture ratio of 3:1, and then injected slowly into the hollow fiber through using the 1.0 mL sterile injection syringe. Each port of the fiber was sealed with a lighter and then the hollow fiber was bent into a U-shape and macerated in the 5 mL milk sample solution. The mixture was sufficiently vortex-mixed for 3 min at a temperature (ca. 30°C). Then, the fiber was removed, both of the ports were cut and 200 μL acetonitrile was injected into one of its port to dissolve the analytes, and then introduced into HPLC system for analyses. Each new hollow fiber was used only once for extraction. Results Optimization of the IL-HF-LPME procedure A series of disparate experimental parameters affecting the extraction efficiency for the IL-HF-LPME procedure have been investigated, such as the type and mixture ratio of extraction solvent, fiber length, the extraction time, sample pH value, salt addition and extraction temperature. The study and optimization of the above-mentioned variables were performed using the one variable at a time method. Finally, the optimized experimental conditions were established. Selection of extraction solvent The selection of an appropriate extraction solvent was very important for the IL-HF-LPME method. In general, the solvent selected should be compatible with the polypropylene fiber so as to be able to easily fill its wall pores. The immiscibility with the water sample and the acceptor phase should also be seriously considered as it acts as a suitable medium between the two phases. In the proposed method, according to the polarity of the analytes, several solvents were examined, including lauric acid dissolved by acetonitrile (concentration was 5%, w/v), [BMIM]PF6, [HMIM]PF6 and [OMIM]PF6. As shown in Figure 1, [OMIM]PF6 and a mixture of [OMIM]PF6 and lauric acid in a ratio of 3:1 provided similar satisfactory results. However, the viscosity of [BMIM][PF6], [HMIM][PF6] and [OMIM][PF6] were 450, 585 and 685 cP, respectively (34), and a high viscosity of extractant could reduce mass diffusion rates of PAHs into the ionic liquid phase during extraction. A mixture of [OMIM]PF6 and lauric acid in a ratio of 3:1 has suitable viscosity and less volatility, so it can impregnate the pores of HF and has form mixed extraction efficiency to PAHs. Consequently, a mixture of [OMIM]PF6 and lauric acid, in a ratio of 3:1, was adopted as the extraction agent in the following experiments. Figure 1. View largeDownload slide Effect of different kinds of extraction solvent on the extraction efficiency. Figure 1. View largeDownload slide Effect of different kinds of extraction solvent on the extraction efficiency. Effect of fiber length The length of hollow fiber determines the volume of extraction solvent, which was a critical parameter in the extraction. For the sake of examining the length of the hollow fiber, the mixed extraction solvent involving varied volumes were subjected to the identical IL-HF-LPME approach. Hence, the length of the hollow fiber from 2 to 10 cm was discussed. The results in Figure 2 illustrated that the maximum extraction efficiency was at 6 cm, and no discernible influence was changed when the fiber length increased from 8 to 10 cm. The short fiber length enhanced a high concentration of the trace analytes in the IL-HF-LPME method. Nevertheless, fiber membranes cannot be too short to offer adequate extraction solvent to facilitate transport of the analytes (35). Thus, it could be seen that the increase of fiber length was not beneficial to the extraction of the analytes. After comprehensive consideration, 6 cm the hollow fiber containing around 20 μL extraction solvent was utilized in the subsequent experiments. Figure 2. View largeDownload slide Effect of fiber length on the extraction efficiency. Figure 2. View largeDownload slide Effect of fiber length on the extraction efficiency. Effect of the extraction time The HF-LPME process mainly rested with the vortex-mixing of the target analytes between the extraction solvent and the sample solution (36), so it was important to find a minimum extraction time without losing sensitivity. To appraise the optimal extraction time, experiments were actualized by changing the time from 1 to 6 min, respectively. As shown in Figure 3, the results suggested that the system essentially reached equilibrium within 3 min, and there was no significant increase in the extraction efficiency at longer extraction time. Accordingly, in accordance with the above analysis results, 3 min was adopted as the optimized extraction time in the subsequent experiments. Figure 3. View largeDownload slide Effect of the extraction time on the extraction efficiency. Figure 3. View largeDownload slide Effect of the extraction time on the extraction efficiency. Effect of pH value Sample pH value was closely related to the solubility of the target analytes, which could change the partition coefficient of the analytes between the sample solution and the extraction solvent. The effect of sample pH value on the IL-HF-LPME method was studied within the range of 4.0–9.0 in present experiment. It was observed from Figure 4 that the maximum extraction efficiency could be obtained without changing the pH. PAHs were a group of organic compounds with strong inert and stable properties, and the pH value of donor phase did not produce an effect on the extraction efficiency (32). Consequently, there was no need to change the pH of the solution in following further experiments. Figure 4. View largeDownload slide Effect of pH on the extraction efficiency. Figure 4. View largeDownload slide Effect of pH on the extraction efficiency. Effect of salt addition As a result of the salting-out effect, addition of salt to the sample solution would increase the ionic strength of the solution and then reduce the solubility of the analytes in the IL-HF-LPME method. The impact of salting strength on the extraction of PAHs was carried out by increasing various dosages of NaCl within the range of 0.2–1.0 g. Figure 5 shows the extraction efficiency of PAHs in diverse amounts of NaCl. As expected (Figure 5), the results revealed that the extraction efficiency was almost unchanged. This phenomenon could be explained by the non-polarity of the PAHs compounds (6). Hence, no addition of salt to the sample solution was employed in the following whole experiments. Figure 5. View largeDownload slide Effect of the amount of salt on the extraction efficiency. Figure 5. View largeDownload slide Effect of the amount of salt on the extraction efficiency. Effect of extraction temperature Extraction temperature could also influence the extraction performance of the developed method, because it would affect both kinetics and thermodynamics of the analytes diffusion process in extraction procedure. The temperature of the sample solution was increased in the investigated range from 20 to 40°C, respectively. The results (Figure 6) revealed that the extraction efficiency increased with an increase temperature until the temperature was 30°C, and then decreased slightly when the temperature of the sample solution was further increased from 30 to 40°C. The mass transfer rates of analytes would increase with the increasing of temperature, thereby increasing the extraction efficiency. Nevertheless, high temperature might raise loss of organic phase and decrease precision. Therefore, the optimum sample temperature was chosen to be 30°C ± 1. Figure 6. View largeDownload slide Effect of extraction temperature on the extraction efficiency. Figure 6. View largeDownload slide Effect of extraction temperature on the extraction efficiency. Discussion Method validation To investigate the good recovery and excellent reproducibility of the developed method, PAHs were spiked into milk samples at three concentrations (5, 10 and 50 ng/mL), and analyzed five times per concentration level under the final optimized conditions. Linear calibration curves were acquired through plotting the peak area against the concentration of the respective compounds. All analytes showed a good linearity over the calibration range from 5 to 1,000 ng/mL with the correlation coefficients values (R2) higher than 0.9990. The limit of detections (LODs), on the basis of signal-to-noise ratio (S/N) of 3, were in the range of 0.14–0.71 ng/mL for the developed method. All the detailed analytical results were revealed in Table I. Table I. Analytical Parameters of the Proposed Method (n = 5) Analytes  Linearity range (ng/mL)  Calibration equations  R2  LODs (ng/mL)  1-OHP  5–1,000  Y = 1.468X − 0.720  0.999  0.71  Pyr  5–1,000  Y = 1.922X + 0.137  1.000  0.25  BaA  5–1,000  Y = 4.072X + 0.183  1.000  0.14  BaP  5–1,000  Y = 2.211X − 0.441  0.999  0.19  Analytes  Linearity range (ng/mL)  Calibration equations  R2  LODs (ng/mL)  1-OHP  5–1,000  Y = 1.468X − 0.720  0.999  0.71  Pyr  5–1,000  Y = 1.922X + 0.137  1.000  0.25  BaA  5–1,000  Y = 4.072X + 0.183  1.000  0.14  BaP  5–1,000  Y = 2.211X − 0.441  0.999  0.19  Analysis of milk samples To appraise the proposed IL-HF-LPME technique, milk samples purchased from a local supermarket in Kunming were selected to separate and determine their PAHs contents under the optimal experimental situations. The results were listed in Table II. Figure 7 showed the typical HPLC–DAD chromatograms of milk samples. As can be seen that 1-OHP was detected in milk samples. The results clarified that the IL-HF-LPME method was a rapid, effective and feasible technique for high selectivity extraction and high sensitivity determination of trace concentrations of four PAHs in milk samples. Table II. Analytical Results for Milk Samples Over 2 Days by the Proposed Method Analyte  Added (ng/mL)  Found (ng/mL) Day 1  Recovery (%)  RSD (%) (n = 5)  Found (ng/mL) Day 2  Recovery (%)  RSD (%) (n = 5)  1-OHP  0  7.23  –  –  6.89  –  –  5  12.17  98.8  3.05  12.03  102.8  3.14  10  17.05  98.2  2.47  16.84  99.5  2.57  50  58.09  101.7  1.98  56.35  98.9  1.24  Pyr  0  nd  –  –  nd  –  –  5  4.98  99.6  2.96  4.83  96.6  2.74  10  9.36  93.6  2.07  10.24  102.4  2.39  50  49.59  99.2  1.59  49.25  98.5  2.45  BaA  0  nd  –  –  nd  –  –  5  4.76  95.2  3.27  4.68  93.6  3.19  10  9.89  98.9  2.19  9.27  92.7  2.56  50  50.43  100.9  2.49  49.91  99.8  1.97  BaP  0  nd  –  –  nd  –  –  5  4.85  97.0  3.09  4.96  99.2  2.83  10  10.19  101.9  1.94  9.53  95.3  2.36  50  49.62  99.2  2.28  50.85  101.7  1.96  Analyte  Added (ng/mL)  Found (ng/mL) Day 1  Recovery (%)  RSD (%) (n = 5)  Found (ng/mL) Day 2  Recovery (%)  RSD (%) (n = 5)  1-OHP  0  7.23  –  –  6.89  –  –  5  12.17  98.8  3.05  12.03  102.8  3.14  10  17.05  98.2  2.47  16.84  99.5  2.57  50  58.09  101.7  1.98  56.35  98.9  1.24  Pyr  0  nd  –  –  nd  –  –  5  4.98  99.6  2.96  4.83  96.6  2.74  10  9.36  93.6  2.07  10.24  102.4  2.39  50  49.59  99.2  1.59  49.25  98.5  2.45  BaA  0  nd  –  –  nd  –  –  5  4.76  95.2  3.27  4.68  93.6  3.19  10  9.89  98.9  2.19  9.27  92.7  2.56  50  50.43  100.9  2.49  49.91  99.8  1.97  BaP  0  nd  –  –  nd  –  –  5  4.85  97.0  3.09  4.96  99.2  2.83  10  10.19  101.9  1.94  9.53  95.3  2.36  50  49.62  99.2  2.28  50.85  101.7  1.96  nd, not detected. Figure 7. View largeDownload slide HPLC–UV chromatograms: (a) milk sample which has not spiked with PAHs, (b) milk sample spiked with PAHs (10 ng/mL) without the IL-HF-LPME method and (c) milk sample spiked with PAHs (10 ng/mL) after using the IL-HF-LPME method. Figure 7. View largeDownload slide HPLC–UV chromatograms: (a) milk sample which has not spiked with PAHs, (b) milk sample spiked with PAHs (10 ng/mL) without the IL-HF-LPME method and (c) milk sample spiked with PAHs (10 ng/mL) after using the IL-HF-LPME method. Comparison of the developed method with other extraction methods According to the view point of sample preparation, extraction efficiency, extraction time and LODs, the proposed IL-HF-LPME method was in comparison with other extraction methods in extracting and determining PAHs. Judging from the analytical results in Table III, the developed method revealed that the LODs were equally matched with great majority of the previously mentioned methods, yet the relative standard deviation (RSD) for the developed technique were lower than other reported methods. The highlight of the proposed method was that the extraction time was shorter than those of previously published methods. The comparison results revealed that the proposed technique was a rapid, simple, sensitive and accurate method which would be developed for the extraction of PAHs in milk samples. Table III. Comparison of Different Methods for the Determination of PAHs Matrix  Method  LODs (ng/mL)  Time (min)  Recovery (%)  RSD (%)  Reference  Fresh milk samples  HF-LPME-GC–MS  0.07–1.4  30  85–110  3.1–6.0  (37)  Milk samples  DI-SPME-GC–MS  0.003–1.5  60  87.6–112  <20  (20)  Water samples  CPE-HPLC/FID  0.26–2.56  60  72.0–98.8  1.52–8.07  (23)  Aqueous samples  IL-LPME-HPLC/FID  –  30  –  2.8–12  (22)  Milk samples  IL-HF-LPME-HPLC/UV  0.14–0.71  5  91.8–101.6  1.19–3.56  This work  Matrix  Method  LODs (ng/mL)  Time (min)  Recovery (%)  RSD (%)  Reference  Fresh milk samples  HF-LPME-GC–MS  0.07–1.4  30  85–110  3.1–6.0  (37)  Milk samples  DI-SPME-GC–MS  0.003–1.5  60  87.6–112  <20  (20)  Water samples  CPE-HPLC/FID  0.26–2.56  60  72.0–98.8  1.52–8.07  (23)  Aqueous samples  IL-LPME-HPLC/FID  –  30  –  2.8–12  (22)  Milk samples  IL-HF-LPME-HPLC/UV  0.14–0.71  5  91.8–101.6  1.19–3.56  This work  Conclusion 1-OHP, Pyr, BaA and BaP in milk samples were concurrently separated and determined by the IL-HF-LPME method integrated with HPLC and DAD. It was demonstrated that a mixture of [OMIM]PF6 and lauric acid, in a ratio of 3:1, was applied as an excellent extraction solvent for the analysis of four PAHs. Compared to other previously published methods, the proposed method in this study not only reduced the extraction time, but also provided higher extraction efficiency. The method proposed in this paper has been proven to be a simple, rapid, low-cost, sensitive and accurate method for the quantitative and routine analysis of four PAHs assay in milk samples. On balance, the IL-HF-LPME method will have a broad potential in the analysis of other PAHs in sample preparation. Funding The work was strongly supported by the Analysis and Testing Foundation of Kunming University of Science and was supported by the National Natural Science Foundation of China (No. 21365026). Conflict of interest statement. The authors claim that there is no conflict of interest between them. 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Published: Jan 1, 2018

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