3.1. Characterisation of MIL
The FTIR spectra of the synthesized MIL and precursor was shown in Fig. 1(a). It can be seen that the FTIR spectrum of synthesized [P6,6,6,14+]2[CoCl4 2-] and precursor [P6,6,6,14+][Cl-] are almost identical, showing MIL and precursor contained the same cationic structure. As shown in Fig. 1(b), [P6,6,6,14+]2[CoCl4 2-] exhibited low absorption in UV region which made it a better alternative and sensitive for the determination of analytes coupled in HPLC. Besides [P6,6,6,14+]2[CoCl4 2-] are immiscible in aqueous phase and it can be quite easily manipulated by an external strong magnetic field.
3.2. Optimization of MIL-DLLME conditions
To obtain high extraction efficiency, extraction conditions were optimized using spiked samples (100 ng mL-1). The experimental parameters affecting the extraction efficiency were carefully investigated, including amount of extraction solvent, the pH and ionic strength of sample solution, the type and volume of dispersant, as well as extraction time and temperature. All experiments were performed in triplicates (n=3). The extraction recovery and enrichment factor (EF) were calculated based on the following equations:
Ca and C0 are the concentration of analyte in the extraction phase and the initial analyte concentration in the sample solution, respectively. Va and V0 are the volumes of extraction phase and sample solution, respectively
3.2.1. Effect of extraction solvent amount
The amount of extraction solvent is an important factor that can influence extraction efficiency. In order to evaluate the influence of extraction solvent amounts on extraction efficiencies of tagets, different amounts of [P6,6,6,14+]2[CoCl42-] (10-30 mg) were tested using the same MIL-DLLME procedure. As shown in Fig.2(a), the recoveries of targets increase with the increase of the volume of [P6,6,6,14+]2[CoCl42-] from 10 to 20 mg, but above 20 mg, the recoveries remained a constant level. The amount of extraction solvent can also determine the enrichment performance because lower amounts generally result in high EFs. Therefore, 20 mg of [P6,6,6,14+]2[CoCl42-] was selected in the following studies because a higher recoveries were obtained and the EFs were acceptable.
3.2.2. Effect of extraction time
DLLME is a time-dependent process. Consequently, the effect of the extraction time was examined within the range of 1-10 min. Results (Fig.2(b)) showed that the recoveries of targets increased with increased extraction time. The extraction recoveries were in rang of 96.2–98.4% at 3 min. After 3 min, the recoveries of targets were constant. Therefore, the extraction time for this method was set at 3 min.
3.2.3. Effect of pH
In general, the pH of sample solution plays an important role in the extraction process because the pH value of the solution determines the present state of analytes. The effects of pH on the extraction were studied within the pH range of 2-10 using hydrochloric acid and sodium hydroxide, and the results are shown in Fig. 2(c). The recoveries of targets remained over 90% in a range of pH 2-6, but the recoveries decreased at a pH > 8.0. In this study, the pH of natural target analytes solution was close to 6.0, thus, the sample solution was used directly without any pH adjustment.
Generally, Hydrophobic are important force in the extraction of MIL. These compounds exist as a neutral form in acid and neutral aqueous solutions, and hydrophobicity interaction was enhanced, which was beneficial to extraction and separation. Under the alkaline conditions, these compounds were decomposed, which result in lower recoverier of targets.
3.2.4. Effect of ionic strength
In order to investigate the influence of the ionic strength, different concentrations of NaCl (from 0% to 7%) were added to targets solution, respectively. Results (Fig.2(d)) indicate that the recoveries of targets increase slightly with the increase of NaCl concentration in range of 0-5%. Generally, the salt addition can increase extraction efficiency, could be attributed to the fact that the dissolution of sodium chloride in water increased the viscosity of the solution, which reduced the solubility of the targets in aqueous phase, and facilitates the transfer of the targets from the aqueous phase to non-aqueous phase. However, the recoveries of targets decrease slightly when the concentrations of NaCl was 7%. The results, the salt addition decreases extraction efficiency, could be attributed to the fact that the dissolution of sodium chloride in water increased the viscosity of the solution, which reduced the diffusion rates that targets diffused into extraction solvent. Furthermore, the addition of salt enhanced the solubility of MIL in water. According to theses facts, the subsequent experiments were carried out with 5% (w/v) NaCl.
3.3. Interference studies
Fruit, vegetable and juice samples contain considerable amounts of organic matter and inorganic ions. In order to assess the possible analytical applications of the proposed method, the effect of concomitant species on the determination of three pyrethroids in real samples was examined under the optimal conditions as described above. The sample solutions containing 100 ng mL-1 of pyrethroids and the added interfering matter were subjected to the proposed method. The tolerance ratio of each interfering matter was taken as the largest amount yielding an error in the determination of the target analytes not exceeding 5% (Table1).
Table 1. Effects of interfering matter on determination of 100.0 ng mL-1 of three pyrethroids.
Interference
|
Interference to pyrethroids ratio (w/w)
|
Saccharose, glucose, fructose
Ascorbic acid
|
400
600
|
Na+, K+, Fe3+, Mg2+, Ca2+
|
1000
|
SO42-, PO43 -, NO3- , HCO3- ,CO32
|
1000
|
3.5. Method evaluation
3.5.1 Analytical performances
A novel MIL-DLLME based on [P6,6,6,14+]2[CoCl42-] coupled with HPLC for the simultaneous determination of three pyrethroids was developed. Under optimal experimental conditions, a series of experiments were performed to obtain linear ranges, precision, the limit of detection (LOD, S/N=3, S/N: signal-to-noise ratio ) and quantification (LOQ, S/N=10). All the experiments were performed in triplicate. As shown in Table.2, good linearities were observed in the range of 10-1000 ng mL-1 with the correlation coefficients (r2) of 0.9986-0.9995 The LOD and the LOQ were found to be 0.75-1.75 ng mL-1 and 2.5-6.0 ng mL-1, respectively. The proposed method showed good precision with the intra-day and inter-day RSDs in the range of 1.73-2.19% (n=6) and 3.02–3.89% (n=3), respectively.
Table 2. Analytical performance for pyrethroids obtained by MIL-DLLME-HPLC-UV.
compound
|
Regression equation
|
LOD
(ng mL-1)
|
LOQ
(ng mL-1)
|
r2
|
EF
|
Beta-cypermethrin(trans)
|
y=1.1242C+0.9308
|
1.0
|
3.5
|
0.9992
|
20
|
Beta-cypermethrin(cis)
|
y=0.7017C-0.7918
|
1.75
|
6.0
|
0.9990
|
20
|
Fenvalerate
|
y=1.5455C-4.0931
|
0.9
|
3.0
|
0.9986
|
20
|
Bifenthrin
|
y=1.5727C+2.2480
|
0.75
|
2.5
|
0.9995
|
20
|
In this procedure, the estimation of uncertainty of the final results and traceability is necessary. According to the Guide to the Expression of Uncertainty in Measurement (GUM). The expanded uncertainty of the target compouds analysis is obtained using the formula (Konieczka & Namieśnik, 2010):
Where U is expanded uncertainty, k is coverage factor, for which 3 is usually chosen to obtain a confidence level of approximately 95%, c is average concentration of the analyte, ur(sample) is relative standard uncertainty of sample mass determination, ur(cal) is relative standard uncertainty of calibration step, ur(true) is relative standard uncertainty of recovery determination, ur(rep) is relative standard uncertainty of repeatability, ur(LOD) is relative standard uncertainty of LOD determination and cdet is the concentration of the target analyte.
Where SDxy is the residual standard deviation, b is the direction coefficient of the calibration curve, p is the number of measurements carried out for given sample, n is the total number of standard samples used for plotting the calibration curve, xsample is the concentration of sample, xm is the mean of all the concentration of a standard solution for which the measurement was made in order to plot a standard curve, xi is the concentration of standard solution. The related parameters were listed in Table 3. The uncertainty of the weight and/or volume of a sample are usually small, so ur(sample) is very often neglected during construction ofthe uncertainty budget(Konieczka & Namieśnik, 2010, L. Wang, et al., 2015) .
Table.3 Calculated values of relative standard uncertainty and expanded uncertainty (U, k=2) for the determination of fenvalerate, beta-cypermethrin and bifenthrin in mushroom.
Compound
|
Concentration
(ng mL-1)
|
|
Relative standard uncertainty
|
U
|
ur(sample)
|
ur(cal)
|
ur(true)
|
ur(rep)
|
ur(LOD)
|
(ng mL-1)
|
Beta-cypermethrin(trans)
|
51.91
|
0.001
|
0.0026
|
0.022
|
0.012
|
0.019
|
3.28
|
Beta-cypermethrin(cis)
|
48.52
|
0.001
|
0.0025
|
0.021
|
0.017
|
0.036
|
4.38
|
Fenvalerate
|
49.93
|
0.001
|
0.0023
|
0.027
|
0.024
|
0.018
|
4.04
|
Bifenthrin
|
49.36
|
0.001
|
0.0025
|
0.025
|
0.025
|
0.015
|
3.80
|
To evaluate the applicability of the present method, food samples were analyzed. It can be seen (Fig.3) that no significant interference peaks were found at the retention positions of target compounds. To evaluate the precision and accuracy of the proposed method, the spiked samples (50.0, 200.0 ng mL-1) were analyzed and the analytical results were showed in Table 4. The recoveries of targets obtained from cucumber, mushroom and lemon samples were in the range of 96.3-103.8%. It can be considered that the current method provides acceptable recoveries and precision for the determination of three pyrethroids in real samples.
Table 4. Recoveries of three pyreghroids in cu and mushroom samples.
Compound
|
Cucumber
|
Mushroom
|
Lemon
|
Spike
(ng mL−1)
|
Found
(ng mL−1)
|
Recovery
(%)
|
Spike
(ng mL−1)
|
Found
(ng mL−1)
|
Recovery
(%)
|
Spike
(ng mL−1)
|
Found
(ng mL−1)
|
Recovery
(%)
|
Beta-cypermethrin
|
0
|
n.d.
|
-
|
0
|
n.d.
|
-
|
0
|
n.d.
|
-
|
(trans)
|
50.0
|
51.37±2.92
|
102.7
|
50.0
|
51.91±3.28
|
103.8
|
50.0
|
51.63± 3.44
|
103.2
|
|
200.0
|
200.2±2.56
|
100.1
|
200.0
|
202.6±2.08
|
101.3
|
200.0
|
201.8±3.07
|
100.9
|
Beta-cypermethrin
|
0
|
n.d.
|
-
|
0
|
n.d.
|
-
|
0
|
n.d.
|
-
|
(cis)
|
50.0
|
48.91±3.78
|
97.8
|
50.0
|
48.52±4.38
|
97.0
|
50.0
|
48.66±4.17
|
97.3
|
|
200.0
|
197.4±3.12
|
98.7
|
200.0
|
192.6±2.88
|
96.3
|
200.0
|
195.7±3.38
|
97.8
|
Fenvalerate
|
0
|
n.d.
|
-
|
0
|
n.d.
|
-
|
0
|
n.d.
|
-
|
|
50.0
|
48.67±3.45
|
97.3
|
50.0
|
49.93±4.04
|
98.7
|
50.0
|
49.18±4.12
|
98.4
|
|
200.0
|
197.1±2.87
|
98.6
|
200.0
|
200.8±3.03
|
100.4
|
200.0
|
198.2±3.73
|
99.1
|
Bifenthrin
|
0
|
n.d.
|
-
|
0
|
n.d.
|
-
|
0
|
n.d.
|
-
|
|
50.0
|
49.42±3.78
|
98.8
|
50.0
|
49.36±3.80
|
97.3
|
50.0
|
49.38±3.26
|
98.8
|
|
200.0
|
197.4±1.98
|
98.7
|
200.0
|
198.6±2.65
|
99.3
|
200.0
|
198.1±2.27
|
99.0
|
a n.d.= not detected.