3.1 Effect of extraction solvent and its volume
The selection of extraction solvent is very important for extraction efficiency. Methanol and acetonitrile are the most commonly used as extraction solvent for target compounds from plant-derived sample. In this study, effect of three solvents including methanol and acetonitrile were evaluated for extraction efficiency, and the results are shown the average recoveries were higher methanol as extraction solvent. Therefore, methanol was selected as the extraction solvent. SCN, SDN, SBA, SBB, ISBA, ISBB are flavonoids with high polarity.Therefore, methanol aqueous solution was investigated as extraction solvent for analyze compounds ,and the results are shown in Fig.1. Consequently, 75% methanol was selected as extraction solvent for the subsequent studies.
<Fig.1>
Comparing the extraction recoveries obtained by using different volumes of extraction solvents (i.e. 20, 30, 40, 50, 60, 70 and 80 mL), it shows that using 50 mL of 75% methanol can obtain satisfactory extraction recoveries, and further increasing the solvent volume will not significantly improve the extraction recoveries, as shown in Fig.2. Thus, 50 mL was selected as the appropriate 75% methanol volume used in the following tests.
<Fig.2>
3.2 Optimization of ILs-DLLME conditions
In order to obtain the optimal conditions of ILs-DDLME, a series of parameters affecting the extraction efficiency were studied, including extraction solvent, dispersive solvent, extraction time, pH value and ionic strength. The recovery was used as an indicator and 0.5 samples (each analyte with 100 ng g-1 added to each sample) were used to optimize the extraction conditions. All experiments were made in triplicate, and the blank control sample was 0.5g sample without any target compound.
3.2.1 Effect of extraction solvent and its volume
Appropriate extractant is the key to improve the analytical sensitivity. The effects of [OMIM] PF6 and [HMIM]OTF on the extraction efficiency of SCN, SDN, SBA, SBB, ISBA, ISBB were investigated. The results showed that [OMIM] PF6 had better extraction efficiency for SCN, SDN, SBA, SBB, ISBA, ISBB with less interference. Therefore, [OMIM] PF6 was selected as the extractant in this paper.
The recoveries increased with the increase of volume of [OMIM] PF6. 20, 40, 60, 80, 100, 120 and 140 μL were investigated. When 100 μL [OMIM] PF6 was added , the extraction recovery reaches the maximum and tends to be stable, as shown in Fig.3, Because of ILs-DLLME has reached equilibrium using 100 μL [OMIM] PF6 as as extraction agent. Therefore, 100 μL [OMIM] PF6 was used as the optimal extraction solvent volume in subsequent experiments.
<Fig.3>
3.2.2 Effect of dispersant and its volume
Dispersion solvent is considered to be another important factor affecting the extraction efficiency(32). The effects of methanol, ethanol, acetonitrile and acetone as dispersion solvents were studied. The results are shown in Fig. 4. When acetonitrile is used, the recovery rate is the highest. Therefore, acetonitrile was selected as the extraction procedure.
<Fig.4>
Because the volume of acetonitrile will affect the solubility of extraction solvent in water, the effect of the volume of dispersion solvent on the recovery was also investigated. In Fig. 5, when 600 μLacetonitrile is used for extraction, the recovery of each analyte is the highest.
<Fig.5>
3.2.3 Effect of sample pH value
The pH value is another main factor affecting ILs-DLLME. The pH value of the sample solution can affect the stability and existing form of the tested compounds by deionizing analytes. Considering the variation of analytes containing pKa between 6.4 and 7.4, the effect of sample pH value in the range of 5.0-8.0 on extraction efficiency was studied.The recovery of analytes is little affected by pH value. Therefore, the proposed experiment was carried out without pH.
<Fig.6>
3.2.4 Effect of extraction time
The establishment of extraction equilibrium takes some time. Ultrasound can accelerate the mass transfer between the two phases, shorten the extraction time and improve the extraction efficiency, so it is widely used to assist ILS DLLME. The effect of ultrasonic time of 1-5 min on the extraction results was investigated. Fig. 6 clearly shows that the optimal extraction time is 2 minutes. When the extraction time is more than 2 minutes, the recovery decreases slightly, mainly because some extraction solvents containing some target compounds enter the sample solution phase under ultrasonic conditions. Therefore, in the preprocessing process, the extraction time is set to 2 minutes.
<Fig.7>
3.2.5 Effect of ionic strength
The effect of ionic strength on extraction efficiency was studied by adding 0,2,4,6 and 8% sodium chloride to the prepared samples. The results showed that the extraction efficiency increased with the increase of salt dosage. When the salt concentration is 4%, the extraction efficiency is the best (see Fig. 7). The results showed that different salt concentrations had no significant effect on the recovery of antibiotics. Therefore, the ionic strength of the sample solution was set to 4% for further experiments.
3.3 Analytical evaluation
3.3.1 Linearity and limit of detection
The proposed method was verified, including the linearity, precision, repeatability, limit of detection (LOD), limit of quantification (LOQ) and recovery of the six target compounds in SMS under the optimized conditions. The values obtained through validation testare shown in Tables 2 and 3.
The linearity of the establishd method for estimated were observed using targets compounds with series concentration. According to peak area (Y) and concentration of spiked sample (X, ng kg-1) drawing standard curve the linear relation between peak area and concentration of the six target compounds. For each level, three replicate extractions were carried out. Good linearity of response (r≥0.9991) within the investigated concentration range was obtained for six targets in SMS.
LOD and LOQ were achieveed founded on signal-to noise ratios of 3 and 10, for six target compounds ranged from 0.16 to 0.74 ng kg-1 and 0.42 to 2.52 ng kg-1, respectively.
<Table 1>
In order to determine the accuracy, three different concentration levels (0.5, 2.0 and 5.0 ng kg-1) were used for recovery experiments under the optimal conditions for analytes, respectively. The recover values and relative standard deviations (RSD) are summarined in Table 3. The average recoveries obtained were in the range of 89.5% to 105.6% and RSDs were in the range of 1.5% to 4.6%, which indicates that the established method exhibited a high extraction efficiency for the six target compounds.
<Table.4>
3.3.2 Real samples analysis
The above method was used to determine the concentration of the six target analytes in SMS, and the results are listed in Table.4. The high-performance liquid chromatograms of six target analytes was shown Fig. 8. SCN, SDN, SBA, SBB, ISBA, ISBB were detected in all SMS, which indicates that these six compounds are commonly in SMS, the content of SBB is the highest, the average content of SBB reached 37.6% among the detected compounds.
<Table.7>
<Fig.8>