3.1. Characterization
The structure of MDES was confirmed based on FT-IR spectra of pure decanoic acid, TBABr and MDES and the results are presented in Fig. 1 (a). As shown in Fig. 1, the broad band at 3423cm-1 related to O–H vibrations of decanoic acid shifted to 3416 cm-1 in the FTIR spectrum of MDES. The shift of the stretching vibration of the O–H group in the MDES confirms the formation of MDES through hydrogen bonding. Peaks at 2926 cm-1 and 2859 cm-1 are assigned to the symmetric and asymmetric vibrational state of C–H bonds in methyl groups. In addition, the peak at 1727 cm-1 is attributed to the C=O stretching in the carboxylic acid and the band at the range of 573 cm-1 and 630 cm-1 corresponds to the vibration of the Fe-O bonds.
Also, the magnetic properties of the MDES were measured by the vibrating sample magnetometer and Fig. 2 confirmed the magnetic properties of the synthesized MDES with the saturation magnetization of 0.1 emu g-1.
3.2. Effect of solution pH
The solution pH affects the existing forms of the analyte. To evaluate the effect of solution pH on extraction efficiency, the solution pH was adjusted in the range of 3-11. It can be seen from Fig.3 that the highest extraction recovery was obtained at pH 7. Hence, the adjustment of solution pH was not performed.
3.3. Selection of extraction solvent
The selection of an appreciated extraction solvent is an important parameter to obtain a high extraction performance. The high extraction efficiency of MDES could be attributed to the hydrophobic and π-π stacking interactions between the analyte and extraction solvent. According to the above-mentioned decanoic acid was selected as the HBD because of its high hydrophobicity. TBABr as the HBA has alkyl chains and acts as one of the main components of the extraction solvent along with a high affinity for target compounds. Therefore, DES based on TBABr and decanoic acid at a molar ratio of 1:2 was investigated.
3.4. Effect of DES volume
DES volume is a primary factor in the performance of the method and is required to achieve a high enrichment factor. Therefore, the effect of the volume of DES was tested based on a series of sample solutions containing various volumes of DES (50-300 µL). According to the outcome of analyses (Fig.4), the highest enrichment factor was obtained by increasing the volume of DES up to 200 µL. The higher volumes led to a decrease in the enrichment factor of the drug due to the dilution effect. Therefore, 200 μL of DES was applied as extraction solvent volume.
3.5. Effect of amount of anhydrous iron (III) chloride
To have an effective magnetic DES, a proper amount of iron chloride was required. The effect of iron chloride amount on the extraction recovery of the drug was assessed from 10 to 50 mg. The results illustrated in Fig. 5, that increasing the amount of anhydrous iron (III) chloride enhanced the extraction recovery up to 40 mg and no significant change was observed over 40 mg. Hence, 40 mg of anhydrous iron (III) chloride was selected to form the magnetized DES in the following microextraction
3.6. Effect of extraction time
Sonication has been applied to improve the mass transfer rate of the analyte between the extraction solvent and the sample solution and also enhanced the formation of MDES. Thus, in the first step, the influence of the extraction time was subjected to in the range of 0-15 min. The results indicated that the extraction recovery was increased in the duration of 2 min with increasing the ultrasonic time due to the increase of the number of microdroplets of extraction solvent. After 2 min, the differences in extraction efficiency with more time are not observed. Hence, an extraction time of 2 min was selected for future experiments. Moreover, the effect of sonication on the formation of MDES was investigated in the range of 0-10 min. The results indicated that the extraction recovery had a significant change up to 3 min. Therefore, a time of 3 min was used to enhance the generation of MDES in the sample solution.
3.7. Effect of desorption condition
To have a high extraction process, the analyte should be recovered by an elution process from MDES. A proper eluent should effectively desorb analyte from the MDES with the lowest volume. For this purpose, acetone, ethanol, and acetonitrile were studied to elute cefixime. The obtained results showed that ethanol had the most efficient affinity for desorption of cefixime. Thus, ethanol was used as the elution solvent. Moreover, the effect of eluent volume was studied to obtain the highest enrichment factor. Under the same experimental conditions, the effect of eluent volume on the extraction recovery was considered from 10 to 1000 µL (Fig.6). The results depicted that 500 µL of ethanol desorbed cefixime quantitative from the MDES. Also, the elution time was varied in the range of 1–7 min by ultrasonication. According to the obtained results, a time of 1 min was chosen for further experiments.
3.8. Method Validation
The analytical characteristics of the proposed method were evinced in terms of the linear ranges (LRs), limit of detection (LOD), limit of quantification (LOQ), correlation coefficients (R2), enrichment factor(EF), extraction recoveries (ER), precision (RSD %) and arranged in Table 1. The linearity of the drug was obtained up to 5000 µg L–1 along with correlation coefficients (R2)>0.9950 in real samples. The repeatability (intra-day RSD %) was accounted for five analyses of spiked samples within one day, whereas the reproducibility (inter-day precision) was determined by spiking samples for five consecutive days. The RSD%s were in the range of 3.3–3.9 % for intra-day analysis and 4.5–5.2 % for inter-day analysis. LODs and LOQs based on S/N of 3 and 10 were 0.5 µg L–1 and 1.6 µg L–1 in water and biological samples, respectively. The EF, and ER of cefixime were 125, and 92.1- 93.8%, respectively.
3.9. Comparisons of the proposed method with other works
The comparison between some main aspects of the analytical performances of HMDES-UA-DLLME with other methods was investigated for cefixime (Table.2). The developed method had a shorter extraction time (6 min) without centrifugation time, which eliminated centrifugation equipment. Also, the method provided appropriate LODs and ERs values. The MDES offered the reduced process of synthesis at ambient temperature without needing special conditions while the synthesis routes of nano sorbents are costly and time-consuming processes. Therefore, the time and cost are retained.
3.9. Real sample analysis
The applicability of the method was checked to determine concentration of cefixime in wastewater, plasma and human urine samples (Table.3). To assess the matrix effects, certain concentrations of the analyte were added, and the relative recoveries (RR %) were investigated.
As can be shown in Table.3, the RR % were from 91.9 to 97.2%. The accuracy (E %) ranged from -8.1 to 5.3% was demonstrated that the analytical method can be used in a wide variety of matrices. Fig. 7 presented typical chromatograms obtained by HPLC-UV of the drug in the standard solutions (50-2500 µg L-1), the blank plasma sample and the extracted cefixime from the plasma sample spiked at 100 and 200 µg L-1 of the analyte. These chromatograms illustrated the quality of the extraction method for the analysis of cefixime due to the absence of interference peaks in the retention time of the peak of the analyte