@2024 Afarand., IRAN

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ISSN: 1027-1457    Scientific Journal of Forensic Medicine 2019;25(3):121-129

Background
Chlorpyrifos is widely used in agriculture as an insecticide and acaricide [1, 2]. … [3–6].
Previous Studies
Urinary specimens in acute poisoning, due to the ease and non-invasiveness of the sampling method, simplification of the matrix of the sample, and concentration of the analyte is considered as a suitable sample in clinical and forensic toxicology [7] ... [8-23]. Although using traditional liquid-liquid extraction methods is one of the most commonly used methods of sample preparation due to their ease and low cost, however, disadvantages, such as the need to use high volumes of organic solvents while being toxic and environmentally friendly, the possibility of emulsification during the extraction process, the needed time, the high volume of sample and low utilization have led to the use of liquid-liquid microextraction [24].
Aim(s)
The aim of this study was to design and optimize a suitable liquid-liquid microextraction method for the analysis of chlorpyrifos in urine using chemometrics for use in forensic and clinical toxicology laboratories.
Research type
This was an experimental study.
Research society, place and time
None declared.
Sampling method and number
None declared.
Used devices&materials
To prepare standard solutions of chlorpyrifos from stock solution made at 1000 μg/ml, concentrations of 0.5, 1, 1.5, 2, 2.5, 3 and 4 μg/ml were prepared in Blanc urine. Chlorpyrifos was added to 3 ml of urine sample in a test tube, at specified concentrations and 10 µl of pyrimifus ethyl (2.5 µg / ml) was added as internal standard. The sample was vortexed for 30 s after mixing. Then, 100 µL of NaCl (2.1 wt.%) and 100 µl of SLS (2.5 wt.%) were added to the sample and vortexed again. Using a micro-syringe (Hamilton), 355 ml of toluene and 780 ml of methanol were then injected into the sample. After forming an opaque solution, the sample was centrifuged for 10 min at 3000 rpm. The organic phase was appeared as the upper phase and separated using a micro-syringe and collected in a microtube (Eppendorf). Then, the sample was dried using nitrogen gas stream and after resolving the residue in 30 µL of acetonitrile, 20 µL of the solution was injected to the HPLC apparatus. The high performance liquid chromatography (HPLC) equipped with a pump (Model 1050) and PDA detector was used to identify and quantify chloropyrifos. The chromatography conditions were: C18 Reversed-Phase Analytical HPLC Columns (250 mm × 4.6mm, 5μm particle size, Perfectsil Target®), mobile phase consisting of acetonitrile-phosphate buffer (63 : 37, v/v; pH = 2.3), and mobile Phase flow rate of 1 ml / min at 25 ° C. … [28]. In this study, the Taguchi model L18 (2 ^ 2 * 3 ^ 6) was used to investigate the factors and evaluate the model obtained by Design-Expert 7.1.3 software. Also, two-way ANOVA was used to investigate the effective factors.
Findings by Text
Among the studied factors in the model, volume (p = 0.0142; F = 9.75) and extractive solvent type (p = 0.0362; F = 5.16), salt concentration (0.0066; F = 13.22), the type (p = 0.005; F = 30.87) and volume (p = 0.0308; F= 6.84) of the diffuser solvent and surfactant (p = 0.0078; F = 12.42) were found effective and the ultrasonication (p = 0.3360; F = 1.04) and pH (p = 0.775; F = 0.10) had no significant effect on extraction efficiency. Among the diffuser solvents, methanol and among the extractive solvents, toluene were identified appropriate. The results of DLLME optimizing using CCD method to increase the outcome of chloropyrifos extraction from the urine and its important factors and levels for CCD are presented in Table 1. A CCD was used to reduce the number of experiments. The total number of trials was 30, with 6 replications at the center (Table 2).The presented models for the responses were evaluated and considering the priority and omitting the important factors, including independent factors and interactions, F = 42.08 indicated the validity of the model (p <0.0001). According to the obtained indices for model evaluation, the selected model was appropriate (Table 3). To illustrate the interaction between the factors, 3D response surface plots were used (figure 1). Figure 2 shows the results of the effects of the factors influencing the increase in outcome. The results showed that the optimum level of 770 µl for diffuser solvent and 1.2 wt% for NaCl, 2.43 wt% for SLS, and 336 µl for toluene and following optimizing, the outcome reached 95.6%. The lower limit of detection (LOD) and the limit of quantitation (LoQ) were calculated 0.082 and 0.25 μg / ml, according to the Excel software's Linest calibration curve, respectively, and R2 coefficient was 0.9996, (Figure 3). The specificity of the method by adding drug and toxin standards indicates that there was no interaction with chloropyrifos peak in the chromatogram within its peak inhibition time (Fig. 4).
Main comparison to the similar studies
The results of the present study are consistent with the study by Hu et al., who showed that the use of SLS through the formation of supramolecules with bilayer micelles increased the rate of substitution of aqueous and organic phases in liquid phase of microextraction and also increased the extraction efficiency of berberine and epiberrin alkaloids from plant rhizome samples [29]. In this study, NaCl 2% (w/v) was identified as an effective factor in increasing the efficiency of the DLLME method under optimized conditions. This finding is consistent with the results of a previous study, in which NaCl 1.36% (w/v) was used for the condensation and extraction of nitrazepam and midazolam drugs from human serum samples under optimized conditions using DLLME-HPLC [30]. [31-35]
Suggestions
Future studies are suggested to consider urinary metabolites.
Limitations
No identification and measurement of the metabolites of this toxin in urine is one of the limitations of this study.
Conclusions
The optimized DLLME-HPLC-PDA method for the analysis of chlorpyrifos in urine samples can be a good alternative to other methods.
Acknowledgments
The authors are thankful to the Vice chancellor for education and research of the Iran Legal Medicine Organization to support this study and also the Director of Legal Medicine Organization of Kurdistan Province and its all respected staff for their cooperation.
Conflict of interest
None declared.
Ethical Permissions
All ethical standards were observed in accordance with the guidelines of the Ethics Committee of the Vice chancellor for education and research of the Legal Medicine Organization and also the Helsinki Declaration.
Funding Sources
This research was funded by the Vice chancellor for education and research of the Legal Medicine Organization.

TABLES and CHARTS

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