Sensitive determination of Fluoxetine and Citalopram antidepressants in urine and wastewater samples by liquid chromatography coupled with photodiode array detector.

A new analyte separation and preconcentration method for the trace determination of antidepressant drugs, Fluoxetine (FLU) and Citalopram (CIT) in urine and wastewaters, was developed based on HPLC-DAD analysis after magnetic solid phase extraction (MSPE). In the proposed method, FLU and CIT were retained on the newly synthetized magnetic sorbent (Fe3O4@PPy-GO) in the presence of buffer (pH 10.0) and then were desorbed into a lower volume of acetonitrile prior to the chromatographic determinations. Before HPLC analysis, all samples were filtered through a 0.45 µm PTFE filter. Experimental parameters such as interaction time, desorption solvent and volume, and pH were studied and optimized in order to establish the detection limit, linearity, enrichment factor and other analytical figures of merit under optimum operation conditions. In the developed method, FLU and CIT were analyzed by diode array detector at the corresponding maximum wavelengths of 227 and 238 nm, respectively, by using an isocratic elution of 60% pH 3.0 buffer, 30% acetonitrile, and 10% methanol. By using the optimum conditions, limit of detections for FLU and CIT were 1.58 and 1.43 ng mL-1, respectively, while the limit of quantifications was 4.82 and 4.71 ng mL-1, respectively. Relative standard deviations (RSD%) for triplicate analyses of model solutions containing 100 ng mL-1 target molecules were found to be less than 5.0 %. Finally, the method was successfully applied to urine (both simulated and real healthy human) and wastewater samples, and quantitative results were obtained in recovery experiments.

Selective electrochemical detection of antidepressant drug imipramine in blood serum and urine samples using an antimony telluride-graphite nanofiber electrode.

A high-performance imipramine (IMPR) sensor has been developed  based on metal chalcogenide-carbon composite materials. The antimony telluride-graphite nanofiber (Sb2Te3-GNF, hereafter SBT-GNF) composite was synthesized by the hydrothermal method and confirmed by X-ray powder diffraction (XRD) pattern. The morphology, crystalline lattice, and chemical states were characterized by HRTEM, SAED, and XPS analysis. The characterizations confirmed the formation of an effective composite, SBT-GNF. The SBT-GNF was fabricated as a disposable sensor electrode with a screen-printed carbon electrode (SPCE) and examined for the detection of IMPR by differential pulse voltammetry (DPV). The electroanalytical results of SBT-GNF are compared with the SBT and GNF, and the rational design of effective composite is discussed. SBT-GNF/SPCE showed a good linear range (0.01­51.8 µM), sensitivity (1.35 ± 0.1 µA µM-1 cm-2), and low LOD (4 ± 2 nM). Moreover, the SBT-GNF/SPCE revealed high selectivity and high tolerance limit against potential interfering compounds in blood serum and urine samples. Therefore, this electrochemical sensor can be applicable for the detection of tricyclic antidepressant drug IMPR in clinical and pharmaceutical analysis.

LC-MS-sMRM method development and validation of different classes of pain panel drugs and analysis of clinical urine samples.

Urine drug testing (UDT) is an important analytical/bio-analytical technique that has inevitably become an integral and vital part of a testing programme for diagnostic purposes. This manuscript presents a tailor-made LC-MS/MS quantitative assay method development and validation for a custom group of 33 pain panel drugs and their metabolites belonging to different classes (opiates, opioids, benzodiazepines, illicit, amphetamines, etc.) that are prescribed in pain management and depressant therapies. The LC-MS/MS method incorporates two experiments to enhance the sensitivity of the assay and has a run time of about 7 minutes with no prior purification of the samples required and a flow rate of 0.7 mL/min. The method also includes the second-stage metabolites for some drugs that belong to different classes but have first-stage similar metabolic pathways that will enable to correctly identify the right drug or to flag the drug that might be due to specimen tampering. Some real case examples and difficulties in peak picking were provided with some of the analytes in subject samples. Finally, the method was deliberated with some randomly selected de-identified clinical subject samples, and the data evaluated from "direct dilute and shoot analysis" and after "glucuronide hydrolysis" were compared. This method is now used to run routinely more than 100 clinical subject samples on a daily basis.

Urothelial toxicity of esketamine in the treatment of depression.

RATIONALE: Ketamine is the first widely used substance with rapid-onset antidepressant action. However, there are uncertainties regarding its potential urothelial toxicity, particularly after repeated application. In the context of rising recreational ketamine use, severe side effects affecting the human urinary tract have been reported. It is assumed that ketamine interacts with bladder urothelial cells and induces apoptosis. OBJECTIVES: This study aimed to assess whether single or repeated doses of esketamine used in an antidepressant indication are associated with urinary toxicity. METHODS: We included male and female inpatients with a current episode of depression and a diagnosis of recurrent depressive disorder, bipolar disorder or schizoaffective disorder according to ICD-10 criteria (n = 25). The esketamine treatment schedule involved a maximum of 3× weekly dosing at 0.25-0.5 mg/kg i.v. or s.c. The primary outcome was the change in urine toxicity markers (leukocytes, erythrocytes, protein and free haemoglobin). Description of demographic, clinical and laboratory data was conducted using means, standard deviations, frequencies and percentages. Changes in urinary toxicity markers over time were evaluated using linear mixed models with gender as a covariate. RESULTS: The participants received an average of 11.4 (SD 8) esketamine treatments, and an average number of 11.2 (SD 8) urine samples were analysed over the course of treatment. Neither urinary leukocyte concentration (F(20; 3.0) = 3.1; p = 0.2) nor erythrocyte concentration (F(20;2.2) = 4.1; p = 0.2) showed a significant trend towards increase during the course of esketamine treatment. Similarly, free haemoglobin and protein concentrations, which were analysed descriptively, did not display a rise during treatment. There was a significant improvement in depression ratings after esketamine treatment (p < 0.001). CONCLUSIONS: This study is, to the best of our knowledge, the first to focus on urothelial toxicity of esketamine used in antidepressant indication and dose. The results indicate that the use of single or repeated doses of esketamine is unlikely to cause urothelial toxicity. The results are in need of confirmation as sample size was small.

Simultaneous determination of some antidepressant drugs and vitamin B12 in pharmaceutical products and urine sample using HPLC method.

Three sensitive, accurate and precise HPLC methods were devolved for the simultaneous determination of vilazodone HCl (VILHC), agomelatine (AGO) or duloxetine HCl (DULHC) and vitamin B12 (cyanocoblamine B12) in bulk, pharmaceutical dosage form and in urine samples. Both similar methods (I and II) were carried out using 0.04 M phosphate buffer (pH 7.0), acetonitrile and methanol in the ratio (30:30:40, v/v) as a mobile phase. Thermo BDS hypersil-C18 column, with 5 µm particle size and 250 × 4.5 mm dimensions, at flow rate 1.0 mL min-1 and UV detection at 277 nm at ambient temperature 25 °C were used. The retention times were 5.12 and 2.54 min for VILHC and vitamin B12, 4.98 and 2.53 min for AGO and vitamin B12, respectively, with linearity range from 0.001 to 200 µg mL-1. However, for the separation of DULHC and B12, UV detection at 230 nm and Agilent Eclipse XDA-C8 (150 × 4.5 mm, 5 µm) column, were used (method III). The retention time of DULHC and vitamin B12 was found to be 4.53 and 1.35 min, respectively, with linearity range from 0.0005 to 200 µg mL-1. The proposed methods were validated as per the ICH guideline with very low LOD and LOQ. The values of %RSD for precision was less than 2% and the value of % recovery were found to be 99.20-100.9% and 99.23-100.67% for determination of the drugs in pure and pharmaceutical formulations, respectively, for all methods confirming that the methods are precise, accurate and selective for separation and determination of VILHC, AGO or DULHC from B12 in tablets and in urine samples without any interference from each other or from common excipients.

Polyoxomolybdate368 /polyaniline nanocomposite as a novel fiber for solid-phase microextraction of antidepressant drugs in biological samples.

A novel solid-phase microextraction fiber was synthesized by coating a stainless steel wire with polyoxomolybdate368 /polyaniline as a sorbent aimed at extraction of amitriptyline, nortriptyline, and doxepin as antidepressant drugs from urine and blood samples. The polyoxomolybdate368 /polyaniline composite coating was applied using electropolymerization process under constant potential. This composition leads to enhanced extraction efficiency of the fiber. Scanning electron microscopy images show that huge three-dimensional structures of polyoxomolybdate368 in composite induced more non-smooth and porous fiber. In order to optimize of the extraction process, a series of variables including concentration of the composite materials, coating thickness, pH, extraction time, salt addition, and stirring rate was investigated and optimum conditions were determined. Analysis of surface morphology and chemical composition was performed. High-performance liquid chromatography was used for separation and evaluation of mentioned antidepressant drugs from the matrixes. The experiments indicated a detection limits of <0.2 ng/L and a linear dynamic range of 0.3-100 ng/L (R2  > 0.994). The relative recovery values were found to be in the range of 92-98%. It was concluded that the purposed fiber is highly efficient in analyzing traces of antidepressant drugs in urine and blood.

A green and low-cost method employing switchable hydrophilicity solvent for the simultaneous determination of antidepressants in human urine by gas chromatography - mass spectrometry detection.

In this study, the use of switchable hydrophilicity solvent with a simple and low-cost lab-made device for the extraction procedure in homogeneous liquid-liquid microextraction is proposed for the first time in the determination of antidepressants in human urine. The antidepressants studied consisted of fluoxetine, amitriptyline, nortriptyline, imipramine, desipramine and sertraline. The optimization of the main parameters that can influence on the extraction efficiency was performed through multivariate approaches. The analytes were separated and identified by gas chromatography coupled to mass spectrometry (GC-MS). The optimal extraction conditions consisted of using N,N-dimethylcyclohexylamine (DMCHA) as the switchable hydrophilicity solvent (SHS), 500 µL of urine sample previously diluted with ultrapure water at 1:1 ratio (v/v), 200 µL of a mixture of SHS:HCl 6 mol L-1 (1:1 v/v), 600 µL of NaOH 10 mol L-1 and 3 min of extraction time. A volume of 40 µL of diphenylamine at concentration of 500 µg L-1 (20 ng) was used as internal standard. The method developed was in-house validated, providing coefficients of determination higher than 0.995 for all analytes, limits of detection (LOD) from 0.02 to 0.88 µg L-1, limits of quantification (LOQ) from 0.05 to 2.92 µg L-1, relative recoveries of 68 to 102%, intra-day precision from 0.5 to 15.9%, inter-day precision from 4.2 to 19.3%, selectivity and robustness. The method proposed was successfully applied in five human urine samples from a Toxicological Information Center located in Porto Alegre (Brazil). The results demonstrated that the µP-SHS-HLLME approach is highly cost-effective, rapid, simple and environmentally-friendly with satisfactory analytical performance.

Enhanced pseudotargeted analysis using a segment data dependent acquisition strategy by liquid chromatography-tandem mass spectrometry for a metabolomics study of liquiritin in the treatment of depression.

An enhanced pseudotargeted method using a segment data-dependent acquisition mode based on ultra-high performance liquid chromatography-tandem mass spectrometry was developed. This segment data dependent acquisition-based pseudotargeted method could improve the detection of co-eluted ions and extend the coverage of analytes. A set of 502 multiple reaction monitoring channels were obtained by this segment strategy, which was twice the number created by the traditional data-dependent acquisition mode. Compared with the untargeted method, the pseudotargeted profiling demonstrated higher sensitivity and higher precision. More than 90% of the metabolites detected by the enhanced pseudotargeted method had relative standard deviations less than 15%. The segment data dependent acquisition-based pseudotargeted method was successfully applied to the metabolomics study of the depressed rats with the treatment of liquiritin. Forty-seven differential metabolites were screened and five metabolic pathways were found to be related to depression including retinol metabolism, phenylalanine, tyrosine, and tryptophan biosynthesis, phenylalanine metabolism, terpenoid backbone biosynthesis, and lysine degradation. The segment data dependent acquisition-based pseudotargeted method widened the coverage of metabolites with good sensitivity and precision, which exhibited great potential in the discovery of differential metabolites in metabolomics studies.

Multidimensional Liquid Chromatography Employing a Graphene Oxide Capillary Column as the First Dimension: Determination of Antidepressant and Antiepileptic Drugs in Urine.

Human mental disorders can be currently classified as one of the most relevant health topics. Including in this are depression and anxiety, which can affect us at any stage of life, causing economic and social problems. The treatments involve cognitive psychotherapy, and mainly the oral intake of pharmaceutical antidepressants. Therefore, the development of analytical methods for monitoring the levels of these drugs in biological fluids is critical. Considering the current demand for sensitive and automated analytical methods, the coupling between liquid chromatography and mass spectrometry, combined with suitable sample preparation, becomes a useful way to improve the analytical results even more. Herein we present an automated multidimensional method based on high-performance liquid chromatography-tandem mass spectrometry using a lab-made, graphene-based capillary extraction column connected to a C8 analytical column to determined five pharmaceutical drugs in urine. A method enhancement was performed by considering the chromatographic separation and the variables of the loading phase, loading time, loading flow, and injection volume. Under optimized conditions, the study reports good linearity with R2 > 0.98, and limits of detection in the range of 0.5-20 µg L-1. Afterward, the method was applied to the direct analysis of ten untreated urine samples, reporting traces of citalopram in one of them. The results suggest that the proposed approach could be a promising alternative that provides direct and fully automated analysis of pharmaceutical drugs in complex biological matrices.

A novel insight into the potential toxicity mechanisms of Zhi-Zi-Hou-Po decoction by dynamic urinary metabolomics based on UHPLC-Q-Exactive Orbitrap-MS.

In recent years, depression occurs frequently. Given the long duration of the disease and the high risk of recurrence, the treatment of depression requires long-term medication. Zhi-Zi-Hou-Po Decoction (ZZHPD) has been used in clinical treatment of depression and related diseases for many years, and the potential toxic damage caused by its long-term use has gradually emerged. Existing research methods that expose toxicity by a one-time administration of large doses cannot provide a reference for clinical safe drug use. In this study, the potential toxicity of ZZHPD in repeated administration was studied by urinary metabolomics with nondestructive sampling. Based on ultra-high performance liquid chromatography-quadruple-Exactive Orbitrap mass spectrometry (UHPLC-Q-Exactive Orbitrap-MS) and chemometrics, 33 differential biomarkers, such as 3-hydroxybutyric acid, indole sulfuric acid, hippuric acid and citric acid, were screened and dynamically tracked. The changes of some endogenous substances showed obvious time dependence. Further analysis of these time-dependent components in combination with network pharmacology revealed that the potential hepatotoxicity and nephrotoxicity of ZZHPD were related to the disorders of amino acid metabolism, energy metabolism, lipid metabolism, nucleotide metabolism and gut microflora metabolism pathway. This study can better grasp the occurrence and development of drug toxicity, and provide reference for rational and safe drug use and potential toxicity prevention of ZZHPD.

Application of LC-ESI-MS/MS Method for Analysis of Escitalopram Oxalate in Human Urine and Pharmaceutical Dosage Forms.

An effective and sensitive liquid chromatographic-electrospray ionization tandem mass-spectrometric (LC-ESI-MS/MS) method was developed and validated for quantification of escitalopram oxalate (ESC-OX), antidepressant drug in spiked human urine and pharmaceutical formulations. In this work, simple liquid-liquid extraction was optimized and used for extraction of cited drug from urine samples. The chromatographic separation was attained within 6 min including re-equilibration time by using gradient elution with 0.1% formic acid in acetonitrile and 0.1% formic acid in water as mobile phase, Zorbax Eclipse RP C18 (50 × 2.1 mm) column was used with a particle size of 1.8 µm; the flow-rate was 0.35 mL min-1. Ion signal m/z 262.0 and 109.0 for ESC-OX product ions were monitored at positive ESI mode. Validation of the method was carried out according to the ICH Q2 (R1) guidelines and EMEA criteria. The method was linear over 79-196,450 pg mL-1 with a regression of 0.9999 and 0.9993 for both standard and urine samples. The LOD was 3.88 and 10.66 pg mL-1 for standard and urine samples, respectively, while lower limit of quantification was 79 pg mL-1.

Analysis of four antidepressants in plasma and urine by gas chromatography-mass spectrometry combined with sensitive and selective derivatization.

A sensitive and selective method was developed for simultaneous determination of four antidepressants (ATDs) in plasma and urine samples by gas chromatography- mass spectrometry (GC-MS) based on an N-nitrosation reaction. In this study, fluoxetine (Flu), nortriptyline (Nor), maprotiline (Map), and paroxetine (Paro) were first derivatized with sodium nitrite to appropriate N-nitrosamines under acidic condition, then the derivatives were easier to detect by GC-MS. The derivatization conditions including the amount of hydrochloric acid, the amount of sodium nitrite, reaction temperature, reaction time and the extraction reagents were optimized. Under the optimal conditions, the limit of detections (LODs) and limit of quantitations (LOQs) were in the range of 0.04-1.38 µg L-1 and 0.14-4.62 µg L-1, respectively. Low, medium, and high concentrations of antidepressants were added in plasma and urine samples, spiked recovery ranged from 85.88%-110.34% for plasma and 80.64%-113.07% for urine, respectively. The derivatization reaction was very quickly, only 5 min was needed for the reaction process, in addition, the proposed method exhibited superior sensitivity and selectivity, it showed sufficient advantages for determination of Flu, Nor, Map, and Paro in plasma and urine of patients.

A fast and simple approach for the quantification of 40 illicit drugs, medicines, and pesticides in blood and urine samples by UHPLC-MS/MS.

A fast and simple approach to overcome challenges in emergency toxicological analysis, using ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) has been developed, for the detection of analytes in blood and urine samples from the following drug classes: analgesics, benzodiazepines, antidepressants, anticonvulsants, drugs of abuse, and pesticides. These substances are relevant in the context of emergency toxicology in Brazil. The sample preparation procedure was relatively easy and fast to perform. The method was fully validated giving limits of in the range of 0.5 and 20 ng mL-1 for blood and urine samples. The intraday and interday precision and accuracy were considered adequate for all analytes once the relative standard deviation (RSD) (%) was lower than 20% for quality control (QC) low and lower than 15% for CQ medium and high. The developed method was successfully applied to 320 real samples collected at the Poison Control Center of São Paulo, and 89.1% have shown to be positive for some of the analytes. This confirms its applicability and importance to emergency toxicological analysis, and it could be very useful in both fields of clinical and forensic toxicology.

Fabric phase sorptive extraction for the isolation of five common antidepressants from human urine prior to HPLC-DAD analysis.

An innovative fabric phase sorptive extraction-high performance liquid chromatography-diode array detection (FPSE-HPLC-DAD) method is developed and validated herein for the simultaneous extraction and analysis of five common antidepressants (venlafaxine, paroxetine, fluoxetine, amitriptyline, clomipramine) in human urine samples. Separation was obtained using a reverse phase column LiChroCART-LiChrospher®100 RP-8e (5 µm, 250 × 4 mm) with diode array detection monitoring at 235 nm. Gradient elution was applied at a flow rate of 1.2 mL/min with a mobile phase that consisted of acetonitrile and ammonium acetate (0.05 M). Fabric Phase Sorptive Extraction was evaluated for the first time with regards to the extraction of the examined antidepressants from human urine and proved to be a rapid, simple, environmentally friendly, efficient, and selective sample preparation approach. Sol-gel graphene sorbent, coated on cellulose FPSE media, were the most efficient among other with different polarities. The absolute recovery values were 25.5% for venlafaxine, 33.9% for paroxetine, 67.0% for fluoxetine, 43.0% for amitriptyline and 29.0% for clomipramine, while relative recoveries were higher than 90%. The developed method provides satisfactory limit of detection 0.15 ng/µL.

Microextraction by packed sorbent followed by ultra high performance liquid chromatography for the fast extraction and determination of six antidepressants in urine.

The growing use of antidepressants in recent years has led to their increasing presence in forensic analyses. In this work, microextraction by packed sorbent followed by ultra-performance liquid chromatography with photodiode array detection provided a fast method for determining the antidepressants mirtazapine, venlafaxine, escitalopram, fluoxetine, fluvoxamine, and sertraline in human urine. The microextraction conditions (viz., type of sorbent, number of draw-eject extraction cycles or strokes, sample volume and pH, and type and volume of washing solution and eluent) were optimized by using an experimental design. The ensuing analytical method was validated in terms of linearity (25-1000 ng/mL urine), limit of detection (lower than 7.1 ng/mL), limit of quantification (25 ng/mL), precision (4.7-15.1% as relative standard deviation), and accuracy (80.4-126.1% as mean recovery for four replicate determinations). The proposed method allowed the six target antidepressants to be determined at concentrations from therapeutic to toxic levels. The application to small volumes (300 µL) of urine afforded fast extraction of the analytes and provided results on a par with those of existing clinical and forensic alternatives.

Extraction of antidepressant drugs in biological samples using alkanol-based nano structured supramolecular solvent microextraction followed by gas chromatography with mass spectrometric analysis.

In the present study, a supramolecular solvent was formed from reverse micelle aggregates of octanol. The proposed supramolecular solvent was used for rapid extraction of some antidepressants drugs including amitriptyline, imipramine, desipramine, maprotiline, sertraline, and doxepin from biological samples. Alkanol-based supramolecular solvents have a unique array of physicochemical properties, making them a very attractive alternative to replace organic solvents in analytical extractions. The parameters affecting the extraction of target analytes (i.e., the volume of tetrahydrofuran and octanol as the major components comprising the supramolecular solvent, chain length of alkanols, sample solution pH, salt addition, and ultrasonic time) were investigated and optimized by factor by factor optimization method. Under the optimum conditions, preconcentration factors of 470, 490, 460, 385, 370, and 430 were obtained for amitriptyline, doxepin, imipramine, desipramine, maprotiline, and sertraline, respectively. The linear ranges and coefficients of determination (R2 ) were obtained in the range of 0.01-100 µg/L and 0.9974-0.9991, respectively. Also the limits of detection (S/N = 3) of 0.003-0.03 µg/L, and precisions (n = 5) of 4.9-8.9% were calculated. Finally, the method was successfully applied for the extraction of antidepressant drugs in biological samples, and relative recoveries in the range of 91-102% were obtained.

Zebrafish (Danio rerio) water tank model for the investigation of drug metabolism: Progress, outlook, and challenges.

Zebrafish (Danio rerio) water tank (ZWT) approach was investigated as an alternative model for metabolism studies based on six different experiments with four model compounds. Sibutramine was applied for the multivariate optimization of ZWT conditions, also for the comparison of the metabolism among ZWT, humans and mice, beyond for the role of CYP2B6 in ZWT. After the optimization, 18 fish and 168 hours of experiments is the minimum requirement for a relevant panel of biotransformation products. A comparison among the species resulted in the observation of the same hydroxylated metabolites, with differences in metabolites concentration ratio. However, the ZWT allowed tuning of the conditions to obtain a specific metabolic profile, depending on the need. In addition, by utilizing CYP2B6 inhibition, a relevant ZWT pathway for the demethylation of drugs was determined. The stereospecificity of the ZWT metabolism was investigated using selegiline and no racemization or inversion transformations were observed. Moreover, the investigation of metabolism of cannabimimetics was performed using JWH-073 and the metabolites observed are the same described for humans, except for the hydroxylation at the indol group, which was explained by the absence of CYP2C9 orthologs in zebrafish. Finally, hexarelin was used as a model to evaluate studies by ZWT for drugs with low stability. As a result, hexarelin displays a very fast metabolization in ZWT conditions and all the metabolites described for human were observed in ZWT. Therefore, the appropriate conditions, merits, and relevant limitations to conduct ZWT experiments for the investigation of drug metabolism are described.

Combination of electromembrane extraction and electro-assisted liquid-liquid microextraction: A tandem sample preparation method.

As a well-known extraction procedure, electromembrane extraction (EME) was combined with electro-assisted liquid-liquid microextraction (EA-LLME) in the present work, which resulted in a promising method. This hyphenated sample preparation method, named EME-EA-LLME, was followed by GC for the determination of two model analytes (clomipramine and imipramine). The effective parameters of both EME and EA-LLME (such as organic solvent, pH of acceptor and sample solutions, voltage and extraction time) were optimized. The proposed EME-EA-LLME procedure demonstrated good linearity with coefficients of determination, R2 ≥ 0.998 over the concentration range of 0.5-750 ng/mL. Limit of detection for both analytes was 0.15 ng/mL. The corresponding repeatability ranged from 6.9 to 12.2% (n = 3). The high enrichment factors were obtained as 770.3 and 561.4 for imipramine and clomipramine, respectively. The advantages of this tandem sample preparation method were low detection limits, simplicity, low cost, and short analysis time (<10 min). Finally, the optimized method was used to extract and determine the analytes in urine and wastewater samples. Overall, the results revealed that the developed EME-EA-LLME procedure had better extraction efficiency in comparison with EME and EA-LLME alone.