Application of magnetic dispersive solid phase extraction combined with solidification of floating organic droplet-based dispersive liquid-liquid microextraction and GC-MS in the extraction and determination of polycyclic aromatic hydrocarbons in honey.

A magnetic dispersive solid phase extraction method combined with solidification of floating organic droplet-based dispersive liquid-liquid microextraction has been validated for the extraction of polycyclic aromatic hydrocarbons from honey samples. For this purpose, a carbonised cellulose-ferromagnetic nanocomposite was used as a sorbent through the magnetic dispersive solid phase extraction. For preparation of the sorbent, first, carbonised cellulose nanoparticles were created by treating cellulose filter paper with concentrated solution of sulfuric acid. Then, the prepared nanoparticles were loaded onto Fe3O4 nanoparticles through coprecipitation. In the extraction process, first, a few mg of the sorbent was added to the diluted honey solution and dispersed in it using vortex agitation. The particles were then separated and the adsorbed analytes were eluted with an organic solvent. The eluent was taken and after mixing with a water-immiscible extraction solvent was used in the following solidification of floating organic droplet-based dispersive liquid-liquid microextraction procedure. By performing the extraction process under the obtained optimum conditions, low limits of detection (0.08-0.17 ng g-1) and quantification (0.27-0.57 ng g-1), satisfactory precision (relative standard deviations ≤ 5.0%), and wide linear range (0.57-500 ng g-1) with great coefficients of determination (r2≥ 0.9986) were obtained.

In vitro anticancer activity of Scrophularia amplexicaulis extracts on MCF-7 and WEHI-164 cell line.

Scrophularia amplexicaulis is an Iranian endemic plant belonging to the Scrophulariaceae family, which is used in traditional medicine to treat many diseases. The aim of this study was to evaluate the in vitro anticancer activity of S. amplexicaulis extracts against human breast carcinoma (MCF-7) and mouse fibrosarcoma (WEHI-164) cell lines. The ground aerial parts of S. amplexicaulis were soxhlet-extracted with n-hexane, dichloromethane and methanol. MTT assay exhibited that dichloromethane and methanol extracts remarkbly inhibited the growth of MCF-7 and WEHI-164 cancer cells in a dose-and time-dependent manner with little cytotoxicity on normal cell line HUVEC. Cell death ELISA, TUNEL assay, and the cleavage of poly ADP-ribose polymerase (PARP) uncovered that the cytotoxic effects of dichloromethane and methanol extracts were attributed to apoptosis in cancerous cells. Furthermore, quantitative real-time PCR revealed significant increases in the mRNA expression levels of p-53, caspase-3, caspase-9, Bax, and also a decrease in Bcl-2 expression. These results suggested that the extracts mainly induced apoptosis via a mitochondria-mediated intrinsic pathway. Notably, dichloromethane extract had higher cytotoxic and apoptotic activities than that of methanol extract, against both cancer cell lines, particularly MCF-7 cells. Our results indicate that S. amplexicaulis may serve as a promising source of potent agents for the treatment of human cancers.

Quantitative analysis of phenobarbital in biological fluids: Analyte enrichment by an electrically-assisted microextraction technique

An electrically-assisted microextraction method called electromembrane extraction, followed by a simple high performance liquid chromatography and ultraviolet detection was developed and validated for determining phenobarbital in biological samples. The major parameters influencing the electromembrane extraction procedure including solvent composition, voltage, pH of acceptor and donor solutions, salt effect, and time of extraction were evaluated and optimized. The drug was extracted from the donor aqueous sample solution (pH 9) to the acceptor aqueous solution (pH 13). The donor and acceptor phases were separated by a hollow fiber dipped in 1-octanol as a supported liquid membrane. A voltage of 40 V during 20 minutes was applied as the driving force. The enrichment factor was obtained >51 which enhanced the sensitivity of the instrument. Limit of detection and limit of quantitation were 7.5 and 25 ng/mL, respectively. The method was linear over the range of 25-1000 ng/mL for phenobarbital (R2 >0.9998) with repeatability (%RSD) between 0.4% and 6.8% (n = 3). The proposed method was successfully applied to human plasma and urine samples with relative recovery of 70-80% and %RSD < 6.8%.

Determination of tricyclic antidepressants in human urine samples by the three-step sample pretreatment followed by HPLC-UV analysis: an efficient analytical method for further pharmacokinetic and forensic studies.

In this work, an efficient sample pretreatment method has been developed by combining salt induced-homogenous liquid-liquid extraction, dispersive solid phase extraction, and dispersive liquid-liquid microextraction based on the solidification of floating organic droplet for the extraction of some widely used tricyclic antidepressant (TCA) drugs (nortriptyline, amitriptyline, desipramine, clomipramine, and imipramine) in human urine samples before their determination by high performance liquid chromatography-ultraviolet detection. In brief, the target analytes are first isolated from urine samples into acetonitrile (ACN) separated by adding a salt. Then the obtained ACN phase is treated with a mixture of appropriate sorbents to remove interferences. Afterward, the purified ACN is mixed with menthol as an extractant and rapidly injected into alkaline HPLC-grade water as a preconcentration step. Next, the obtained solution is placed in an ice bath and menthol collects on top of the solution after solidification. The solidified drop is then withdrawn and injected into separation system after dissolving in 10 µL ACN. Under the optimum experimental conditions, extraction recoveries and enrichment factors of the selected drugs ranged from 69-84 % and 345-420, respectively. The limits of detection and quantification were obtained at the ranges of 0.22-0.31, and 0.71-1.1 µg L-1, respectively. The relative standard deviations of the proposed method were ≤ 6 % for intra- (n=6) and inter-day (n=4) precisions at a concentration of 10 µg L-1 (each drug). Finally, the suggested approach was applied to determine of TCA drugs in different patients' urine samples. The method could be applied in further TCAs pharmacokinetic and forensic studies.

Combination of dispersive solid phase extraction and deep eutectic solvent-based air-assisted liquid-liquid microextraction followed by gas chromatography-mass spectrometry as an efficient analytical method for the quantification of some tricyclic antidepressant drugs in biological fluids.

A dispersive solid phase extraction coupled with deep eutectic solvent-based air-assisted liquid-liquid microextraction has been developed and applied to the extraction and preconcentration of some tricyclic antidepressant drugs in the human urine and plasma samples prior to their determination by gas chromatography-mass spectrometry. In this method, a sorbent (C18) is first added into an alkaline aqueous sample and dispersed by vortexing. By this action, the analytes are adsorbed onto the sorbent. Then, the sorbent particles are isolated from the aqueous solution by centrifugation. Afterward, a deep eutectic solvent, prepared from choline chloride and 4-chlorophenol is used to desorb the analytes from the sorbent. Subsequently, the supernatant solution is removed and added into an alkaline deionized water placed into a test tube with a conical bottom. The resulting mixture is rapidly sucked into a glass syringe and then injected into the tube. This procedure is repeated for several times and a cloudy solution consisting of fine droplets of deep eutectic solvent dispersed into the aqueous phase is formed. After centrifuging the obtained cloudy solution, the tiny droplets of the extractant, containing the extracted analytes, settle at the bottom of the tube. Finally, an aliquot of the extractant is taken and injected into the separation system for quantitative analysis. Several significant factors affecting the performance of the proposed method are evaluated and optimized. Under optimum extraction conditions, the method shows low limits of detection in the ranges of 5-10, 8-15 and 32-60 ng L-1 in deionized water, urine, and plasma, respectively. Enrichment factors are observed to be between 325 to 385 in deionized water, 155 to 185 in urine, and 64 to 72 in plasma. Extraction recoveries are in the range of 65-77 (in deionized water), 62-74 (in urine), and 64-72% (in plasma). The relative standard deviations of the proposed method are ≤ 6% for intra- (n = 6) and inter-day (n = 4) precisions at a concentration of 200 ng L-1 of each analyte. Finally, the applicability of the introduced method is investigated by analyzing the selected drugs in different biological fluids. In the proposed method, for the first time, a deep eutectic solvent composed of safe, cheap, and biodegradable compounds was synthesized and used (at µL-level) as an elution and extraction solvent, simultaneously which led to omit the consumption of toxic organic solvents. This represents a significant advantage in the era of green chemistry. In addition, the introduced method is sensitive, simple in operation, rapid, and efficient.

Quantitation of zolpidem in biological fluids by electro-driven microextraction combined with HPLC-UV analysis.

In this study, for the first time, an electro-driven microextraction method named electromembrane extraction combined with a simple high performance liquid chromatography and ultraviolet detection was developed and validated for the quantitation of zolpidem in biological samples. Parameters influencing electromembrane extraction were evaluated and optimized. The membrane consisted of 2-ethylhexanol immobilized in the pores of a hollow fiber. As a driving force, a 150 V electric field was applied to facilitate the analyte migration from the sample matrix to an acceptor solution through a supported liquid membrane. The pHs of donor and acceptor solutions were optimized to 6.0 and 2.0, respectively. The enrichment factor was obtained >75 within 15 minutes. The effect of carbon nanotubes (as solid nano-sorbents) on the membrane performance and EME efficiency was evaluated. The method was linear over the range of 10-1000 ng/mL for zolpidem (R2 >0.9991) with repeatability ( %RSD) between 0.3 % and 7.3 % (n = 3). The limits of detection and quantitation were 3 and 10 ng/mL, respectively. The sensitivity of HPLC-UV for the determination of zolpidem was enhanced by electromembrane extraction. Finally, the method was employed for the quantitation of zolpidem in biological samples with relative recoveries in the range of 60-79 %.

A Survey on Phytochemical Composition and Biological Activity of Zygophyllum fabago from Iran.

Purpose: Zygophyllum fabago L. (Z. fabago) is a widespread perennial herb which is used as a medicinal plant in traditional medicine of Iran, Turkey and China. The present study was a survey on phytochemical constituents and biological activities of this plant. Methods: Methanolic extract of the roots was fractionated over a C-18 pre-packed cartridge (Sep-pak) and chromatographic separation was performed on a reversed-phase preparative HPLC. Structural elucidation of the isolated compounds was carried out using UV, 1H-NMR and 13C-NMR spectral analyses. Furthermore, the chemical compositions of the essential oil of the aerial parts were identified by GC-MS analysis. Antiproliferative and antioxidant activities of all extracts from aerials were determined by MTT and DPPH assays, respectively. Results: Phytochemical investigation on the plant roots led to the isolation and identification of two the 60% methanol-water Sep-pak fraction, a prenylated flavone glycoside, 6-C-prenyl-7-O-[ ß -D-4'''-O-acetyl-glucopyranosyl-(1'''→2'')-ß-D-glucopyranosyl] apigenin, which was named as a Zygocaperoside and also, other flavonoid, was named as the Isorhamnetin -3-O glucoside. None of the extracts showed antiproliferative effect against cancerous cells. However, among the extracts, methanolic extract indicated antioxidant activity. Moreover, essential oils of flowers and leaves of plant have high amounts of sesquiterpene hydrocarbons and diterpenoides. Conclusion: The results of present study introduce Z. fabago roots as a new source of flavonoid glycosides and suggest it as an appropriate candidate for further pharmacological studies.

Electromembrane extraction of tartrazine from food samples: Effects of nano-sorbents on membrane performance.

In the present study, for the first time electromembrane extraction followed by high-performance liquid chromatography coupled with ultraviolet detection was developed and validated for the determination of tartrazine in some food samples. The parameters influencing electromembrane extraction were evaluated and optimized. The membrane consists of 1-octanol immobilized in the pores of a hollow fiber. As a driving force, a 30 V electrical field was applied to make the analyte migrate from sample solution with pH 3, through the supported liquid membrane into an acceptor solution with pH 10. Best preconcentration (enrichment factor >21) was obtained in extraction duration of 15 min. Effects of some solid nano-sorbents like carbon nanotubes and molecularly imprinted polymers on membrane performance and electromembrane extraction efficiency were evaluated. The method provided the linearity in the range 25-1000 ng/mL for tartrazine (R(2) > 0.9996) with repeatability range (RSD) between 3.8 and 8.5% (n = 3). The limits of detection and quantitation were 7.5 and 25 ng/mL, respectively. Finally, the method was applied to the determination and quantification of tartrazine from some food samples with relative recoveries in the range between 90 and 98%.

Electromembrane extraction of gonadotropin-releasing hormone agonists from plasma and wastewater samples.

In the present study, for the first time electromembrane extraction followed by high performance liquid chromatography coupled with ultraviolet detection was optimized and validated for quantification of four gonadotropin-releasing hormone agonist anticancer peptides (alarelin, leuprolide, buserelin and triptorelin) in biological and aqueous samples. The parameters influencing electromigration were investigated and optimized. The membrane consists 95% of 1-octanol and 5% di-(2-ethylhexyl)-phosphate immobilized in the pores of a hollow fiber. A 20 V electrical field was applied to make the analytes migrate from sample solution with pH 7.0, through the supported liquid membrane into an acidic acceptor solution with pH 1.0 which was located inside the lumen of hollow fiber. Extraction recoveries in the range of 49 and 71% within 15 min extraction time were obtained in different biological matrices which resulted in preconcentration factors in the range of 82-118 and satisfactory repeatability (7.1 < RSD% < 19.8). The method offers good linearity (2.0-1000 ng/mL) with estimation of regression coefficient higher than 0.998. The procedure allows very low detection and quantitation limits of 0.2 and 0.6 ng/mL, respectively. Finally, it was applied to determination and quantification of peptides in human plasma and wastewater samples and satisfactory results were yielded.

Inhibition of Growth and Induction of Apoptosis in Fibrosarcoma Cell Lines by Echinophora platyloba DC: In Vitro Analysis.

Echinophora platyloba DC plant (Khousharizeh) is one of the indigenous medicinal plants which is used as a food seasoning and medicine in Iran. The objective of this study was to examine the in vitro cytotoxic activity and the mechanism of cell death of crude methanolic extracts prepared from Echinophora platyloba DC, on mouse fibrosarcoma cell line (WEHI-164). Cytotoxicity and viability of methanolic extract was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and dye exclusion assay. Cell death ELISA was employed to quantify the nucleosome production result from nuclear DNA fragmentation during apoptosis and determine whether the mechanism involves induction of apoptosis or necrosis. The cell death was identified as apoptosis using terminal deoxynucleotidyl transferase- (TdT-) mediated dUTP nick end labeling (TUNEL) assay. Our results demonstrated that the extract decreased cell viability, suppressed cell proliferation, and induced cell death in a time- and dose-dependent manner in WEHI-164 cells (IC50 = 196.673 ± 12.4 µg/mL) when compared with a chemotherapeutic anticancer drug, Toxol. Observation proved that apoptosis was the major mechanism of cell death. So the Echinophora platyloba DC extract was found to time- and dose-dependently inhibit the proliferation of fibrosarcoma cell possibly via an apoptosis-dependent pathway.