Liquid Extraction Method Research Articles

Residual detection of naproxen, methyltestosterone and 17α-hydroxyprogesterone caproate in aquatic products by simple liquid-liquid extraction method coupled with liquid chromatography-tandem mass spectrometry.

In the present study, we aimed to develop a reliable screening method based on liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for the detection and quantification of naproxen, methyltestosterone and 17α-hydroxyprogesterone caproate residues. The target analytes were extracted from samples of eel, flatfish and shrimp using acetonitrile with 1% acetic acid, followed by liquid-liquid purification with n-hexane. Chromatographic separation was achieved on a reversed-phase analytical column using 0.1% formic acid containing 10 mm ammonium formate in distilled water (A) and methanol (B) as mobile phases. All the matrix-matched calibration curves were linear (R2 ≥ 0.99) over the concentration range of the tested analytes. Recovery at three spiking levels (0.005, 0.01 and 0.02 mg/kg) ranged from 68 to 117% with intra- and inter-day precisions <10%. Five market samples for each matrix (eel, flatfish and shrimp) were collected and tested for method application. In summary, the proposed method is feasible to screen and quantify the analytes with high selectivity in aquatic food products meant for human consumption.

Salt-assisted liquid–liquid extraction coupled with reversed-phase dispersive liquid–liquid microextraction for sensitive HPLC determination of paraquat in environmental and food samples

In this study, salt-assisted liquid–liquid extraction (SALLE) method is coupled with reversed-phase dispersive liquid–liquid microextraction (RP-DLLME) as a novel method for the determination of paraquat in various samples. Firstly, paraquat was extracted from the aqueous phase into the acetonitrile and then it pre-concentrated into the aqueous phase using the RP-DLLME method. Influential parameters of SALLE such as extracting solvent, volume of extracting solvent, sample pH, salt and its concentration were optimized. Also, effective parameters of RP-DLLME including immiscible solvent and its volume, and water volume as extracting solvent were investigated. Under the optimized conditions recoveries and relative standard deviations were in the range of 80.0–96.0 and 3.5–7.5, respectively. The limit of detection and limit of quantification of method were 0.02 and 0.09 µg/mL, respectively. The proposed method successfully was applied for the determination of paraquat in food and environmental samples.

Rapid screening of sulfonamides in dietary supplements based on extracted common ion chromatogram and neutral loss scan by LC-Q/TOF-mass spectrometry

There is an increasing amount of dietary supplements that are adulterated with diuretics and anti-diabetic drugs; this has become a global problem due to the wide distribution of dietary supplements and the serious negative health effects of the adulterants. In this study, a rapid screening method was developed for detection and confirmation of 35 sulfonamides in supplements by ultra-high performance liquid chromatography quadrupole/time of flight mass spectrometry. For effective extraction of sulfonamides from dietary supplements, four extraction protocols including HLB and WAX solid-phase extraction, Quick Easy Cheap Effective Rugged and Safe method, and pH-controlled liquid–liquid extraction were evaluated, and pH-controlled liquid–liquid extraction method was shown to be the most effective with high recovery efficiency and low matrix effect. Rapid separation of 35 sulfonamides was achieved with the UHPLC C18 column (150 × 2.1 mm, 1.7 um) within 7 min using ammonium acetate aqueous solution (pH 8) and acetonitrile as the mobile phase. From the MS/MS spectra of sulfonamides, common ions (m/z 77.9650 [SO2N]- and m/z 79.9812 [SO2NH2]-) and neutral molecule loss fragments (HCl and SO2) were observed according to their structural characteristics. Extracted common ion chromatograms and neutral loss scan of these characteristic fragments could effectively apply for rapid screening of sulfonamides in various types of supplements. A reduced mass tolerance window of ±5 ppm was useful for detecting targeted and untargeted sulfonamides and could avoid false positive and false negative results. Overall calibration curves within dynamic range for all targets were shown to be linear with a correlation coefficient R2 > 0.995 and limits of detection ranged from 0.04 to 11.18 ng/g for all sulfonamides. The established method was successfully applied for screening and confirmation of sulfonamides in various supplements. The developed method will be helpful for the identification of sulfonamide diuretics and anti-diabetics in dietary supplements, promoting public health and consumer safety.

Effects of Pure and Mixed Autochthonous Torulaspora delbrueckii and Saccharomyces cerevisiae on Fermentation and Volatile Compounds of Narince Wines.

The cultivar of Narince is a native white grape variety of Vitis vinifera, grown in Tokat city, the Mid-Black Sea Region of Anatolia. In this study, the effects of pure and mixed autochthonous Torulaspora delbrueckii-214 and Saccharomyces cerevisiae-1088 cultures on the fermentation behavior and aroma compounds of Narince wines were investigated. Volatile compounds formed in wines were extracted using a liquid–liquid extraction method and determined by GC-MS-FID. Narince grape must was fermented in duplicate, under the following three conditions. Two pure cultures of T. delbrueckii-214 and S. cerevisiae-1088 and a mixture of T. delbrueckii-214 and S. cerevisiae-1088 (1:1). The presence of the non-Saccharomyces T. delbrueckii-214 yeast slowed down the fermentation and produced a lower level of ethanol and a higher levels of glycerol and volatile acid. Only the pure culture of T. delbrueckii-214 was unable to finish fermentation. On the other hand, mixed culture fermentation improved the aroma intensity and complexity of wine due to increased levels of higher alcohols and esters. According to sensory analysis, wine fermented with mixed culture was the most preferred wine followed by wine inoculated with pure S. cerevisiae-1088. This study confirms the role of T. delbrueckii in wine aroma and the potential of non-Saccharomyces use in winemaking.

Adsorption and determination of polycyclic aromatic hydrocarbons in water through the aggregation of graphene oxide

Abstract In this study, a simple method was developed for the aggregation of graphene oxide (GO) with the addition of NaCl to concentrate and separate polycyclic aromatic hydrocarbons (PAHs) from water samples, and this method was used as a environmentally friendly method for the determination of PAHs. We demonstrate the uniform dispersion of GO sheets in aqueous samples for the fast high-efficiency adsorption of PAHs. Aggregation occurred immediately upon elimination of electrostatic repulsion by adding NaCl to neutralize the excessive negative charges on the surfaces of the GO sheets. The aggregates of GO and PAHs were separated and treated with hexane to form a slurry. The slurry was filtered and subjected to gas chromatography–mass spectroscopy (GC-MS) analysis. Based on a 40-mL sample volume, limits of detection of 10–30 ng L-1 were obtained for 16 PAHs, with correlation coefficients (R2) exceeding 0.99. The method yielded good recovery, ranging from 80 to 111% and 80 to 107% for real spiked water samples at 100 and 500 ng L-1, respectively. Compared to traditional solid-phase extraction and liquid–liquid extraction methods, this method is free of organic reagents in the pretreatment procedure and uses only 1 mL hexane for sample introduction.

Optimization and in Silico Analysis of a Cold-Adapted Lipase from an Antarctic Pseudomonas sp. Strain AMS8 Reaction in Triton X-100 Reverse Micelles

A moderate yield of a purified enzyme can be achieved by using the simple technique of reverse micellar extraction (RME). RME is a liquid–liquid extraction method that uses a surfactant and an organic solvent to extract biomolecules. Instead of traditional chromatographic purification methods, which are tedious and expensive, RME using the nonionic surfactant Triton X-100 and toluene is used as an alternative purification technique to purify a recombinant cold-adapted lipase, AMS8. Various process parameters were optimized to maximize the activity recovery of the AMS8 lipase. The optimal conditions were found to be 50 mM sodium phosphate buffer, pH 7, 0.125 M NaCl, and 0.07 M Triton X-100 in toluene at 10 °C. Approximately 56% of the lipase activity was successfully recovered. Structural analysis of the lipase in a reverse micelle (RM) was performed using an in silico approach. The predicted model of AMS8 lipase was simulated in the Triton X-100/toluene reverse micelles from 5 to 40 °C. The lid 2 was slightly opened at 10 °C. However, the secondary structure of AMS8 was most affected in the non-catalytic domain compared to the catalytic domain, with an increased coil conformation. These results suggest that an AMS8 lipase can be extracted using Triton X-100/water/toluene micelles at low temperature. This RME approach will be an important tool for the downstream processing of recombinant cold-adapted lipases.

Simultaneous Determination and Pharmacokinetics of Peimine and Peiminine in Beagle Dog Plasma by UPLC-MS/MS after the Oral Administration of Fritillariae ussuriensis Maxim and Fritillariae thunbergii Miq Powder.

A simple and high sensitive ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for the simultaneous determination of peimine and peiminine in beagle dog plasma after the oral administration of Fritillariae ussuriensis Maxim and Fritillariae thunbergii Miq powder. Chromatographic separation was achieved on an ACQUIT UPLC® BEH C18 column (1.7 μm, 2.1 × 100 mm) in a gradient elution way with a mobile phase consisting of acetonitrile and water containing 0.1% formic acid at a flow rate of 0.4 mL/min. The plasma samples were prepared by a liquid–liquid extraction (LLE) method with ethyl acetate. The analytes were detected with a triple quadrupole tandem mass spectrometry (MS) in multiple reaction monitoring (MRM) mode and a positive ion electrospray ionization (ESI) of the transitions at m/z 432.4→414.4 for peimine and m/z 430.3→412.3 for peiminine. The method was linear for two analytes over the investigated range with all determined correlation coefficients exceeding 0.9900. The lower limit of quantification (LLOQ) was 0.988 ng/mL for peimine and 0.980 ng/mL for peiminine. The mean extraction recoveries of peimine and peiminine at three quality control samples (QC) levels were ranged from 82.56 to 88.71%, and matrix effects ranged from 92.06 to 101.2%. The intra-day and inter-day precision and accuracy were within the acceptable limits at LLOQ and QC levels. The method was effectively and successfully applied to the pharmacokinetics of peimine and peiminine after oral administration of powder to beagle dogs. The obtained results may be help to guide the clinical application of Fritillaria ussuriensis Maxim and Fritillaria thunbergii Miq.

Liquid–Liquid Extraction from Frozen Aqueous Phases Enhances Efficiency with Reduced Volumes of Organic Solvent

Liquid–liquid extraction is widely employed for the separation and analysis of mixtures of compounds, both in the laboratory and in industry. Although a large volume of organic solvent is required to maximize extraction efficiency, the principles of sustainability and green chemistry dictate that the consumption of organic solvents should be minimized. This paper proposes a new liquid–liquid extraction method, realizing high extraction efficiencies with only a small volume of organic solvent. Since extraction efficiency is a function of the ratio of organic and aqueous solvent volumes, attaining high extraction efficiency usually requires a large volume of the organic phase. In general, it is difficult to increase this ratio by reducing the volume of the aqueous phase, except by heating—which damages, and potentially decomposes, the solutes. However, if the aqueous phase is frozen, a freeze-concentrated solution containing the solutes is formed; the volume of the freeze-concentrated solution can be contro...

Preparation of PSf-g-BN15C5/NWF composite membrane with sponge-like pore structure for lithium isotopes adsorptive separation

Polysulfone-graft-monoazabenzo-15-crown-5 ether (PSf-g-BN15C5) was synthesized via nucleophilic substitution reaction and used to prepare PSf-g-BN15C5/non-woven fabrics (NWF) composite membrane for lithium isotopes adsorptive separation by non-induced phase separation (NIPS). The influences of polymer concentration, additive and coagulation bath temperature on membrane properties were systematically explored. A solid–liquid dynamic extraction method was employed to investigate the lithium isotopes separation of the composite membrane. Results showed that the PSf-g-BN15C5/NWF composite membrane with a sponge-like pore structure, the porosity of 69% and the pure water flux of 1433 L/m2/h were obtained under the conditions: PSf-g-BN15C5 polymer concentration of 14 wt%, DMF as solvent and water as a non-solvent in coagulation bath at 60 °C. Moreover, it was found that the lithium isotopes equilibrium separation factor obtained from the membrane with immobilization amount (IA) of 0.81 mmol/g was up to 1.052 ± 0.002, which was much higher than that obtained from the traditional liquid–liquid extraction system of H2O-LiI/BN15C5-CHCl3 (1.007 ± 0.002) and the solid–liquid static extraction using PSf-g-BN15C5 polymer (1.014 ± 0.002) as extractant. In brief, the composite membrane has a great potential application in the development of a green-friendly adsorptive separation method for lithium isotopes.

An improved Ultra-High Performance Liquid chromatography-tandem mass spectrometry method for simultaneous quantitation of cytochrome P450 metabolites of arachidonic acid in human plasma

This study aims to develop a straightforward, sensitive UHPLC–MS/MS method to quantify 15 eicosanoids derived from arachidonic acid in human plasma. Tert-Butyl methyl ether was used on the liquid–liquid extraction method and significantly reduced the expense and time. The method showed excellent linearity for all analytes, with regression coefficients higher than 0.99 over a wide range of concentrations from 0.01 ng mL−1 to 100 ng mL−1. The recovery rates were over 65.00%, and the matrix effects ranged from 8.42% to 40.00%. The limits of detection ranged from 6 pg mL−1 to 10 pg mL−1, and all of the limits of quantification were 20 − 33 pg mL−1. For the broad concentration range, the RE% for accuracy and precision were less than ± 15%. Moreover, trans-4-{4-[3-(4-Trifluoromethoxyphenyl)-ureido] cyclohexyloxy} benzoic acid (t-TUCB) pretreatment extended the window of detection for as much as 30 days. Eicosanoid signaling is altered in various neurological diseases, including pain, Alzheimer’s disease and major depressive disorder. Therefore, this rapid, robust quantitative profiling of 15 eicosanoids in plasma could provide a distinct eicosanoid fingerprint for precision medicine in these patients.

Decolonization potential of 0.02% polyhexanide irrigation solution in urethral catheters under practice-like in vitro conditions

BackgroundLong-term use of indwelling urethral catheters is associated with high risk of urinary tract infection (UTI) and blockage, which may in turn cause significant morbidity and reduce the life of the catheter. A 0.02% polyhexanide irrigation solution has been developed for routine mechanical rinsing together with bacterial decolonization of suprapubic and indwelling urethral catheters.MethodsUsing a practice-like in vitro assay and standard silicon catheters, artificially contaminated with clinically relevant bacteria, experiments were carried out to evaluate the bacterial decolonization potential of polyhexanide vs. 1) no intervention (standard approach) and 2) irrigation with a saline (NaCl 0.9%) solution. Swabbing and irrigation was used to extract the bacteria.ResultsIrrigation with polyhexanide reduced the microbial population vs. the control catheters by a factor of 1.64 log10 (swab extraction) and by a factor of 2.56 log10 (membrane filtration). The difference in mean microbial counts between the two groups (0.90) was statistically significant in favor of polyhexanide when the liquid extraction method was used (p = 0.034). The difference between the two groups using the swab extraction method did not reach statistical significance.ConclusionsThe saline and polyhexanide solutions are able to reduce bacterial load of catheters, which shows a combined mechanical and antimicrobial effect. Further research is required to evaluate the long-term tolerability and efficacy of polyhexanide in clinical practice.

Direct Liquid–Liquid Lipid Extraction Method for Biodiesel Production from Sewage and Petrochemical Industry Sludges

Municipal wastewater treatment plant sludge (sewage sludge) has gained importance in biodiesel production due to its high lipid content. The oily sludges from petrochemical industry wastewater treatment plants (WWTPs) have high concentrations of phospholipids, free fatty acids, neutral lipids and can also be used as efficient feedstocks for biodiesel production. Lipid extraction is the first step of biodiesel production from sludges. Standard drying lipid extraction method necessitates expensive sludge dewatering/drying steps, holding almost 50% of overall biodiesel production cost, to remove high water content in sludge. The aim of this study was to explore lipid extraction from municipal and petrochemical industry WWTP sludges by using the novel direct liquid–liquid lipid extraction method, which does not require expensive sludge dewatering/drying steps, and to compare it to standard drying method. The study also investigated the effect of acid pre-treatment on lipid yields of the sludge samples. In both methods, hexane was used as an organic solvent. Acid pre-treatment increased the lipid yields noticeably. The highest lipid yield of 31.2% (dry sludge) was obtained from pre-acidified petrochemical industry WWTP sludge by using liquid–liquid lipid extraction method. Compared to standard drying method, direct liquid–liquid lipid extraction method resulted with 53% higher lipid and 56% higher biodiesel yields for petrochemical industry WWTP sludge sample. However, liquid–liquid lipid extraction method caused to a very slight increase in the biodiesel yield of primary municipal WWTP sludge samples.

Determination of Hexavalent Chromium Fractions in Plastics Using Laboratory-Based, High-Resolution X-ray Emission Spectroscopy.

Cr(VI) is a well-known human carcinogen with many water-soluble moieties. Its presence in both natural and man-made substances poses a risk to public health, especially when contamination of groundwater is possible. This has led the European Union and other jurisdictions to include Cr(VI) in restriction of hazardous substances regulations. However, for several important industrial and commercial purposes, analytical capability to characterize Cr(VI) is known to be insufficient for regulatory purposes. For example, advanced X-ray spectroscopies, particularly synchrotron-based X-ray absorption fine structure (XAFS) studies, have shown that species interconversion and under-extraction can be difficult to prevent in many existing liquid extraction protocols when applied to plastics, mining ores and tailings, and paint sludges. Here, we report that wavelength dispersive X-ray fluorescence spectroscopy taken at energy resolutions close to the theoretical limit imposed by the core-hole lifetime, generally called X-ray emission spectroscopy (XES) in the synchrotron community, can be used in the laboratory setting for noninvasive, analytical characterization of the Cr(VI)/Cr ratio in plastics. The selected samples have been part of ongoing efforts by standards development organizations to create improved Cr(VI) testing protocols, and the present work provides a direct proof-of-principle for the use of such extremely high-resolution laboratory WDXRF as an alternative to liquid extraction methods for regulatory compliance testing of Cr(VI) content. As a practical application of this work, we report a value for the Cr(VI) mass fraction of the new NIST Standard Reference Material 2859 Restricted Elements in Polyvinyl Chloride.

Extractability-mediated stability bias and hematocrit impact: High extraction recovery is critical to feasibility of volumetric adsorptive microsampling (VAMS) in regulated bioanalysis

Volumetric absorptive microsampling (VAMS), a new microsampling technique, was evaluated for its potential in supporting regulated bioanalysis. Our initial assessment with MK-0518 (raltegravir) using a direct extraction method resulted in 45–52% extraction recovery, significant hematocrit (Ht) related bias, and more importantly, unacceptable stability (>15% bias from nominal concentration) after 7-day storage. Our investigation suggested that the observed biases were not due to VAMS absorption, sampling techniques, lot-to-lot variability, matrix effect, and/or chemical stability of the compound, but rather the low extraction recovery. An effort to improve assay recovery led to a modified liquid–liquid extraction (LLE) method that demonstrated more consistent performance, minimal Ht impact (Ht ranged from 20 to 65%), and acceptable sample stability. The same strategy was successfully applied to another more hydrophilic model compound, MK-0431 (sitagliptin). These results suggest that the previously observed Ht effect and “instability” were in fact due to inconsistent extractability, and optimizing the extraction recovery to greater than 80% was critical to ensure VAMS performance. We recommend adding Ht-independent recovery as part of feasibility assessment to de-risk the long-term extractability-mediated stability bias before implementing VAMS in regulated bioanalysis.

Ocular Pharmacokinetics of 25-Hydroxyvitamin D3 After Weekly Supplementation in Rabbits Using Ultra Performance Liquid Chromatography-Tandem Mass Spectrometer.

The protective role of vitamin D supplementation has recently been shown to be present in various ocular inflammatory diseases. The oral supplementation of vitamin D may take time to achieve adequate levels in intraocular fluids. Therefore, the present study was performed to understand the ocular pharmacokinetics of 25-hydroxyvitamin D3 (25D3) in aqueous humor after weekly supplementation of 25D3 in rabbits. A total of 21 rabbits were fed orally with 25D3 (7.22µg/kg/week) for 8weeks and 9th dose was given at the end of 8weeks. The blood and aqueous humor samples were collected from ear vein and though anterior chamber paracentesis, respectively. The serum and aqueous humor samples were spiked with deuterium labeled internal standard and were extracted using liquid extraction method. Furthermore, the samples were derivatized and 25D3 estimation was performed using ultra performance liquid chromatography-tandem mass spectrometer (UHPLC-MS/MS). The 25D3 supplementation significantly increased the 25D3 levels in serum (78.5 ± 21.6ng/ml) (mean ± SD) (p < 0.0001) and in aqueous humor (991.3 ± 180.6pg/ml) (mean ± SD) (p < 0.0001) compared to baseline levels. The maximum concentration was achieved in serum after the 10th hour of supplementation of 1st and 9th dose, while the same was observed at the 24th hour in aqueous humor. The oral supplementation of 25D3 was found to significantly increase 25D3 levels in aqueous humor; however, the time required to achieve 25D3 concentration in aqueous humor was higher as compared to that in serum. Therefore, weekly oral supplementation of 25D3 may have a beneficial role in ocular diseases.

Subcritical water extraction of bioactive components from ginseng roots (Panax ginseng C.A. Mey)

The feasibility of applying subcritical water extraction (SWE), which is considered to be an environmentally friendly and efficient extraction technology for the extraction of bioactive components from ginseng roots, was evaluated by comparing it with conventional water (WE) and ethanol (EE) extraction methods SWE was conducted at different temperatures ranging from 120 to 200 °C, and WE and EE were performed by solid–liquid heating extraction methods using water or 70% (v/v) aqueous ethanol as a solvent, respectively. SWE showed significantly and markedly higher extraction yields of total sugar (TS), total protein (TPro), phenolic components (TP), and more potent antioxidant activities than WE and EE. The optimized temperature for TS, TP, and antioxidant activities was 200 °C, and that for TPro was 180 °C. Although vanillin–perchloric acid colorimetric quantification showed that SWE yielded more total ginsenosides (TG) than WE and EE, ultrafast liquid chromatography tandem mass spectroscopy (UFLC–MS/MS) analysis revealed that SWE induced extensive hydrolysis of the ginsenosides, except for Rg2. At 160 °C, SWE yielded 9.7- and 6.2-fold more Rg2 than WE and EE, respectively. In comparison, the extraction yields of R1, Rb1, Rb2, Rb3, Rc, Rd, Re, Rg1, and Ro decreased significantly with the increase of SWE temperature. These findings suggested that SWE is a promising environmentally friendly and efficient technology for extracting bioactive polysaccharides, proteins, TP, and Rg2 from ginseng roots, but it has potency to induce extensive hydrolysis of ginsenosides, such as Rb1 and Re.

PLGA-based nanofibers with a biomimetic polynoradrenaline sheath for rapid in vivo sampling of tetrodotoxin and sulfonamides in pufferfish.

Nanomaterials have shown great potential for application in microextraction due to their distinguishing nanoscale architectures and superior physicochemical properties. Herein, novel poly(lactic-co-glycolic acid) (PLGA) solid-phase microextraction (SPME) fibers, which were incorporated with a self-assembled graphene oxide (GO)-coated γ-Al2O3 composite (Al2O3@GO), were fabricated on stainless steel wires via an electrospinning method. The as-spun nanofibers were further sheathed through the self-polymerization of noradrenaline (NA), an agonist found in oysters, to provide a compatible biointerface and antifouling capacity. Acting as the coating substrate of the as-prepared fibers, PLGA is known for its prominent biocompatibility and biodegradability, while the adsorptive Al2O3@GO particles helped to increase their loading capacity. The modified PLGA-based electrospun nanofibers exhibited much higher extraction efficiency compared with the thicker polydimethylsiloxane (PDMS) coatings (165 μm) and polyacrylate (PA) coatings (85 μm). Due to the fine biointerface of the PLGA-based nanofibers, a rapid extraction equilibrium was observed and sampling with the custom-made Al2O3@GO-PLGA@PNA fibers could be accomplished within 15 min. The fibers were then successfully employed for simultaneous in vivo sampling of tetrodotoxin (TTX) and sulfonamides (SAs) in the dorsal-epaxial muscle of living pufferfish (Takifugu obscurus), and satisfactory sensitivities with limits of detection (LODs) in the range of 0.52-2.30 ng g-1 and comparable accuracies to the conventional liquid extraction (LE) method were achieved. In vivo sampling of target pharmaceuticals with the modified nanofibers showed their feasibility for further metabolomics and pharmacokinetics studies in biotissues.

Rapid detection of methamphetamine in human fingernails by liquid-liquid extraction method and one-step methamphetamine test strip.

Methamphetamine cannot be detected through conventional urine screening tests or other analytical methods in methamphetamine abusers who have not used the drug for some time. In some instances, detection of methamphetamine in fingernails can be a good alternative. We aimed to determine the sensitivity and specificity of the one-step methamphetamine test strip used in the detection of methamphetamine in urine in the detection of methamphetamine in fingernails. We took 72 fingernail samples, including 60 samples from methamphetamine abusers and 12 samples as controls from their relatives who had no history of methamphetamine use. The liquid-liquid extraction method was used on fingernail samples, and the resultant solution was tested with one-step methamphetamine test strip. We analysed participants' demographics including age, gender, duration of methamphetamine abuse and strip test results. The mean (SD) age of the participants was 25 (4.33) years. The mean (SD) duration of methamphetamine abuse was 10 (4.5) months. Of the 72 participants, 61 (84.7%) had positive and 11 (15.3%) had negative strip test results. All 60 methamphetamine abusers had positive test results. A positive or negative history of methamphetamine abuse was taken as the gold standard. The sensitivity and specificity of the test was 100% and 91.6%, respectively. Performing liquid-liquid extraction on fingernails and using the strip test for detection of methamphetamine is a simple, inexpensive, rapid and accessible method, and its high sensitivity and specificity make it appropriate for screening. This method may be preferred over other urine and blood methamphetamine detection methods when the patient has not used the drug for a few days.

In vitro antioxidant and anticancer activities of leaf extracts of Rhododendron arboreum and Rhododendron campanulatum from Uttarakhand region of India

Background: Rhododendron arboreum and Rhododendron campanulatum are two important trees of Uttarakhand, known for their ethnopharmacological importance. In the present study, the aqueous and methanolic extracts of leaves of both the plants were analyzed for phytochemical, antioxidant, and antiproliferative activities against cancer cell line and repression of vascular endothelial cell growth factor (VEGF) and hypoxia-inducible factor-1 (HIF-1α) transcription. Materials and Methods: Aqueous and methanolic leaf extracts of both the plants were prepared through pressurized liquid extraction method. Various assays were performed to analyze phytochemical and antioxidant potential. The antiproliferative activity was determined through 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxy anilide salt (XTT) assay. Further, the effect of plant extracts on the modulation of HIF-1α and VEGF mRNA expression was analyzed through quantitative real-time polymerase chain reaction. Results: A significantly high total phenol content, total flavonoid content, free radical scavenging, and reducing power activities were observed in both the plant extracts. High-performance thin-layer chromatography analysis indicated the presence of ascorbic acid, quercetin, gallic acid, and hesperidin in different extracts. XTT reduction assay confirmed the antiproliferative activities of aqueous extracts against cancer cell line, HeLa. Aqueous extract of R. arboreum (RAA), at a concentration of 31.25 μg/ml, inhibited 60.12% and 25.41% proliferation of HeLa and Vero cell lines, respectively. An equal concentration of aqueous extract of R. campanulatum (RCA) inhibited the growth by 48.04% and 15.17% for HeLa and Vero cell lines, respectively. Further, the RAA and RCA have downregulated the expression of two key angiogenic factors responsible for tumor neovascularization, HIF-1α and VEGF to a great extent. Conclusion: The present study affirms that the leaf extracts of R. arboreum and R. campanulatum have enormous potential to be developed as an effective natural antioxidant and anticancer drug. Abbreviations used: PLE: Pressurized liquid extraction; XTT: 2,3-Bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxy anilide salt; TPC: Total phenol content; TFC: Total flavonoid content.

Antagonistic Properties of Methanol Extract of Elaeocarpus sphaericus Leaves against in vitro Growth, Germ Tube Formation, Adhesion and Biofilm of Candida Species

The extracts of Elaeocarpus sphaericus (Roxb.) have shown antimicrobial activities against many pathogenic microbes including fungi. However, the effects of E. sphaericus have not been investigatd against key virulence attributes of Candida. In the present study, methanol extract of E. sphaericus leaves was analyzed against planktonic cells, hyphal growth, adhesion and biofilm formation of two most important fungal pathogens, Candida albicans and Candida glabrata. Methanol extract of E. sphaericus leaves was prepared by pressurized liquid extraction method. Inhibitory concentration of methanol extract of E. sphaericus leaves for both pathogens was determined by broth microdilution assay. Germ tube formation assay was performed to study the effect of the extract on hyphal transition. Effect of methanol extract of E. sphaericus leaves on adhesion and biofilm formation was investigated in microtiter plate using 2,3-bis(2-methoxy-4-nitro-sulfophenyl)-2H-tetrazolium-5-carboxanilide reduction assay. IC50 values of the extract for C. albicans and C. glabrata were found between 0.625-1.25 mg/ml. Serum-induced germ tube development in C. albicans was inhibited in presence of 5 mg/ml methanol extract . The extract remarkably inhibited in vitro adhesion and biofilm formation by C. albicans and C. glabrata in microtiter plates. The results of this study showed anticandida potential of methanol extract of E. sphaericus leaves.