Liquid-liquid Extraction Research Articles

Efficient Online TurboFlow Method for Simultaneous Determination of 60 Emerging Per- and Polyfluoroalkyl Substances in Milk.

Emerging perfluoroalkyl and polyfluoroalkyl substances (PFASs) have received much attention in recent years. Milk has usually low concentrations of PFASs, but it remains an important PFAS exposure route due to its high consumption. In this study, a method for simultaneous determination of 60 PFASs in milk was developed by online TurboFlow liquid chromatography-tandem mass spectrometry. A pH-dependent cold-induced liquid-liquid extraction was developed for precleanup and enrichment. Method quantification limits ranged from 0.003 to 0.500 ng/mL. For PFASs without corresponding isotope-labeled internal standards, Tanimoto coefficient and logP were introduced for the selection of internal standards. The matrix spiked recoveries ranged from 47 to 190%. The method was applied for PFAS analysis in 35 milk samples. PFBS, perfluorohexanesulfonic acid, perfluorooctanesulfonic acid, PFNS, PFPeA, perfluorooctanoic acid, PFNA, and 6:2 FTS were detected. The developed online TurboFlow method was simple, reliable, efficient, and applicable for trace analysis of PFASs in milk.

Styrene divinyl benzene beads (SM-2) functionalized with triisobutylphosphine sulfide for selective uptake of palladium(II) from nitric acid medium

Resins impregnated with organic extractants regarded as effective adsorbent materials due to their ability for selective sorption. These resins consist of a polymeric material that is infused with accessible organic extractants. Furthermore, these resins merge the unique properties and the distinctive characteristics of both liquid-liquid extraction and solid-liquid sorption processes. In this work, the impregnation of Styrene divinyl benzene beads (SM-2 beads) with triisobutylphosphine sulfide (TIBPS = CYANEX 471X) was prepared. A SM-2 bead functionalized with CYANEX 471X (SM-2-TIBPS beads) was characterized using scanning electron microscope (SEM) and Fourier-transform infrared spectroscopy (FT-IR) techniques and investigated for recovery and separation of palladium ions from a 3 M nitric acid solution. Various parameters were studied, as shaking time effect, nitric acid concentration, SM-2-TIBPS beads weight, Pd(II) concentration and temperature. The Pd(II)/SM-2-TIBPS sorption system has an endothermic nature and was fitted with a Langmuir isotherm model with a 0.9860 regression factor. The kinetics modeling was fitted with PSO and IPD models with regression factors of 0.9889 and 0.9959, respectively. The maximum monolayer sorption capacity of SM-2-TIBPS was found to be 17.47 ± 0.9 mg/g. The sorption of Pd(II) from different media at 3 M takes the following sequence: HNO3 > HCl > H2SO4. Desorption of about 76.38 ± 2.4% was achieved with (1 M + 1 M) of thiourea + HCl after one desorption stage. Effect of other interfering ions, as Pt(II), Pb(II), Eu(III), Cu(II), Mo(VI), Co(III), Zr(IV), Fe(III), Cd(II), and Ce(III), indicates high separation factors between Pd(II) and other investigated metal ions, which ranged from 9.4 to 35.

Rapid Liquid Chromatography-Tandem Mass Spectrometry Method for Determination of Total and Free Testosterone in Human Serum and Its Application to Monitoring Biomarker Response of Elite Athletes.

Total testosterone (TT) and free testosterone (FT) are important biochemical markers for anabolism of the human body, and can also serve as early screening indicators for overtraining syndrome (OTS). Presently, there is no fast and reliable serum TT and FT determination method in the field of sport science that can meet the requirements of sports research. Thus, a rapid and accurate determination method for serum TT and FT to fill the gap is needed urgently in sports training. Herein, a simple and reliable liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous determination of TT and FT in serum was developed and fully validated, followed by the application of professional athletes in training monitoring. Efficient pretreatments based on only one-step liquid-liquid extraction (LLE) for TT and one-step LLE after a 20 min ultrafiltration for FT were adopted in this study, and the isotope internal standard of testosterone-13C3 was used to ensure the reliability of the whole procedure. A linear range of four orders of magnitude with 0.02-100 ng/mL can meet the concentration range requirement between a higher limit for male TT and a lower limit for female FT. The accuracy, precision, stability, and matrix effect were all within the limits of the guidelines. The serum TT and FT levels of 200 professional athletes (98 male athletes and 102 female athletes) were investigated by this method. Serum TT, FT, and FT/TT levels of professional athletes were significantly higher than the general population, and serum TT levels were significantly higher by LC-MS/MS than by a chemiluminescence immunoassay. In conclusion, the LC-MS/MS method for TT and FT measurement developed in this study is time-saving and easy to operate, which can be used as a reliable method for the determination of serum TT and FT in sports training, offering valuable information for monitoring anabolism of athletes and screening OTS in the early stage.

Critical analysis of analytical techniques developed for statins in biological fluids, environmental and fermentation samples.

Statins are the most prescribed drug for regulating the high cholesterol level in the blood, which can lead to severe complications, such as cardiovascular diseases and other health complications. A wide range of analytical techniques have been employed for the quantification of statins from various origins, including fermentation derived (lovastatin, pravastatin, and compactin), semi-synthetic (simvastatin), and synthetic (atorvastatin, rosuvastatin, and fluvastatin) routes. The presence of more than one structural form and structural analogue generated in the biosynthesis pathway, as well as reaction intermediates and macromolecules in the clinical sample, complicates the quantification of statins. Furthermore, significant concentrations of statins in environmental samples pose serious health and ecology hazards, and estimating statins in those diluted samples is extremely difficult. On the other hand, the: cost, accurate estimation of the desired one from other structural forms, sample complexity, time, limits of detection and quantification, were major criteria distinguishing the usability of each technique. As a result, the current manuscript focuses on analytical techniques such as molecular spectroscopy (normal and derivatives UV-Visible spectrophotometer), chromatography (TLC, HP-TLC, HPLC, GC, swing column, micellar, and supercritical fluid), mass spectroscopy (HPLC-MS/MS and GC-MS/MS), sequential flow injection, capillary electrophoresis, and cyclic voltammetry, as well as their: optimal operating conditions, limits of detection and quantification, advancements, and limitations. Furthermore, various online and offline sample preparations (precipitation, solid phase extraction, liquid-liquid extraction, and micellar extraction) have been highlighted as an essential pretreatment technique to avoid the interference caused by structural analogues and other macromolecules. The greener and more sustainable concepts used in analytical approaches for the quantification statins are also highlighted with current advancements.

Optimizing Membrane Performance using Various Filler Materials in Membrane Fabrication for Enhancing Gas Separation Efficiency: A Review

The membrane technology has been receiving significant interest in both research and industrial applications, particularly in the separation of CO2/CH4. These methods, as published, aim to overcome the limitations of pure polymeric membranes and offer an alternative approach in separation techniques where membrane technology can overcome the constraints of conventional methods such as Solid Phase Extraction (SPE) and Liquid-Liquid Extraction (LLE). Fabricating membranes using fillers as adsorbents can enhance membrane separation performance due to their porous nature. Hence, the selection of suitable fillers is crucial to maximize membrane efficiency in separating analytes. This paper will review 3 types of fillers-metal-organic frameworks, carbonaceous nanomaterials and mesoporous molecular sieves-from various research papers published in recent years. HIGHLIGHTS Additives introduced to polymeric membrane to increase the membrane performances in terms of selectivity and permeability. Short review of materials used for fabrication of mixed matrix membrane. Modification of selected additives used to study its effects on membrane performances. Recent advancement of hybrid additives materials, combining MOF and carbonaceous materials. GRAPHICAL ABSTRACT

Development and Validation of an Improved HPLC-MS/MS Method for Quantifying Total and Unbound Lenalidomide in Human Plasma.

This study aimed to develop a fully validated HPLC-MS/MS method for quantifying total and unbound lenalidomide concentrations in human plasma. Unbound concentrations were measured using plasma ultrafiltrate prepared with Amicon® Centrifugal Filters. Lenalidomide and lenalidomide-d5 (internal standard) were extracted from 50 μL of human plasma using liquid-liquid extraction. Chromatography was conducted with a Halo® C18 column using 0.1% formic acid and methanol (20:80, v/v) as the mobile phase. The mass spectrometer was operated in a positive ion mode with an electrospray ionization interface and multiple reaction monitoring modes. Calibration curves were linear over the range of 5 to 1000 ng/mL (r2 > 0.996) for both the total and unbound lenalidomide. For total lenalidomide concentrations, between-run precision (coefficients of variation) and accuracy were 1.70-7.65% and 94.45-101.10%, respectively. For unbound concentrations, inter-day precision and accuracy were 1.98-10.55% and 93.95-98.48%, respectively. We developed a highly reproducible, sensitive, and efficient bioanalytical method using a smaller volume of plasma sample (50 μL) with a relatively short run time (2.5 min). The proposed analytical method was successfully applied to measure total and unbound lenalidomide concentrations at various time points in multiple myeloma patients with renal impairment.

Evaluating the detection of barbiturates in dried blood spots: A comparative analysis using gas chromatography-mass spectrometry, gas chromatography-tandem mass spectrometry, and liquid chromatography-tandem mass spectrometry with different extraction methods

Rapid and accurate characterization and quantitation of blood barbiturates and their combination drugs are very important for the clinical treatment of acute barbiturate poisoning. A comparison of dried blood spot (DBS) and traditional liquid-liquid extraction (LLE) in the pre-treatment stage, as well as a comparison of gas chromatography-mass spectrometry (GC–MS), gas chromatography-tandem mass spectrometry (GC–MS/MS), and liquid chromatography-tandem mass spectrometry (LC-MS/MS) as instrumental analysis methods, revealed differences in the analysis results of barbiturates and their combination drugs under different conditions. Based on these findings, we introduce a DBS-GC–MS/MS method. The developed and validated method showed good selectivity, sensitivity (LOD: 0.1 μg mL−1, LOQ: 0.2 μg mL−1), linearity (R2>0.9992), trueness (<15 %, except for carbamazepine, at 29.4 %), and precision (<15 %). Recovery was also good for most target compounds, but significant matrix effects were evident. Compared with the LLE method, the DBS method has the benefits of easy sample collection, storage, and transport, as well as simple pre-treatment and reduced reagent and energy consumption. Compared to LC-MS/MS, GC–MS/MS requires no switching between positive and negative ion modes and uses the MRM detection mode, meaning that more information about the sample compounds can be obtained in less analysis time. Using actual sample analysis, we have demonstrated the advantages of the DBS-GC–MS/MS method for the qualitative and quantitative analysis of barbiturates and poisoning events due to combinations of these drugs. Comparison of the three instruments and the two treatment methods revealed their analysis characteristics. From the perspective of practical application, the broad practical value and advantages of DBS should be embraced in more applications, and future analytical laboratory development should continue to recognize GC–MS/MS as a useful supplement to LC-MS/MS.

Introducing Phosphate Ester into DAPhen by Propyl Enhanced the Selectivity for UO22+ over Th4.

A new type of phenanthroline carboxamide(DAPhen)-phosphate ester ligand (L1/L2) was synthesized for the selective separation of U(VI) over Th(IV). Liquid-liquid extraction experiments showed that the introduction of phosphate ester could increase the extraction ability of ligands for U(VI), especially L2, which showed high selectivity for the separation of U(VI) over Th(IV). The slope analysis indicated that L1 could form 1:1 and 1:2 complexes with U(VI) and 1:1 complexes with Th(IV). NMR titration revealed that the DAPhen unit of ligands combined with one U(VI) to form 1:1 complexes, and then the phosphate ester unit of the 1:1 complexes further combined with another U(VI) to form 1:2 complexes. Ligands provide only the DAPhen unit to Th(IV) to form 1:1 complexes. The crystal structures found 1:2 complexes of L1 and U(VI), 1:1 complexes of L2 and U(VI), and 1:1 complexes of L1 and Th(IV). The larger stability constant (log β) of the 1:1 complexes of L2 with U(VI) than that of the 1:1 complexes of L1 with U(VI) showed that the binding ability of U(VI) with the DAPhen unit of L2 is stronger than that of U(VI) with the DAPhen unit of L1. This study provides new ideas for designing extractants with excellent properties.

Application of salt-assisted liquid extraction in bioanalytical methods

This review provides a comprehensive analysis of bioanalytical methods employed for the quantification of drug molecules in various biological matrices, including human plasma, urine, breast milk, and mouse plasma. The study not only examines traditional sample preparation techniques such as protein precipitation (PP), liquid-liquid extraction (LLE), and solid-phase extraction (SPE), but also delves into the relatively new and innovative salting-assisted liquid-liquid extraction (SALLE). It offers a thorough comparison of analytical methods utilizing SALLE, focusing on key parameters such as analysis time, calibration range, and the type and quantity of salts and organic solvents used. This review aims to serve as an essential resource for researchers and practitioners in selecting the most suitable bioanalytical methods for pharmacokinetic studies and drug monitoring, ultimately enhancing data quality and analytical efficiency in both clinical and research settings.

Development of a Versatile High-through-put Oligonucleotide LC-MS Method to Accelerate Drug Discovery.

Liquid chromatography-mass spectrometry (LC-MS) is an effective tool for high-throughput quantification of oligonucleotides that is crucial for understanding their biological roles and developing diagnostic tests. This paper presents a high-throughput LC-MS/MS method that may be versatilely applied for a wide range of oligonucleotides, making it a valuable tool for rapid screening and discovery. The method is demonstrated using an in-house synthesized MALAT-1 Antisense oligonucleotide (ASO) as a test case. Biological samples were purified using a reversed liquid-liquid extraction process automated by a liquid handling workstation and analyzed with ion-pairing LC-MS/MS. The assay was evaluated for sensitivity (LLOQ = 2nM), specificity, precision, accuracy, recovery, matrix effect, and stability in rat cerebrospinal fluid (CSF) and plasma. Besides some existing considerations such as column selection, ion-pairing reagent, and sample purification, our work focused on the following four subtopics: 1) selecting the appropriate Multiple Reaction Monitoring (MRM) transition to maximize sensitivity for trace-level ASO in biological samples; 2) utilizing a generic risk-free internal standard (tenofovir) to avoid crosstalk interference from the oligo internal standard commonly utilized in the LC-MS assay; 3) automating the sample preparation process to increase precision and throughput; and 4) comparing liquid-liquid extraction (LLE) and solid-phase extraction (SPE) as sample purification methods in oligo method development. The study quantified the concentration of MALAT-1 ASO in rat CSF and plasma after intrathecal injection and used the difference between the two matrices to evaluate the injection technique. The results provide a solid foundation for further internal oligonucleotide discovery and development.

Development of a UHPLC-MS/MS Method for the Determination of Moxidectin in Rat Plasma and Its Application in Pharmacokinetics.

The aim of the present study was to establish a simple and reliable ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method and apply it for the determination of pharmacokinetics of moxidectin-loaded microspheres (MOX-MS) in rats. Plasma samples were processed using a simplified liquid-liquid extraction method and were separated using an Agilent Zorbax Eclipse Plus C18 column (50 mm × 2.1 mm, 1.8 μm) with a mobile phase consisting of a 10 mM ammonium formate solution with 0.1% formic acid (A) and acetonitrile (B) at a flow rate of 0.4 mL/min for 5 min. Avermectin B1a was used as an internal standard (IS). The sample was injected at a volume of 10 μL with a column temperature of 35 °C and detected in a positive ion mode. A good linear response across the concentration range of 1.00-200 ng/mL (r2 > 0.99) and a lower limit of quantification (LLOQ) of 1.00 ng/mL were achieved. The extraction recovery of moxidectin exceeded 94.1%, the matrix effect was between 91.2% and 96.2%, the accuracy ranged from 100.1 to 103.6%, and the relative standard deviation (RSD) did not exceed 15% for the intra- and inter-day accuracy and precision. The pharmacokinetic results showed that MOX-MS significantly decreased Cmax, prolonged T1/2, and improved bioavailability. The developed method significantly reduced the assay volume, shortened detection time, simplified sample processing methods and saved assay costs, which may contribute to the development of the new antiparasitic drug.

Revealing the backbone structures of land-based fulvic acids derived from river and soil through n-butylsilane reduction.

While ultra-high-resolution mass spectrometry has enabled the identification of the molecular composition of dissolved organic matter (DOM), elucidating its molecular structure remains a challenging endeavor. Here, two fulvic acids (FAs), one from river and the other from forest soil, were subjected to reduction using an optimized n-butylsilane (n-BS) reduction method. The reduction products were purified through a combination of liquid-liquid extraction and silica gel column chromatography, resulting in the separation into saturates, aromatics, and polar products. The polar products were analyzed by high-resolution mass spectrometry (HRMS), and the saturates and aromatics were analyzed using gas chromatography-mass spectrometry (GC-MS). HRMS results showed that the number of oxygen atoms and double-bond equivalent (DBE) values of FA decreased after reduction. GC-MS results revealed that a total of 270 hydrocarbon monomers were identified from the reduction products of a single sample, with the highest carbon number of cycloalkanes reaching C33. For the first time, steranes and hopanes were detected in the reduction products, potentially serving as evidence for the existence of carboxyl-rich alicyclic molecule (CRAM) precursors. Additionally, a significant number of polycyclic aromatic hydrocarbons were identified, and the potential sources of various compounds were preliminarily inferred based on their isomers. This study extends the knowledge of the possible backbone structure of the DOM and provides a new potential tool for investigating the origin and transformation mechanisms of DOM.

Innovative Techniques for Pharmaceutical Waste Management: Enhancing Drug Recovery and Environmental Sustainability

The pharmaceutical sector is a major component of current healthcare, manufacturing and distributing drugs, biological substances, and medical equipment. Despite its advantages, the sector creates enormous waste, including materials for packaging, production by-products, expired or unused drugs, and other residues, creating health and environmental issues. Appropriate pharmaceutical waste handling and medication recovery strategies are vital for limiting these problems. This article aims to investigate and evaluate multiple techniques for recovering pharmaceuticals from pharmaceutical waste, highlighting the significance of sustainable waste management in the pharmaceutical sector. The paper emphasizes the need to use modern methods such as liquid-liquid extraction, membrane crystallization, solid-liquid extraction, and adsorption to recover drugs from pharmaceutical waste. Liquid-liquid extraction exhibits excellent adaptability and efficiency for varied Active Pharmaceutical Ingredients (APIs), whereas membrane crystallization provides low-energy solutions for thermally sensitive compounds. Solid-liquid extraction is useful for recovering APIs from solid dosage forms, while adsorption approaches exploit substances like activated carbon for organic component recovery. Each process has particular benefits and disadvantages, with the selection of methodology based on waste properties and recovery objectives. It emphasizes the promise of these technologies for high extraction yields, purity, and environmental sustainability, supporting effective pharmaceutical waste management procedures. Additionally, difficulties such as cost-effectiveness, scalability, and regulatory compliance are addressed, pointing to opportunities for future research and development to improve the efficacy of drug recovery procedures. In conclusion, using advanced techniques to recover pharmaceuticals from pharmaceutical waste offers a viable way to implement sustainable waste recovery procedures and lessen the pharmaceutical industry's negative environmental effects.

Quantitation of the DNA-dependent protein kinase inhibitor peposertib (M3814) and metabolite in human plasma by LC-MS/MS.

The DNA-dependent protein kinase (DNA-PK) is an abundant nuclear protein that mediates DNA double-strand break repair by nonhomologous end joining (NHEJ). As such, DNA-PK is critical for V(D)J recombination in lymphocytes and for survival in cells exposed to ionizing radiation and clastogens. Peposertib (M3814) is a small molecule DNA-PK inhibitor currently in preclinical and clinical development for cancer treatment. We have developed a high-performance liquid chromatography-mass spectrometry method for quantitating peposertib and its metabolite in 0.1 mL human plasma. After MTBE liquid-liquid extraction, chromatographic separation was achieved with a Phenomenex Synergi polar reverse phase (4μm, 2× 50 mm) column and a gradient of 0.1% formic acid in acetonitrile and water over an 8min run time. Mass spectrometric detection was performed on an ABI SCIEX 4000 with electrospray, positive-mode ionization. The assay was linear from 10 to 3000 ng/mL for peposertib and 1-300 ng/mL for the metabolite and proved to be both accurate (97.3%-103.7%) and precise (<8.9%CV) fulfilling criteria from the Food and Drug Administration (FDA) guidance on bioanalytical method validation. This liquid chromatography-tandem mass spectroscopy (LC-MS/MS) assay will support several ongoing clinical studies by defining peposertib pharmacokinetics.

Development and Validation of a New HPLC Bio Analytical Internal Standard Method for the Analysis of Two Immunology Drugs (Lamivudine and Dolutegravir) in Human Plasma

Bioanalytical methods are useful for the quantitative analysis of drugs and their metabolites in biological fluids. Bio-analytical internal standards methods plays vital role in areas of human clinical pharmacology. The aim of this work is to development and validation of bio-analytical internal standard method for determination of two immunology drugs Lamivudine (LMVD) and Dolutegravir (DTGR) in plasma with Abacavir(ABVR) drug as internal standard (IS). Liquid-liquid extraction with Diethyl ether and methanol in the ratio of 50:50 (v/v) was used for the extraction of drugs from the biological matrix. The optimized chromatography conditions consist of Water, Acetonitrile and Methanol in the ratio of 40:25:35 (v/v) as a mobile phase with Ascentis, C18 Column (250 X 4.6 mm, 5μ) as stationery phase. Isocratic elution with 1.2 ml flow separates theLMVD at 3.5 min, DTGR at 9.8 min and ABVR at 6.8 min. The method was validated as per ICH guidelines. The relative standard deviation (%RSD) were found to be &lt;5%for precision studies. Hence the method was found to be suitable fort the analysis of LMVD and DTGR in spiked human plasma.

Liquid-Liquid Extraction Solvent Selection for Comparing Illegal Drugs in Whole Blood and Dried Blood Spot with LC-MS/MS.

This study focused on the simultaneous detection of amphetamine, 3,4-methyl enedioxy methamphetamine, morphine, benzoylecgonine, and 11-nor-9-carboxy- tetrahydrocannabinol (Δ9-THC-COOH) in whole blood and DBS. It is aimed to select a solvent mixture for liquid-liquid extraction (LLE) technique employing LC-MS/MS. The obtained DBS results were compared with the whole blood samples results. A simple, rapid, and reliable LC-MS/MS method was developed and validated for all analytes in whole blood and DBS. LC was performed on a Hypersil Gold C18 column an initial step with a gradient of 0.01 % formic acid, 5 mM ammonium format buffer in water, and acetonitrile at 0.3 ml/min with 7.5-min runtime. A methanol:acetonitrile (40:60 v/v) mixture was selected for both matrices. LOQ values were 10-25 ng/mL; linear ranges were LOQ-500 ng/ml for all analytes; correlation coefficients were greater than 0.99, and all calibrator concentrations were within 20%. Analytical recovery in blood and DBS ranged from 84.9-113.2% of the expected concentration for both intra and-inter day. Analytes were stable for 1, 10, and 30 days after three freeze/thaw cycles. It was determined that the variances of the results obtained with the two matrices in the comparison study were equal for each analyte, and the results were highly correlated (r=0.9625). A sensitive, accurate, and reliable chromatographic method was developed to determine amphetamine, MDMA, morphine, benzoylecgonine, and cannabis, by performing the same preliminary steps with whole blood and dried blood spots. It was observed that the results obtained in these two matrices were compatible and interchangeable when statistically compared.

Direct ion chromatographic method for speciation micro analysis of arsenic forms in industrial samples with “rich” matrix composition

The speciation analysis of arsenic has consistently been a subject of great interest. However, it remains challenging to analyze complex matrix samples that contain both arsenic and interfering components. In this case, it can be hard to choose the right combinations of different instrumental methods, or a separation method followed by detection, which is usually done using a spectral approach (hybrid methods). In the production control of copper electrorefining, the determination of the concentration of As (III) and As (V) helps to improve the quality of the cathode copper produced. This work investigated the possibility of directly determining both arsenic forms and total As in an electrolyte bath using ion chromatography (IC) with conductometric detection. The use of the ion chromatographic approach for the determination of As(V) in complex matrix samples such as copper electrolyte must take into account the presence of potential interferences from anions such as sulphates, sulfites, selenites, selenates, etc. The results revealed that the method is accurate and precise, with As(V) quantification limits of 15 µg.L-1 and detection limits of 5 µg.L-1. This method is suitable for assessing various types of arsenic in the production of electrolytic copper, with the aim of replacing the current technique that requires liquid-liquid extraction and ICP-OES detection. This led to the following improvements: Enhanced efficiency: The method eliminates the need for extensive and time-consuming sample preparation for the initial separation of arsenic forms. At the same time, the method's characteristics are comparable to those of ICP-OES with liquid-liquid extraction, which is often used in the speciation analysis of arsenic. The method is environmentally friendly as it avoids the use of organic and poisonous extractants. The method can simultaneously analyze other anions (PO43-, SO42-, F-, Cl-, etc.) with arsenates with appropriate calibration.

Fast determination of organophosphate esters and their metabolites in human matrices by a straightforward cold-induced strategy coupled with HPLC-MS/MS.

Organophosphate esters (OPEs) are commonly used chemicals and are also regarded as emerging environmental pollutants. Recently, it has been proved that metabolites of OPEs (mOPEs) could also cause health concerns. However, analytical methods for the concurrent measurement of OPEs and mOPEs in human matrices are still complicated. In this study, a convenient and efficient analytical method combining a cold-induced strategy and HPLC-MS/MS was developed to simultaneously determine 18 OPEs and 10 mOPEs in human serum, urine, and human milk. In brief, after the sample was extracted with acetonitrile, a "one-step" treatment combining purification and enrichment was accomplished by cold-induced liquid-liquid extraction (CI-LLE), and analytes were then quantified by HPLC-ESI-MS/MS. The ratio of acetonitrile/water, and the temperature and time set in the CI-LLE procedure were optimized for achieving the highest enrichment factors. Under thebest conditions, linearity, limits of detection (LODs), recovery, precision, and matrix effects of OPEs/mOPEs were verified. LODs of OPEs/mOPEs in serum, urine, and human milk were 0.1-113pg/mL, 0.1-22pg/mL, and 0.2-22pg/mL, respectively. Average recoveries ranged from 80 to 123%, with relative standard deviations lower than 15% for most analytes. The matrix effect test showed slight signal enhancement or inhibition, and the use of isotopically labeled internal standards (ISs) could compensate for the effects. In real sample analysis, both OPEs and mOPEs showed high detecting frequency, which indicated their ubiquity in humans.

Evaluation of antibacterial and anticancer properties of secondary metabolites isolated from soil Bacillus spp focusing on two strains of Bacillus licheniformis and Bacillus siamensis

BackgroundBacillus strains are well recognized for their inherent production of bioactive compounds that exhibit antibacterial and anticancer properties. This study aims to evaluate the antimicrobial and anticancer effects of the secondary metabolite isolated from Bacillus licheniformis and Bacillus siamensis strain.Material and methodWe developed and purified a new soil-derived Bacillus strain to study its metabolites on cancer cells and bacteria. After evaluating the antimicrobial effects of the selected strains’ secondary metabolites by well diffusion, growth conditions and temperature optimised using liquid-liquid extraction, secondary metabolites isolated, and active compounds identified using GC-MS. Evaluation of PC-3 and HPrEpC cytotoxicity. AV/PI staining and comet assay assessed necrosis and apoptosis. Real-time PCR measured apoptotic gene expression. Finally, the scratch test measured cell movement.ResultsBacillus strain metabolites exhibit dual-purpose antimicrobial and anticancer properties. Bacillus licheniformis isolate 56 and S2-G12 isolate 60 demonstrated the greatest antibacterial activity. Among all Bacillus isolates, isolates 56 (Bacillus licheniformis) and 60 (Bacillus siamensis strain) had the highest antibacterial activity. Crude extracts obtained from strains 56 and 60 decreased PC-3 cell viability in a dose-dependent manner. At 200 µg/mL, the survival rate of cells treated with strain 56 and 60 crude extract was 23% and 25%, respectively (p < 0.001). The treatment of PC-3 cells with strains 56 and 60 crude extract led to considerable apoptosis (46.2% and 50.09%, respectively) compared to the control group. After treatment with the crude extract from strains 56 and 60 at an IC50 concentration, a significant number of PC-3 cells showed comet formation, indicating DNA fragmentation. Metabolites extracted from strain 56 and 60 enhanced caspase 3, caspase 8, and Bax genes expression and reduced Bcl-2 expression (p < 0.001). Cell migration was also prevented.ConclusionOur findings show that the secondary metabolites of B. licheniformis and B. siamensis have antibiotic and anticancer properties. However in vivo studies are necessary to confirm these findings.

A-043 A Fast, Novel and Analytically Sensitive Clinical Research Method for the Analysis of Free Testosterone using UPLC-MS/MS

Abstract Background The aim of this work was to develop an analytically sensitive, rapid clinical research method for the analysis of free testosterone in serum using a small sample volume. Laboratories have found this difficult, partly due to the inherent low concentrations (less than 3% of total testosterone is free), technical challenges and variability in results have been noted with equilibrium dialysis, which is considered the gold standard. Methods Testosterone certified reference material (Merck, UK) was used to create calibrators in 52.75 mM HEPES buffer adjusted to pH 7.4 to mimic the ionic environment of serum. Precision was evaluated using in-house QC materials prepared in serum (Golden West Biologicals, USA and BioIVT, UK). 200 µL serum was incubated at 37°C with 400 µL pH-adjusted 52.75 mM HEPES buffer for 2 hours, mixing at 800 r.p.m. with Rapid Equilibrium Analysis (RED) inserts and plates (Thermo Scientific™, UK). Testosterone-13C3 (Merck, UK) was spiked into resultant diasylate, then liquid-liquid extraction (LLE) was used to concentrate and clean the sample. The ACQUITY™ UPLC™ I-Class System with the Waters™ ACQUITY UPLC BEH C18, 1.7µm, 2.1 x 100 mm Column using a water/methanol/ammonium fluoride gradient and the Waters Xevo™ TQ Absolute Mass Spectrometer were used for quantification. An accelerated protocol for equilibrium dialysis, using a 2-hour incubation and fast mixing step was used (4 hours or longer is more typical historically) though it is essential to control the pH of the dialysis buffer and, particularly, the temperature of the chamber. Results No significant carryover or matrix effects were demonstrated with linearity from 0.5-650 pg/mL. Addition of high concentrations of endogenous substances (albumin, bilirubin, creatinine, cholesterol, triglycerides and uric acid) did not affect quantification. Analytical sensitivity investigations indicated the analytical sensitivity would allow precise quantification (≤20%CV and ≤15% bias) at 0.50 pg/mL. Coefficients of variation (CV) for total precision and repeatability on 5 analytical runs for low, mid and high QCs at approximately 2.79, 8.80 and 134 pg/mL respectively were all ≤ 8.4% (n=25). Accuracy was assessed by analyzing 45 male external quality assurance samples (NEQAS, UK) ranging in concentration from 50.1-268.6 pg/mL and the Bland-Altman agreement showed a small negative bias of -7.6% compared with the all-laboratory trimmed mean (ALTM), though excellent agreement overall. When LC-MS/MS determined concentrations were processed against Vermeulen equation determined concentrations (total testosterone and SHBG concentrations were provided by the scheme, a concentration of 45 g/L albumin was assumed), again good agreement was shown by the Bland-Altman bias of -7.7%. Conclusions A novel clinical research method using a fast equilibrium dialysis procedure with LLE and LC-MS/MS analysis with only 200 µL of serum has been demonstrated for clinical research use. Sub pg/mL analytical sensitivity, excellent linearity and precision, with minimal matrix effects and a strong agreement with an EQA scheme were established.