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In healthcare role of biological analyte like uric acid plays a crucial role. Among various antioxidants, uric acid is significant antioxidant that helps in regulating blood-pressure and also reduces the antioxidant stress as well. In this proposed research work different concentration of uric acid were taken for detection using optical absorbance based spectroscopy. After the characterization it was found that as the concentration of uric acid increases, absorbance associated with peak absorbing wavelength also increases. The concentration of uric acid was taken 2, 4 and 8 mg/dl respectively for testing and absorbance found to be 0.75, 1.8 and 2.2 respectively. The sensitivity of device found to be 1 mg/dl.

Here, silica microspheres were decorated with two-dimensional metal–organic frameworks (2D MOFs) nanosheets and ionic liquids, and evaluated as the mixed-mode stationary phase for chromatographic separation. The ionic liquids were used to assist the synthesis of 2D MOFs nanosheets, and also acted as adhesives among the nanosheets and silica. In contrast with the 2D MOFs-based column without ionic liquids and commercial columns, the prepared column exhibited enhanced chromatographic separation performance for partially hydrophilic compounds such as alkaloids, sulfonamides and antibiotics, etc. In addition to excellent chromatographic repeatability and stability, it has also been verified that the composites could be easily and repeatedly prepared. The relative standard deviation of the retention time of the same type of analyte between the three batches of materials was ranging from 0.21% to 1.7%. In short, these results indicated that the synthesized composites were promising separation material for liquid chromatography, which made it possible to broaden the application of 2D MOFs in the field of chromatography.

We propose a concept for ligase detection by conversion of aggregation-based homogeneous analysis into surface-tethered electrochemical assay through streptavidin (SA)-biotin interaction. Sortase A (SrtA) served as the model analyte and two biotinylated peptides (bio-LPETGG and GGGK-bio) were used as the substrates. SrtA-catalyzed ligation of the peptide substrates led to the generation of bio-LPETGGGK-bio. The ligation product (bio-LPETGGGK-bio) induced the aggregation and color change of SA-modified gold nanoparticles (AuNPs) through the SA-biotin interactions, which could be assayed by the colorimetric method. Furthermore, we found that the bio-LPETGGGK-bio could trigger the assembly of tetrameric SA proteins with the formation of the (SA-bio-LPETGGGK-bio)n assemblies through the same interactions. The above results were further confirmed by atomic force microscopy and fluorescent imaging. The insulated assemblies were in-situ fabricated at the SA-modified gold electrode, thus hindering the electron transfer of [Fe(CN)6]3−/4− and leading to an increase in the electron-transfer resistance. The capability of the method for the detection of SrtA both in vitro and Staphylococcus aureus (S. aureus) has been demonstrated. SrtA with a concentration down to 1 pmol/L has been determined by the electrochemical analysis, which is lower than that achieved by the colorimetric assay (50 pmol/L). By integrating the advantages of homogeneous reaction and heterogeneous detection, the strategy serves as an ideal means for the fabrication of various sensing platforms by adopting biotin-labeled and sequence-specific peptide or nucleic acid substrates.

The feasibility of light-addressed detection and manipulation of pH gradients inside an electrochemical microfluidic cell was studied. Local pH changes, induced by a light-addressable electrode (LAE), were detected using a light-addressable potentiometric sensor (LAPS) with different measurement modes representing an actuator-sensor system. Biosensor functionality was examined depending on locally induced pH gradients with the help of the model enzyme penicillinase, which had been immobilized in the microfluidic channel. The surface morphology of the LAE and enzyme-functionalized LAPS was studied by scanning electron microscopy. Furthermore, the penicillin sensitivity of the LAPS inside the microfluidic channel was determined with regard to the analyte’s pH influence on the enzymatic reaction rate. In a final experiment, the LAE-controlled pH inhibition of the enzyme activity was monitored by the LAPS., Deutsche Forschungsgemeinschaft, Bundesministerium für Forschung und Technologie

The uncontrollable synthesis of Prussian blue (PB) and its weak stability toward OH– are great challenges affecting its electrochemical biosensing application. Herein we utilize the unique properties of chitosan (CS) to realize the facile and controllable synthesis of a CS-PB nanocomposite and combine it with the urease-catalyzed deposition of polydopamine (PDA) for amplifying the electrochemical signal inhibition of PB to develop a novel immunosensing method for protein detection. The immunosensor was constructed on a CS-PB modified electrode, and a urease-functionalized silica nanoprobe was prepared for tracing its sandwich immunoassay toward the model analyte of carcinoembryonic antigen. Besides the electrochemical impedance effect of the quantitatively captured nanoprobes, their enzymatic reaction can release numerous OH– to destroy the PB crystals and also induce the PDA deposition onto the immunosensor. These caused drastic electrochemical signal inhibition to PB. Based on the above multi-signal amplification mechanism, the method exhibits a very low detection limit of 0.042 pg mL–1 along with a very wide linear range of six-order of magnitude. In addition, the CS-PB based immunosensor has excellent specificity, repeatability, stability and reliability. Thus this PB nanocomposite and the proposed electrochemical immunosensing method reveal a promising potential for future applications.

Ganoderma (lingzhi) is a famous herbal medicine and edible supplement in oriental countries for a long history. In this study, a simple micellar electrokinetic chromatography (MEKC) method was established for the analysis of nucleosides and bases, the major bioactive components in Ganoderma for the first time. By optimizing the borate concentration, the sodium dodecyl sulfate (SDS) concentration and the pH value of running buffer, 10 nucleosides and bases achieved an ideal separation. In real sample analysis, the developed method was successfully used to determine the 10 target analytes in 23 batches of Ganoderma samples from different regions. Results indicated that contents of 10 investigated analytes in each sample showed obvious variation. The principal components analysis (PCA) and hierarchical cluster analysis (HCA) analysis classified the samples into three groups, and the HCA tree visualized the relationships which was mainly contributed by geographical partition. The results indicated geographical origin to be an important factor that affect the accumulation of nucleosides and bases in Ganoderma. In summary, this study provides a simple and practical strategy for quality assessment and cultivation reference of Ganoderma.

Background Hemolysis is the most common type of preanalytical interference. Cut-offs based on the hemolysis index level can be established using different approaches. The Working Group for Preanalytical Phase of the European Federation of Laboratory Medicine has developed a protocol for hemolysis management based on cut-offs estimated from biological variation (BV) and the use of interpretative comments. We developed and assessed the implementation of the protocol in our laboratory. Methods Hemolysates from whole blood were prepared following the Meites method, and pooled serum samples with known Hb concentrations were prepared. For each analyte (42 ), interferograms were generated and used to establish cut-offs: desirable analytical quality specification and reference change value. This protocol was assessed, both pre- and post-implementation, according to expert rules in the Laboratory Information System. Results Among the analytes evaluated, we selected those that showed the highest degree of hemolysis interference: lactate dehydrogenase (LDH), aspartate aminotransferase, direct bilirubin, potassium, and folic acid. The cut-offs for LDH and direct bilirubin were the lowest. Only 28.16% of all LDH values were adequately reported in the pre-implantation retrospective study, but this percentage improved in the post-implementation stage. Conclusions The development and implementation of a harmonized protocol for hemolysis management based on BV cut-offs and result reporting significantly improve hemolysis detection and lead to a decrease in the number of hemolyzed samples over time.

Hypothesis Due to the intrinsic nature of the surface-enhanced Raman scattering (SERS), the detection of molecules with weak binding affinities toward metal substrates is critical for development of a universal SERS sensing platform. We hypothesized the physical trapping of small pesticide molecules for active hot spot generation using tyramine-mediated crosslinking chemistry and silver nanoparticles (Ag NPs) enhances SERS detection sensitivity. Experiments Tyramine-mediated crosslinking chemistry for sensor application was validated by ultraviolet–visible absorption spectroscopy, scanning electron microscopy, dynamic light scattering, and Raman spectroscopy. SERS sensing platform using tyramine-mediated crosslinking reaction was systematically studied for detection of 1,4-dyethylnylbenzene as a model analyte. This sensor system was applied to detect two other pesticides, thiabendazole and 1,2,3,5-tetrachlorobenzene, which have different binding affinities toward metal surfaces. Findings The SERS signal of 1,4-dyethylnylbenzene obtained using this sensor system was 3.6 times stronger than that obtained using the Ag colloidal due to the nanogap of approximately 1.3 nm within the generated hot spots. This sensor system based on tyramine-mediated crosslinked Ag NPs was evaluated as a promising tool to achieve a solution based sensitive detection of various pesticide molecules that cannot be adsorbed on the surfaces of typical SERS substrates such as metal nanoparticles.

The solid-phase microextraction technique quantifies analytes without considerably affecting the sample composition. Herein, a proof-of-concept study was conducted to demonstrate the use of coated probe electrospray ionization (coated-PESI) and coated blade spray (CBS) as ambient mass spectrometry approaches for monitoring drug biotransformation. The ability of these methods was investigated for monitoring the dephosphorylation of a prodrug, i.e., combretastatin A4 phosphate (CA4P), into its active form, i.e., combretastatin A4 (CA4), in a cell culture medium supplemented with fetal bovine serum. The CBS spot analysis was modified to achieve the same extraction efficiency as liquid-liquid extraction and obtain results in 7 min. Low quantities of target analytes were extracted using coated-PESI, indicating that this technique can be used to perform extractions with only negligible target analyte depletion. Because coated-PESI does not consume any sample, it is the preferred technique in the case of a limited sample volume. Although coated-PESI only extracts small quantities of analytes, it uses the desorption solvent volume of 5–10 pL, resulting in high sensitivity, thus allowing the detection of compounds after only 1 min of extraction. The biotransformation of CA4P into CA4 via phosphatases occurs within the simple matrix, and the proposed sample preparation techniques are suitable for monitoring the biotransformation.

Uric acid is the primary end product of human purine metabolism and has been regarded as a key parameter in urine and blood for monitoring physiological conditions. This paper presents a paper-based biosensor for a quantitative determination of uric acid using electrochemical detection. The working electrode of the biosensor is modified with graphene oxide (GO) and 5-amino-1,3,4-thiadiazole-2-thiol (ATT) by electropolymerizing ATT on the surface of graphene oxide. In this study, cyclic voltammetry (CV) measurements required only 200 μL of analyte solution. The experimental results showed that the oxidation peak current increased as the concentration of uric acid become higher and exhibited a linear relationship in the concentration range of 0.1–10 mM, indicating that this proposed biosensor has high sensitivity. In addition, this biosensor has good selectivity to detect uric acid because ATT has a specific binding with it. In human blood and body fluids, nitrites may be the only factor that can interfere with the detection of uric acid using this proposed biosensor. Nevertheless, uric acid can be discriminated from nitrite in the CV measurement due to different oxidation potentials. Thus, this proposed paper-based biosensor is a promising tool for detecting uric acid in biological samples.

The file attached to this record is the author's final peer reviewed version. The Publisher's final version can be found by following the DOI link. Estimations of accurate and reliable NMR chemical shift values, coupling patterns and constants within a reasonable timeframe remain significantly challenging, and the unavailability of reliable software strategies for the prediction of low-field (e.g., 60 MHz) spectra from those acquired at higher operating frequencies hampers their direct comparison. Hence, this study explored the applications of accessible software options for predicting these parameters in the 1H NMR profiles of analytes as a function of magnetic field strength; this was performed for individual analytes and also for complex biofluid matrices featured in metabolomics investigations. For this purpose, results from the very first successful experimental acquisition and simulation of the 1H NMR profiles of intact human salivary supernatant samples on a 60 MHz benchtop spectrometer were evaluated. Using salivary metabolite concentrations determined at 400 MHz, it was demonstrated that simulation of the low-field spectra of five biomolecules with the most prominent 1H resonances detectable allowed multiple component fits to be applied to experimental spectra. Hence, these salivary 1H NMR profiles could be successfully predicted throughout the 45–600 MHz operating frequency range. With the exception of propionate resonance multiplets, which revealed more complex coupling patterns at low field and required more astute computational and fitting options, valuable quantitative metabolomics data on salivary acetate, formate, methanol and glycine could be attained from low-field spectrometres. These studies are both timely and pertinent in view of the recent advancement of low-field benchtop NMR facilities for diagnostically significant biomarker tracking in biofluids. Experiments performed with added ammonium chloride to facilitate the release of salivary metabolites from biopolymer binding sites provided evidence that a small but nevertheless significant proportion of propionate, but not lactate, was bound to such sites, an observation of much relevance to biomolecule quantification in salivary metabolomics investigations.

Calixarenes are well-known supramolecular host molecules with versatile applications. Over the past decades, hundreds of selective and sensitive detections of several analytes have been reported by employing calixarenes as the chemosensor agent. The detection and quantification of metal ions and anions are crucial as heavy metal ions are harmful to living organisms, while monitoring anions is pivotal in the environmental samples. On the other hand, detecting and quantifying biomolecules and neutral molecules are critical due to their irreplaceable role in human health. In this review, we summarized the application of calixarenes as the supramolecular chemosensor agent for detecting metal ions, anions, biomolecules, and neutral molecules through fluorescent spectroscopy to give brief information on the design and development of the chemosensor field. This review updates the world with the application of calixarene derivatives as fluorescent chemosensors and challenges researchers to design and develop better chemosensor agents in the future.

Introduction: In this work, we used a thread-paper microfluidic device (μTPAD) system, where a threaded part for the handling of the whole blood samples and a paper part for the reaction of plasma with immobilized bioreagents integrated into woman pad as a wearable sensing device namely as smart women pad. The μTPAD as a wearable smart woman pad is developed for the detection of pH and urea in mensuration blood as real samples. Methods: This combined device was constructed to cover the elements required, that is, separation of red blood cell, conditioning, analyte reaction, and colorimetric detection. The color change in sensing areas was measured in the RGB values via a smartphone using the Color Grab after a smart woman pad was used. The thread allowed red blood cell sampling and separation, while the paper microfluidic device was used for conditioning, biorecognition, and colorimetric transduction of pH and urea as analytes. Results: The time needed for analysis was measured as 110 s using the equilibrium method for both analytes, with a limit of detection (LOD) of 72.55 μg/mL for urea, with precision around 1.68%, while for pH around 0.80%. The smart woman pad allowed rapid detection of pH and urea in menstruation blood as real samples for monitoring of the kidney functions, and the results showed an agreement with the conventional methods that have been generally used in the clinical laboratory. Conclusion: The smart woman pad has the potential to be used as a wearable device to monitor the health status of the user via its blood mensuration analysis.

As a neglected member of the platinum group elements, osmium, the metal with the highest density in the earth, is very suitable for the preparation of a peroxidase with high catalytic activity and stability, and can also be associated with the development of a sensor. In this study, we accessed Os nano-hydrangeas (OsNHs) with one-pot synthesis and utilized them in a bifunctional immunosensor that can present both catalytic chromogenic and tinctorial signal for nanozyme-linked immunosorbent assay (NLISA) and lateral flow immunoassay (LFIA) for use in folic acid (FA) detection. In the OsNHs-NLISA, the linear range is from 9.42 to 167.53 ng mL−1. The limit of detection (LOD) is 4.03 ng mL−1 and the IC50 value is 39.73 ng mL−1. In OsNHs-LFIA, the visual cut-off value and limit of detection (v-LOD) are 100 ng mL−1 and 0.01 ng mL−1, respectively. Additionally, the outcome from the specificity and spiked sample analysis offered recovery from the spiked milk powder sample ranging from 93.9 to 103.6% with a coefficient of variation under 4.9%, compared with UPLC-MS/MS for a correlation of R2 = 0.999 and admirable validation. The promising application of the OsNHs can also be used in other bioprobes, and this bifunctional immunosensor analysis mode is suitable for diversified analytes.

Colorimetric detection of glucose by using hybrid nanostructures is a rapidly growing research hotspot. In this work, we established a simple route for synthesis of a class of multi-metal hybrid nanostructure materials and investigated their peroxidase-like performance for colorimetric detection of glucose. The hybrid nanomaterial (Fe3O4@Au-Pt) incorporated ferrosoferric oxide nanoparticals (Fe3O4 NPs) and the heterodimers composed of gold (Au) and platinum (Pt), which presents excellent morphology and structure. On the basis of our research, we constructed an easy and sensitive colorimetric sensor for the detection of glucose and hydrogen peroxide (H2O2), and the results indicated that the Fe3O4@Au-Pt hybrid nanomaterial possessed preferable peroxidase-like activity in comparison with other nanozyme materials and showing prominent selectivity for glucose detection. For H2O2, the sensor has a linear range of 0.05-120 μM and a relative lower limit of detection (LOD) of 0.018 μM. For glucose, the linear range is 0.05-140 μM with the LOD of 0.025 μM. It is envisioned that these hydrophilic hybrid nanostructures will be widely applied in sensing target analytes, biomedical diagnosis, and therapeutic applications in the future by taking advantage of their specific structure and excellent catalytic performance.

Herein, several components including mesoporous silica nanoparticles (MSNs) as a reservoir to load iron(III) ions for triggering a color change and a gold nanoparticle (AuNP) as an imaging agent prepared and stabilized using poly(tannic acid) (PTA) were combined together to successfully construct a kind of colorimetric assay for detection of protein. Iron(III) ion-loaded MSNs with an amine-modified surface were capped with the probe-labelled PTA-based shell@core AuNP (AuNP@PTA) through electrostatic interaction, forming probe-labelled PEGylated AuNP@PTA@Fe3+-loaded MSNs. The stronger interaction of the target analyte and the probe molecule would pull the gatekeeper AuNP@PTA away from the surface of the MSNs. The released iron(III) ion would coordinate with PTA, leading to AuNP aggregation for a visual color change. In this study, isozyme carbonic anhydrase II (CA II) and 4-sulfonamidebenzoic acid (SABA) were selected as the protein analyte and probe molecule. The SABA-labelled PEGylated AuNP@PTA@Fe3+-loaded MSNs showed good recognition specificity and sensitivity towards CA II with a visual detection limit of 50 nM, and the limit of detection (LOD) for the AuNP@PTA@Fe3+-loaded MSNs was about 15 nM using a signal-to-noise ratio of 3 with a UV-vis technique. Such proposed nanocomposites exhibited great potential as sensors in medical diagnostics and biological systems without the need for any separation process.

Summary Objective The objective of the present study is to develop and validate a simple, rapid stability indicating high performance liquid chromatography method for the simultaneous quantification of metformin, empagliflozin, linagliptin in bulk and pharmaceutical dosage form. Materials and methods The chromatographic separation was achieved on C18 X-bridge phenyl column (250 × 4.6 mm, 5 μm particle size). The pharmaceutical analytes were quantified by diode array detector in HPLC, eluted with acetonitrile and triethylamine (70:30) as mobile phase, monitored at 240 nm over a runtime of 7 min. Results The method was linear in the range of 50–750 μg/mL (r2 = 0.999) for metformin, 0.5–7.5 μg/mL (r2 = 0.999) for empagliflozin, 0.25–3.75 μg/mL (r2 = 0.999) for linagliptin. The percentage recoveries of these 3 drugs were within acceptable limits (99.2–100.8). The method was found to be precise as % RSD Conclusion The validated (as per ICH guidelines) rapid method can be routinely used in quality control lab for the quantification of metformin, empagliflozin and linagliptin in raw materials as well as in pharmaceutical dosage form.

Biological nanopores for single-pore sensing have the advantage of size homogeneity, structural reproducibility, and channel amenability. In order to translate this to clinical applications, the functional biological nanopore must be inserted into a stable system for high-throughput analysis. Here we report factors that control the rate of pore insertion into polymer membrane and analyte translocation through the channel of viral DNA packaging motors of Phi29, T3 and T7. The hydrophobicity of aminol or carboxyl terminals and their relation to the analyte translocation were investigated. It was found that both the size and the hydrophobicity of the pore terminus are critical factors for direct membrane insertion. An N-terminus or C-terminus hydrophobic mutation is crucial for governing insertion orientation and subsequent macromolecule translocation due to the one-way traffic property. The N- or C-modification led to two different modes of application. The C-terminal insertion permits translocation of analytes such as peptides to enter the channel through the N terminus, while N-terminus insertion prevents translocation but offers the measurement of gating as a sensing parameter, thus generating a tool for detection of markers. A urokinase-type Plasminogen Activator Receptor (uPAR) binding peptide was fused into the C-terminal of Phi29 nanopore to serve as a probe for uPAR protein detection. The uPAR has proven to be a predictive biomarker in several types of cancer, including breast cancer. With an N-terminal insertion, the binding of the uPAR antigen to individual peptide probe induced discretive steps of current reduction due to the induction of channel gating. The distinctive current signatures enabled us to distinguish uPAR positive and negative tumor cell lines. This finding provides a theoretical basis for a robust biological nanopore sensing system for high-throughput macromolecular sensing and tumor biomarker detection.

The safety of animal-origin foods has a major influence on human health. Although the use of veterinary drug additives (VDAs) significantly promotes the normal growth and development of animals against diseases, the excessive consumption of VDAs is always the most worrying part. Thus, realizing a direct and prompt detection of solid VDAs is highly desirable. In this study, we incorporated a functional unit of triazolo [4,5-c] pyridine (TP) in polymer semiconductors. The theoretical simulation demonstrates that TP can effectively form hydrogen bonding with the VDA molecules. Furthermore, highly sensitive polymeric field-effect transistors (PFETs) are fabricated through the configurational design of low-dimensional polymer networks (LDPNs). The conducting channel and hydrogen bonding sites of the LDPN-based PFETs could be directly exposed to the analytes, thereby effectively capturing VDA molecules and converting the stimuli into electrical signals. The obtained PFET-based solid sensors showed highly sensitive and stable detection of melamine powders at a low operation voltage of −5 V. The detection limit realized is lower than the international standards in animal feeds (2.5 ppm). These results demonstrate the promising applications of PFETs for high-efficiency and low-power-consumption sensors for solid detection or biological diagnosis.

We introduce a new approach for glucose oxidase (GOx) immobilization on graphene field-effect transistors (gFETs) to fabricate highly sensitive glucose sensors. The strategy relies on the electropolymerization of a layer of the copolymer poly(3-amino-benzylamine-co-aniline) (PABA) on graphene-based transistors. The synthesized polymer film provides the suitable electrostatic charge and non-denaturing environment for enzyme immobilization without the need of any chemical primer. Then, the local pH changes triggered by the enzyme-catalyzed oxidation produce a shift in the Dirac potential of the gFETs to more negative values, which is evidenced by the differences in the graphene channel conductivity and constitutes the signal transduction mechanism of the sensing devices. The assembled biosensors revealed a low LOD of 4.1 μM and were capable of detecting glucose in the range from 10 to 1000 μM in a flow configuration. Moreover, they showed sensitivity of −24.9 μA per decade of glucose concentration and a fast response time, with an average value of 190 seconds, while allowing the operation at small gate-source and drain-source voltages. Finally, the biosensors were able to successfully monitor the analyte in urine samples, showing their potential towards the fabrication of point-of-care glucose testing devices.

Funder: Please see main manuscript file., The detection of proteins is of central importance to biomolecular analysis and diagnostics. Typical immunosensing assays rely on surface-capture of target molecules, but this constraint can limit specificity, sensitivity, and the ability to obtain information beyond simple concentration measurements. Here we present a surface-free, single-molecule microfluidic sensing platform for direct digital protein biomarker detection in solution, termed digital immunosensor assay (DigitISA). DigitISA is based on microchip electrophoretic separation combined with single-molecule detection and enables absolute number/concentration quantification of proteins in a single, solution-phase step. Applying DigitISA to a range of targets including amyloid aggregates, exosomes, and biomolecular condensates, we demonstrate that the assay provides information beyond stoichiometric interactions, and enables characterization of immunochemistry, binding affinity, and protein biomarker abundance. Taken together, our results suggest a experimental paradigm for the sensing of protein biomarkers, which enables analyses of targets that are challenging to address using conventional immunosensing approaches.

In this work, an innovative colorimetric assay method for the determination of carcinoembryonic antigen (CEA) is developed with aptamer probes utilized as recognition element. DNA hybridization chain reaction (HCR) is used as signal amplification technique, and peroxidase-mimicking hemin/G-quadruplex-assisted catalytic oxidation of 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) is deployed as signal reporting mechanism. The detection principle was firstly verified by using gel electrophoresis analysis and absorbance measurements. After condition optimization, a detection limit was theoretically determined as 24.8 ng/mL. Furthermore, the method exhibited good selectivity and satisfactory recovery rates (92.2~108.6%) in serum samples. Moreover, the sensing scheme is easily extended for the detection of other analytes via similar target-aptamer recognition principle. To sum up, this is an enzyme- and label-free, cost-effective yet signal-amplifiable assay scheme for the determination of tumor markers with promising simplicity and selectivity, practical utility, and potential universality. This article is protected by copyright. All rights reserved.

In this study, a fully automated two-dimensional liquid chromatography (2D-LC) system was used for the investigation of the clean-up effect and was compared with a traditional Quick Easy Cheap Effective Rugged and Safe (QuEChERS) liquid chromatography (LC) method. The focus of those investigations was on negative electrospray ionization (ESI) mode. For that purpose, matrix fingerprinting profiles were created. The results allowed a comparison of both methods regarding the estimation of the number and the polarity of detected compounds. Moreover, the results of the present study were compared with the results generated in positive ESI mode (presented in a previous study). Furthermore, the two methods were compared with regard to matrix effects (ME) of 321 analytes in positive ESI mode and 96 analytes in negative ESI mode. In general, fewer compounds could be detected when 2D-LC and/or the negative ESI mode was used. Especially, very polar compounds with m/z values >1000 could be separated and could not be detected anymore when 2D-LC was applied. Furthermore, the best results were obtained for most analytes when 2D-LC was used, although the extent of ME seemed to be higher with 2D-LC.

Surface plasmon resonance (SPR) is used to infer information about a sample that is in contact with an Au-coated glass slide coupled to the SPR prism. Shifts in the angle of the "SPR minimum reflection" can be related to changes in the refractive index (and/or thickness) of the sample that is in contact with the Au film, which can then be used to determine the concentration of an analyte in that sample. Here, we show that by depositing a layer of poly(N-isopropylacrylamide-co-acrylic acid) [p(NIPAm-co-AAc)] microgel on the SPR's Au film, with a subsequent layer of Au deposited on top of the microgels, the sensitivity of SPR to changes in solution properties can be enhanced. We investigated the sensitivity of the SPR to changes in the temperature of water in contact with the SPR's Au film as a function of the microgel immobilization density and the thickness of the Au layer deposited on the microgel layer. The data revealed that the SPR's Au film densely coated with microgels, with 5 nm of Au deposited, exhibited the maximal enhancement. The plasmon coupling effect between the additional Au film on the microgels and the SPR's Au film was further confirmed by 3D finite difference time domain simulations.

A simple, selective and rapid ultra performance liquid chromatography-tandem mass spectrometry method was developed and validated for the simultaneous estimation of dolutegravir, lamivudine and tenofovir in bulk and tablet dosage form. Chromatographic separation was attained on Acquity Ethylene Bridged Hybrid (BEH) C18 column (50 × 2.1 mm, 3.5 µm), using a mixture of acetonitrile and 0.1% formic acid in water (60:40, v/v) as a mobile phase at a flow rate of 0.12 mL/min. The total run time of analysis was 3.5 min. The analytes were detected using tandem mass spectrometry, operating in positive ionization and multiple reaction monitoring modes. The method's linearity was determined to be in the range of 10–150 ng/mL with r2 > 0.99. The proposed method was validated as per the International Council for Harmonization (ICH) guidelines, and the results were found well within the acceptance limits. The method was successfully applied for the simultaneous quantification of all the three analytes in the combined tablet dosage form.

Determining trace chromium in complex samples usually requires a sample preparation technique to isolate and extract the analytes from matrices and enrich them for trace analysis. Several methods h...

An extremely sensitive methodology for the determination of Ru was developed by coupling photochemical vapor generation (PVG) analyte introduction with inductively coupled plasma mass spectrometry (ICPMS). PVG was undertaken with a thin-film flow-through photoreactor in a medium comprising 8 M formic acid in the presence of 10 mg L⁻¹ Co²⁺ and 25 mg L⁻¹ Cd²⁺. The volatile product (presumably ruthenium pentacarbonyl) was generated in a flow injection mode, yielding an overall efficiency of 29% at a sample flow rate of 1.4 mL min⁻¹. The presence of both Co²⁺ and Cd²⁺ sensitizers enhanced PVG efficiency by 3,200-fold, permitting a 31 s irradiation time. Although enhanced efficiency (≈40%) could be obtained with increased Co²⁺ concentration, this was not suitable for routine use due to co-generation of cobalt carbonyl. Excellent repeatability (

Biomolecule detection based on surface-enhanced Raman scattering (SERS) for application to biosensors and bio-imaging requires the fabrication of SERS nanoprobes that can generate strong Raman signals as well as surface modifications for analyte-specific recognition and binding. Such requirements lead to disadvantages in terms of reproducibility and practicality, and thus, it has been difficult to apply biomolecule detection utilizing the advantages of the SERS phenomenon to actual clinically relevant analysis. To achieve reproducible and practical SERS signal generation in a biomolecule-specific manner without requiring the synthesis of nanostructures and their related surface modification to introduce molecules for specific recognition, we developed a new type of SERS probe formed by enzyme reactions in the presence of Raman reporters. By forming unique plasmonic structures, our method achieves the detection of biomolecules on chips with uniform and stable signals over long periods. To test the proposed approach, we applied it to a SERS-based immunohistochemistry assay and found successful multiplexed protein detection in brain tissue from transgenic mice.

A rapid and sensitive immunoassay for the simultaneous detection of thiabendazole (TBZ) and carbendazim (CBZ) was developed by using magnetic nanoparticles (MNPs) and two-color upconversion nanoparticles (UCNPs). The anti-thiabendazole and anti-carbendazim antibody were linked to NaYF4:Yb, Tm (emission at 474 nm) and NaYF4:Yb, Er (emission at 542 nm) UCNPs as multicolor signal probe, respectively. The thiabendazole and carbendazim coating antigen were connected to magnetic microspheres as capture probe, respectively. Based on a competitive format, capture probes compete with analytes to combine corresponding signal probes. Under excitation at 980 nm, the formed competitive immune-complex fluorescent signal at 474 and 542 nm indicates the concentration of thiabendazole and carbendazim, respectively. Under the optimal conditions, the 50% inhibiting concentration (IC50) and limit of detection (LOD, IC10) were 2.21 and 0.083 ng/mL for thiabendazole and 1.18 and 0.075 ng/mL for carbendazim, respectively. The average recoveries of thiabendazole and carbendazim in foods, including oyster mushroom, shiitake mushrooms, oranges and orange juice, ranged from 83.6 to 100.9% with the coefficient of variance (CV) of 1.0–9.8% for thiabendazole and ranged from 89.6 to 105.9% with the CV of 2.5 to 9.1% for carbendazim. This multiplexed analysis format has potential applications for rapid, efficient detection of multiple food risk factors.

Microscale-based separations are increasingly being applied in the field of metabolomics for the analysis of small-molecule metabolites. These methods have the potential to provide improved sensitivity, less solvent waste, and reduced sample-size requirements. Ion-pair free microflow-based global metabolomics methods, which we recently reported, were further compared to analytical flow ion-pairing reagent containing methods using a sample set from a urea cycle disorder (UCD) mouse model. Mouse urine and brain homogenate samples representing healthy, diseased, and disease-treated animals were analyzed by both methods. Data processing was performed using univariate and multivariate techniques followed by analyte trend analysis. The microflow methods performed comparably to the analytical flow ion-pairing methods with the ability to separate the three sample groups when analyzed by partial least-squares analysis. The number of detected metabolic features present after each data processing step was similar between the microflow-based methods and the ion-pairing methods in the negative ionization mode. The observed analyte trend and coverage of known UCD biomarkers were the same for both evaluated approaches. The 12.5-fold reduction in sample injection volume required for the microflow-based separations highlights the potential of this method to support studies with sample-size limitations.

A method for analyzing fluoroquinolone antibiotics, viz. enrofloxacin and ciprofloxacin, in chicken meat has been developed with the aim of reducing the total analysis time. Certified reference materials (CRMs) for the chicken meat powder, i.e. KRISS CRM 108-03-003 and KRISS CRM 108-03-004, were used for method development. Isotope dilution ultra-performance liquid chromatography–mass spectrometry (ID-UPLC-MS) was employed for sample analysis. The liquid chromatography (LC) conditions were optimized with an “in-sample addition” method using a trace amount of ethylenediaminetetraacetic acid (EDTA), in which EDTA was not added to the mobile phase but to the sample alone. The optimal EDTA amount was determined, and its influence on the separation of the target analyte was investigated. After the feasibility of the “in sample-addition” approach was confirmed, we attempted to improve the previously established sample pretreatment method by varying the conditions for liquid–liquid extraction and solid phase extraction. The developed method was validated using raw chicken meat for recovery evaluation. The “in-sample addition” method afforded highly improved peak shapes without adversely affecting the LC/MS method performance, and the quantification values of enrofloxacin and ciprofloxacin corresponded well with those obtained using the established method. Consequently, the total analysis time for the determination of enrofloxacin and ciprofloxacin in chicken meat CRM could be reduced by simplifying the sample pretreatment procedure. The developed method delivered better reliability compared to the previously established analytical method. The method will be used for stability monitoring of the chicken meat CRM and the development of other meat CRMs for fluoroquinolone analysis and improved food safety applications.

Background Even though the use of β-agonists in livestock has been banned in many countries, β-agonists abuse is still out of control owing to time-consuming and complicated determination techniques. Objective This study applied ambient thermal desorption electrospray ionization mass spectrometry (TD-ESI/MS) as a new strategy for rapidly screening seven β-agonists. As it does not require pretreatment, this method allows on-site and real-time detection. Method Samples were collected directly using a simple metal probe. Analytes on the probe were desorbed and ionized by TD-ESI, after which analyte ions were detected using a triple quadrupole mass analyser. Results The results showed that a thermal desorption temperature of 320°C and nebulizer pressure of 3 psi demonstrated the highest sensitivity. Excellent linear relationships were obtained in concentrations ranging from 10 to 400 μg/kg. The limit of detection of the method was 1–10 μg/kg with RSDs of 7.2–14.6%. A positive sample was verified using HPLC coupled with triple quadrupole tandem mass spectrometry, consistent with the results from TD-ESI/MS. Conclusion This method was the first to apply TD-ESI/MS to rapid analyse β-agonists in livestock hair and was proven to be accurate, fast, and environmentally friendly. Because qualitative screening for each analysis was completed within approximately 1 min, the method could contribute to preventing non-compliance in the flow of animal-derived food to the market. Highlights This method was a promising method for the rapid screening of β-agonists in livestock hair at any time of breeding without imposing any harm to animal which proven to be a rapid, in situ, and non-destructive detection technique without any pretreatment.

Coffee is the most popular beverage after water and is consumed worldwide. Its consumption in high amount may pose health risks. Moreover, most of the market's available coffee beans products do not mention their proper amount of caffeine. To develop an efficient and reliable method of caffeine extraction in commercial coffee brands, automated solvent extraction was used along with a sensitive, rapid and precise method for simultaneous analysis of caffeine using ultra-high performance liquid chromatography coupled with an ID-X-Orbitrap Mass spectrometer (UHPLC-IDX-Orbitrap-MS). The results showed comparable values to previously published studies. The lowest average amount of caffeine (µg/g) was in the Colombian brand (6.7) and Brazil-CF brand (7.1). The highest average amount of caffeine (µg/g) was in Brazil-C brand (10) and Kenyan brand (10.4). For the rest of coffee beans extract, the caffeine amount was similar varying between 7.7 and 9.7 µg/g. The results demonstrated that the proposed procedure is highly reproducible (with an RSD error of less than 20% for most of the analytes) and the recovery levels of the target compound (i.e., caffeine) varied in the range of 45–88%. Moreover, the limit of detection of caffeine was equal to 1 ng mL−1. This was a pilot study to examine caffeine extraction from different coffee sources and a future study will identify all active compounds.

Aflatoxin B1 (AFB1), as the main metabolite, found in aflatoxin contaminated food is highly toxic and strictly controlled in many countries. Aiming to construct a simplified, highly sensitive, and accurate quantitative detection platform of AFB1 in ordinary conditions, a one-step competitive quantum dot-labeled immunosorbent assay (cQLISA) was developed using biocompatible CdSe/ZnS core/shell quantum dots (QDs). The quantitative detection of AFB1 can be achieved by adding food sample and QD-AFB1 antibody simultaneously, with an analytical range between 1 and 40 ng/mL. The half maximal inhibitory concentration (IC50) of this method is 542 pg/mL and the limit of detection (LOD) is 56 pg/mL for standard samples, demonstrating a twofold improvement in sensitivity compared with that of the commercial enzyme-linked immunosorbent assay (ELISA) test kit. The recovery rates of negative corn spike-in samples range from 87.01 to114.7% (CV

Novel psychoactive substances (NPS) continue to represent a threat to public health and safety. The number of new drugs in the latest emergent synthetic opioid class—the 2-benzylbenzimidazole analogs—also called the nitazenes—has begun to dominate the current new synthetic opioid (NSO) subclass of NPS. We describe a liquid chromatography–tandem quadrupole mass spectrometry method for the quantification of nine analogs and/or metabolites of drugs in this series: isotonitazene, metonitazene, protonitazene, etonitazene, clonitazene, flunitazene, N-desethyl isotonitazene, 5-amino isotonitazene and 4ʹ-hydroxy nitazene in human whole blood, urine, and tissue. Samples were prepared for analysis using a basic liquid–liquid extraction. Chromatographic separation was achieved using a C-18 analytical column. Multiple reaction monitoring mode was used for detection. The calibration range for the analytes was 0.5–50 ng/mL (except for 5-amino isotonitazene, which was 1.0–50 ng/mL). The limit of detection was 0.1 ng/mL, and the limit of quantitation was 0.5 ng/mL. The method had no carryover or interferences. Ionization enhancement was observed but did not affect quantitation. All analytes passed the method validation assessment. Authentic human samples suspected of containing NSOs were obtained from a medical examiner and coroner offices, as well as partnering forensic toxicology laboratories. Isotonitazene was confirmed in 92 blood samples, and its metabolites were confirmed across various matrices. Metonitazene (n = 35), flunitazene (n = 5), protonitazene (n = 3), etodesnitazene (n = 2) and butonitazene (n = 1) were also detected in cases. These newly emerging 2-benzylbenzimidazole analogs were commonly found in combination with NPS benzodiazepines and opioids (e.g., flualprazolam, fentanyl). Nitazene analogs are potent esoteric drugs that may not be identified during routine toxicological screening, and specialized assays based on sensitive instrumentation are needed to accurately characterize these NSOs.

A surface plasmon resonance-based biosensor is investigated theoretically and mathematically. The proposed sensor is a multilayered structure of titanium disilicide and graphene along with Ag metal. These layers are stacked over a BK7 prism. The thickness of the metal, titanium disilicide, and graphene layers is 45 nm, 2 nm, and 1.7 nm, respectively. Titanium disilicide is an air-stable two-dimensional (2D) material. The performance of the biosensor is analyzed using the attenuated total reflection (ATR) method. The proposed configuration exhibits a maximum sensitivity of 183.4°/RIU, being 47.45% higher than that of the conventional sensor. The designed sensor has wide applications in chemical, medical, and biological analyte sensing.

In this study, cyclofructan (CF)-, cyclodextrin (CD)-, and polysaccharide-based chiral stationary phases (CSPs) were exploited in high-performance liquid chromatography (HPLC) for the chiral separations of different clinically and pharmaceutically important compounds. In particular, R-naphthylethyl carbamate CF6 (RN–CF6), 3,5-dimethylphenyl carbamate CF7 (DMP–CF7), neutral beta cyclodextrin (β–CD), 3,5-dimethylphenyl carbamate β–CD (DMP–β–CD), and cellulose tris-(3,5-dimethylphenylcarbamate) (Cellulose–Tris DMP) columns were utilized under isocratic elution. The performance of these CSPs as chiral separation media was evaluated by use of nine analytes: acidic, basic, and amphiprotic. A possible correlation between the functional groups of these analytes and the chiral-recognition ability of each chiral column was also examined. The enantioseparations were optimized by varying different parameters, such as mobile phase additives, column temperature, and flow rate. Finally, a comparison was made between all CSPs, and it was expressed in terms of resolution (RS), efficiency (N), selectivity (α), retention factors (k1′, k2′) and analysis time (tR1, tR2). It was observed that RN–CF6 was the most suitable and efficient CSP for the chiral separation of various types of analytes, including acids, primary and tertiary amines, alcohols, and many neutral compounds. It was the only CSP that provided baseline enantioseparation of thyroxine (RS = 1.6) and cetirizine (RS = 2.0).

Tobacco use is the leading preventable cause of premature disease and death in the United States. Approximately, 34 million U.S. adults currently smoke cigarettes. We developed a method for automated sample preparation and liquid chromatography-tandem mass spectrometry quantitation of 14 tobacco-related analytes: nicotine (NICF), cotinine (COTF), trans-3′-hydroxycotinine (HCTF), menthol glucuronide (MEG), anabasine (ANBF), anatabine (ANTF), isonicoteine (ISNT), myosmine (MYOS), beta-nicotyrine (BNTR), bupropion (BUPR), cytisine (CYTI), varenicline (VARE), arecaidine (ARD), and arecoline (ARL). The method includes automated solid-phase extraction using customized positive-pressure functions. The preparation scheme has the capacity to process a batch of 96 samples within 4 h with greater than 88% recovery for all analytes. The 14 analytes, separated within 4.15 min using reversed-phase liquid chromatography, were determined using a triple-quadrupole mass spectrometer with atmospheric-pressure chemical ionization and multiple reaction monitoring in negative and positive ionization modes. Wide quantitation ranges, within 1.2–72,000 ng/mL, were established especially for COTF, HCTF, MEG, and NICF to quantify the broad range of biomarker concentrations found in the U.S. population. The method accuracy is above 90% while the overall imprecision is below 7%. Finally, we tested urine samples from 90 smokers and observed detection rates of over 98% for six analytes with urinary HCTF and MEG concentrations ranging from 200–14,100 and 60–57,100 ng/mL, respectively. This high throughput analytical process can prepare and analyze a sample in 9 min and along with the 14-compound analyte panel can be useful for tobacco-exposure studies, in smoking-cessation programs, and for detecting changes in exposure related to tobacco products and their use.

The Vitamin D External Quality Assessment Scheme (DEQAS) distributes human serum samples four times per year to over 1000 participants worldwide for the determination of total serum 25-hydroxyvitamin D [25(OH)D)]. These samples are stored at −40 °C prior to distribution and the participants are instructed to store the samples frozen at −20 °C or lower after receipt; however, the samples are shipped to participants at ambient conditions (i.e., no temperature control). To address the question of whether shipment at ambient conditions is sufficient for reliable performance of various 25(OH)D assays, the equivalence of DEQAS human serum samples shipped under frozen and ambient conditions was assessed. As part of a Vitamin D Standardization Program (VDSP) commutability study, two sets of the same nine DEQAS samples were shipped to participants at ambient temperature and frozen on dry ice. Twenty-eight laboratories participated in this study and provided 34 sets of results for the measurement of 25(OH)D using 20 ligand binding assays and 14 liquid chromatography–tandem mass spectrometry (LC–MS/MS) methods. Equivalence of the assay response for the frozen versus ambient DEQAS samples for each assay was evaluated using multi-level modeling, paired t-tests including a false discovery rate (FDR) approach, and ordinary least squares linear regression analysis of frozen versus ambient results. Using the paired t-test and confirmed by FDR testing, differences in the results for the ambient and frozen samples were found to be statistically significant at p

In the present study, a peptide nanotube functionalized polydopamine (p-Dop) based molecularly imprinted (MIP) sensor system was constructed, characterized, and studied for the impedimetric sensing of an organophosphorus pesticide, malathion (MLT). Electropolymerization in the presence of a template (MLT) was utilized as a convenient and effective strategy to generate imprinted p-Dop films on peptide nanotubes (PNTs) modified graphite electrodes (PGEs). Upon the removal of template, the adsorption of MLT on the specific cavities formed in the MIP film was tracked using electrochemical impedance spectroscopy (EIS). To attain optimal sensor response, experimental conditions, such as film thickness, analyte/functional monomer ratio, and desorption/adsorption time, were analyzed. The obtained MIP(p-Dop)-PNT-PGE sensor exhibited high sensitivity for electrochemical MLT analysis with a wide dynamic detection range of 13 pg mL−1 – 1.3 µg mL−1 and a LOD of 1.39 pg mL−1. The combination of a bio-inspired p-Dop-based MIP with the EIS technique allowed excellent sensitivity and selectivity toward MLT sensing which also yielded high recoveries in real samples. The success of this research strategy in real samples revealed its potential for various future environmental applications.

An efficient, sensitive and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) chiral analysis method was established for determination of chloroquine and hydroxychloroquine enantiomers in rat liver microsomes. Effects of polysaccharide chiral stationary phases and basic additives on chiral separations of two analytes were discussed in detail. Amylose tris(3, 5-dimethylphenylcarbamate)-coated chiral stationary phase showed the best separation performance for them with acetonitrile-diethylamine-ethanol-diethylamine mixture (90:0.1:10:0.1, v/v/v/v) among four chiral stationary phases. Then, multiple reaction monitoring mode was selected as the data acquisition for determination of two pairs of enantiomers. The proposed LC-MS/MS chiral analysis method was validated in terms of linearity, accuracy, precision, and specificity. Good linearity with correlation coefficient over 0.998 was obtained in the concentration range of 0.05-5 μM. Limits of quantification for chloroquine and hydroxychloroquine enantiomers were 5.0 and 1.0 nM, respectively. The recoveries ranged from 81.14% to 111.09%. The intra-day and inter-day relative standard deviation were less than 6.5%. Moreover, concentrations of chloroquine and hydroxychloroquine enantiomers in rat liver microsomes were determined through the proposed LC-MS/MS analysis method. After incubated with rat liver microsomes for 10 min, the enantiomeric factor of hydroxychloroquine decreased from 0.50 to 0.45 (p

Objectives Automated storage and retrieval modules (SRM), as part of total lab automation (TLA) systems, offer tremendous practical and economic benefits. In contrast to manual storage systems, SRMs indicate continuous motion of samples and may leave samples prone to temperature fluctuations. This study investigates analyte stability in serum and heparin plasma within an automated storage module. Methods The stability of 28 common biochemistry analytes was investigated using 57 freshly obtained routine serum samples and 42 lithium-heparin plasma samples. Following baseline measurement, samples were stored at 2–8 °C in the automated SRM of the Accelerator a3600 TLA and reanalyzed at fixed time points (2, 4, 8, 12, 24, 48 and 72 h) on the Abbott Architect c16000 chemistry analyzer. The concentration at each time point was expressed as %-difference to the baseline value and mean results were compared to the criteria for desirable bias derived from the biological variation database. Results Nine of the analytes exceeded the bias criterion within 72 h after initial measurement in either serum samples, plasma samples or both. Lithium-heparin plasma samples showed increasing values for phosphor, potassium and lactate dehydrogenase (LDH), which were only considered stable for respectively 24, 12 and 4 h, glucose was considered stable for 8 h. Electrolyte concentrations and LDH activity significantly increased in serum samples beyond 48 h. Bicarbonate should not be performed as add-on test at all. Conclusions The presented data indicate that the conditions within an SRM have no clinical impact on sample stability and allow stable measurement of routine analytes within 72 h, comparable to manual storage facilities.

Diffusion-ordered nuclear magnetic resonance spectroscopy (DOSY), dubbed chromatographic NMR spectroscopy, can be used to simultaneously distinguish and identify the structures of components in a mixture according to their different diffusion coefficients. In order to improve the resolution of DOSY on the diffusion dimension, a lot of matrices have been developed to expand the application of this technique in mixture analysis. However, there is no matrix to detect the mixture of ionic liquids (ILs). Herein, we introduced a new matrix, poly(sodium-p-styrenesulfonate) (PSSNa), which can be used to fully separate the signals of a mixture of different ILs. The mixture of three imidazolium ILs of 1-butyl-3-methylimidazolium bromide, 1-allyl-3-vinylimidazolium bromide and 1-carboxymethyl-3-methylimidazolium chloride could be fully distinguished by virtue of their different interactions with PSSNa. We also investigated the influences of PSSNa amount, IL concentration and solution pH value on the signal resolution of mixtures. This work provides a scientific reference for the analysis of the other IL analytes.

A new liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) method has been developed and validated for alfuzosin quantification in human plasma using amlodipine as an internal standard (IS). The analyte and IS were extracted using a mixture of diethyl ether-dichloroethane (70 : 30, v/v). The samples were separated on a Waters XBridge HILIC column, where a tandem mass spectrometer with a turbo ion spray interface was used for analytes detection. The chromatographic run time of the method was 4.0 min. The method was linear over the concentration range of 0.20–30 ng/mL for alfuzosin. On evaluating the selectivity of the method, no interference was observed for the two MRM transitions of alfuzosin and MRM transition for amlodipine at their respective retention times. The mean matrix effect for alfuzosin was excellent at 99.9%. The method was carefully tested for linearity, system suitability, precision and accuracy. Intra- and inter-day accuracy and precision were evaluated at three quality control levels (LQC, MQC and HQC). Stability of alfuzosin was evaluated under diverse storage conditions. The percentage accuracy for 1 : 2 diluted samples was 93.2% of their nominal concentration with the coefficient of variation of 5.6%. Hence, a highly precise, accurate, rapid, selective and sensitive LC-ESI-MS/MS method has been developed and validated for the quantification of alfuzosin in human plasma using amlodipine as the internal standard.

Aisha Javed,1 Shah Rukh Abbas,1 Muhammad Uzair Hashmi,1 Noor Ul Ain Babar,2 Irshad Hussain2 1Department of Industrial Biotechnology, Atta-ur-Rahman School of Applied Biosciences, National University of Science and Technology, Islamabad, 44000, Pakistan; 2Department of Chemistry, Syed Babar Ali School of Science and Engineering, Lahore University of Management Sciences, Lahore, 54792, PakistanCorrespondence: Shah Rukh AbbasDepartment of Industrial Biotechnology, Atta-ur-Rahman School of Applied Biosciences, National University of Sciences and Technology, H-12, Islamabad, PakistanTel +92 3355449622; +92 51-9085-6125Email sabbas@asab.nust.edu.pkBackground: Mycobacterium tuberculosis’ rapid detection is still a formidable challenge to have control over the lethal disease. New diagnostic methods such as LED fluorescence microscopy, Genexpert, Interferon Gamma Release Assay (IGRA) are limited on efficacy spectrum owing to their high cost, time-intensive and laborious nature, in addition their low sensitivity hinders their robustness and portability. Electroanalytical methods are now being considered as an excellent alternative, being currently employed for efficient detection of the analytes with the potential of being portable. This report suggests label-free electrochemical detection of Mycobacterium tuberculosis (Mtb) via its marker, insertion sequence (IS6110).Methods: In this pursuit, graphene oxide-chitosan nanocomposite (GO-CHI), a biocompatible matrix, having a large electroactive area with an overall positively charged surface, is fabricated and characterized. The obtained GO-CHI nanocomposite is then immobilized on the ITO surface to form a positively functionalized electrochemical sensor for the detection of Mtb. DNA probe, specific for the IS6110, was electrostatically anchored on a positively charged electrode surface and the resistance of charge transfer was investigated for the sensitive and specific (complementary vs non-complementary) detection of Mtb by cyclic voltammetry and differential pulse voltammetry techniques.Results: The cyclic voltammetry was found to be diffusion controlled facilitating the absorption of analyte on the electrode surface. The label-free “genosensor” was found to detect a hybridization efficiency with a limit of detection of 3.4 pM, and correlation coefficient R2=0.99 when analysed over a range of concentrations of DNA from 7.86 pM to 94.3pM. The genosensor was also able to detect target DNA from raw sputum samples of clinical isolates without DNA purification.Conclusion: This electrochemical genosensor provides high sensitivity and specificity; thus offering a promising platform for clinical diagnosis of TB and other infectious diseases in general.Keywords: label-free detection, DNA genosensor, Mycobacterium detection, tuberculosis, graphene oxide nanocomposites, GO nanocomposites electrochemical sensing

Summary Objectives The main aim of the study was to develop an economical, insightful, accurate and simple RP-HPLC-DAD method with high precision and good sensitivity for concurrent determination of Tenofovir disoproxil fumarate, Doravirine and Lamivudine in blended bulk form and their combined tablet form. Material and methods A method with Ascentis C18 (150 × 4.6 mm, 5 μm) column, mobile phase ratio of 0.1% ortho phosphoric acid and Acetonitrile in 70:30 (v/v), 1 mL/min flow rate and detection wavelength of 260 nm was highly proficient in effective separation of all three drugs. The developed method was validated in accordance with ICH specifications. Results The retention times of Doravirine, Lamivudine and Tenofovir disoproxil fumarate observed were 2.4, 2.9, and 3.6 min, respectively. The linear responses were observed for Doravirine, Lamivudine and Tenofovir disoproxil fumarate in the range of 12.5–75 μg/mL, 75–225 μg/mL and 75–225 μg/mL, respectively. The limit of detection and quantification values were calculated to be 0.36 μg/mL and 0.11 μg/mL for Lamivudine, 0.55 μg/mL and 1.66 μg/mL for Tenofovir disoproxil fumarate and 0.03 μg/mL and 0.09 μg/mL for Doravirine. The % RSD values of the intra-day and inter-day precision were calculated in the range of 0.134–1.749. The mean percentage recovery of all three analytes was in the range of 98.85–100.18%. The statistical results of the validation parameters ensured that the method was accurate, specific, and precise with good sensitivity. Investigation of analytes under different stressed conditions ensures the stability of analytes reflecting the stability indication of the method. The developed method has high proficiency in separation of Tenofovir disoproxil fumarate, Doravirine and Lamivudine. The degradation products generated due to stress conditions also separated with good resolution. Conclusion The current method is a stability-indicating assay method consisting of appropriate specificity, accuracy, precision and sensitivity. The developed method has a good potential to be adopted by the pharmaceutical industrial sector.

Background To establish a simple, sensitive, accurate, precise, efficient, economical RP-UPLC method for simultaneous estimation of Doravirine, Lamivudine and Tenofovir disoproxil fumarate in bulk and their combined pharmaceutical formulations. Optimization of Chromatographic separation was achieved on analytical column HSS C18 (100 × 2.1 mm, 1.8 μ) maintained at temperature 30 °C and mobile phase consisting of 0.01 N Potassium dihydrogen orthophosphate buffer (pH-4.8) and acetonitrile in the ratio 60:40 v/v and at a flow rate 0.3 mL/min in isocratic mode. The injection volume was set as 1 µl detection wavelength is 260 nm. The proposed method validation was done as per International Council on Harmonization Q2 (R1) guidelines. Results Doravirine, Lamivudine and Tenofovir disoproxil fumarate were eluted at retention times of 1.2, 1.5, and 1.8 min respectively. The proposed method was identified an excellent linearity over concentration range of 12.5–75.0 µg/mL for Doravirine and 37.5–225.0 µg/mL for Lamivudine and 37.5–225.0 µg/mL for Tenofovir disoproxil fumarate. The percentage relative standard deviation for intra-day and inter-day precision of the present method was less than 2% for Doravirine, Lamivudine and Tenofovir disoproxil fumarate. Accuracy of the present method was evaluated by recovery studies which were in the range of 99.62–99.88% for Doravirine and 98.78–99.44% for Lamivudine and 99.67–100.52% for Tenofovir disoproxil fumarate. The limit of detection and limit of quantification were found to be 0.249 µg/mL and 0.756 µg/mL for Doravirine and 0.24 µg/mL and 0.727 µg/mL for Lamivudine and 0.797 µg/mL and 2.966 µg/mL for Tenofovir disoproxil fumarate. Forced degradation studies were carried out under various stress conditions like acid, base, peroxide, thermal, photo and neutral conditions. Conclusions The present method makes sure about no degraded impurity peak interference at the retention time of analyte peak hence can be applied for quality control investigation of Doravirine, Lamivudine and Tenofovir disoproxil fumarate in bulk and pharmaceutical formulations.

Colorimetric reporter enzymes are useful for generating eye-readable biosensor readouts that do not require a device to interpret, an attractive property for applications in remote or developing parts of the world. The use of cell-free gene expression further facilitates such applications via amenability to lyophilization and incorporation into materials like paper. Currently, detection of multiple analytes simultaneously with these systems requires multiple reactions or a device. Here we evaluate seven enzymes and 15 corresponding substrates for functionality in a particular cell-free expression system known as PURE. We report eight enzyme/substrate pairs spanning four enzymes that are compatible with PURE. Of the four enzymes, three pairings exhibit no cross-reactivity. We finally show that at least one pairing can be used to create a third color when both are present, highlighting the potential use of these reporters for multiplex sensing.