High-performance Liquid Chromatography Detection Research Articles

Development of a 3D Printed Chemiluminescence Smartphone Detector for High Performance Liquid Chromatography

A novel chemiluminescence detector for high-performance liquid chromatography (HPLC) was designed that incorporates a smartphone as photodetector, signal processor, and data storage unit. All detector components, including flow cell, flow cell holder, mixers, and housing, were fabricated by 3D printing and integration of the smartphone and 3D printed components provides a portable, low-cost, and user-friendly device. The detector was applied in the determination of carbamazepine using HPLC through a chemiluminescence reaction with tris(2,2′-bipyridyl)ruthenium(II) and cerium sulfate. Design and fabrication of the flow cell using two fabrication methods—3D printing and laser cutting—along with the integration of a mixer and the optimization of smartphone parameters were investigated. To expand the applicability of the detector for other analytes and different chemiluminescence reactions, detection of three piperazine derivatives by reaction with tris(2,2′-bipyridyl)ruthenium(II) and four phenethylamine compounds via reaction with acidic potassium permanganate reagent was studied. The linear dynamic range, limit of detection, and limit of quantification of the detector for the determination of carbamazepine were measured as 3.0–30.0 mg/L, 1.0 mg/L, and 3.0 mg/L, respectively, using the Samsung S20 smartphone device. Intraday and interday precision was evaluated for carbamazepine, with relative standard deviation (RSD) values for intraday precision ranging from 1.7% to 6.2% (n = 3) and interday precision RSD values ranging from 3.2% to 4.8% (n = 9).

Simultaneous extraction and deglycosylation for flavonoid analysis in Ginkgo biloba products using a two-phase deep eutectic solvent system

Total flavonoid content is one of the most important quality indicators of Ginkgo biloba leaves and its products, but the complex pretreatment procedures and the excessive use of organic solvents complicate the detection process. In response, an innovative and efficient two-phase deep eutectic solvent (DES) system—composed of an acidic hydrophilic DES and a hydrophobic DES—was developed to quantify flavonoids for quality control of G. biloba products. High-performance liquid chromatography (HPLC) detection conditions were optimized, followed by systematic screening and design of the two-phase DES system through adjustments to polarity, hydrophilicity, and acidity. Optimal pretreatment conditions were established through operating condition optimization. This approach enabled simultaneous extraction, deglycosylation, and enrichment of all flavonoids from G. biloba samples into the hydrophobic phase in a single step. Method validation showed strong linearity with a correlation coefficient (R2 > 0.9995). The detection (LOD) and quantification (LOQ) limits were between 0.35 and 0.37 mg/kg, and 0.92 and 0.96 mg/kg, respectively. The method also exhibited high sensitivity and accuracy, with a recovery rate of 96.91–100.39%. A greenness assessment with the ComplexGAPI tool confirmed this method’s eco-friendly advantages over conventional techniques, aligning well with green chemistry and economic principles. Ultimately, this technique effectively measured flavonoid levels in different G. biloba products. The method developed in this work not only enhances the efficiency of simultaneous extraction and deglycosylation by modifying the polarity, hydrophilicity, and acidity of the two-phase DES system, but also offers valuable insights for the advancement of environmentally friendly analysis technology for flavonoids.

Comparing ergosterol identification by HPLC with fungal serology in human sera

BackgroundErgosterol, a predominant sterol in fungal cell membranes, holds promise as a specific marker for detecting fungal presence in human samples. This study investigated the performance of ergosterol detection compared to serological tests in identifying the presence of fungi in human sera. MethodsEighty-four non-duplicate human sera were analyzed by high performance liquid chromatography (HPLC) for ergosterol detection. Results were compared to serological tests for Aspergillus antigen, Candida antigen, Cryptococcus antigen, Aspergillus antibody and Candida antibody performed on the same patient sera. ResultsOut of the 84 serum samples, 51 (60.7 %) were positive for ergosterol. Among the 33 serology-positive sera, 26 (78.8 %) were also ergosterol-positive. In contrast, 26 out of 51 (51 %) serology-negative sera (including 20 negative controls) tested negative for ergosterol. Seven out of 33 (21.2 %) serology-positive sera were ergosterol-negative, while 25 out of 51 (49 %) serology-negative sera were ergosterol-positive. Compared to serological tests, HPLC detection of ergosterol had a sensitivity of 78.8 %, specificity of 51 %, positive predictive value of 51 %, negative predictive value of 78.8 % and overall accuracy of 61.9 %. ConclusionsErgosterol detection may serve as a useful supplementary tool for identifying fungi in human sera, acting as a broad-spectrum diagnostic marker. However, further research with larger sample sizes and clinical comparisons is needed to validate these findings.

Elastin-like polypeptide-functionalized nanobody for column-free immunoaffinity purification of aflatoxin B1.

A column-free immunoaffinity purification (CFIP) technique for sample preparation of aflatoxin B1 (AFB1) was developed using an AFB1-specific nanobody (named G8) and an elastin-like polypeptide (ELP). The reversible phase transition between liquid and solid in response to temperature changes was exhibited by the ELP which was derived from human elastin. The G8 was tagged with ELPs of various lengths (20, 40, 60, and 80 repeat units) at the C-terminus using recursive directional ligation (RDL). Coding sequences were then subcloned into pET30a at the multiple cloning sites. Bioactive recombinant proteins were produced by expressing them as inclusion bodies in Escherichia coli BL21 (DE3), then dissolved and refolded. Analysis by indirect competitive enzyme-linked immunosorbent assay (icELISA) and transition temperature (Tt) measurement confirmed that the refolded G8-ELPs preserved the ability to recognize AFB1 as well as phase transition when the temperature rose above Tt. To establish the optimal conditions for cleaning AFB1, the effects of various parameters on recovery were investigated. The recovery in ELISA tests was 95 ± 3.67% under the optimized CFIP workflow. Furthermore, the CFIP-prepared samples were applied for high-performance liquid chromatography (HPLC) detection. The recovery in the CFIP-HPLC test ranged from 54 ± 1.86% to 98 ± 3.58% for maize, rice, soy sauce, and vegetable oil samples. To the best of our knowledge, this is the first report combining the function of both nanobody and ELP to develop a cleanup technique for small molecules in a complex matrix. The CFIP for the sample pretreatment was easy to use and inexpensive. In contrast to conventional immunosensitivity materials, the reagent utilized in the CFIP was entirely biosynthesized without any chemical coupling reactions. This suggests that the nanobody-ELP may serve as a useful dual-functional reagent for the development of sample cleaning or purification methods.

Fusarium andiyazi, a Pathogenic Species Associated with Rice Bakanae Disease in Malaysia.

Rice is the main staple food crops for the Malaysian population. Rice is also susceptible to bakanae diseases caused by some Fusarium species and reducing yield, and quality of rice also profit. In this study, several rice fields were surveyed to collect Fusarium isolates associated with bakanae disease. The morphological features of Fusarium andiyazi isolates found on infected rice plants were identified in this investigation. For biological species identification, MAT-1 (Mating type idiomorphs) bearing isolates were crossed with MAT-2 isolates. Crossing was succeeded between cross of two different mating type bearing field isolates. Consequently, there is a possibility of exchange of genetic material within the F. andiyazi population in Malaysia. The identity of the isolates was further determined up to the species level by comparing DNA sequences and phylogenetic analysis of two genes. The phylogenetic analyses of the joined dataset of translation elongation factor 1-alpha (TEF1-α) and RNA polymerase subunit II (RPB2) revealed that all the isolates were F. andiyazi. In pathogenicity tests, F. andiyazi were found to be pathogenic on the susceptible rice cultivars MR211 and MR220. Inoculated rice seedling produced typical bakanae symptom like elongation, thin and yellow leaves. F. andiyazi was further confirmed as pathogenic species by Ultra High-Performance Liquid Chromatography (UPLC) detection of Gibberellic acid (GA3) and Fusaric acid. In this study, F. andiyazi strains have been identified as the responsible pathogen for causing rice bakanae disease in Malaysia and it is the first report of F. andiyazi, as a pathogenic species on rice in Malaysia.

Construction of magnetic recoverable porous electron-rich organic frameworks for efficiently enrichment of phenylurea herbicides from water and milk samples prior to HPLC detection

Porous electron-rich organic frameworks have attracted an increased attention in the adsorption and removal of pollutants due to their abundant electron-rich nitrogen atoms, which can effectively interact with positively charged substance. In this study, a porous electron-rich organic framework (Car-POF) and positively charged amino-functionalized magnetic nanoparticles (Fe3O4-NH2) were used to construct a magnetic electron-rich Fe3O4-NH2@Car-POF for the enrichment of some phenylurea herbicides from water and milk samples prior to high performance liquid chromatographic detection. The adsorption capacity of Fe3O4-NH2@Car-POF for the phenylureas ranged from 14.93 to 28.83 mg g−1. The LODs were observed in the range of 0.05–0.20 ng mL−1 and 0.5–1.5 ng mL−1, and LOQs in the range of 0.17–0.66 ng mL−1 and 1.7–5.0 ng mL−1 for water and milk samples with RSD less than 9.0. The adsorption studies with cationic and anionic dyes revealed that Fe3O4-NH2@Car-POF is favorable for the adsorption of positively charged compounds.

Fabrication of sea urchin shaped polyaniline-modified magnetic microporous organic network for efficient extraction of non-steroidal anti-inflammatory drugs from animal-derived food samples

In this work, a novel polyaniline-modified magnetic microporous organic network (MMON-PANI) composite was fabricated for effective magnetic solid phase extraction (MSPE) of five typical nonsteroidal anti-inflammatory drugs (NSAIDs) from animal-derived food samples before high performance liquid chromatography (HPLC) detection. The core-shell sea urchin shaped MMON-PANI integrates the merits of Fe3O4, MON, and PANI, exhibiting large specific surface area, rapid magnetic responsiveness, good stability, and multiple binding sites to NSAIDs. Convenient and effective extraction of trace NSAIDs from chicken, beef and pork samples is realized on MMON-PANI via the synergetic π-π, hydrogen bonding, hydrophobic, and electrostatic interactions. Under optimal conditions, the MMON-PANI-MSPE-HPLC-UV method exhibits wide linear ranges (0.2–1000 μg L−1), low limits of detection (0.07–1.7 μg L−1), good precisions (intraday and inter-day RSDs < 5.4 %, n = 3), large enrichment factors (98.6–99.9), and less adsorbent consumption (3 mg). The extraction mechanism and selectivity of MMON-PANI are also evaluated in detail. This work proves the incorporation of PANI onto MMON is an efficient way to promote NSAIDs enrichment and provides a new strategy to synthesize multifunctional MON-based composites in sample pretreatment.

CdTe@ZnS quantum dots for rapid detection of organophosphorus pesticide in agricultural products

Organophosphorus pesticides (OPPs) constitute the most widely employed class of pesticides. However, the prevalent use of OPPs, while advantageous, raises concerns due to their toxicity, posing serious threats to food safety. Chemical sensors utilizing quantum dots (QDs) demonstrate promising applications in rapidly detecting OPPs residues, thereby facilitating efficient inspection of agricultural products. In this study, we employ an aqueous synthesis approach to prepare low toxic CdTe@ZnS QDs with stable fluorescence properties. To mitigate the risk of imprecise measurements stemming from the inherent susceptibility of fluorescence to quenching, we have adopted the principle of fluorescence resonance energy transfer (FRET) for the construction of the turn-on quantum dot sensor. With a detection limit for chlorpyrifos as low as 10 ppb (10 μg/L), the QDs sensor exhibits notable resistance to interference from various pesticides. Application of this system to detect organophosphorothioate pesticides in apples produced results consistent with those obtained from high-performance liquid chromatography (HPLC) detection, affirming the promising application prospects of this sensing system for the rapid detection of OPPs residues.

A Review on Extraction and Chromatographic Techniques for Organophosphate Pesticidal Residue Analysis in Paddy and Rice Bran

Organophosphorus pesticides (OPPS) are extensively employed in agricultural techniques and may expose people to them via food intake. We offer an analytical approach for detecting OPPS in paddy, rice grains and or rice bran that combines solid-phase extraction with a high-performance liquid chromatography detector. Acetonitrile extraction was used before SPE–HPLC–MSMS to isolate specific residues, which helped decrease matrix disruptions and identify a broad array of numerous residues in rice grains. Accordingly, ranges of quantitative determination ranged from 0.01mg/kg to more than 1.0 mg/kg, with coefficients extending from 0.995 to more than 0.999. At three stimulating intensities, high recuperation was reported, suggesting excellent precision. The relative SD of all residues confirmed the technique’s accuracy. Using this, various OPPS were quantified, ranging from just below to maximum permissible limits to exceeding the limit. According to a sample study of different rice grain varieties accessible in the international market. This study focuses on the techniques of organophosphate pesticide residue extraction from paddy and rice ban. The study further discusses chromatographic techniques for the same.

Split-ring resonator with interdigital Split electrodes as detector for liquid and ion chromatography

The analysis of food and drugs as well as the monitoring of chemical processes and bioreactors by high-performance liquid chromatography and ion chromatography requires universal, cost-effective, and sensitive detectors. Therefore, this work presents a split-ring resonator that detects changes in the electrical and dielectric properties of the eluate from liquid chromatography or ion chromatography by monitoring the amplitude of the transmitted signal using an envelope detector. The used split-ring resonator consists of a simple printed circuit board featuring two microstrip lines. One is formed to a ring with interdigital split electrodes. The interdigital split electrodes forming the sensitive area significantly enhance the sensitivity to changes in relative permittivity, dielectric losses and electrical conductivity of the eluate In addition, the split-ring resonator is a small, inexpensive and easy-to-use detector that uses several dielectric parameters simultaneously for the measurement. Furthermore, the split-ring resonator measures these parameters at a significantly lower frequency than optical detectors and therefore measures different changes in frequency-dependent permittivity. For demonstrating feasibility of a split-ring resonator as a detector for high-performance liquid chromatography and ion chromatography, various anion and cation standards, basic and acidic amino acids, sugars, as well as sugar alcohols were separated and detected. A conductivity detector for ion chromatography and a refractive index detector for high-performance liquid chromatography were used as reference detectors.

Methodology for high-performance liquid chromatography detection of latanoprost and latanoprost free acid

AbstractA gradient high-performance liquid chromatography (HPLC) method has been developed to determine the concentrations of latanoprost (LP) and latanoprost free acid (LPA) in aqueous solutions. It is novel due to a combination of its simplicity, speed, and detection capability in aqueous solutions for both active drug (LPA) and prodrug (LP). This method is applicable for the research and development of novel drug delivery devices and quality control assays for experimental and commercial laboratory settings, as it allows for a high sample throughput. Samples were chromatographed across a C18 + 2.7 µm 4.6 × 7.5 mm reversed-phase column with gradient elution using a mobile phase of aqueous acetic acid (pH 3.1) and acetonitrile with 0.1% acetic acid. UV spectrophotometry was used to monitor the eluents at 210 nm. Drug concentrations from 1.0 to 150 μg mL−1 were tested, with good linearity observed across the range. LPA had a signature peak at approximately 4.82 min (SD &lt; 0.08) and LP at 9.27 min (SD &lt; 0.07). For both drug and pro-drug, LOD and LOQ were 1.0 and 2.5 μg mL−1, respectively. This assay which accurately measures both prodrug and drug in a single injection, has significant applicability in determining the release kinetics of novel LP drug delivery systems.

A Multi-step High-Performance Liquid Chromatography (HPLC) Technique for the Extraction and Purification of Medical-Grade Cyclosporine A

Cyclosporine A (CsA), a potent immunosuppressive drug, is used to treat autoimmune diseases and prevent organ transplant recipients from rejecting their new organs. Maintaining CsA's uniformity and purity is essential to maintaining its therapeutic efficacy and safety. Impurities and contaminants in CsA formulations can exacerbate side effects and patient toxicity. Purification processes can improve the safety profile of the drug and reduce the likelihood of adverse effects by eliminating impurities. Using High-performance liquid chromatography (HPLC) methods, CsA has been isolated and refined from crude in recent years. However, after the procedure, the CsA purity was not at its best. To solve this issue, this work employs a multi-step HPLC method to increase purity levels to above 95%. When exploring the HPLC detection conditions of CsA, we found that the method from the Chinese pharmacopoeia: acetonitrile-water-tert-butyl methyl ether-phosphoric acid (430 : 520 : 50 : 1) was a better mobile phase solution, our further research, including the substitution of tert-butyl methyl ether with other buffer solvents, showed no significant improvement. At the same time, we established that the key factor affecting its separation was temperature. When the temperature is lower than 70 ℃, the HPLC separation effect worsens, the retention time increases, and the peak width becomes longer. Finally, the utilization of acetonitrile-water-tert-butyl methyl ether-phosphoric acid (430 : 520 : 50 : 1) as the mobile phase with a column temperature of 70 °C, petroleum ether: acetic acid Ethyl ester (70 : 30) as the mobile phase, and filling the φ 40 mm × 500 mm chromatography column using a 40~60 μm silica gel so that a 10 : 1 height-to-diameter ratio of the packed part is obtained, under normal temperature conditions, resulted in an average cyclosporine A yield of 80.1%. The corresponding average purity was 97.2%, of which the first isolation yield was 82.3%, and the purity was 98.1%.

The Effect of Bacterial AHL on the Cyclic Adenosine Monophosphate Content in Plants According to High-Performance Liquid Chromatography.

Cyclic adenosine monophosphate (cAMP) is an important second messenger in cells, mediating various stimulation signals such as the growth and development of organisms and stress and participating in regulating various biological processes of cells. This article explores the quantitative determination of cAMP in plants using High-Performance Liquid Chromatography (HPLC) and applies this method to analyzing the changes in cAMP content during the process of plant response to the bacterial quorum sensing signal N-acyl homoserine lactone (AHL). Research has shown that the optimal detection conditions for HPLC are as follows: the chromatographic column is Venusil MP C18 (2), the mobile phase is methanol-water (0.1% trifluoroacetic acid) (v:v, 10:90), the detection wavelength is 259 nm, the column temperature is 35 °C, and the flow rate is 0.8 mL/min. The precision of the standard sample of this method is 98.21%, the precision of the sample is 98.87%, and the recovery rate is 101.067%. The optimal extraction conditions for cAMP in Arabidopsis are to use 15% methanol ultrasonic extraction for 10 min, followed by a 40 °C water bath for 4 h. Bacterial AHL signal processing can significantly stimulate an increase in cAMP levels in Arabidopsis leaves and roots. The establishment of HPLC detection methods for the cAMP content in plants is of great significance for in-depth research on the signal transduction mechanisms of plant-bacterial interactions.

Transcriptome and comparative chloroplast genome analysis of Taxus yunnanensis individuals with high and low paclitaxel yield

Paclitaxel is a potent anti-cancer drug that is mainly produced through semi-synthesis, which still requires plant materials as precursors. The content of paclitaxel and 10-deacetyl baccatin III (10-DAB) in Taxus yunnanensis has been found to differ from that of other Taxus species, but there is little research on the mechanism underlying the variation in paclitaxel content in T. yunnanensis of different provenances. In this experiment, the contents of taxoids and precursors in twigs between a high paclitaxel-yielding individual (TG) and a low paclitaxel-yielding individual (TD) of T. yunnanensis were compared, and comparative analyses of transcriptomes as well as chloroplast genomes were performed. High-performance liquid chromatography (HPLC) detection showed that 10-DAB and baccatin III contents in TG were 18 and 47 times those in TD, respectively. Transcriptomic analysis results indicated that genes encoding key enzymes in the paclitaxel biosynthesis pathway, such as taxane 10-β-hydroxylase (T10βH), 10-deacetylbaccatin III 10-O-acetyltransferase (DBAT), and debenzoyl paclitaxel N-benzoyl transferase (DBTNBT), exhibited higher expression levels in TG. Additionally, qRT-PCR showed that the relative expression level of T10βH and DBAT in TG were 29 and 13 times those in TD, respectively. In addition, six putative transcription factors were identified that may be involved in paclitaxel biosynthesis from transcriptome data. Comparative analysis of plastid genomes showed that the TD chloroplast contained a duplicate of rps12, leading to a longer plastid genome length in TD relative to TG. Fifteen mutation hotspot regions were identified between the two plastid genomes that can serve as candidate DNA barcodes for identifying high-paclitaxel-yield individuals. This experiment provides insight into the difference in paclitaxel accumulation among different provenances of T. yunnanensis individuals.

Silver nanoparticle-functionalized melamine-formaldehyde aerogel for online in-tube solid-phase microextraction of polycyclic aromatic hydrocarbons followed by HPLC-DAD analysis

Based on the π-metal interaction between silver nanoparticles (AgNPs) and aromatic compounds, AgNPs were in-situ grown to melamine-formaldehyde (MF) aerogel for improving the extraction performance to polycyclic aromatic hydrocarbons (PAHs). The AgNPs/MF aerogel was regulated through varing the concentration of reactants, and characterized by scanning electron microscopy, X-ray photoelectron spectroscopy and X-ray powder diffraction. As a new extraction coating, the AgNPs/MF aerogel was coated to stainless-steel wires for in-tube solid-phase microextraction (IT-SPME). The extraction effects of MF aerogels before and after the modification of AgNPs were compared, and the AgNPs greatly improved the extraction ability for PAHs reaching to 166.4 %. Combining IT-SPME with high performance liquid chromatographic detection, an online analytical system was constructed. Furthermore, the sampling volume and rate, concentration of organic solvent, and desorption time were optimized factor by factor. The online analytical method with low detection limits (0.003–0.010 μg L−1) and efficient enrichment factors (1998–3237) for PAHs was established, which fastly detected trace level of PAHs in drinking and environmental water samples. Compared with other methods, the method was comparable or better in the detection limit and linear range, indicating prospective application of the AgNPs/MF aerogel for sample preparation.

Amperometric Inkjet-Printed Thyroxine Sensor Based on Customized Graphene and Tunned Cyclodextrins as the Preconcentration Element.

The determination of thyroid hormones has practical clinical significance for the diagnosis of hyperthyroidism and hypothyroidism diseases. Considering this aspect, a wide range of analytical methods for the detection of analytes, including immunoassay, chemiluminescence, mass spectroscopy and high-performance liquid chromatography, among others, has been developed. This type of analysis provides feasible results. Nevertheless, it requires qualified staff, special facilities and is time-consuming. For this reason, this paper relies on the fabrication of an electrochemical device developed with inkjet printing technology for the free detection of Thyroxine (T4). To manufacture our electrochemical device, several aspects were considered from the use of materials that amplify electrical signals, to finding a supramolecular scaffold that possess affinity towards the target analyte and the need of preconcentrating the analyte on the electrode's surface. For this task, printed devices were modified with a hybrid nanomaterial consisting of reduced graphene oxide (rGO) tuned with Au nanoparticles (Au-NPs) and an entrapment agent and different thiolated cyclodextrins (x-CD-SH) as carrying agents. Analytes were preconcentrated via supramolecular chemistry due to the formation of an inclusion complex between the cyclodextrin and hormones. Morphological and electrochemical characterization of the final device was carried out to ensure the proper workability of the electrode, achieving excellent response, sensitivity and limit of detection (LOD).

Determination of Aflatoxin B1 in Grains by Aptamer Affinity Column Enrichment and Purification Coupled with High Performance Liquid Chromatography Detection.

Aflatoxin B1 (AFB1) is a highly teratogenic and carcinogenic secondary metabolite produced by Aspergillus. It is commonly detected in agricultural products such as cereals, peanuts, corn, and feed. Grains have a complex composition. These complex components severely interfere with the effective extraction and separation of AFB1, and also cause problems such as matrix interference and instrument damage, thus posing a great challenge in the accurate analysis of AFB1. In this study, an aptamer affinity column for AFB1 analysis (AFB1-AAC) was prepared for the enrichment and purification of AFB1 from grain samples. AFB1-AAC with an AFB1-specific aptamer as the recognition element exhibited high affinity and specificity for AFB1. Grain samples were enriched and purified by AFB1-AAC, and subsequently analyzed by high performance liquid chromatography with post-column photochemical derivatization-fluorescence detection (HPLC-PCD-FLD). The average recoveries of AFB1 ranged from 88.7% to 99.1%, with relative standard deviations (RSDs) of 1.4-5.6% (n = 3) at the spiked levels of 5.0-20.0 μg kg-1. The limit of detection (LOD) for AFB1 (0.02 μg kg-1) was much below the maximum residue limits (MRLs) for AFB1. This novel method can be applied to the determination of AFB1 residues in peanut, corn, and rice.

Preconcentration of Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) in Clinical Samples by Various Hollow Fiber–Liquid Phase Microextraction (HF-LPME) Configurations With High-Performance Liquid Chromatography (HPLC) Detection

Hollow fiber liquid-phase microextraction is commonly used in analytical chemistry, providing high clean-up and pre-concentration factors. However, a major issue with this method is the long extraction time. The objective of this project is to reduce the extraction time by inducing different types of fluid motion in the sample solution. Three methods were employed: agitation using a magnetic stir bar in the horizontal position (conventional method), utilization of an air pump, and agitation using a magnetic stir bar in the vertical position for the pre-concentration and determination of non-steroidal anti-inflammatory drugs (naproxen, ibuprofen, and mefenamic acid) in biological samples via high-performance liquid chromatography with ultraviolet absorbance detection. The results demonstrated a significant reduction in extraction time and limit of detection when the air pump was employed compared to the conventional method of stirring with a magnetic stir bar in the horizontal position. Moreover, the mentioned parameters were even lower when stirring solutions were accomplished using a vertically-positioned magnetic stir bar, surpassing the method utilizing the air pump for agitation. Furthermore, the relative standard deviations were also lower when a vertically-positioned magnetic stir bar was used compared to the conventional method with a horizontally-positioned magnetic stir bar, while those of the air pump-assisted agitation method fell between the values of the aforementioned methods.

Equilibrium dialysis with HPLC detection to measure substrate binding affinity of a non-heme iron halogenase.

Determination of substrate binding affinity (Kd) is critical to understanding enzyme function. An extensive number of methods have been developed and employed to study ligand/substrate binding, but the best approach depends greatly on the substrate and the enzyme in question. Below we describe how to measure the Kd of BesD, a non-heme iron halogenase, for its native substrate lysine using equilibrium dialysis coupled with High Performance Liquid Chromatography (HPLC) for subsequent detection. This method can be performed in anaerobic glove bag settings. It requires readily available HPLC instrumentation for ligand quantitation and is adaptable to meet the needs of a variety of substrate affinity measurements.

Preparation of branched polyethyleneimine-assisted boronic acid-functionalized magnetic MXene for the enrichment of catecholamines in urine samples.

Herein, a magnetic borate-functionalized MXene composite with multiple boronic affinity sites was fabricated by embedding Fe3 O4 nanoparticles with 4-formylphenylboronic acid functionalized Ti3 C2 Tx nanosheets and served as sorbent for the simultaneous extraction of catecholamines (CAs) in urine samples. The morphology and structure of the magnetic materials were investigated using scanning microscopy, vibrating sample magnetometer, X-ray photoelectron spectrometer, and X-ray diffraction. The introduction of polyethyleneimine can amplify the bonded boronic acid groups, thereby effectively improving the adsorption capacities for CAs based on the multiple interactions of boronic affinity, hydrogen bonding, and metal coordination. The adsorption performance was investigated using the kinetics and isotherms models, and the main parameters that influence the extraction efficiency were optimized. Under the most favorable magnetic solid-phase extraction condition, a sensitive method for the analysis of CAs in urine samples was developed by combining magnetic solid-phase extraction conditions with high-performance liquid chromatography detection. The findings illustrated that the proposed approach possessed a wide linearity range of 0.05-250ng/mL with an acceptable correlation coefficient (R2 ≥0.9984) and detection limits of 0.010-0.015ng/mL for the target CAs. The research not only provides a notable composite with multiple boronic affinity sites but also offers an effective and feasible measure for the detection of CAs in biological samples.