MOF-derived porous carbon microspheres Ni@C-acid as solid-phase microextraction coating for extraction of polycyclic aromatic hydrocarbons from tea infusions.

The improvement of the stability and adsorption properties of materials on targets in sample pre-treatment has long been an objective. Extensive efforts have been made to achieve this goal. In this work, metal-organic framework Ni-MOF precursors were first synthesized by solvothermal method using polyvinylpyrrolidone (PVP) as an ideal templating agent, stabiliser and nanoparticle dispersant. After carbonization and acid washing, the nanoporous carbon microspheres material (Ni@C-acid) was obtained. Compared with the material without acid treatment (Ni@C), the specific surface area, pore volume, adsorption performance of Ni@C-acid were increased. Thanks to its excellent characteristics (high stability, abundant benzene rings), Ni@C-acid was used as fiber coatings in headspace solid-phase microextraction (HS-SPME) technology for extraction and preconcentration of polycyclic aromatic hydrocarbons (PAHs) prior to gas chromatography-flame ionization detector (GC-FID) analysis. The experimental parameters of extraction temperature, extraction time, agitation speed, desorption temperature, desorption time and sodium chloride (NaCl) concentration were studied. Under optimal experimental conditions, the wide linear range (0.01-30 ng mL-1), the good correlation coefficient (0.9916-0.9984), the low detection limit (0.003-0.011 ng mL-1), and the high enrichment factor (5273-13793) were obtained. The established method was successfully used for the detection of trace PAHs in actual tea infusions samples and satisfied recoveries ranging from 80.94-118.62 % were achieved. The present work provides a simple method for the preparation of highly stable and adsorbable porous carbon microsphere materials with potential applications in the extraction of environmental pollutants.

A multiplex UPLC-MS/MS method for the quantification of three PD-L1 checkpoint inhibitors, atezolizumab, avelumab, and durvalumab, in human serum.

BACKGROUND AND AIM: To support pharmacokinetic studies, a multiplex UPLC-MS/MS assay was developed and validated to quantify PD-L1 checkpoint inhibitors atezolizumab, avelumab, and durvalumab in serum. METHODS: A bottom-up sample pre-treatment procedure was developed to determine atezolizumab, avelumab, and durvalumab in serum. This procedure consisted of (1) precipitation of the monoclonal antibody with ammonium sulfate, (2) reduction with dithiothreitol, (3) denaturation with methanol, and (4) tryptic digestion of the protein. The unique signature peptides resulting after sample pre-treatment of the antibodies were measured using UPLC-MS/MS with a total run time of 11 minutes. The clinical application was evaluated by analyzing 114 atezolizumab patient samples. RESULTS: The developed method was found to be accurate and precise for all three analytes over a concentration range of 3.00-150 µg/mL. No endogenous interference was present in serum samples. Cross-interference experiments showed no cross-analyte interference and acceptable cross-internal standard interference. In addition, no substantial carry-over was observed. The stable isotopically labeled signature peptides were most effective in compensating for matrix effects. Recovery based on back-calculated concentrations of calibration standards and quality control samples was found to be high. The analytes were stable for at least three freeze-thaw cycles, for 42 hours at processing conditions, for at least two days at 2-8°C in the final extract, for five days before re-injection analysis at 4°C, and long-term for at least 11 months at -70°C. The assay was tested for its applicability in clinical practice. For this purpose, 114 atezolizumab patient samples were measured. CONCLUSION: A multiplex UPLC-MS/MS assay was developed and validated to quantify atezolizumab, avelumab, and durvalumab in human serum. The applicability of this method was demonstrated by the analysis of clinical atezolizumab samples. The method is suitable to support clinical pharmacokinetic studies involving atezolizumab, avelumab, or durvalumab.

Pesticide residues in animal-derived food: Current state and perspectives.

Pesticides are widely used in the cultivation and breeding of agricultural products all over the world. However, their direct use or indirect pollution in animal breeding may lead to residual accumulation, migration, and metabolism in animal-derived foods, posing potential health risks to humans through the food chain. Therefore, it is necessary to detect pesticide residues in animal-derived food using simple, reliable, and sensitive methods. This review summarizes sample extraction and clean-up methods, as well as the instrumental determination technologies such as chromatography and chromatography-mass spectrometry for residual analysis in animal-derived foods, including meat, eggs and milk. Additionally, we perspectives on the future of this field. This information aims to assist relevant researchers in this area, contribute to the development of ideas and novel technical methods for residual detection, metabolic research and risk assessment of pesticides in animal-derived food.

Evaluation of multiple immunoassay formats for detection of anti-drug antibodies to zinpentraxin alfa.

Zinpentraxin alfa (rhPTX-2; PRM-151) is currently being developed for the treatment of fibrotic diseases such as idiopathic pulmonary fibrosis and myelofibrosis. Notably, because it is administered chronically and has an endogenously expressed counterpart, clinical studies of zinpentraxin alpha must include immunogenicity assessments. Since the typical homogenous bridging ELISA assay does not adequately measure anti-drug antibodies (ADAs) against zinpentraxin alfa, additional assay formats have been developed to evaluate immunogenicity of this therapeutic. Here, we present the evaluation of four distinct assay formats that were used to measure zinpentraxin alpha ADA: step-wise bridging, direct binding, total ADA, and the semi-homogeneous formats, based on multiple parameters including assay sensitivity, precision, and drug tolerance. This paper presents the full details of method development for each of the aforementioned assay formats including evaluation of sample pre-treatment, determination of cut point, and assessment of assay performance by analyzing a subset of clinical samples. Overall, the semi-homogenous ADA assay format with no sample pre-treatment was selected for the measurement of zinpentraxin alpha immunogenicity as it provided the desired sensitivity, drug tolerance, and reproducibility. Our study emphasizes the importance of assay format evaluation during drug development and the necessity to select the most suitable assay format and sample pre-treatment method by which to evaluate therapeutic drug immunogenicity.

A method for detection of anti-drug antibodies to a biotherapeutic (CSL112) with endogenous counterpart (apolipoprotein A-I) using a novel sample pre-treatment electrochemiluminescence assay.

BACKGROUND: There are numerous challenges encountered during clinical testing for an immunogenic response to a plasma-derived therapeutic. Distinguishing between antibodies that recognize endogenous versus therapeutic protein can be particularly difficult. This study focused on CSL112 (human plasma-derived apolipoprotein A-I; apoA-I), which is in clinical development for reducing the risk of recurrent major adverse cardiovascular events following acute myocardial infarction. AIM: To develop and validate a high-throughput, highly sensitive and specific assay to detect antibodies to CSL112 that can be used for immunogenicity assessment in large clinical studies. RESULTS: We developed a clinical anti-drug antibody (ADA) assay utilizing an immunoglobulin purification step that improved specificity and drug tolerance, demonstrating that measurement of pre-existing or treatment emergent ADAs was highly dependent on assay format. The Sample Pre-treatment Electrochemiluminescence (ECL; SPECL) assay incorporates a protein A extraction of serum samples before a bridging assay is performed on an ECL platform. The assay is qualitative, sensitive (lower limit of quantification <39 ng/mL) and has a drug tolerance of 0.5 mg/mL in line with U.S. Food and Drug Administration requirements for clinical immunogenicity assays for therapeutic proteins. Importantly, the SPECL assay demonstrated the absence of antibodies to both apoA-I and CSL112 both prior to drug exposure and after repeated dosing across multiple trials (n = 970 subjects). CONCLUSION: The SPECL method has been validated and applied to support the CSL112 preclinical and clinical development program and has broader application to similar protein therapeutics. Attributes of the methodology include high drug tolerance, high sensitivity, selectivity, and precision. This format is amenable to automation providing the high throughput and reduced variability required to support large scale clinical studies that span extended time periods.

Cavitandos naturales y de síntesis: desafíos y respuestas en química y tecnología farmacéutica / Natural and synthetic cavitands: challenges in chemistry and pharmaceutical technology

La química supramolecular es la base para numerosas aplicaciones en síntesis, catálisis, separaciones quirales, diseño de sensores químicos, así como en procesos de señalización, vehiculización y trasporte de fármacos. Ciertas supramoléculas pueden combinarse y auto-ensamblarse dando lugar a entidades con geometría y topología exclusiva.Se denominan cavitandos al conjunto de supramoléculas que tiene una cavidad central capaz de reconocer y alojar en su interior una variedad de especies. Entre los cavitandos de síntesis sobresalen los éteres corona, calixarenos, y los cucurbiturilos, presentando indudables cualidades ventajosas las porfirinas y las ciclodextrinas. Las ciclodextrinas ofrecen numerosas ventajas comparados con otros cavitandos, ya que ya que son productos naturales obtenidas a partir del almidón, lo cual las hace muy atractivas desde el punto de vista de la química sostenible. Se producen a escala industrial a través de un proceso enzimático relativamente sencillo y a un precio razonable. Los efectos secundarios o tóxicos a que pueden dar lugar son prácticamente inexistentes, por lo que se utilizan en la industria farmacéutica, cosmética y alimentaria.En este trabajo se revisa la utilidad las ciclodextrinas en el campo de la tecnología farmacéutica, incrementando la solubilidad y estabilidad de fármacos y mejorando sus propiedades farmacocinéticas. (AU)

Supramolecular chemistry involves non-covalent interactions and specific molecular recognition of molecules/analytes by host molecules or supramolecules. These events are present in synthesis, catalysis, chiral separations, design of sensors, cell signaling processes and drug transport by carriers. The typical behavior of supramolecules is derived from their ability to build well-structured self-assembled and self-organized entities.Cavitands are a particular group of supramolecules possessing a cavity able to include a variety of compounds thanks to host-guest non-covalent interactions developed among cavitands and analytes. Some typical cavitands are crown ethers, calixarenes, cucurbiturils, porphyrins and cyclodextrins. The two latter families are natural product cavitands that are generally considered models for molecular recognition of cations and organic and inorganic guest molecules, being attractive host molecules from the sustainability point of view. The natural cyclodextrins (𝛼-, 𝛽- and 𝛾-CD) are obtained with reasonable cost by enzymatic treatment of starch under adequate temperature conditions. They are profusely used in pharmaceutical, food and cosmetic industries due to their very low toxicity and side effects.This review is focused on the relevance and applications of cyclodextrins in pharmaceutical technology for their ability to increase solubility and stabilize drug molecules, thereby enhancing their bioavailability. The association of cyclodextrins with diverse nanostructured materials, i.e. carbon nanotubes, magnetic nanoparticles, silica and molecularly imprinted polymers, allows to synergize the properties of cyclodextrins and these nanostructured materials to reach highly specific molecular recognition of analytes. (AU)

Affimer sandwich probes for stable and robust lateral flow assaying.

Lateral flow assays (LFAs) widely deployed for on-site diagnosis have predominantly utilized antibodies as recognition molecules. Antibodies with limited thermal stability deteriorate the performance of the LFA over time. Herein, we demonstrate a stable and robust LFA by utilizing thermally stable peptide-based 12-14 kDa affimers as recognition molecules, in lieu of conventional protein-based antibodies to analyze complex samples with a significantly improved shelf life at room temperature. The model system studied here is that of interleukin-8 (IL8) biomarker for validating the efficacy of the proposed approach, using a pair of affimer probes that demonstrates dual functionality of capturing and reporting. Affimers immobilized on the test zone of LFA serve as capture probes for IL8-affimer-MB complexes. Whereas affimers conjugated with the MBs that enable extraction of IL8 from the sample matrix serve as reporters for visual detection. The MB complexes captured at the test zone resulted in brownish test bands that enable concentration-dependent detection of IL8. The assay yielded sensitive visual detection of IL8 at ng/mL levels (~ 0.1 ng/mL and 1 ng/mL in buffer and human plasma, respectively), within 20 min, using sample volumes of ~ 100 µL. Importantly, the stability of affimer-incorporated LFA improved significantly in contrast to antibody-incorporated LFA over time, even when stored at 4 °C. Therefore, the proposed affimer-based LFA in conjunction with MBs offer stable and reliable detection of biomarkers at clinically relevant concentration ranges in complicated matrices, even without requiring cold storage, hence, offering a promising avenue for on-site diagnosis in resource-limited settings.

Metal organic frameworks as advanced adsorbent materials for separation and analysis of complex samples.

Sample pre-treatment is of great significance for study protein phosphorylation and glycosylation. Due to the low concentration and suppression of matrices, the direct detection of phosphoprotein and glycoprotein by mass spectrometry (MS) are still facing many challenges. The demand of efficient and specific enrichment of phosphoprotein and glycoprotein promotes the sample preparation methods based on metal-organic frameworks (MOFs). In this review, recent advances in MOFs-based samples pre-treatment, including the enrichment of protein, endogenous peptides, glycopeptides, phosphopeptides and glycans are summarized and discussed. In addition, the design and synthesis of different types of MOFs according to different enrichment mechanisms and principles are discussed. Furthermore, the potential problems of sample pre-treatment in proteomics is prospected.

Optimized sample pre-treatment procedure for the simultaneous UPLC-MS/MS quantification of ipilimumab, nivolumab, and pembrolizumab in human serum.

Ipilimumab, nivolumab, and pembrolizumab are immune checkpoint inhibiting monoclonal antibodies. Their efficacy has been proven to be correlated with clearance, and hence, bioanalytical assays to study their pharmacokinetics are of pivotal importance. We present the first kit-free sample pre-treatment procedure of only three hours for the Ultra-Performance Liquid Chromatography - tandem Mass Spectrometry (UPLC-MS/MS) simultaneous quantification of ipilimumab, nivolumab, and pembrolizumab in human serum. The conventional bottom-up sample pre-treatment steps for protein MS bioanalysis including pre-digestion purification, denaturation, reduction, alkylation, and digestion were optimized in terms of sensitivity and reproducibility. In the final, optimal sample pre-treatment procedure, samples were purified by protein precipitation with saturated ammonium sulfate solution, reduced with dithiothreitol, denatured with methanol, and digested with trypsin. The method was then validated according to European Medicines Agency (EMA) and the United States Food and Drug Administration (FDA) guidelines for bioanalytical method validation, and 4-6-20 acceptance criteria were applied. This method was selective, accurate, and precise within the range of 3-200 µg/mL for all analytes. The validated developed assay was applied to determine ipilimumab, nivolumab, and pembrolizumab concentrations in patient serum, and the results were compared to enzyme-linked immunosorbent assay (ELISA) results.

QuEChERS-based analytical methods developed for LC-MS/MS multiresidue determination of pesticides in representative crop fatty matrices: Olives and sunflower seeds.

Oilseed crops are greatly extended all over the world. Their high fat content can interfere during pesticide multiresidue analysis through liquid chromatography-tandem mass spectrometry (LC-MS/MS). This work aimed at overcoming this issue by developing and validating two QuEChERS-based methods for LC-MS/MS determination of 42 pesticides in two fatty food matrices: olives and sunflower seeds. Optimization of the extraction method was achieved following a 26-2 fractional factorial design in a highly cost-effective way. Validation of the multi-residue methods demonstrated improved limits of detection, below the established maximum residue levels (MRLs) for almost all compounds, good precision, and trueness, in compliance with SANTE guidelines. Application of these methods to the analysis of real samples from the Iberian Peninsula showed the presence of some pesticides of relevant environmental concern, including four compounds contained in the Pesticide Action Network International list of highly hazardous pesticides, found at levels between 0.03 ng/g and 104 ng/g.

Tip-tip filtration ameliorates single-phase extraction methods for plasma large-scale lipidomics analysis.

We evaluated the performance of three different single-phase extraction methods to be used before untargeted lipidomics analysis by Liquid Chromatography High-Resolution Mass Spectrometry. Lipids were extracted from a pool of healthy human donors' plasma in triplicates and run in both positive and negative ESI. The most satisfactory results were attained using methanol/chloroform (2:1, v/v) mixture. Eventually, we evaluated whether a filtration of the samples could be beneficial to yield cleaner and more mass-friendly extracts. Instead of using syringes, we set up a method we called tip-tip filtration, which requires the usage of a filtrating pipette tip. This way of purification led to superior results than the solvent extraction method alone. This additional procedure not only increased reproducibility but also allowed the same lipid coverage. In addition, it permitted to spare time and money, as tip-tip filtration is not particularly expensive nor time-consuming and hopefully it may be useful to increase analytical column lifetime.

Critical review of solid phase extraction for multiresidue clean-up and pre-concentration of antibiotics from livestock and poultry manure.

The release of antibiotics into the environment from agricultural industries has received tremendous attention in recent years. Nonpoint source contamination of the terrestrial environment by these compounds can result from fertilisation of agricultural soils with manure. The presence of antibiotics and their metabolites in manure may pose a threat to agro-ecosystems. This may result in the emergence of antibiotic resistance bacteria in humans through the food chain and this is a major concern globally at the moment. Therefore, monitoring of manure for antibiotic residues is of vital importance in order to assess the risks of environmental pollution to human health by these drugs. Several sample pre-treatment techniques have been developed for the extraction of antibiotic residues from complex matrices including manure over the years. Despite new developments in recent years in separation science where the common trend is miniaturisation and green approaches, solid-phase extraction is still the most widely used technique in the extraction of antibiotics from agricultural wastes such as manure. In view of this, the aim of this review was to give a critical overview of studies that have been conducted in the past 6 years on the extraction of antibiotic residues from manure employing solid-phase extraction based on Oasis HLB and Strata-X. Adsorption mechanisms of these sorbents were also briefly discussed.

Cavitandos naturales y de síntesis: desafíos y respuestas en química y tecnología farmacéutica / Natural and synthetic cavitands: challenges in chemistry and pharmaceutical technology

La química supramolecular es la base para numerosas aplicaciones en síntesis, catálisis, separaciones quirales, diseño de sensores químicos, así como en procesos de señalización, vehiculización y trasporte de fármacos. Ciertas supramoléculas pueden combinarse y auto-ensamblarse dando lugar a entidades con geometría y topología exclusiva.Se denominan cavitandos al conjunto de supramoléculas que tiene una cavidad central capaz de reconocer y alojar en su interior una variedad de especies. Entre los cavitandos de síntesis sobresalen los éteres corona, calixarenos, y los cucurbiturilos, presentando indudables cualidades ventajosas las porfirinas y las ciclodextrinas. Las ciclodextrinas ofrecen numerosas ventajas comparados con otros cavitandos, ya que ya que son productos naturales obtenidas a partir del almidón, lo cual las hace muy atractivas desde el punto de vista de la química sostenible. Se producen a escala industrial a través de un proceso enzimático relativamente sencillo y a un precio razonable. Los efectos secundarios o tóxicos a que pueden dar lugar son prácticamente inexistentes, por lo que se utilizan en la industria farmacéutica, cosmética y alimentaria. (AU)

Supramolecular chemistry involves non-covalent interactions and specific molecular recognition of molecules/analytes by host molecules or supramolecules. These events are present in synthesis, catalysis, chiral separations, design of sensors, cell signaling processes and drug transport by carriers. The typical behavior of supramolecules is derived from their ability to build well-structured self-assembled and self-organized entities.Cavitands are a particular group of supramolecules possessing a cavity able to include a variety of compounds thanks to host-guest non-covalent interactions developed among cavitands and analytes. Some typical cavitands are crown ethers, calixarenes, cucurbiturils, porphyrins and cyclodextrins. The two latter families are natural product cavitands that are generally considered models for molecular recognition of cations and organic and inorganic guest molecules, being attractive host molecules from the sustainability point of view. The natural cyclodextrins (𝛼-, 𝛽- and 𝛾-CD) are obtained with reasonable cost by enzymatic treatment of starch under adequate temperature conditions. They are profusely used in pharmaceutical, food and cosmetic industries due to their very low toxicity and side effects.This review is focused on the relevance and applications of cyclodextrins in pharmaceutical technology for their ability to increase solubility and stabilize drug molecules, thereby enhancing their bioavailability. The association of cyclodextrins with diverse nanostructured materials, i.e. carbon nanotubes, magnetic nanoparticles, silica and molecularly imprinted polymers, allows to synergize the properties of cyclodextrins and these nanostructured materials to reach highly specific molecular recognition of analytes. The exploitation of these benefits for analytical sample pre-treatment and chiral chromatographic separations are described. (AU)

Sample pre-treatment procedures for the omics analysis of human gut microbiota: Turning points, tips and tricks for gene sequencing and metabolomics.

The connection between gut microbiota and human health is becoming increasingly relevant and the number of groups working in this field is constantly growing. In this context, from high-throughput gene sequencing to metabolomics analysis, the omics technologies have contributed enormously to unveil the secret crosstalk between us and our microbes. All the omics technologies produce a great amount of information, and processing this information is time-consuming and expensive. For this reason, a correct experimental design and a careful pre-analytical planning are crucial. To study the human gut microbiota, faeces are the sample of choice. Faecal material is complex, and procedures for collecting and preserving faeces are not well-established. Furthermore, increasing evidence suggests that multiple confounding factors, such as antibiotics consumption, mode of delivery, diet, aging and several diseases and disorders can alter the composition and functionality of the microbiota. This review is focused on the discussion of critical general issues during the pre-analytical planning, from patient handling to faeces sampling, including collection procedures, transport, storage conditions and possible pre-treatments, which are critical for a successful research in omics with a special attention to metabolomics and gene sequencing. We also point out that the adoption of standard operating procedures in the field is needed to guarantee accuracy and reproducibility of results.

Analysis of mobile chemicals in the aquatic environment-current capabilities, limitations and future perspectives.

Persistent and mobile water contaminants are rapidly developing into a focal point of environmental chemistry and chemical regulation. Their defining parameter that sets them apart from the majority of regularly monitored and regulated contaminants is their mobility in the aquatic environment, which is intrinsically tied to a high polarity. This high polarity, however, may have severe implications in the analytical process and thus the most polar of these mobile contaminants may not be covered by widely utilized trace-analytical methods, and thus, alternatives are required. In this review, we infer the physical and chemical properties of mobile water contaminants from a set of almost 1800 prioritized REACH chemicals and discuss the implications these substance properties may have on four integral steps of the analytical process: sampling and sample storage, sample pre-treatment, separation and detection. We discuss alternatives to widely utilized trace-analytical methods, examine their application range and limitations, highlight potential analytical techniques on the horizon and emphasize research areas we believe still offer the most room for further improvement. While we have a comprehensive set of analytical methods to cover a large portion of the known mobile chemicals, these methods are still only infrequently utilized. Graphical abstract.

LC-MS/MS measurement of leukocyte cystine; effect of preanalytic factors.

Cystinosis is an autosomal recessive disorder characterized by the accumulation of cystine in lysosomes, causing irreversible damage to organs, especially the kidneys. Intracellular leukocyte cystine concentrations are used to diagnose cystinosis and to monitor cysteamine treatment. The aim of this study was to develop and validate a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method without derivatization capable of measuring leukocyte intracellular cystine concentrations. During development, the effects of using three different protein precipitation agents were evaluated in terms of sensitivity and the matrix effect, with 12% trichloroacetic acid providing the highest sensitivity. The effects of different blood collection tubes were also assessed in terms of recovery, matrix effect, and protein content. Compared to other methods, our method was quicker (run time of 3 min), was linear over the range 0.078-100 µM, and had lower limits of detection (0.0192 µM) and quantification (0.0582 µM). The intra-day and inter-day reproducibility %CVs were ≤10%. and the method had excellent recovery rates (94%-106%). Other parameters including matrix selectivity, injection carryover, leukocyte lysate stability were also validated and met the acceptance criterias of European Medicines Agency (EMA) Guideline. The assay was successfully applied to quantify cystine leukocyte concentration in healthy and cystinosis patients.

Automated continuous-flow in-syringe dispersive liquid-liquid microextraction of mono-nitrophenols from large sample volumes using a novel approach to multivariate spectral analysis.

Continuous magnetic stirring-assisted dispersive liquid-liquid extraction followed by dispersive backextraction as a novel pre-treatment technique for adaptable and milliliter volumes of environmental samples has been developed. The procedure was automated using the technique "Lab-In-Syringe". The void of the automated syringe pump was used as size-adaptable extraction chamber. By a flow channel in the syringe piston, continuous flow through the syringe void was enabled. 1-Octanol was used as an extractant and dispersed by the action of a magnetic stirring bar, which was placed inside the syringe and driven by an external rotating magnetic field. Extract washing and dispersive backextraction in an alkaline aqueous acceptor phase were carried out after the preceding extraction from the acidified water sample. Analyte determination was achieved using multivariate spectrum analysis. The method was applied to determine priority pollutants, mono-nitrophenols, in surface water and enabled to reach limits of detection for o-, m-, p-nitrophenol (λ = 418, 390, 400 nm, respectively) of 0.14, 0.26, and 0.02 µmol L-1 (19.5, 36.2, and 2.8 µg L-1), respectively. Under optimized conditions, relative standard deviations were generally less than 5% and enrichment factors of o-, m-, p-nitrophenol 19, 25, and 21, respectively, were achieved using sample volumes of up to 24 mL. Average recoveries of o-, m-, p-nitrophenol from spiked surface water were 94, 82, and 92%, respectively. The concentration of humic acid was found 6-times reduced with respect to the analyte. In addition, adding spectral background modeling allowed nitrophenol determination with precision adequate for routine analysis.

Determination of Kanamycin by High Performance Liquid Chromatography.

Kanamycin is an aminoglycoside antibiotic widely used in treating animal diseases caused by Gram-negative and Gram-positive infections. Kanamycin has a relatively narrow therapeutic index, and can accumulate in the human body through the food chain. The abuse of kanamycin can have serious side-effects. Therefore, it was necessary to develop a sensitive and selective analysis method to detect kanamycin residue in food to ensure public health. There are many analytical methods to determine kanamycin concentration, among which high performance liquid chromatography (HPLC) is a common and practical tool. This paper presents a review of the application of HPLC analysis of kanamycin in different sample matrices. The different detectors coupled with HPLC, including Ultraviolet (UV)/Fluorescence, Evaporative Light Scattering Detector (ELSD)/Pulsed Electrochemical Detection (PED), and Mass Spectrometry, are discussed. Meanwhile, the strengths and weaknesses of each method are compared. The pre-treatment methods of food samples, including protein precipitation, liquid-liquid extraction (LLE), and solid-phase extraction (SPE) are also summarized in this paper.

Approaches to Resolve False Reporting in Neutralizing Antibody Assays Caused by Reagent Leaching from Affinity Capture Elution Solid Phase.

Insufficient drug tolerance presents a major challenge in the development of neutralizing antibody (NAb) assays for biotherapeutics. Sample pre-treatment using solid-phase extraction with acid dissociation (SPEAD) is widely reported to improve drug tolerance. In this paper, a case study is presented in which SPEAD was used in conjunction with a competitive ligand binding NAb assay format. A significant degree of biotin-drug conjugate leaching was observed resulting in the reporting of both false positive and false negative results in NAb assay. Mitigation steps have been evaluated to address drug/biotin-drug conjugate leaching. These steps included assessment of the streptavidin-coated plate in conjunction with biotin-drug conjugates at various biotin molar challenge ratios (MCR). In addition, an alternative method based on covalent capture of the drug on an aldehyde-activated plate was assessed. Both approaches were compared for the degree of drug/biotin-drug conjugate leaching during the second elution step of the SPEAD procedure. Moreover, the impact of various conditions on the assay performance was assessed, including elution pH, sample incubation time, and biotin MCR. For the covalent drug capture method, capture conditions were evaluated. Optimized conditions in both streptavidin capture and covalent capture methods enabled a significant reduction of drug/biotin-drug conjugate leaching. A streptavidin high binding capacity approach using biotin-drug conjugate with a MCR of 50:1 was chosen as the optimal method yielding a NAb assay with a fit for purpose sensitivity (153 ng/mL) and a drug tolerance of up to 50 µg/mL with 500 ng/mL PC.

Bead-extraction and heat-dissociation (BEHD): A novel way to overcome drug and matrix interference in immunogenicity testing.

Biological therapeutics are foreign antigens and can potentially induce immune response resulting in the formation of anti-drug antibodies (ADA), which in turn may lead to a wide range of side effects. Neutralizing Ab (NAb) is a subset of ADA that can bind to the pharmacological activity regions of therapeutic to inhibit or complete neutralize its clinical efficacy. A cell-based functional NAb assay is preferred to characterize its neutralization activity. However, cell-based NAb assays are often vulnerable to drug interference, as well as interference from numerous serum factors, including but not limited to growth factors and disease-related cytokines. Bead Extraction with Acid Dissociation (BEAD) has been successfully applied to remove circulating drug and/or other interfering factors from human serum samples, thereby enriching for ADA/NAb. However, the harsh acid used in the extraction procedure can cause irreversible denaturing of NAb and lead to underestimated NAb measurement. Herein we describe a new approach when acid-dissociation is not optimal for a PEGylated domain antibody (Ab). We further demonstrate that heating at 62 °C can not only dissociate drug/ADA/NAb immune complex but also selectively and irreversibly denature domain Ab drug due to much lower thermal stability of the domain Ab, when compared to that of full antibodies. The irreversible denaturing of the drug favors the formation of an immune complex between ADA/NAb and the added biotinylated drug thus increasing the recovery of ADA/NAb from samples. We call this new procedure Bead Extraction with Heat Dissociation (BEHD), which can potentially be applied to other NAb assays that have poor compatibility with acid dissociation.