Development of an ELISA with acidification treatment for an antibody conjugate incorporating Exatecans.

The successful development of Sacituzumab Govitecan and Trastuzumab Deruxtecan has made camptothecin derivatives one of the most popular payloads for antibody-drug conjugates (ADCs). Camptothecin and its derivatives all exist in a pH-dependent equilibrium between the carboxylate and lactone forms. Such transformation may lead to differences in the ratio of the two molecular forms in calibration standards and biological matrix (bio-matrix) samples, thereby leading to inaccurate conjugated antibody results. In this study, we reported an enzyme-linked immunosorbent assay (ELISA) free of the aforementioned influence for the detection of the Exatecans-conjugated antibody (conjugated SM001) in cynomolgus monkey serum. The assay was developed by first acidifying all samples with glacial acetic acid (HAc), then performing neutralization and thereafter capturing conjugated SM001 with anti-Exatecan monoclonal antibody (mAb) and detecting it with biotinylated Nectin4 (hNectin4-Bio) and horseradish peroxidase-labeled streptavidin (SA-HRP). Results showed that all tested performance parameters met the acceptance criteria. The conjugated SM001 concentrations obtained were in parallel to but slightly lower than total antibody (TAb) throughout the pharmacokinetic (PK) study, revealing that the assay strategy implemented for conjugated SM001 measurement worked well for the elimination of interference triggered by the heterogeneous existence of the lactone and carboxylate forms of Exatecan (lactone-Exatecan and carboxylate-Exatecan).

A novel LC-MS/MS method for the determination of favipiravir ribofuranosyl-5'-triphosphate (T-705-RTP) in human peripheral mononuclear cells.

Favipiravir is a broad-spectrum antiviral that is metabolised intracellularly into the active form, favipiravir ribofuranosyl-5'-triphosphate (F-RTP). Measurement of the intracellular concentration of F-RTP in mononuclear cells is a crucial step to characterising the pharmacokinetics of F-RTP and to enable more appropriate dose selection for the treatment of COVID-19 and emerging infectious diseases. The described method was validated over the range 24 - 2280 pmol/sample. Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood and lysed using methanol-water (70:30, v/v) before cellular components were precipitated with acetonitrile and the supernatant further cleaned by weak anion exchange solid phase extraction. The method was found to be both precise and accurate and was successfully utilised to analyse F-RTP concentrations in patient samples collected as part of the AGILE CST-6 clinical trial.

Observations from a decade of oligonucleotide bioanalysis by LC-MS.

There is a growing need for efficient bioanalysis of oligonucleotide therapeutics. This broad class of molecules presents numerous challenges relative to traditional small molecule therapeutics. Methodologies including ligand-binding assays or polymerase chain reaction may be fit-for-purpose in many instances, but liquid chromatography coupled to mass spectrometry (LC-MS) often delivers the best balance of sensitivity and selectivity. Over the last decade, we have engaged with many such molecules and derived insights into challenges and solutions. Herein, we provide four case studies illustrating challenges we have encountered. These issues include low or variable analyte recovery, poor resolution from related species, chromatographic abnormalities or challenging sensitivity. We present a summary of considerations, based on these experiences, to assist others working in the area.

Derivatization-free determination of chiral plasma pharmacokinetics of MDMA and its enantiomers.

3,4-Methylenedioxymethamphetamine (MDMA) is an entactogen with therapeutic potential. The two enantiomers of MDMA differ regarding their pharmacokinetics and pharmacodynamics but the chiral pharmacology of MDMA needs further study in clinical trials. Here, an achiral and an enantioselective high performance liquid chromatography-tandem mass spectrometry method for the quantification of MDMA and its psychoactive phase I metabolite 3,4-methylenedioxyamphetamine (MDA) in human plasma were developed and validated. The analytes were detected by positive electrospray ionization followed by multiple reaction monitoring. The calibration range was 0.5-500 ng/mL for the achiral analysis of both analytes, 0.5-1,000 ng/mL for chiral MDMA analysis, and 1-1,000 ng/mL for chiral MDA analysis. Accuracy, precision, selectivity, and sensitivity of both bioanalytical methods were in accordance with regulatory guidelines. Furthermore, accuracy and precision of the enantioselective method were maintained when racemic calibrations were used to measure quality control samples containing only one of the enantiomers. Likewise, enantiomeric calibrations could be used to reliably quantify enantiomers in racemic samples. The achiral and enantioselective methods were employed to assess pharmacokinetic parameters in clinical study participants treated with racemic MDMA or one of its enantiomers. The pharmacokinetic parameters assessed with both bioanalytical methods were comparable. In conclusion, the enantioselective method is useful for the simultaneous quantification of both enantiomers in subjects treated with racemic MDMA. However, as MDMA and MDA do not undergo chiral inversion, enantioselective separation is not necessary in subjects treated with only one of the enantiomers.

Improved Catalytic Activity of Spherical Nucleic Acid Enzymes by Hybridization Chain Reaction and Its Application for Sensitive Analysis of Aflatoxin B1.

Conventional spherical nucleic acid enzymes (SNAzymes), made with gold nanoparticle (AuNPs) cores and DNA shells, are widely applied in bioanalysis owing to their excellent physicochemical properties. Albeit important, the crowded catalytic units (such as G-quadruplex, G4) on the limited AuNPs surface inevitably influence their catalytic activities. Herin, a hybridization chain reaction (HCR) is employed as a means to expand the quantity and spaces of G4 enzymes for their catalytic ability enhancement. Through systematic investigations, we found that when an incomplete G4 sequence was linked at the sticky ends of the hairpins with split modes (3:1 and 2:2), this would significantly decrease the HCR hybridization capability due to increased steric hindrance. In contrast, the HCR hybridization capability was remarkably enhanced after the complete G4 sequence was directly modified at the non-sticky end of the hairpins, ascribed to the steric hindrance avoided. Accordingly, the improved SNAzymes using HCR were applied for the determination of AFB1 in food samples as a proof-of-concept, which exhibited outstanding performance (detection limit, 0.08 ng/mL). Importantly, our strategy provided a new insight for the catalytic activity improvement in SNAzymes using G4 as a signaling molecule.

A novel fluorescent sensor for selective rifampicin detection based on the bio-inspired molecularly imprinted polymer-AgInS2/ZnS quantum dots.

A fluorescent sensing material based on the ternary core-shell quantum dots with outstanding optical properties and a bio-inspired molecularly imprinted polymer (MIP) as a recognition element has been prepared for selective detection of rifampicin (RFP). Firstly, AgInS2/ZnS core/shell quantum dots (ZAIS QDs) were prepared by a hydrothermal process. Then, the fluorescent sensor was prepared by coating these QDs by a dopamine-based MIP layer. The fluorescence of MIP@ZAIS QDs was quenched by RFP probably due to the photoinduced electron transfer process. The quenching constant was much higher for MIP@ZAIS QDs than the non-imprinted polymer@QDs, indicating that MIP@ZAIS QDs could selectively recognize RFP. Under the optimized conditions, the sensor had a good linear relationship at the RFP concentration range of 5.0 to 300 nM and the limit of detection was 1.25 nM. The respond time of the MIP@ZAIS QDs was 5 min, and the imprinting factor was 6.3. It also showed good recoveries ranging from 98 to 101%, for analysis of human plasma samples. The method is simple and effective for the detection of RFP and offers a practical application for the rapid analysis of human plasma samples.

Towards clinical adherence monitoring of oral endocrine breast cancer therapies by LC-HRMS-method development, validation, comparison of four sample matrices, and proof of concept.

Oral endocrine therapies (OET) for breast cancer treatment need to be taken over a long period of time and are associated with considerable side effects. Therefore, adherence to OET is an important issue and of high clinical significance for breast cancer patients' caregivers. We hypothesized that a new bioanalytical strategy based on liquid chromatography and high-resolution mass spectrometry might be suitable for unbiased adherence monitoring (AM) of OET. Four different biomatrices (plasma, urine, finger prick blood by volumetric absorptive microsampling (VAMS), oral fluid (OF)) were evaluated regarding their suitability for AM of the OET abemaciclib, anastrozole, exemestane, letrozole, palbociclib, ribociclib, tamoxifen, and endoxifen. An analytical method was developed and validated according to international recommendations. The analytical procedures were successfully validated in all sample matrices for most analytes, even meeting requirements for therapeutic drug monitoring. Chromatographic separation of analytes was achieved in less than 10 min and limits of quantification ranged from 1 to 1000 ng/mL. The analysis of 25 matching patient samples showed that AM of OET is possible using all four matrices with the exception of, e.g., letrozole and exemestane in OF. We were able to show that unbiased bioanalytical AM of OET was possible using different biomatrices with distinct restrictions. Sample collection of VAMS was difficult in most cases due to circulatory restraints and peripheral neuropathy in fingers and OF sampling was hampered by dry mouth syndrome in some cases. Although parent compounds could be detected in most of the urine samples, metabolites should be included when analyzing urine or OF. Plasma is currently the most suitable matrix due to available reference concentrations.

A highly sensitive Lock-Cas12a biosensor for detection and imaging of miRNA-21 in breast cancer cells.

The expression levels of microRNA (miRNA) vary significantly in correlation with the occurrence and progression of cancer, making them valuable biomarkers for cancer diagnosis. However, their quantitative detection faces challenges due to the high sequence homology, low abundance and small size. In this work, we established a strand displacement amplification (SDA) approach based on miRNA-triggered structural "Lock" nucleic acid ("Lock" DNA), coupled with the CRISPR/Cas12a system, for detecting miRNA-21 in breast cancer cells. The "Lock" DNA freed the CRISPR-derived RNA (crRNA) from the dependence on the target sequence and greatly facilitated the extended detection of different miRNAs. Moreover, the CRISPR/Cas12a system provided excellent amplification ability and specificity. The designed biosensor achieved high sensitivity detection of miRNA-21 with a limit of detection (LOD) of 28.8 aM. In particular, the biosensor could distinguish breast cancer cells from other cancer cells through intracellular imaging. With its straightforward sequence design and ease of use, the Lock-Cas12a biosensor offers significant advantages for cell imaging and early clinical diagnosis.

A Review of Advances in Bioanalytical Methods for the Detection and Quantification of Olanzapine and Its Metabolites in Complex Biological Matrices.

Schizophrenia is a serious mental disorder that significantly affects the social and professional life of patients, causing distortion of reality and loss of identity and cognitive abilities. Psychopharmacological treatment is an integral part of modern psychiatry, and the introduction of new "atypical" antipsychotic drugs has brought significant progress in the treatment of this disorder. One of these drugs is olanzapine, which has an effective effect on the productive symptoms of schizophrenia while having an almost minimal potential to cause extrapyramidal syndrome. However, its effectiveness is confronted with frequent side effects, referred to as "metabolic disorders". Therefore, to ensure the effectiveness of treatment and to minimize the side effects caused by olanzapine, it is recommended to monitor the drug level during therapy. This article reviews the bioanalytical methodologies that enable efficient extraction and sensitive analysis of olanzapine. We considered the advantages and disadvantages of different sample pretreatment methods, including traditional and novel strategies. The analytical conditions required for the separation and detection of olanzapine and its metabolites were analyzed using chromatographic methods combined with various detectors.

Advancements in Brain Research: The In Vivo/In Vitro Electrochemical Detection of Neurochemicals.

Neurochemicals, crucial for nervous system function, influence vital bodily processes and their fluctuations are linked to neurodegenerative diseases and mental health conditions. Monitoring these compounds is pivotal, yet the intricate nature of the central nervous system poses challenges. Researchers have devised methods, notably electrochemical sensing with micro-nanoscale electrodes, offering high-resolution monitoring despite low concentrations and rapid changes. Implantable sensors enable precise detection in brain tissues with minimal damage, while microdialysis-coupled platforms allow in vivo sampling and subsequent in vitro analysis, addressing the selectivity issues seen in other methods. While lacking temporal resolution, techniques like HPLC and CE complement electrochemical sensing's selectivity, particularly for structurally similar neurochemicals. This review covers essential neurochemicals and explores miniaturized electrochemical sensors for brain analysis, emphasizing microdialysis integration. It discusses the pros and cons of these techniques, forecasting electrochemical sensing's future in neuroscience research. Overall, this comprehensive review outlines the evolution, strengths, and potential applications of electrochemical sensing in the study of neurochemicals, offering insights into future advancements in the field.

Recent advance on microextraction sampling technologies for bioanalysis.

The contents of target substances in biological samples are usually at low concentration levels, and the matrix of biological samples is usually complex. Sample preparation is considered a very critical step in bioanalysis. At present, the utilization of microextraction sampling technology has gained considerable prevalence in the realm of biological analysis. The key developments in this field focus on the efficient microextraction media and the miniaturization and automation of adaptable sample preparation methods currently. In this review, the recent progress on the microextraction sampling technologies for bioanalysis has been introduced from point of view of the preparation of microextraction media and the microextraction sampling strategies. The advance on the microextraction media was reviewed in detail, mainly including the aptamer-functionalized materials, molecularly imprinted polymers, carbon-based materials, metal-organic frameworks, covalent organic frameworks, etc. The advance on the microextraction sampling technologies was summarized mainly based on in-vivo sampling, in-vitro sampling and microdialysis technologies. Moreover, the current challenges and perspective on the future trends of microextraction sampling technologies for bioanalysis were briefly discussed.

A review of analytical parameters in 'rapid' liquid chromatographic methods for bioanalysis: Can we do better?

Rapid bioanalysis is beneficial to many applications. However, how 'rapid' a method is, or could be, is often an unanswered question. In this statistical review, the authors have assessed multiple pre-analytical (i.e. sample preparation), and analytical method parameters specifically for liquid chromatography to assist researchers in developing and validating 'rapid' bioanalytical methods. We restricted the search to urine and plasma matrices only. Data were extracted from over 2,000 recent studies and evaluated to assess how these parameters affected the 'on-instrument' analysis time. In addition to methods using ultra-violet (UV) detection, there were a large number of mass spectrometric (MS) methods, allowing additional review of the differences between high- and low-resolution MS on analysis time. We observed that most (N = 922, 70 %) methods used 5 or 10 cm columns, and that whilst uptake of ultra-high performance (U)HPLC columns was good, the use of sub-5 cm columns and/or flow rates in excess of 1 mL/min was incredibly rare (N = 25, 3 %). The detector of choice for quantitative (U)HPLC-MS remains the triple quadrupole, although a number of groups report the use of high-resolution MS for such methods.

The tightest self-assembled ruthenium metal-organic framework combined with proximity hybridization for ultrasensitive electrochemiluminescence analysis of paraquat.

Metal-organic frameworks (MOFs), as porous materials, have great potential for exploring high-performance electrochemiluminescence (ECL) probes. However, the constrained applicability of MOFs in the realm of ECL biosensing is primarily attributed to their inadequate water stability, which consequently impairs the overall ECL efficiency. Herein, we developed a competitive ECL biosensor based on a novel tightest structural ruthenium-based organic framework emitter combining the proximity hybridization-induced catalytic hairpin assembly (CHA) strategy and the quenching effect between the Ru-MOF and ferrocene for detecting paraquat (PQ). Through a simple hydrothermal synthesis strategy, ruthenium and 2,2'-bipyrimidine (bpm) are head-to-head self-assembled to obtain a novel tightest structural Ru-MOF. Due to the metal-ligand charge-transfer (MLCT) effect between ruthenium and the bpm ligand and the connectivity between the internal chromophore units, the Ru-MOF exhibits strong ECL emissions. Meanwhile, the coordination-driven Ru-MOF utilizes strong metal-organic coordination bonds as building blocks, which effectively solves the problem of serious leakage of chromophores caused by water solubility. The sensitive analysis of PQ is realized in the range of 1 pg/mL to 1 ng/mL with a detection limit of 0.352 pg/mL. The tightest structural Ru-MOF driven by the coordination of ruthenium and bridging ligands (2,2'-bipyrimidine, bpm) provides new horizons for exploring high-performance MOF-based ECL probes for quantitative analysis of biomarkers.

Advancing Personalized Medicine by Analytical Means: Selection of Three Metabolites That Allows Discrimination between Glaucoma, Diabetes, and Controls.

This paper aimed at devising an intelligence-based method to select compounds that can distinguish between open-angle glaucoma patients, type 2 diabetes patients, and healthy controls. Taking the concentration of 188 compounds measured in the aqueous humour (AH) of patients and controls, linear discriminant analysis (LDA) was used to identify the right combination of compounds that could lead to accurate diagnosis. All possibilities, using the leave-one-out approach, were considered through ad hoc programming and in silico massive data production and statistical analysis. Our proof of concept led to the selection of four molecules: acetyl-ornithine (Ac-Orn), C3 acyl-carnitine (C3), diacyl C42:6 phosphatidylcholine (PC aa C42:6), and C3-DC (C4-OH) acyl-carnitine (C3-DC (C4-OH)) that, taken in combination, would lead to a 95% discriminative success. 100% success was obtained with a non-linear combination of the concentration of three of these four compounds. By discarding younger controls to adjust by age, results were similar although one control was misclassified as a diabetes patient. Methods based on the consideration of individual clinical chemical parameters have limitations in the ability to make a reliable diagnosis, stratify patients, and assess disease progression. Leveraging human AH metabolomic data, we developed a procedure that selects a minimal number of metabolites (3-5) and designs algorithms that maximize the overall accuracy evaluating both positive predictive (PPV) and negative predictive (NPV) values. Our approach of simultaneously considering the levels of a few metabolites can be extended to any other body fluid and has potential to advance precision medicine. Artificial intelligence is expected to use algorithms that use the concentration of three to five molecules to correctly diagnose diseases, also allowing stratification of patients and evaluation of disease progression. In addition, this significant advance shifts focus from a single-molecule biomarker approach to that of an appropriate combination of metabolites.

Greenness assessment of microextraction techniques in therapeutic drug monitoring.

Aim: In this study, we evaluated the greenness and whiteness scores for microextraction techniques used in therapeutic drug monitoring. Additionally, the cons and pros of each evaluated method and their impacts on the provided scores are also discussed. Materials & methods: The Analytical Greenness Sample Preparation metric tool and white analytical chemistry principles are used for related published works (2007-2023). Results & conclusion: This study provided valuable insights for developing methods based on microextraction techniques with a balance in greenness and whiteness areas. Some methods based on a specific technique recorded higher scores, making them suitable candidates as green analytical approaches, and some others achieved high scores both in green and white areas with a satisfactory balance between principles.

Combinatorial Effect of Tricomponent Dual-Rim Nanoring Building Blocks: Label-Free SERS Detection of Biomolecules.

Detecting weakly adsorbing molecules via label-free surface-enhanced Raman scattering (SERS) has presented a significant challenge. To address this issue, we propose a novel approach for creating tricomponent SERS substrates using dual-rim nanorings (DRNs) made of Au, Ag, and CuO, each possessing distinct functionalities. Our method involves depositing different metals on Pt nanoring skeletons to obtain each nanoring with varying surface compositions while maintaining a similar size and shape. Next, the mixture of these nanorings is transferred into a monolayer assembly with homogeneous intermixing on a solid substrate. The surface of the CuO DRNs has dangling bonds (Cu2+) that facilitate the strong adsorption of carboxylates through the formation of chelating bonds, while the combination of Au and Ag DRNs significantly enhances the SERS signal intensity through a strong coupling effect. Notably, the tricomponent assemblies enable the successful SERS-based analysis of biomolecules such as amino acids, proteins, nucleobases, and nucleotides.

A simple UHPLC-MS/MS method for determination of SET2, a selective antagonist of TRPV2 receptor, in rat plasma samples.

Targeting the transient receptor potential vanilloid 2 channels (TRPV2) in order to alleviate or reverse the course of several diseases including multiple cancers, cardiovascular, immunological, or neurological disorders have been a matter of focus for several years now. SET2, a selective TRPV2 inhibitor, represents an innovative molecule which came into recognition in 2019 and seems to be a promising therapeutic modality in cancer and cardiac diseases. Drug discovery and bioanalysis in clinical environment demands simple, excellent, highly reliable, fast, sensitive, and selective analytical approaches which enable unambiguous identification and quantification of demanded molecule. Here, a targeted ultra-high-performance liquid chromatography - tandem mass spectrometry with electrospray ionization was developed for the quantification of SET2 in plasma samples. The developed method enabled analysis of approx. 15 samples within one hour. Simplicity of the whole analytical procedure can be emphasized by a very simple sample pretreatment based only on the protein precipitation with organic acid (here, 2 M tricholoroacetic acid). The validation procedure was characterized by promising validation parameters and excellent sensitivity what was documented by the limit of detection value at pg.mL-1 concentration level. Analytical validation reported intra- and interday accuracy < 15 % for all quality control samples concentration levels. Similarly, excellent level of intra- (0.1 - 4.8 %) and interday (0.5 - 3.3 %) precision for the tested quality control samples was obtained. The applicability of the developed method was proven by quantifying SET2 concentration levels in plasma samples obtained from Wistar rats that were administered this drug intraperitoneally at a dose of 25 mg/kg. We expect that our new analytical method represents a very attractive tool that could be easily implemented in pharmacokinetics studies and/or therapeutic drug monitoring. Moreover, its applicability was confirmed by the new practicability evaluation metric tool.