Kenichiro Todoroki

Nucleic acid aptamers are single-stranded oligonucleotides that recognize diverse molecular targets with high affinity and specificity. Owing to their small size, amenability to chemical synthesis, and facile functionalization, aptamers are emerging as versatile alternatives or complements to antibodies in pharmaceutical sciences. This review summarizes recent advances from 2020 to 2025 in the pharmaceutical applications of aptamers, focusing on four areas: therapeutics, biosensing, bioanalysis, and process analysis. In therapeutics, the U.S. Food and Drug Administration (FDA) approval of Izervay (avacincaptad pegol) in 2023 marked a significant milestone, renewing interest in aptamer-based drug development. Aptamers are being explored for cancer therapy, immunotherapy, and neurodegenerative disease treatment, with several candidates in clinical trials. In diagnostics, aptamer-based biosensors (aptasensors) enable sensitive detection of tumor markers, disease biomarkers, and infectious disease agents, demonstrating particular utility in point-of-care applications. For bioanalysis of biopharmaceuticals, anti-idiotype aptamers serve as capture molecules for antibody drug quantification, offering advantages in batch-to-batch consistency and stability. In process analytical technology, aptamers are being applied to real-time monitoring of cell culture conditions and quality control of antibody drug manufacturing. These advances position nucleic acid aptamers as increasingly important tools in pharmaceutical sciences.

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Bioanalysis of the immune checkpoint inhibitor nivolumab is crucial for pharmacokinetic studies. However, conventional ligand-binding assays (LBAs) often suffer from the lot-to-lot variability of capture antibodies. Here, we developed a high-affinity anti-nivolumab DNA aptamer as a robust alternative reagent. An integrated strategy combining Fast Protein Liquid Chromatography-based screening, next-generation sequencing, aptamer search algorithm, and computational modeling enabled rapid aptamer identification and optimization. The anti-nivolumab aptamer, mutant 2-3, discovered through in silico modification exhibited a dissociation constant (KD) of 33 nM. Using this aptamer, an enzyme-linked aptamer assay (ELAA) successfully quantified nivolumab in 400-fold diluted plasma samples over a range of 0.02–5.0 µg/mL, demonstrating good precision with a coefficient of variation less than 11.9%. This novel analytical method provides a sufficient quantitative range for the bioanalysis of nivolumab, thus offering a robust, specific, and cost-effective alternative for future pharmacokinetic studies.

1. In the development and appropriate use of therapeutic monoclonal antibodies (mAbs) and antibody–drug conjugates (ADCs), bioanalysis of drug concentrations in blood is a core-enabling platform th...

Carnitine (CAR) is an essential compound for animals and plays several physiological roles related to energy production. These functions are possessed by only the L-forms, while the D-forms are known to inhibit the uptake of the L-forms thus causing a CAR deficiency. Therefore, it is required to determine DL-CAR in various samples such as foods, supplements and drugs by an enantioselective analytical method. In this study, we developed a chiral liquid chromatography-tandem mass spectrometry (LC-MS/MS) method with the combination of derivatization. The CAR enantiomers derivatized with 4-nitrophenylhydrazine were successfully separated on a CHIRALPAK ZWIX (-) column (resolution = 1.64) and detected with a high sensitivity and selectivity by MS/MS. The developed method was well validated and applied to the analysis of 8 kinds of foods and an L-CAR supplement. Only the L-forms were found in all the tested food samples and were abundant in meat, milk and yogurt. The system was also applicable for a supplement sample and could detect the D-CAR contamination at the 2.5% level, suggesting its potential for use in the purity test of CAR preparations.

We report the misuse of a novel synthetic glucocorticoid, 9α-fluoro-6α-methylprednisolone (9F6MP) for the first time in camel racing and, to the best of our knowledge, human or other animal sports. During routine post-race drug testing of cameline plasma samples using liquid chromatography-tandem mass spectrometry we encountered an unknown peak with the same selected reaction monitoring traces as a dexamethasone formate adduct but at a different retention time. The product ion mass spectrum of the unknown peak in negative ion mode was identical to dexamethasone. However, significant differences were observed in positive ion mode. Based on mass spectral analysis, we postulated the unknown peak to be a 6-methyl-16-nor isomer of dexamethasone. Following the procurement of a commercial 9F6MP reference material, the unknown peak was successfully identified as this substance. Interestingly, previous research predicted a high potential for glucocorticoid and anti-inflammatory activity for 9F6MP. However, the therapeutic use of 9F6MP in camels has not been approved by any authorities, and any toxicities and side-effects potentially caused by 9F6MP have not been thoroughly evaluated. Therefore, the misuse of 9F6MP should be strictly controlled for the sake of animal welfare and the integrity of camel racing. The information described in this case report will be beneficial for other anti-doping laboratories in both human and animal sports for the purpose of doping control.

Several years have passed since the dynamics of d-amino acids (AAs) in the living body of mammals have drawn attention as potential biomarkers of several diseases. So far, researchers have developed an enantiomer-separable method using liquid chromatography and mass spectrometry (LC-MS). Using these excellent methods for dl-AA analysis, much knowledge was obtained about d-AAs. In our previous study, (S)-2,5-dioxopyrrolidin-1-yl-1-(4,6-dimethoxy-1,3,5-triazin-2-yl)pyrrolidine-2-carboxylate (DMT-(S)-Pro-OSu: DMT(S)), which was a chiral derivatization reagent for LC-MS analysis, showed high sensitivity that enabled detection of dl-AAs at the low concentration level of biological samples. However, DMT(S) required five different elution conditions and two columns for 19 dl-AA analysis with resolution (Rs) > 1.5. Herein, we report the development of novel derivatization reagents, which have a modified structure based on DMT(S) to enhance enantiomeric separation. A comparison of the separation efficiency of developed reagents and DMT(S) was higher for DMT(S) and the isopropoxy-modified reagent (i.e., DiPT(S)). In addition, a multiple regression model for the prediction of Rs was also developed. Our binary mobile phase system prediction model showed high accuracy with only five condition test data sets. The resulting analytical method, using parallel derivatization (DMT(S) and DiPT(S)) and an elution condition predicted by the developed model, enabled simultaneous analysis of 19 dl-AAs in 45 min LC run by stepwise elution. These techniques contribute to the efficient and speedy development of an analysis method with chiral derivatization for dl-AAs and other chiral metabolites.

This study introduces F-trap pyrene Ox, a reagent peak-free pre-column fluorescence derivatization reagent for sensitive analysis of amines and amino acids. By oxidizing the thioether moiety of previously reported F-trap pyrene to sulfone, we significantly enhanced reactivity toward amino groups, achieving complete derivatization in 10 min at 90 °C. The reagent incorporates a fluorophilic perfluoroalkyl tag that enables selective removal of excess unreacted reagent through fluorous solid-phase extraction (F-SPE) with 75 % aqueous acetonitrile as eluent, achieving >99.4 % removal rate while maintaining excellent recovery. Method validation for aliphatic amines demonstrated good linearity (r2 > 0.995) over 50 fmol to 50 pmol, excellent precision (intra-day RSD ≤ 6.7 %), and detection limits 4-times lower than F-trap pyrene. Additionally, we developed an online column-switching F-SPE system that automates the removal process. The method successfully analyzed 13 aliphatic amines and 16 amino acids without interference from reagent peaks, offering significant advantages for sensitive analysis of biologically active amines in complex matrices.