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The increase in the use of therapeutic monoclonal antibodies (mAbs) and antibody-drug conjugates (ADCs) has made the detailed bioanalysis of these drugs essential not only for planning optimal therapeutic programs for clinical practice, but also for evaluating the biological equivalencies in the development of other biosimilars. The ligand binding assays that are widely in use now are being replaced rapidly by the highly accurate, sensitive, and selective analytical method using a mass spectrometer. This review will discuss the progress in and challenges observed during the development of a mass spectrometry-based bioanalytical method for therapeutic mAbs and ADCs.

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In this study, an on-line supercritical fluid extraction (SFE) and supercritical fluid chromatography (SFC) method was developed for the rapid determination of oleanoic acid and ursolic acid in Chaenomelis Fructus. After optimization of the conditions, the two triterpenoid acids was obtained by SFE using 20% methanol as a modifier at 35°C in 8 min. They were resolved on a Shim-pack UC-X Diol column (4.6 × 150 mm, 3 μm) in 14 min (0 – 10 min, 5 – 10%; 10 – 14 min, 10% methanol in CO₂) with a backpressure of 15 MPa at 40°C. The on-line SFE-SFC method could be completed within 40 min (10.79 mg/g dry plant, R_s = 2.36), while the ultrasound-assisted extraction and HPLC method required at least 90 min (3.55 mg/g dry plant, R_s = 1.92). This on-line SFE-SFC method is powerful to simplify the pre-processing and quantitative analysis of natural products.

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In this work, bismerthiazol was firstly assayed by a fast and portable method employing protein-capping gold nanoculsters as probes. The luminescent intensity of the nanoclusters showed a correlative response towards bismerthiazol from 5 to 4000 μg/mL with a linear relation in the range of 5 – 100 μg/mL. As little as 5 μg/mL of bismerthiazol could be quantified. The high affinity of bismerthiazol to interact with the soybean protein-capped gold nanoclusters contributed to the excellent selectivity of this method over other common pesticides. The recoveries in several cabbage samples were 101 – 135%, indicating good performance in practical applications. By comparison to previous reported approaches, this method bears advantages including simple operation, fast response, visual readout and good selectivity.

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The degradation of polycyclic aromatic hydrocarbons (PAHs) can generate AhR-binding compounds, exhibiting genotoxicity and carcinogenicity. In this investigation, aryl hydrocarbon receptor (AhR) from carp and anthracene (Ant) were coupled as antigen to establish an indirect competition ELISA (ic-ELISA) with an AhR-Ant antibody. A standard curve was determined for the ic-ELISA concerning detection range and limit. Also, the specificity, stability and the recovery of the ic-ELISA were checked. Results indicate that the ratio of antibody to antigen titer is 1:64000. The resulting standard curve is Y = 21.326 × X + 1.8213. The detection range lies within 10 – 1000 ng mL⁻¹ and the limiting concentration is 2.43 ng mL⁻¹. The cross reaction ratio (CR) between Ant and naphthalene (Nap), Ant and phenanthrene (Phe) or Ant and fluoranthene (Flu) were 5.7, 19.1 and less than 0.1%, respectively. The range of the coefficient of variance (C.V) amounts was from 4.2 up to 9.5% and the recovery range was from 90 to 115%. These results show that the AhR-Ant ic-ELISA is sensitive, and can be used as a technical support to quantify Ant in the environment.

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A simple enzyme-based nanohybrid material was fabricated via immobilizing ascorbic acid oxidase (AO) on the surface of flower-like electrodeposited gold nanoparticles (dpAu) and reduced graphene oxide (rGO) modified glassy carbon electrodes (GCEs). The composite material was used for stereoselective interaction with ascorbic acid (AA) and isoascorbic acid (IAA). Herein, AO was applied as a stereoselective selector, and the dpAu/rGO nanohybrid not only acted as a supporter for high loading of AO, but also served as the nanomaterial for signal amplification. The results showed obvious peak current differences between AA and IAA, indicating that this strategy could be employed to recognize AA and IAA. Under the optimum conditions, the sensor exhibited a good linear response to AA and IAA in a linear range of 1.0 × 10⁻⁴ – 5.0 × 10⁻³ M. This approach with the merits of simplicity and rapid response provided a promising perspective for identification of AA and IAA.

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Surface-initiated anionic-ring-opening multibranching polymerization was employed to prepare a hyperbranched polyglycerol (HPG) functionalized silica stationary phase for hydrophilic interaction liquid chromatography (HILIC). The obtained stationary phase was characterized by Fourier-transform infrared spectrometry (FT-IR) and thermogravimetric analysis (TGA). The chromatographic properties of the prepared stationary phase were systematically investigated. The abundance and multitude distribution of hydroxyl groups in HPG endowed the stationary phase with improved hydrophilicity and enhanced separation performance compared with the stationary phase functionalized with monolayer of hydroxyl groups. The stationary phase showed excellent retention of various polar compounds, such as nucleosides, necleobases, phenols and sulfanilamides, indicating great potential in the separation of complex biosamples.

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Resveratrol is one of representative ingredient in red wine, but its quantification is a challenge because of a complex and abundant matrix. In this study, two sample pretreatments, direct dilution and QuEChERS extraction, coupling LCMS analysis were examined for resveratrol quantification. Similar recoveries of 106.4 to 93.7% were obtained for direct dilution and QuEChERS, respectively. With the aid of condition optimization, QuEChERS extraction could concentrate the resveratrol from red wines to improve the detection sensitivity with a LOD value of 2.5 ng/mL, which is four-times greater than the direct dilution approach. As a result, the QuEChERS method can provide a high linearity within the concentration range of 5 – 500 ng/mL, in which direct dilution produced the linear calibration curve within the concentrations of 25 – 500 ng/mL. A high consistency was obtained for both approaches in which intra-day precisions were within 0.5 to 7.2% (n = 3), and the inter-day precisions were within 7.8 to 16.0% (n = 9). Overall, the sample pretreatment of QuEChERS can effectively reduce the matrix effect, which leads LCMS to quantify the low resveratrol abundance of 8.0 ppb in each red wine sample, which is not achieved with the direct dilution approach.

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Reduced imine cage (RCC3) was first adopted for the preparation of porous organic cage embedded C18 amide silica stationary phase for high performance liquid chromatography. The prepared stationary phase was characterized by scanning electron microscope (SEM) and Fourier transformation infrared spectrum (FT-IR). Its chromatographic performance under reversed-phase mode was investigated in detail and compared with that of an ODS column. Multiple interactions, including hydrophobic interaction, π-π interactions, electrostatic interactions and hydrogen bonding, were involved due to the synergism of the C18 chain and RCC3. The column showed typical methylene selectivity and enhanced aromatic selectivity for nonpolar analytes while demonstrating high selectivity for polar analytes. In addition, the stationary phase showed the capability of separation of polar and hydrophilic compounds under per aqueous liquid chromatography mode (PALC), providing a green and economical way for the separation of polar and hydrophilic compounds. These results indicated the great application potential of the prepared stationary phase in the analysis of complex samples.

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The fluorescent probe has become an important method for the detection of heavy metal ions. In the present work, a new and simple fluorescent probe, Cu-P, for detecting copper ion (Cu²⁺) was designed and synthesized. The probe has shown high sensitivity and selectivity toward Cu²⁺. The detection limit was 13 nM (based on the 3σ/slope). A significant color change from yellow to pink was observed; thus, the probe Cu-P could serve as a “naked-eye” indicator for Cu²⁺. Furthermore, the proposed probe was used to detect Cu²⁺ in real water and soil extract samples, with the result being satisfactory. Therefore, our proposed probe would provide a promising method for the detection of Cu²⁺ in the environment.

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A non-enzymatic fluorescence method for the determination of hydrogen peroxide (H₂O₂) was investigated. This method is based on the hydroxylation reaction of terephthalate (TP) by hydroxyl radical formed from reaction between H₂O₂ and Fe(II), resulting in the formation of a strongly fluorescent 2-hydroxyterephthalate (HTP). Under optimized conditions, a 3 nM detection limit and 1.0% precision at 200 nM were obtained. This was sensitive enough to determine the concentrations of H₂O₂ in coastal marine environments. The slopes of the calibration curve in seawater were nearly the same as those in Milli-Q water, suggesting that the fluorescent intensity was not affected by coexisting sea salts. However, the presence of nitrite at more than 10 μM interfered with the formation of HTP. The developed method was successfully applied to determine the concentrations of H₂O₂ in Osaka Bay on the research vessel. The results obtained in Osaka Bay demonstrated that H₂O₂ was photochemically produced via the photolysis of dissolved organic matter supplied from the river with fresh water at the surface layer.

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Seawater-extractable organic matter (SWEOM) has a potential to serve as an Fe complexing agent in an Fe-fertilizer intended to restore and preserve macroalgal forests. To better understand the restoration technique, structural alterations in SWEOM that occurred during a 90-day period of incubation in seawater were determined. Up to 82% of the total eluted SWEOM occurred during the initial 30 days; a small amount of SWEOM then continued to elute during the next 60 days. Spectroscopic analyses showed that the high-molecular-weight SWEOM fraction (HMW) altered in aliphatic-rich materials, while the low-molecular-weight fraction (LMW) became significantly enriched in aromatic structures. The structural alterations in the HMW and LMW could be caused by the action of anaerobic microorganisms. In addition, approximately 30 and 60% of the gametophytes were converted to eggs in the presence of HMW and LMW with Fe, respectively.

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The Ar–N₂ mix gas effect can easily improve the sensitivity of ICPMS; however, this effect discriminates against Sr. In this study, it was found that Ar–N₂ mixed gases introduced into nebulizing gas enhanced the sensitivity of online solid-phase extraction (SPE) with inductively coupled plasma mass spectrometry (ICPMS) for radioactive strontium quantification. An ultrasonic nebulizer (USN) improved the Ar–N₂ mixture gases effect of Sr and the mix gases (with USN) enhanced 3.7-times the signal intensity of Sr in normal pure Ar gas (with USN) in an online SPE-ICPMS. By adapting the gas-loading means from a nebulizing gas unit via USN, no careful tuning was necessary for the plasma turning. With this signal enhancement, a 0.06 pg/L detection limit (0.3 Bq/L) was achieved for radioactive strontium (⁹⁰Sr) in online SPE-ICPMS within 30 min. In addition, environmental paddle water in the Fukushima Nuclear Power Plant was measured and the valued correspond to that obtained by radiometry.

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The determination of dissolved silica in seawater was investigated by an ion-exclusion chromatography with a post-column derivatization/silicomolybdenum yellow method. To determine dissolved silica in seawater accurately, the experimental conditions, such as the volume of a reaction coil, the eluent concentration and the sample dilution factor, were optimized. This is the first report that has examined the details of the experimental condition for the measurement of dissolved silica in seawater by ion-exclusion chromatography with silicomolybdenum-yellow detection. The developed method was compared with the widely used continuous flow analysis and an ion-exclusion chromatography isotope dilution inductively coupled plasma mass spectrometry, which is a primary method of measurement. The analytical results by the three methods were in good agreement, considering the expanded uncertainty with a coverage factor, 2. The validity of the three methods was confirmed with each other. The developed method can give a quantitative value with less than 1% of the expanded uncertainty, and can be used for the determination of dissolved silica in candidate reference materials having a seawater matrix.

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The binding constant of receptors for small molecules, proteins, or antibodies is usually determined based on the concentrations of the ligand, receptor, and their complexes. The binding constant is used as a measure of the affinity between the ligand and the receptor. In the present study, we introduce a procedure to determine the binding constant of a cell-shaped cavity formed on a polymer by molecular imprinting for a whole cell. To determine the binding constant, we clarified the numbers of cavities and cells, based on the fluorescence of a single cell, and defined their concentrations. We successfully determined the binding constant of the complementary cavity for a whole cell (1.1 × 10⁵ M⁻¹). This is the first report to describe the binding constant of a complementary cavity for a whole cell.

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A high-precision analytical system to observe the variations in the amount fractions of atmospheric oxygen with a very small uncertainty was developed. The system comprises a magneto-pneumatic oxygen analyzer and three automatic pressure controllers. The drift of the analyzer’s signal intensity can be reduced when the amount fractions of oxygen in the sample and reference gases are similar because the temperature coefficient of the analyzer linearly depends on the difference between these amount fractions. The repeatability of oxygen determination and the long-term stability of the system were tested to assess the applicability of the analyzer to field-based measurements for continuous atmospheric observations. The standard deviation of the average for 10-min measurements in the 5-day long-term stability test was 0.7 μmol mol⁻¹ after a temperature correction. This indicates that the system can continuously measure the amount fractions of oxygen in the atmosphere for a few days without interruption for any calibration and/or compensation for the signal drift.

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Monitoring of trace water in industrial gases is strongly recommended because contaminants cause serious problems during use, especially in the semiconductor industry. An ultra-sensitive trace-water sensor was developed with an in situ-synthesized metal–organic framework as the sensing material. The sample gas is passed through the sensing membrane and efficiently and rapidly collected by the sensing material in the newly designed gas collection/detection cell. The sensing membrane, glass paper impregnated with copper 1,3,5-benzenetricarboxylate (Cu-BTC), is also newly developed. The amount and density of the sensing material in the sensing membrane must be well balanced to achieve rapid and sensitive responses. In the present study, Cu-BTC was synthesized in situ in glass paper. The developed system gave high sensing performances with a limit of detection (signal/noise ratio = 3) of 9 parts per billion by volume (ppbv) H₂O and a 90% response time of 86 s for 200 ppbv H₂O. The reproducibility of the responses within and between lots had relative standard deviations for 500 ppbv H₂O of 0.8% (n = 10) and 1.5% (n = 3), respectively. The long-term (2 weeks) stability was 7.3% for 400 ppbv H₂O and one-year continuous monitoring test showed the sensitivity change of <∼3% before and after the study. Furthermore, the system response was in good agreement with the response achieved in cavity ring-down spectroscopy. These performances are sufficient for monitoring trace water in industrial gases. The integrated system with light and gas transparent structure for gas collection/absorbance detection can also be used for other target gases, using specific metal–organic frameworks.

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Therapeutic peptides and diagnostic agents with their molecular size below the renal clearance threshold suffer from short blood circulation time. Here, we report a novel design of peptide-based ligand with a strong binding affinity to human serum albumin (HSA), which can be used as a tag to extend the blood circulation of small-size molecules. We designed ligands with dual alkyl groups connected with a negatively charged spacer. The ligands showed both higher binding affinity to HSA and a higher retention in mice blood than that of a single alkylated peptide.

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A sensitive capillary electrophoresis method was developed for the determination of aminothiol (cysteine, homocysteine, and glutathione) total levels in human blood plasma. Analytes were derivatized with Ellman’s reagent (5,5′-dithiobis(2-nitrobenzoic acid)) after reduction with dithiothreitol. Liquid–liquid extraction was applied to purify the samples and concentrate the analytes. Total analysis time was 7.5 min using a silica capillary (50 μm i.d.; effective separation length 23.5 cm). Electrophoretic separation was performed using 50 mM citric acid with 20 mM triethanolamine (pH 3) containing 2% Ficoll 400. Detection limit was 0.8 μM for glutathione and 0.3 μM for both cysteine and homocysteine. Accuracy was 94 – 107%, repeatability and reproducibility were ca. 2.7 – 3.5 and 2.5 – 6.5%, respectively.

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