Journal of Pharmaceutical and Biomedical Analysis Open最新文献

A three-dimensional (3D) HPLC system was designed/developed for the discriminative determination of aliphatic chiral amino acids, namely, alanine (Ala), valine (Val), isoleucine (Ile), allo-Ile (aIle) and leucine (Leu), in human physiological fluids. These aliphatic amino acid enantiomers are expected to be new physiologically active molecules and/or biomarkers in mammals. Among these aliphatic amino acids, the structural chain isomers of Leu (Ile/aIle/Leu) have similar chemical-physical properties, and the analytical method for these aliphatic amino acids is required to be highly enantio- and chemo-selective. In the present study, a reversed-phase column (Singularity RP18, first dimension) and a mixed-mode column (Singularity MX-103, second dimension) were utilized to separate these aliphatic amino acids as their scalemic mixtures, then chiral separations were performed using a Pirkle-type enantioselective column (Singularity CSP-001S) in the third dimension. By using the 3D-HPLC system, these aliphatic amino acid enantiomers were completely discriminated, and the analysis of these aliphatic chiral amino acids in the human plasma and urine was successfully carried out. The obtained amounts of the aliphatic amino acids (and %D value, the percentage of D-form to D + L forms) in the human plasma were 2.1 µM (0.5%) for D-Ala, and trace/not-detected for the other D-forms. In human urine, the values were 102.0 µM (24.7%) for D-Ala, 2.0 µM (3.4%) for D-Val, 2.3 µM (10.8%) for D-aIle (%D value of D-aIle was calculated using D-aIle and L-Ile) and 3.3 µM (5.5%) for D-Leu. The present 3D-HPLC system is a powerful and well validated tool for the simultaneous determination of aliphatic amino acid enantiomers, and further biological and clinical studies are expected.

A highly sensitive nanoprobe HAPPF@FeSe₂NPs was developed for real-time detection of hyaluronidase (HAase) as tumor marker. Hyaluronic acid (HA) was first functionalized with adipic acid dihydrazide (ADH) via amide formation. Carboxyl-terminated polylactide (PLA) was then grafted to HA-ADH using EDC/NHS as coupling agents, yielding an HA-ADH-PLA (HAP) graft copolymer. Iron selenide nanoparticles (FeSe₂NPs) were surface coated with HAP by ultrasonic assisted self-assembly. Finally, the resulted HAP@FeSe₂NPs were surface-modified with fluorescein isothiocyanate (FITC) and polyethyleneimine (PEI) to obtain HAPPF@FeSe₂NPs fluorescent nanoprobe. Analyses by FTIR, XRD, XPS, EDX, TEM, DLS, ¹H NMR and TGA confirmed the successful synthesis of the nanoprobe, and fluorescence spectroscopy proved its good detection performance. Moreover, cell imaging results demonstrated the ability of the nanoprobe to target tumor cells. MTT assay and hemolysis tests confirmed its good cyto- and hemo-compatibility. Therefore, it is concluded that HAPPF@FeSe₂NPs nanoprobe could be used in targeted tumor cell imaging for early diagnosis of cancers.

The removal of host cell proteins (HCPs) remains a challenge in the downstream processing of monoclonal antibodies (mAbs). It is critical to monitor residual HCPs since they can have an impact on product stability and safety. The enzyme-linked immunosorbent assay (ELISA) only quantifies the total amount of HCPs and does not indicate the identity or quantity of any specific HCP. Mass spectrometry-based proteomics applications demonstrate an advantage in quantitatively profiling individual HCPs. However, technical reproducibility, dynamic range, and ensuring an acceptable statistical significance of scoring measurements are critical hurdles that ought to be overcome. In this paper, we describe a sensitive and reproducible shotgun proteomics approach that includes an efficient mAb depletion strategy and addresses the shortcomings of such workflows. Our methodology is potentially a valuable complement or alternative to ELISA. Additionally, it can facilitate the purification process development and the evaluation of ELISA kits upon process changes.

The aim of this study is to develop a molecularly imprinted polymer (MIP) sensor via photopolymerization for selective and sensitive analysis of entacapone (ENT), a catecholamine-o-methyl transferase (COMT) inhibitor. ENT is used for Parkinson’s disease treatment with levodopa (LEV) and carbidopa (CAR), and it is available in a triple combination tablet dosage form. For the MIP-based electrochemical sensor (4-AP@MIP/GCE) design, 4-aminophenol (4-AP) was selected as the functional monomer. Surface characterization of the 4-AP@MIP/GCE sensor was performed by scanning electron microscopy, electrochemical characterization by CV, and electrochemical impedance spectroscopy (EIS). The linear concentration range was between 1.0 pM and 10.0 pM under optimum conditions for ENT determination. The limit of detection (LOD) of 0.24 pM and the limit of quantification (LOQ) of 0.80 pM were calculated for ENT analysis using 4-AP@MIP/GCE. The accuracy was proven by performing a recovery study on the tablet sample that contains ENT, LEV, and CAR, and the recovery was found to be 99.32%. The selectivity of the 4-AP@MIP/GCE sensor for ENT has been proven by interference studies on LEV, CAR, and their binary mixtures. Looking at the results of the study, it has been proven that the 4-AP@MIP/GCE sensor is a highly selective, sensitive, and specific quantitative option for the analysis of ENT.

In the last decade, the availability of new and versatile synthetic strategies for the preparation of substituted 4,4’-bipyridyl derivatives based on chemo- and regioselective functionalization of the 4,4’-bipyridine core has encouraged studies for exploring the bioactivity of these compounds in the fields of drug discovery and medicinal chemistry. In substituted 4,4’-bipyridines, chirality may emerge from restricted rotation induced by sterically hindered atoms or functional groups located around the 4,4’-biaryl bond (chiral axis). The first atropisomeric substituted 4,4’-bipyridine was prepared in 2008, and no asymmetric synthesis to produce pure atropisomers of chiral 4,4’-bipyridine derivatives has been available so far. Thus, in the last few years, our groups developed methods to separate atropisomers of a wide series of 4,4’-derivatives by high-performance liquid chromatography (HPLC) using polysaccharide-based chiral stationary phases (CSPs). In the frame of our interest in this field, we reported herein the synthesis of two new chiral carboxylic acids containing an axially chiral 4,4’-bipyridyl unit as source of chirality, and their HPLC enantioseparation on polysaccharide-based CSPs. In particular, the impact of analyte and CSP structures on the enantioseparation outcomes as well as mechanisms and noncovalent interactions underlying the enantioseparation were explored by using electrostatic potential analysis and molecular dynamics (MD) simulations.

Since 1950, the use of perfluoroalkyl substances (PFAS) increased due to their heat resistance, lipophobic and hydrophobic properties; therefore, these compounds are widely employed to make waterproof and heat resistant coatings, such as food packaging or work wear. However, these chemicals represent a risk to the environment due to their stability to degradation. Moreover, these compounds properties represent a risk also for humans; many studies correlated their concentrations in biological matrices to pathologies, such as hypertension, diabetes, and cancer. To this concern, the analytical detection in different biological matrices plays a crucial role to assess the presence of such analytes in different body districts. We performed a literature search in different scientific databases to review articles reporting the application of PFAS analysis for human exposure monitoring and for possible association with pathologies. The search resulted in 58 studies investigating PFAS presence in conventional matrices, such as blood and urine, and unconventional matrices. Although the solid-phase extraction was preferred for all the considered matrices, liquid-liquid extraction and dilute and shot demonstrated to be suitable extraction approach. The most used instrumental technique was the LC-MS/MS equipped with C18 chromatographic column, electrospray injection source operating in negative mode, and multiple reaction monitoring spectrometric acquisition. The untargeted detection of PFAS was attempted using an LC-HRMS method to elucidate possible new compound structures. Notably, the instruments and laboratory tools may represent an important contamination source due to the PFAS presence in their constituents. The development of an analytical method able to reach low limits of detection (LOD) and suitable for different biological matrices is crucial to study both PFAS health effects and a possible pharmacokinetics. For this purpose, current knowledge about PFAS analytical methods in biological matrices applied to human biomonitoring and pathology studies is reviewed to raise awareness of these chemicals’ activities.

Post-translational modifications (PTMs) of therapeutic monoclonal antibodies (mAbs) can impact the efficacy of a drug. Methionine oxidation can alter the overall hydrophobicity of an antibody, thereby inducing conformational changes and affecting its biological activity. To ensure high quality, safety, and efficacy of mAbs, routine monitoring of PTMs such as methionine (Met) oxidation is essential. Met oxidation in the fragment crystallizable (Fc) region of immunoglobulin-G1 (IgG1) is a potential critical quality attribute because it impacts not only the interaction with the neonatal Fc receptor and protein A but also the half-life of mAbs in serum circulation. Although bottom-up mass spectrometry provides high site specificity, it may have limited application in quality control workflows, and its complicated sample preparation could result in procedure-induced oxidation. In this study, we describe the development and characterization of a rapid and robust middle-down hydrophobic interaction chromatography method for monitoring Met oxidation in the Fc region of IgG1. Additionally, we assessed a comprehensive method validation package and demonstrated the specificity, linearity, precision, and accuracy of the new method within a range of 3.8–37.7%. The relative quantitative data provided by this method may be used in a regulated workflow to support process and formulation development as well as in the later stages of drug development and batch release and stability studies.

A highly-selective three-dimensional (3D) HPLC system has been designed/developed for the determination of acidic amino acid enantiomers, i.e., aspartic acid (Asp) and glutamic acid (Glu). The 3D-HPLC system consisted of a reversed-phase column (Singularity RP18, 1.0 ×250 mm) in the first dimension, a mixed-mode column (Singularity MX-001, 1.5 ×250 mm) in the second dimension and a Pirkle-type enantioselective column (Singularity CSP-011S, 1.5 ×250 mm) in the third dimension. By using this system, the amounts of Asp and Glu enantiomers in 6 tissues (cerebrum, heart, lung, liver, kidney and testis) and 2 physiological fluids (plasma and urine) were determined in the control C57BL/6J mice and B6DDO^-/- mice lacking D-aspartic acid oxidase activity. In the control C57BL/6J mice, a high amount of D-Asp (466.15 nmol/g) was observed in the testis, and relatively high amounts of D-Asp were observed in the cerebrum, lung, liver and kidney (21.40–92.87 nmol/g). In the heart and 2 physiological fluids, the amounts of D-Asp were low (trace–7.75 nmol/g or mL). In the B6DDO^-/- mice, the amounts of D-Asp drastically increased in all the tissues and physiological fluids. In contrast to the results of D-Asp, the amounts of D-Glu did not change depending on the alteration of the DDO activity, and the amounts were extremely low and not detectable in most of the tested tissues and physiological fluids. Although the amounts of D-Glu were low, it is noteworthy that clear localization in the testis (8.60–9.53 nmol/g) was demonstrated in both the C57BL/6J and B6DDO^-/- mice.

We have developed a novel aptamer discovery method that rapidly and efficiently yields anti-idiotypic DNA aptamers for trastuzumab using fast protein liquid chromatography (FPLC) separation and large amounts of DNA. The use of large amounts of oligo DNA allowed us to obtain more aptamer candidates without PCR amplification within only two days. Quartz crystal microbalance (QCM) measurements confirmed that the obtained anti-trastuzumab aptamer had a high affinity with a K_D of 120.0 nM and 97.7 nM at pH 6.0 and 7.4, respectively. Molecular docking simulations suggested that this sequence has high specificity and binding affinity to multiple sites in the complementarity-determining region of trastuzumab. The K_D increased to 11.4 nM due to avidity expression after dense immobilization in the QCM sensor cell, indicating that this anti-trastuzumab aptamer is applicable as a capture molecule in the ligand binding assay.

The assessment of fat acid parameters (acid value, saponification value, ester value, iodine value, composition of monounsaturated fatty acids, polyunsaturated fatty acids, and total unsaturated fatty acids) in edible oils, including red fruit oil, delivers essential indices to guarantee their quality. This index also holds true for excipients as well as for traditional medicines. NMR spectroscopy is an alternative tool to the conventional methods for the determination of these quality parameters, offering attractive advantages. Here, the approach reported in the literature based on the ¹H NMR quantitative method is illustrated, highlighting the application procedure strategy and suggested sample processing. Chemometric applications on ¹H NMR spectra are also discussed. Furthermore, this review can support the role of ¹H NMR and chemometrics in routine analysis for oil quality control.