Journal of pharmaceutical and biomedical analysis最新文献

With recent advances in quantitative high-resolution mass spectrometry (HRMS), there is growing interest in developing liquid chromatography (LC)-HRMS methods that can simultaneously quantify numerous critical impurities in a peptide or protein drug. This approach is attractive as it could reduce the total number of methods and instruments required during product development and quality control testing, while taking advantage of the technique's high specificity and sensitivity. To investigate the feasibility of this approach for peptide drugs, an LC-HRMS method was validated for the quantification of six peptide-related impurities in teriparatide, the 34-amino acid active ingredient in Forteo. External calibration curves were constructed to correlate the peak area ratio of impurity-to-teriparatide to a known impurity abundance. The method displayed good specificity, sensitivity, linearity, accuracy, repeatability, intermediate precision, and robustness. The lower limits of quantification were 0.02 % or 0.03 % of teriparatide, below the regulatory reporting threshold of 0.10 %. It was found that quantification using three isotopic peaks per peptide did not provide a significant benefit over quantification with one isotopic peak. The method was validated successfully without the impractical inclusion of an isotopically-labeled internal standard for each impurity. Future studies will be conducted to determine the method's longer-term reproducibility.

Luteolin is one of the bioactive components from the compound Anoectochilus roxburghii (Wall.) Lindl. oral liquid (CAROL), which was reported to have excellent hepatoprotective and anti-inflammatory activities. However, the enrichment and quantitation of luteolin from CAROL is challenging due to the low content and complex aqueous matrix. In this study, a bifunctional monomer surface molecularly imprinted polymer (MIP) with metal-organic frameworks (MOFs) as cores was prepared for the selective adsorption of luteolin from the aqueous system CAROL. Compared with conventional MIPs, this unique nanocomposite adsorbent (MOF@MIPs) has the advantages of short kinetic equilibrium time, good selectivity, and high adsorption capacity in aqueous solution. The theoretical maximum adsorption capacity of MOF@MIPs for luteolin was 36.99 mg/g. After adsorption enrichment of luteolin from CAROL using MOF@MIPs, liquid chromatography-tandem mass spectrometry was applied to analyze the target. The corresponding linearity range for analyte was 10-6000 ng/mL with good linearity (R² =0.9992), and the added recoveries varied from 85.70 % to 99.25 %. The present method has been successfully employed for the analysis of luteolin in five different batches of CAROL. Notably, we found no significant difference in the content of luteolin between these batches, which proved that the composition was stable between batches. The novel structure MIPs are suitable for the specific recognition of template molecules in aqueous solution. Therefore, this study provides a technical reference for the special identification and determination of trace components in complex samples, while the novel MOF@MIP nanocomposite can also provide valuable references for the extraction and purification methods of specific substances in traditional Chinese medicine and expand the application environment of MIPs material.

The pathogenesis of cold-dampness syndrome (CDS) is closely related to intestinal inflammation and immune disorders induced by cold-dampness pathogen. CDS is the root cause of a variety of chronic inflammatory and immune diseases. Jingfang granule (JF) was widely used to treat a variety of diseases closely related to CDS. JF is well known for its clinical effect of dispelling cold and eliminating dampness, but the pharmacological effect and mechanism of JF on the improvement of CDS are still unclear. This study aimed to explore the efficacy and mechanism of JF in improving CDS from the perspective of intestinal immunity. In this study, mass spectrometry (CyTOF), metabolomics, network pharmacology, proteomics and molecular biology experiments were performed to investigate the therapeutic effects and underlying mechanisms of JF on intestinal inflammation and immune disorders in CDS mice. These results showed that JF could improve the clinical symptoms and increase the thymus index of CDS mice. Most strikingly, JF ameliorated intestinal inflammation and immune disorders in CDS mice, as indicated by increased frequency of TH1, CD8 + Tem, CD8 + TEFF, gdT and iNK cells and decreased frequency of Naive B cells, M1-macrophages, DCs and eosinophils. Metabolomics results showed that JF reversed the content of docosahexaenoic acid, arachidonic acid, linoleic acid, inosine and hypoxanthine in CDS mice. Correlation analysis showed that these metabolites were strongly correlated with a variety of intestinal immune cells, indicating that there was a certain regulatory effect between them. Then, 271 JF targets, 316 metabolite targets and 18374 disease targets were integrated to obtain 75 common targets and 138 pathways (such as PI3K/AKT and MAPK pathway, etc). Furthermore, molecular docking, proteomics and western blotting demonstrated that PI3K/AKT signaling pathway might be the key molecular mechanism by which JF regulated intestinal immune disorders in CDS mice. These results suggested that JF may act on the PI3K/AKT pathways to further regulate the levels of metabolites to exert intestinal immunomodulatory effects. In summary, we confirmed the beneficial effects of JF on intestinal immune disorders in CDS mice.

Phosphorylated small molecule metabolites play crucial roles in physiological processes such as glycogen metabolism and inflammation regulation. However, their high polarity, structural similarity, poor chromatographic separation, and weak mass spectrometric signals make their accurate quantification challenging, thereby hindering the study of related metabolic mechanisms and diseases. To address these challenges, we developed a novel derivatization reagent, DMQX (5-diazomethane quinoxaline), and combined it with liquid chromatography-mass spectrometry (LC-MS). This approach achieved baseline separation of five groups of isomers and enabled the quantification of 24 phosphorylated metabolites, providing comprehensive coverage of these metabolites in biological pathways. We applied this method to quantify 21 endogenous phosphorylated metabolites in HepG2 cells with and without vesicular stomatitis virus infection, demonstrating the potential of this analytical approach for advancing the study of metabolic mechanisms through quantitative analysis of phosphorylated metabolites in biological samples.

Cyclin D-dependent kinase 4/6 inhibitors palbociclib, ribociclib and abemaciclib, in combination with aromatase inhibitors anastrozole and letrozole or oestrogen receptor degrader fulvestrant, are being assessed as candidates for therapeutic drug monitoring. An ideal bioanalytical method for their determination in patient plasma samples is therefore of high interest, as there is no routine reference method yet available in the clinical practice. In this work, three sample preparation approaches - dispersive liquid-liquid microextraction (DLLME), solid-phase extraction (SPE), and newly developed phospholipid removal (PLR) for LC-MS determination of these six drugs are comprehensively assessed. The methods are validated in the clinically relevant linear ranges with remarkable precision (RSD ≤6.9 %) and accuracy (bias -13.6 - 11.8 %). To compare the procedures in a real-world setting, they are applied on 38 samples from breast cancer patients. The differences between paired results are below 20 % for more than 92 % of the repeats and the RSD is ≤13.1 % between the corresponding results. Statistical comparison of the results reveals excellent overall agreement between the methods (Lin's concordance correlation coefficient ≥0.9969, maximal Bland-Altman bias 6.3 %). DLLME proved to be the most ecologically acceptable method due to the high degree of miniaturisation (AGREEprep score 0.44), PLR enabled very high sample throughput and cost-effectiveness (BAGI 72.5), while SPE showed the best analytical performance (redness score 100). All three methods are suitable for their designated purpose, and the choice of the ideal method can be made based on the scope of application, available funds and equipment and desired ecological footprint.

Daratumumab is a fully human immunoglobulin G1 monoclonal antibody employed for treating relapsed/refractory multiple myeloma and light-chain amyloidosis. Quantifying monoclonal antibodies in serum presents challenges due to interference from biological matrices. This research aimed to develop and verify an liquid chromatography tandem-mass spectrometry (LC-MS/MS) approach for quantifying serum daratumumab, employing immunoglobulin G purification without alkylation, and to assess its applicability in patients with multiple myeloma receiving intravenous daratumumab. The chromatographic peaks of the daratumumab-derived peptides and internal standard were well-delineated from the serum digests, with an overall run time of 14 min. The calibration curves for serum daratumumab were linear across over 1-1000 μg/mL. The inter- and intra-day accuracy varied between 92.4 % and 108.4 %, with a coefficient-of-variation below 10 %. In patients receiving intravenous daratumumab, serum concentrations ranged from 181.8 to 975.3 µg/mL. Bland-Altman analysis revealed no significant bias, and Passing-Bablok regression demonstrated a good agreement between the LC-MS/MS method and enzyme-linked immunosorbent assay.

Levosimendan is a positive inotrope and vasodilator used in patients with acute and chronic decompensated heart failure. It is metabolized into OR-1855 (inactive metabolite), which is further acetylated into OR-1896 (active metabolite having a prolonged half-life, hence a sustained effect). Levosimendan represents a valuable alternative to traditional inotropes with broad clinical applications in critically ill patients with cardiogenic shock, advanced heart failure and post-cardiac surgery. However, while levosimendan demonstrates dose-dependent hemodynamic effects, its pharmacokinetics has not yet been investigated in adult critically ill patients, a vulnerable population characterized by complex and unstable conditions that may significantly alter drug disposition. Therefore, pharmacokinetics studies of levosimendan and metabolites in critically ill patients require a reliable and sensitive quantification method. We developed and validated a highly sensitive method using ultra-high-performance liquid-chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) for the quantification of levosimendan, OR-1855 and OR-1896 in human plasma. To achieve the required analytical sensitivity, plasma sample preparation included protein precipitation with acetonitrile, subsequent supernatant's evaporation to dryness under nitrogen, and reconstitution of the solid residues with a solution of H₂O:MeOH 4:1, followed by a 40 µL-aliquot injection into the LC column. Chromatographic separation of the three analytes was achieved in a 6-minute run in gradient mode, using an Acquity UPLC BEH C18 1.7 µm, 2.1 × 150 mm column. The method was extensively validated according to international bioanalytical assay guidelines, on a clinically relevant concentration range of 0.1-100 ng/mL, for each analyte. Considering these very low concentrations, the assay showed excellent performances in terms of trueness (94.3-105.3 %), repeatability (1.9-7.2 %) and intermediate fidelity (2.3-9.7 %). Of note, during our ex vivo studies on whole blood samples stability, acetylation of the metabolite OR-1855 into the active OR-1896 metabolite was observed in the presence of red blood cells. The UHPLC method is being applied for a prospective observational pharmacokinetics study of levosimendan in patients undergoing extracorporeal membrane oxygenation support. The benefit of levosimendan therapeutic drug monitoring in intensive care patients remains to be assessed.

Osimertinib (AZD9291) is a widely used tyrosine kinase inhibitor for the treatment of non-small cell lung cancer patients with activating EGFR mutations. However, the correlation between dose and efficacy has been debated for several years. For this reason, there is a need for standardized methods for routine analysis, clinical studies on pharmacokinetics and dose-response relationships, and greater understanding of preanalytical conditions, such as sample storage stability. The objective of this study was to develop and validate a sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous quantification of osimertinib and its two metabolites, AZ7550 and AZ5104, in human plasma and to investigate long-term storage stability of the analytes. Samples were prepared by protein precipitation and separated on a Kinetex EVO C18 column (2.1 × 150 mm, 2.6 µm). Electrospray ionization in positive mode and multiple reaction monitoring were used to monitor the ion transitions. The validated concentration ranges were from 1.25 to 3000 ng/mL. Interassay precisions and accuracies were all ≤ 15 %. Linearity, dilution integrity, and carry-over were also examined and satisfied the validation criteria. Stability was examined under different conditions, and the analytes were found to be stable for more than 3 years at -80°C (< 15 % decline). Finally, the analytical method was successfully applied in a clinical setting on plasma samples from 30 patients with non-small cell lung cancer in treatment with osimertinib, demonstrating its suitability for use in clinical studies and its potential for therapeutic drug monitoring.

Unsaturated fatty acids (UFAs) play a crucial physiological role in human body. However, the concentration-related changes and prognostic significance of UFAs in epilepsyremain unclear. An optimized ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) approach was developed to measure six key UFAs: oleic acid (OA), linoleic acid (LA), arachidonic acid (AA), α-linolenic acid (ALA), docosahexaenoic acid (DHA), and eicosapentaenoic acid (EPA). Subsequently, the levels of these six UFAs were determined in 40 healthy individuals and 49 epilepsy patients. The diagnostic value of UFAs and clinical examination indicators were assessed using statistical analysis and the support vector machine (SVM) algorithm. The results showed that the UPLC-MS/MS method successfully quantified the levels of OA, LA, AA, ALA, EPA, and DHA in both the healthy individuals and epilepsy patients. Compared with the healthy group, the levels of ALA, AA, and DHA were significantly elevated in the epilepsy group (P < 0.05). Pearson correlation analysis revealed a strong positive correlation among the UFAs in the epilepsy group. The orthogonal partial least squares-discriminant analysis (OPLS-DA) model showed that DHA and EPA were more important than cholesterol in distinguishing between two groups, although the separation was not complete. The SVM model achieved better separation, with an area under the curve (AUC) of 0.95 when including the six UFAs. The EPA/DHA ratio was identified as a key feature, with a significant contribution to the model's performance. Removing the six UFAs from the model reduced the AUC to 0.91, highlighting the predictive value of UFAs for epilepsy. In conclusion, ALA, AA, and DHA, are altered in epilepsy patients. The EPA/DHA ratio was found to be a key predictive indicator for epilepsy. The use of UFAs in conjunction with clinical examination data improved the predictive power of the SVM model, suggesting that UFAs have potential as biomarkers for epilepsy.

Carbohydrates are essential biomolecules that play a vital role in various biological processes across humans, plants, and bacteria. Despite their ubiquity, the structural elucidation of carbohydrates, particularly oligo- and polysaccharides, remains a significant challenge due to their complex and heterogeneous nature. The high-performance anion exchange chromatography (HPAEC) or called ion chromatography (IC) coupled with pulsed amperometric detection (PAD) has emerged as a powerful tool for highly effective separation and highly specific detection of glycans. The introduction of mass spectrometry (MS) into HPAEC-PAD systems has further advanced glycan analysis by enabling detailed structural elucidation, including branching, linkage patterns, and sequence determination. The use of suppressor technology allows for the coupling of HPAEC with MS by converting non-volatile salts in the mobile phase into volatile ones. This review highlights the current advancements in HPAEC-PAD/MS for oligo- and polysaccharide structural analysis, discussing the strengths and limitations of different suppressor systems, the role of MS in glycan analysis, and the emerging applications of this technology in the field of glycomics. With continued innovation, HPAEC-PAD/MS is poised to become an essential tool for the detailed characterization of polysaccharides, supporting advancements in pharmaceutical, biomedical, and biotechnological research.