A combination of multiple LC-MS approaches for the comprehensive characterization of cysteine-linked ADCs.

Antibody-drug conjugates (ADCs) represent the forefront of the next generation of biopharmaceuticals. An ADC typically comprises an antibody covalently linked to a cytotoxic drug via a linker, resulting in a highly heterogeneous product. This study focuses on the analysis of a custom-made cysteine-linked ADC. Initially, we developed a LC-MS-based characterization workflow using brentuximab vedotin (Adcetris®), encompassing native intact MS, analysis of reduced chains and subunits under denaturing condition, peptide mapping and online strong cation exchange chromatography coupled with UV and mass spectrometry detection (SCX-UV-MS) applied for brentuximab vedotin first time reported. Subsequently, we applied this in-depth characterization workflow to a custom-made cysteine-linked ADC. The measured drug-to-antibody ratio(DAR) of this ADC is 6.9, further analysis shown that there is a small amount of unexpected over-conjugation. Over-conjugation sites were successfully identified using multiple UHPLC-MS based characterization techniques. Also, one competitively cysteine-conjugated impurity was observed in native intact MS results, by combing native intact MS, reduced chains, subunit analysis and peptide mapping results, the impurity conjugation sites were also identified. Since this molecule is at early development stage, this provides important information for conjugation process improvement and link-drug material purification. SCX-UV-MS approach can separate the custom-made cysteine-linked ADC carrying different payloads and reduce the complexity of the spectra. The integrated approach underscores the significance of combining the SCX-UV-MS online coupling technique with other characterization methods to elucidate the heterogeneity of cysteine-linked ADCs.

Improved method for quantification of intact oxaliplatin by ultra high performance liquid chromatography-inductively coupled plasma mass spectrometry: Applications to clinical and speciation studies.

Interest is increasing in the use of different liquid chromatography techniques coupled online to mass spectrometry for the quantification of platinum anticancer drugs in human plasma to inform cancer chemotherapy. We developed, validated and studied the application of a method for quantification of intact oxaliplatin in human plasma using ultra high performance liquid chromatography hyphenated to inductively coupled plasma mass spectrometry (UHPLC-ICP-MS). Plasma samples were processed instantly after collection from patients to preserve oxaliplatin speciation by methanol-deproteinization, and storage of diluted supernatants (plasma:methanol 1:2 v/v) at -80 °C. UHPLC separation of intact oxaliplatin and internal standard (carboplatin) was achieved using a C18 column and linear gradient mobile phase (Mobile phase A: water-methanol (97:3 v/v), 0.075 mM sodium dodecyl sulfate, 9.79 nM thallium adjusted to pH 2.5 with trifluoromethanesulfonic acid; Mobile phase B: 100 % methanol (v/v)) with ICP-MS detection to monitor platinum and thallium at m/z 195 and 205, respectively. The limit of quantification was 50 nM in methanol-deproteinized diluted plasma (1:2 v/v). Linearity was established for calibration standards ranging from 50 to 500 nM made in methanol-deproteinized diluted plasma (1:2 v/v), and for dilution of higher concentration samples in blank matrix containing internal standard (final dilution 1:29 v/v). Intra-day and inter-day accuracy ranged from 96.8 to 103 % of nominal concentration and precision from 0.62 to 2.49 % coefficient of variation. Recovery was complete and a matrix effect confirmed the requirement for matrix-matched standards. Intact oxaliplatin was stable during storage for at least 473 days, and during analysis, in methanol-deproteinized diluted plasma (1:2 v/v). The method was applied to determining the plasma concentrations of intact oxaliplatin in patients undergoing cancer chemotherapy, and studies of oxaliplatin degradation in vitro. This improved method based on UHPLC-ICP-MS will allow more specific, efficient and reliable quantification of intact oxaliplatin in human plasma.

Determination of eleven components in rat plasma by UPLC-MS/MS and GC-MS for pharmacokinetic studies after oral administration of Citri Reticulatae Pericarpium extract.

Citri Reticulatae Pericarpium (CRP) is used as common health-care food and traditional Chinese medicine (TCM), which exerts pharmacological effects, such as anti-cardiovascular, anti-tumor, anti-oxidant, anti-inflammatory, anti-virus, hepatoprotective, blood pressure-lowering and neuroprotective. In this study, reliable, and sensitive ultra-high performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) and gas chromatography-mass spectrometry (GC-MS) methods were developed and validated for the determination of eleven active components in rat plasma after oral administration of the CRP extract. The results of this method exhibited that the specificity, linearity (r > 0.999), precision and accuracy (the coefficient of variation (CV) < 11.5 %), recovery (52.9-107.9 %), matrix effects (63.8-107.5 %), and stability (CV < 10.8 %) met all requirements for the quantitation of plasma samples. The pharmacokinetic results showed that the Tmax of flavone glycosides was less than 0.7 h, and that of polymethoxyflavones and volatile components were within 1-7 h. Meanwhile, the area-under-the-curve (AUC) and concentration maximum (Cmax) of hesperidin, nobiletin, tangeretin, and D-limonene were higher than those of the other components, suggesting that the plasma exposure levels of these constituents were higher in CRP. The present research lays a foundation for elucidating the therapeutic material basis and provides a reference for further scientific research and clinical application of CRP.

Effect of crosslinker/porogen ratio on sponge-nested polymer monoliths for solid-phase extraction.

Polymer monoliths can be polymerised within different molds, but limited options are available for the preparation of free-standing polymer monoliths for analytical sample preparation, and in particular, solid-phase extraction (SPE). Commercial melamine-formaldehyde sponges can be used as supports for the preparation of polymer monoliths, due its flexibility, giving various shapes to monoliths. Herein, the crosslinker/porogen ratio of highly porous sponge-nested divinylbenzene (DVB) polymer monoliths has been evaluated. Monoliths prepared using different crosslinker/porogen ratios were applied to the extraction of bisphenol F, bisphenol A, bisphenol AF, and bisphenol B. Monoliths containing 50 wt % DVB and 50 wt % porogens presented the highest recovery of bisphenols. Under the optimised conditions, the developed method showed a linear range between 2.5 µg L-1 and 150 µg L-1 for BPA and BPAF, and between 5 µg L-1 and 150 µg L-1 for BPB and BPF. The limits of detection (LOD, S/N = 3) and limits of quantification (LOQ, S/N = 10) ranged from 0.36 µg L-1 to 1.09 µg L-1, and from 1.20 µg L-1 to 3.65 µg L-1, respectively. The recoveries for spiked bisphenols (10 µg L-1) in tap water and water contained in a polycarbonate containers were between 82 % and 114 %.

Basal release of 6-cyanodopamine from rat isolated vas deferens and its role on the tissue contractility.

6-Cyanodopamine is a novel catecholamine released from rabbit isolated heart. However, it is not known whether this catecholamine presents any biological activity. Here, it was evaluated whether 6-cyanodopamine (6-CYD) is released from rat vas deferens and its effect on this tissue contractility. Basal release of 6-CYD, 6-nitrodopamine (6-ND), 6-bromodopamine, 6-nitrodopa, and 6-nitroadrenaline from vas deferens were quantified by LC-MS/MS. Electric-field stimulation (EFS) and concentration-response curves to noradrenaline, adrenaline, and dopamine of the rat isolated epididymal vas deferens (RIEVD) were performed in the absence and presence of 6-CYD and /or 6-ND. Expression of tyrosine hydroxylase was assessed by immunohistochemistry. The rat isolated vas deferens released significant amounts of both 6-CYD and 6-ND. The voltage-gated sodium channel blocker tetrodotoxin had no effect on the release of 6-CYD, but it virtually abolished 6-ND release. 6-CYD alone exhibited a negligible RIEVD contractile activity; however, at 10 nM, 6-CYD significantly potentiated the noradrenaline- and EFS-induced RIEVD contractions, whereas at 10 and 100 nM, it also significantly potentiated the adrenaline- and dopamine-induced contractions. The potentiation of noradrenaline- and adrenaline-induced contractions by 6-CYD was unaffected by tetrodotoxin. Co-incubation of 6-CYD (100 pM) with 6-ND (10 pM) caused a significant leftward shift and increased the maximal contractile responses to noradrenaline, even in the presence of tetrodotoxin. Immunohistochemistry revealed the presence of tyrosine hydroxylase in both epithelial cell cytoplasm of the mucosae and nerve fibers of RIEVD. The identification of epithelium-derived 6-CYD and its remarkable synergism with catecholamines indicate that epithelial cells may regulate vas deferens smooth muscle contractility.

Analysis of Raffinose Metabolism-Related Sugar Components in Seeds.

Raffinose family oligosaccharides (RFOs) are synthesized from sucrose and subsequent addition of galactose moieties which was provided by galactinol. Galactinol is synthesized from UDP-galactose and myo-inositol. RFOs accumulate at late stage of seed development and play important roles in seed longevity. RFOs are major components in seeds of many plant species. Here, we document a methodology for extraction and quantitative analysis of raffinose metabolism-related soluble sugars or the derivative alcohols in plant seeds. This protocol, based on high-performance liquid chromatography (HPLC), achieves the efficient separation and accurate quantification of sucrose, myo-inositol, galactinol, and raffinose within 25 min of retention time.

[Applications of high performance liquid chromatography-mass spectrometry in proteomics].

Proteomics profiling plays an important role in biomedical studies. Proteomics studies are much more complicated than genome research, mainly because of the complexity and diversity of proteomic samples. High performance liquid chromatography-mass spectrometry (HPLC-MS) is a fundamental tool in proteomics research owing to its high speed, resolution, and sensitivity. Proteomics research targets from the peptides and individual proteins to larger protein complexes, the molecular weight of which gradually increases, leading to sustained increases in structural and compositional complexity and alterations in molecular properties. Therefore, the selection of various separation strategies and stationary-phase parameters is crucial when dealing with the different targets in proteomics research for in-depth proteomics analysis. This article provides an overview of commonly used chromatographic-separation strategies in the laboratory, including reversed-phase liquid chromatography (RPLC), hydrophilic interaction liquid chromatography (HILIC), hydrophobic interaction chromatography (HIC), ion-exchange chromatography (IEC), and size-exclusion chromatography (SEC), as well as their applications and selectivity in the context of various biomacromolecules. At present, no single chromatographic or electrophoretic technology features the peak capacity required to resolve such complex mixtures into individual components. Multidimensional liquid chromatography (MDLC), which combines different orthogonal separation modes with MS, plays an important role in proteomics research. In the MDLC strategy, IEC, together with RPLC, remains the most widely used separation mode in proteomics analysis; other chromatographic methods are also frequently used for peptide/protein fractionation. MDLC technologies and their applications in a variety of proteomics analyses have undergone great development. Two strategies in MDLC separation systems are mainly used in proteomics profiling: the "bottom-up" approach and the "top-down" approach. The "shotgun" method is a typical "bottom-up" strategy that is based on the RPLC or MDLC separation of whole-protein-sample digests coupled with MS; it is an excellent technique for identifying a large number of proteins. "Top-down" analysis is based on the separation of intact proteins and provides their detailed molecular information; thus, this technique may be advantageous for analyzing the post-translational modifications (PTMs) of proteins. In this paper, the "bottom-up" "top-down" and protein-protein interaction (PPI) analyses of proteome samples are briefly reviewed. The diverse combinations of different chromatographic modes used to set up MDLC systems are described, and compatibility issues between mobile phases and analytes, between mobile phases and MS, and between mobile phases in different separation modes in multidimensional chromatography are analyzed. Novel developments in MDLC techniques, such as high-abundance protein depletion and chromatography arrays, are further discussed. In this review, the solutions proposed by researchers when encountering compatibility issues are emphasized. Moreover, the applications of HPLC-MS combined with various sample pretreatment methods in the study of exosomal and single-cell proteomics are examined. During exosome isolation, the combined use of ultracentrifugation and SEC can yield exosomes of higher purity. The use of SEC with ultra-large-pore-size packing materials (200 nm) enables the isolation of exosomal subgroups, and proteomics studies have revealed significant differences in protein composition and function between these subgroups. In the field of single-cell proteomics, researchers have addressed challenges related to reducing sample processing volumes, preventing sample loss, and avoiding contamination during sample preparation. Innovative methods and improvements, such as the utilization of capillaries for sample processing and microchips as platforms to minimize the contact area of the droplets, have been proposed. The integration of these techniques with HPLC-MS shows some progress. In summary, this article focuses on the recent advances in HPLC-MS technology for proteomics analysis and provides a comprehensive reference for future research in the field of proteomics.

Comprehensive evaluation and screening of phytochemical compounds and their hypolipidemic activities of lotus leaf based on HPLC-Q-TOF-MS and spectral-effect analysis.

This study aimed to identify and quantify the primary components in lotus leaf and to explore the hypolipidemic components through spectral-effect relationships and chemometric methods. Utilizing a data-dependent acquisition-diagnostic fragment ion/characteristic neutral loss screening strategy (DFI-NLS), a reliable HPLC-Q-TOF-MS analysis was conducted, identifying 77 compounds, including 36 flavonoids, 21 alkaloids, 3 terpenoids, 11 organic acids, 4 phenols, 1 lignin and 1 unsaturated hydrocarbon. A straightforward HPLC-DAD method was developed for the simultaneous determination of seven major components in lotus leaf, and quercetin-3-O-glucuronide (Q3GA) was identified as the most abundant component. The HPLC fingerprints of 36 lotus leaf sample batches were assessed using chemometric approaches such as principal component analysis and hierarchical cluster analysis. The hypolipidemic effect of these samples was analyzed by measuring total cholesterol (TC) and total triglycerides (TG) levels in palmitic acid (PA) and oleic acid (OA)-induced lipid modeling in HepG-2 cells, employing partial least squares regression and grey relation analysis to investigate the spectral-effect relationship of the lotus leaf. The in vivo hypolipidemic effect of these compounds was assessed using an egg yolk powder-induced high-fat zebrafish model. The findings indicated that peak No.11 (Q3GA) in the chemical fingerprint was significantly associated with hypolipidemic activity, suggesting it as a potential hypolipidemic compound in lotus leaf. In summary, this study facilitates the exploration of the phytochemical compounds and their bioactive properties in the lotus leaf.

Pharmacokinetic profile of phenytoin in dried blood spot with high-performance liquid chromatography-photodiode array.

Phenytoin is a first-line antiepileptic drug with narrow therapeutic range and follows non-linear pharmacokinetics. Pharmacokinetics of phenytoin have been studied in plasma matrix before, however, there were several disadvantages. This study aimed to obtain partial validation data of the analytical method and the pharmacokinetic profile of phenytoin in Dried Blood Spot (DBS) of six healthy subjects. DBS has the advantage of only requiring small sample volumes and could be transported more efficiently. Phenytoin along with carbamazepine as the chosen internal standard was analyzed with a reversed-phase high performance-liquid chromatography system and a photodiode array detector at 205 nm. The results of partial validation, which evaluated the linearity, within-run accuracy, and precision, were within the criteria acceptance range. The pharmacokinetic profile showed that average AUC0-t was 83.81 ± 37.32 µg.h/mL and AUC0-∞ was 83.65 ± 38.89 µg.h/mL with an average ratio of 93%. Previous study quantifying phenytoin in the plasma matrix found the average AUC0-t was 39.41 ± 8.57 µg.h/mL and AUC0-∞ was 42.94 ± 9.55 µg.h/mL. Despite the difference between parameters of phenytoin analyzed in DBS and plasma matrices, the pharmacokinetic profiles obtained from both matrices were similar indicated by comparable concentration-time curves, thus, proving that DBS matrix can be used interchangeably with the plasma matrix as a more comfortable and effective alternative to phenytoin quantification in blood.

The behavior of pharmaceutically active compounds and contrast agents during wastewater treatment - Combining sampling strategies and analytical techniques: A critical review.

Increasing consumption of pharmaceuticals and the respective consequences for the aquatic environment have been the focus of many studies over the last thirty years. Various aspects in this field were investigated, considering diverse pharmaceutical groups and employing a wide range of research methodologies. Various questions from the perspectives of different research areas were devised and answered, resulting in a large mix of individual findings and conclusions. Collectively, the results of the studies offer a comprehensive overview. The large variety of methods and strategies, however, demands close attention when comparing and combining information from heterogeneous projects. This review critically examines the application of diverse sampling techniques as well as analytical methods in investigations concerning the behavior of pharmaceutically active compounds (PhACs) and contrast agents (CAs) in wastewater treatment plants (WWTPs). The combination of sampling and analysis is discussed with regard to its suitability for specific scientific problems. Different research focuses need different methods and answer different questions. An overview of studies dealing with the fate and degradation of PhACs and CAs in WWTPs is presented, discussing their strategic approaches and findings. This review includes surveys of anticancer drugs, antibiotics, analgesics and anti-inflammatory drugs, antidiabetics, beta blockers, hormonal contraceptives, lipid lowering agents, antidepressants as well as contrast agents for X-ray and magnetic resonance imaging.

Analytical method development and dermal absorption of gallic acid, a hair dye ingredient.

Gallic acid (GA) is a phenolic compound known as 3,4,5-trihydroxybenzoic acid. GA is used as a hair dye ingredient. It is limited to be below 4.0% in Korea. Dermal absorption rate of GA has not been reported yet. In this study, an analytical method for GA was developed and validated using high-performance liquid chromatography (HPLC) in various matrices of swab, stratum corneum (SC), skin (dermis + epidermis), and receptor fluid (RF). HPLC analysis showed acceptable linearity (r2 = 0.999-0.9998), accuracy (90.3-112.8%), and precision (0.7-13.6%) in accordance with validation guidelines by Korea Ministry of Food and Drug Safety (MFDS). The dermal absorption rate of GA was determined using Franz diffuse cells. GA (4.0%) was applied to mini pig skin of 10 µl/cm2. After 30 min application, the GA was wiped out and receptor fluid sampling was continued until 24 h. After 24 h, skin was wiped off with swab and SC was collected using tape stripping. All samples were extracted with ethanol and analyzed using the validated method. The total dermal absorption rate of GA was determined to be 2.6 ± 1.3% (24 h).

Effect of Vehicles and Irrigation Solutions and Methods on Removal of Double Antibiotic Paste from Root Canals: Insights from High-performance Liquid Chromatography Analysis.

INTRODUCTION: In regenerative endodontics, eradicating antibiotic residues from root canals is imperative, given their detrimental effects on human apical papilla stem cells. Previous antibiotic removal studies lacked precision in identifying types and quantities of residual antibiotics. High-performance liquid chromatography (HPLC) enhances sensitivity and specificity, enabling accurate detection and quantification of residual drugs. Using HPLC analysis, this study explored the influence of vehicles and irrigation solutions and methods on double antibiotic paste (DAP) removal from root canals. METHODS: Two DAP formulations, each containing 5 mg/mL ciprofloxacin and metronidazole, were created using distinct vehicles: macrogol and propylene glycol (MP) or hydroxypropyl methylcellulose (HPMC). Subsequently, 5 µL of DAP was applied to 200 simulated immature teeth with open apices (n = 100 per formulation) and cultured for 28 days at 37°C. Samples were then divided into 11 groups (n = 20 per group), and canals were irrigated with 17% ethylenediaminetetraacetic acid or 10% citric acid, employing a positive pressure syringe or passive ultrasonic irrigation. The irrigation solution and dentin sample from each tooth were evaluated via HPLC for ciprofloxacin and metronidazole quantification. RESULTS: Citric acid exhibited significantly superior efficacy in antibiotic removal from root canals, with no observable effect of irrigation methods on drug removal. The HPMC-based DAP formulation significantly enhanced ciprofloxacin removal compared with MP-based DAP. CONCLUSIONS: For antibiotic paste removal from root canals, citric acid is effective, and HPMC is a preferable vehicle over MP. Overall, HPLC is a valuable method for detecting, removing, and quantifying residual antibiotics in root canals.

Stability of ascorbic acid in human aqueous humor.

OBJECTIVE: The stability of ascorbic acid (AA) in the human aqueous humor (AqH) remains unclear. This study aimed to investigate the stability of AqH AA under varying conditions (27, 4, - 20, and - 80 °C) without acidification. RESULTS: Rapid AA degradation occurred at 27 °C. At 4 °C, a significant 12.2% degradation was observed after 24 h. Storage at - 20 °C resulted in a notable 37.5% degradation after 28 days, whereas storage at - 80 °C resulted in 10.7% degradation after 28 days. Unacidified AqH samples recorded early decomposition at 27 °C and 4 °C. In conclusion, it is recommended to conduct measurements within 28 days for samples stored at - 80 °C.

Fabrication of fluorinated triazine-based covalent organic frameworks for selective extraction of fluoroquinolone in milk.

A novel fluorinated triazine-based covalent organic frameworks (F-CTFs) was designed and synthesized by using melamine and 2,3,5,6-tetrafluoroterephthalaldehydeas as organic ligands for selective pipette tip solid-phase extraction (PT-SPE) of amphiphilic fluoroquinolones (FQs). The competitive adsorption experiment and mechanism study were carried out and verified that this F-CTFs possesses favorable adsorption affinity for FQs. The abundant fluorine affinity sites endowed the F-CTFs high selectivity to FQs extraction through F-F interactions. The adsorption capacity of F-CTFs can reach up to 109.1 mg g-1 for enrofloxacin. The detailed characterization of the F-CTFs adsorbent involved the application of various techniques to examine its morphology and structure. Under optimized conditions, a method combining F-CTF-based PT-SPE with high-performance liquid chromatography (PT-SPE-HPLC) was established, which exhibited a broad linear range, excellent precision, and an impressively low limit of detection, and could be used for the determination of six FQs in milk, with LODs as low as 0.0010 µg mL-1. The recovery rates during extraction varied between 92.1% and 111.4%, exhibiting RSDs below 6.8% at different spiked concentrations.

Functionalized graphene oxide as a nanocarrier for delivering oridonin to improve anti-breast cancer cell activity.

In this study, a targeted nanocarrier was developed by functionalizing graphene oxide with polyethyleneimine and folic acid, intended for loading oridonin. The nanocarrier was successfully synthesized and characterized using an ultraviolet spectrum, Fourier transform infrared spectroscopy and scanning electron microscopy. The nanocarrier demonstrated a remarkable oridonin loading capacity, reaching 424.8 µg/mg, as determined by ultra-high performance liquid chromatography. In vitro drug release experiments exhibited a pH-dependent release profile, with a higher cumulative release in an acidic environment. The release mechanism followed the Ritger-Peppas equation model. Cytotoxicity assays indicated minimal toxicity of the nanocarrier. Enhanced cellular uptake by MCF7 cells was observed for carriers functionalized with folate and polyethyleneimine. These findings highlight the potential of functionalized graphene oxide as a promising carrier for oridonin delivery in biomedical applications.

Chromatographic Methods and Sample Pretreatment Techniques for Aldehydes, Biogenic Amine, and Carboxylic Acids in Food Samples.

This review paper critically examines the current state of research concerning the analysis and derivatization of aldehyde, aromatic hydrocarbons and carboxylic acids components in foods and drinks samples, with a specific focus on the application of Chromatographic techniques. These diverse components, as vital contributors to the sensory attributes of food, necessitate accurate and sensitive analytical methods for their identification and quantification, which is crucial for ensuring food safety and compliance with regulatory standards. In this paper, High-Performance Liquid Chromatography (HPLC) and Gas Chromatographic (GC) methods for the separation, identification, and quantification of aldehydes in complex food matrices were reviewed. In addition, the review explores derivatization strategies employed to enhance the detectability and stability of aldehydes during chromatographic analysis. Derivatization methods, when applied judiciously, improve separation efficiency and increase detection sensitivity, thereby ensuring a more accurate and reliable quantification of aldehyde aromatic hydrocarbons and carboxylic acids species in food samples. Furthermore, methodological aspects encompassing sample preparation, chromatographic separation, and derivatization techniques are discussed. Validation was carried out in term of limit of detections are highlighted as crucial elements in achieving accurate quantification of compounds content. The discussion presented by emphasizing the significance of the combined HPLC and GC chromatography methods, along with derivatization strategies, in advancing the analytical capabilities within the realm of food science.

Alterations in pH of Coffee Bean Extract and Properties of Chlorogenic Acid Based on the Roasting Degree.

Factors influencing the sour taste of coffee and the properties of chlorogenic acid are not yet fully understood. This study aimed to evaluate the impact of roasting degree on pH-associated changes in coffee bean extract and the thermal stability of chlorogenic acid. Coffee bean extract pH decreased up to a chromaticity value of 75 but increased with higher chromaticity values. Ultraviolet-visible spectrophotometry and structural analysis attributed this effect to chlorogenic and caffeic acids. Moreover, liquid chromatography-mass spectrometry analysis identified four chlorogenic acid types in green coffee bean extract. Chlorogenic acid isomers were eluted broadly on HPLC, and a chlorogenic acid fraction graph with two peaks, fractions 5 and 9, was obtained. Among the various fractions, the isomer in fraction 5 had significantly lower thermal stability, indicating that thermal stability differs between chlorogenic acid isomers.

Improved HPLC method with automated pre-column sample derivatisation for serum pegylated L-asparaginase activity measurement in paediatric acute lymphoblastic leukaemia patients.

Therapeutic drug monitoring of pegylated L-asparaginase (ASNase) ensures the drug effectiveness in childhood acute lymphoblastic leukaemia (ALL) patients. The biological drug property with variable immunogenic host clearance, and the prescription of its generic formulation urge the need for a reliable assay to ensure an optimal treatment and improve outcome. This study aimed to optimise an existing isocratic reversed-phase high performance liquid chromatography (RP-HPLC) method with an automated pre-column sample derivatisation and injection program, and a computational algorithm for measuring serum pegylated ASNase activity in children with ALL. Nath et al.'s method in 2009 was adopted and modified using a pegylated ASNase. A set of Microsoft Excel macros was developed for the serum drug activity computation. An Agilent InfinityLab LC Series 1260 Infinity II Quaternary System with fluorescence detection was employed with an Agilent Poroshell 120 EC-C18 4.6×100 mm, 2.7 µm analytical column. System flow rate was optimised to 2.0 mL/min with 40×10-6/bar pump compressibility. The O-phthaldialdehyde (OPA) solution composition was optimised to 1 % o-phthaldialdehyde, 0.8 % 2-mercaptoethanol, 7.13 % methanol, and 1.81 % sodium tetraborate. The pre-column derivatisation program mixed 0.1 µL sample with 25 µL OPA solution before the automated injection. Method validation was according to the ICH guidelines. Total analysis time was 15 min, with L-aspartic acid eluted at 0.96 min and internal standard at 4.7 min. The calibration curves showed excellent linearity (R ≥0.9999). Interday precision for the drug activity at 0.1 IU/mL, 0.5 IU/mL, and 1 IU/mL were 4.15 %, 3.05 %, and 3.09 % (n = 6). Mean %error for the drug activity at 0.1 IU/mL, 0.5 IU/mL, and 1 IU/mL were 0.90±4.41 %, -1.37±3.04 %, and -3.03±3.02 % (n = 6). Limit of quantitation was 0.03 IU/mL. Majority of the patients' serum drug activity fell within the assay calibration range. Our improved method is automated, having shorter analysis time with a well-maintained separation resolution that enables a high-throughput analysis for application.

[Determination of six bisphenols in human urine by ultra-high performance liquid chromatography-tandem mass spectrometry].

OBJECTIVE: To develop and validate a solid phase extraction-ultra-high performance liquid chromatography-tandem mass spectrometry method for the determination of six bisphenols(bisphenol S, bisphenol F, bisphenol A, 2, 2'-methylenediphenol, bisphenol AF, bisphenol AP) in urine. METHODS: After enzymolysis of urine sample, the target substances were quickly purified and extracted by WAX solid phase extraction column. On ACQUITY BEH C_(18) column(2.1 mm×100 mm, 1.7 µm), the mobile phase of water and methanol was used to separate. Finally, multi-reaction detection was carried out under electrospray negative ion scanning, and quantification was carried out by internal standard method. RESULTS: The correlation coefficients(r) of the target compounds were all more than 0.998 in the range of 0.1-50.0 ng/mL, the linearity was good, and the detection limits were all lower than 0.1 ng/mL. The recoveries of the three standard concentrations(0.5, 5.0 and 50.0 ng/mL) were all between 80% and 120%, and the relative standard deviation was less than 20%(n=5). The standard reference material was detected and the concentration was within the reference range. CONCLUSION: This method can be used to detect six bisphenols in urine quickly and accurately, is suitable for the trace analysis of bisphenol compounds in human urine.

Exploring the gut microbiota-hippocampus-metabolites axis dysregulation in sepsis mice.

Background: Sepsis is commonly associated with a sudden impairment of brain function, thus leading to significant rates of illness and mortality. The objective of this research was to integrate microbiome and metabolome to reveal the mechanism of microbiota-hippocampus-metabolites axis dysfunction in a mouse model of sepsis. Methods: A mouse model of sepsis was established via cecal ligation and puncture. The potential associations between the composition of the gut microbiota and metabolites in the hippocampus of mice with sepsis were investigated by combining 16S ribosomal RNA gene sequencing and ultra-high-performance liquid chromatography tandem mass spectrometry. Results: A total of 140 differential metabolites were identified in the hippocampal tissues of mice with sepsis when compared to those of control mice. These differential metabolites in mice with sepsis were not only associated with autophagy and serotonergic synapse, but also involved in the metabolism and synthesis of numerous amino acids. At the phylum level, the abundance of Bacteroidota was increased, while that of Firmicutes (Bacillota) was decreased in mice with sepsis. At the genus level, the abundance of Alistipes was increased, while that of Lachnospiraceae_NK4A136_group was decreased in mice with sepsis. The Firmicutes (Bacillota)/Bacteroidota (F/B) ratio was decreased in mice with sepsis when compared to that of control mice. Furthermore, the F/B ratio was positively correlated with 5'-methylthioadenosine, PC (18:3(9Z,12Z,15Z)/18:0) and curdione, and negatively correlated with indoxylsulfuric acid, corticosterone, kynurenine and ornithine. Conclusion: Analysis revealed a reduction in the F/B ratio in mice with sepsis, thus contributing to the disturbance of 5'-methylthioadenosine, curdione, PC (18:3(9Z,12Z,15Z)/18:0), corticosterone, ornithine, indoxylsulfuric acid and kynurenine; eventually, these changes led to hippocampus dysfunction. Our findings provide a new direction for the management of sepsis-induced hippocampus dysfunction.