Reversed-phase ultrahigh-performance liquid chromatography-mass spectrometry (RP-UHPLC/MS) method is optimized for the quantitation of a large number of lipid species in biological samples, primarily in human plasma and serum. The method uses a C18 bridged ethylene hybrid (BEH) column (150 × 2.1 mm; 1.7 μm) for the separation of lipids from 23 subclasses with a total run time of 25 min. Lipid species separation allows the resolution of isobaric and isomeric lipid forms. A triple quadrupole mass spectrometer is used for targeted lipidomic analysis using multiple reaction monitoring (MRM) in the positive ion mode. Data are evaluated by Skyline software, and the concentrations of analytes are determined using internal standards per each individual lipid class.
- Keywords
- High-throughput lipidomics, Mass spectrometry, Plasma, Quantitation, Reversed-phase, Serum, Ultrahigh-performance liquid chromatography,
- MeSH
- Chromatography, Reverse-Phase * methods MeSH
- Mass Spectrometry methods MeSH
- Liquid Chromatography-Mass Spectrometry MeSH
- Humans MeSH
- Lipidomics * methods MeSH
- Lipids * analysis MeSH
- High-Throughput Screening Assays methods MeSH
- Software MeSH
- Tandem Mass Spectrometry methods MeSH
- Chromatography, High Pressure Liquid methods MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Names of Substances
- Lipids * MeSH
Ultrahigh-performance supercritical fluid chromatography-mass spectrometry (UHPSFC/MS) method is optimized for the high-throughput quantitation of lipids in human serum and plasma with an emphasis on robustness and accurate quantitation. Bridged ethylene hybrid (BEH) silica column (100 × 3 mm; 1.7 μm) is used for the separation of 17 nonpolar and polar lipid classes in 4.4 min using the positive ion electrospray ionization mode. The lipid class separation approach in UHPSFC/MS results in the coelution of all lipid species within one lipid class in one chromatographic peak, including two exogenous internal standards (IS) per lipid class, which provides the optimal conditions for robust quantitation. The method was validated according to European Medicines Agency and Food and Drug Administration recommendations. UHPSFC/MS combined with LipidQuant software allows a semiautomated process to determine lipid concentrations with a total run time of only 8 min including column equilibration, which enables the analysis of 160 samples per day.
- Keywords
- High-throughput lipidomics, Mass spectrometry, Plasma, Quantitation, Serum, Ultrahigh-performance supercritical fluid chromatography, Validation,
- MeSH
- Mass Spectrometry methods MeSH
- Humans MeSH
- Lipidomics * methods MeSH
- Lipids * analysis blood MeSH
- Chromatography, Supercritical Fluid * methods MeSH
- Chromatography, High Pressure Liquid methods MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Names of Substances
- Lipids * MeSH
Herein, we aim to establish a straightforward and versatile reversed-phase liquid chromatography/mass spectrometry (RP-LC/MS) methodology for analyzing a wide range of polar and mid-polar metabolites utilizing a single instrument, column, and mobile phase. We present a comprehensive evaluation of three C18 columns compatible with aqueous solutions using 19 mobile phases in terms of the number of detected metabolites, chromatographic performance, and MS response. The RP-LC/MS platform utilizes the HSS T3 column with a mobile phase consisting of 0.2 % formic acid, acetonitrile, and propan-2-ol, effectively separating polar and mid-polar metabolites through various mobile phase gradients. Our developed method outperforms the conventional hydrophilic interaction liquid chromatography metabolomic method, yielding a higher number of detected metabolites and better chromatographic performance. The RP-LC/MS platform demonstrates excellent intrabatch and interbatch retention time repeatability (<0.8 %). Furthermore, the determined concentrations of metabolites show strong agreement with certified and published concentrations of metabolites in the SRM 1950 plasma sample. We successfully annotate 71 polar metabolites, 36 acylcarnitines, 23 endocannabinoids, 42 oxylipins, and 16 fatty acids in plasma, placenta, and brain samples. The developed RP-LC/MS approach represents a robust and adaptable technique for the targeted or untargeted analysis of polar and mid-polar metabolites employing a single chromatographic column and mobile phase. This is achieved through the simple modification of the gradient program and MS conditions. Consequently, this methodology offers a highly valuable tool for conducting comprehensive, large-scale metabolomic investigations on a variety of biological samples.
- Keywords
- Liquid chromatography, Mass spectrometry, Metabolomics, Reversed-phase, Targeted analysis, Untargeted analysis,
- MeSH
- Chromatography, Reverse-Phase * methods MeSH
- Endocannabinoids analysis blood metabolism MeSH
- Mass Spectrometry * methods MeSH
- Carnitine analogs & derivatives analysis blood MeSH
- Humans MeSH
- Fatty Acids analysis MeSH
- Metabolomics methods MeSH
- Oxylipins analysis blood MeSH
- Pregnancy MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Pregnancy MeSH
- Female MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Names of Substances
- acylcarnitine MeSH Browser
- Endocannabinoids MeSH
- Carnitine MeSH
- Fatty Acids MeSH
- Oxylipins MeSH
BACKGROUND: Electrochemistry offers a range of powerful techniques for solving analytical problems, each with its own advantages and limitations that can significantly affect the information obtained. These variations lead to diverse requirements for newly developed methods. Applying multiple electrochemical techniques simultaneously can optimize information extraction from a sample, aiding in the selection of the best analytical approach. RESULTS: In this study, we present the first investigation of the oxidation of the coccidiostat nicarbazin, along with a comparative evaluation of two established electrochemical methods with complementary strengths: differential pulse voltammetry (DPV) and capillary electrophoresis coupled with amperometric detection (CE-AD). We thoroughly examine the oxidative determination of nicarbazin in poultry feed samples using acetonitrile based media. DPV allows for rapid and efficient analysis, while CE-AD excels in handling complex samples with electrochemically active species due to its high separation efficiency. Both methods exhibit limits of detection (LODs) in the low micromolar range. To provide a comprehensive understanding of the nicarbazin oxidation process, the final reaction products were analyzed by mass spectrometry, identifying 4-nitroaniline and (4-nitrophenyl)formamide as key product compounds. SIGNIFICANCE AND NOVELTY: This pioneering research on the anodic detection of nicarbazin includes a detailed analysis of the components in the studied equimolar mixture. The developed protocols, combined with straightforward sample preparation, enable the successful determination of nicarbazin levels below those allowed by EU regulations.
BACKGROUND: Determination of critical quality attributes (CQAs) of pharmaceutical monoclonal antibodies (mAbs) is an essential part of quality control. Commonly, for each CQA, a separate analytical method and setup is required, making assessment of multiple CQAs time-consuming and labour-intensive. This typically involves offline purification and diverse protein digestion steps, in combination with multiple liquid-chromatographic modes. We developed an integrated, fully online multidimensional platform for direct analysis of mAbs in cell culture fluid (CCF) at an intact, subunit and peptide level from a single injection. RESULTS: This paper focuses on the online middle-up and bottom-up workflows. The platform combines Protein A affinity chromatography (ProtA), immobilized enzyme reactors (IMERs), reversed-phase liquid chromatography (RPLC) and high-resolution mass spectrometry (MS) for characterization of mAbs. Online ProtA was used to isolate mAbs directly from CCF. Subsequent online digestion of isolated mAb was accomplished by IMERs featuring either the proteases IdeS or trypsin. Between ProtA and IMERs, buffer exchange and pH adjustment were achieved using a strong cation-exchange (SCX) trap column. RPLC-MS analysis of F(ab)'2 and Fc/2 fragments obtained after IdeS digestion provided information on mAb glycoform compositions and the potential presence of PTMs and subunit variants. RPLC-MS/MS analysis of trypsin-digested peptides provided over 95 % coverage of the mAb's amino acid sequence, but also identification and localization of modifications related to e.g. oxidation and deamidation. Comparisons with established offline methods were made. The overall capacity of the system to perform intact, middle-, and bottom-up analyses in parallel from a single injection is demonstrated for an industrially-relevant mAb in CCF. SIGNIFICANCE: The developed multidimensional platform enables the simultaneous characterization of multiple fractions from a single ProtA-isolated mAb band at intact, middle-up, or bottom-up level using various LC modes at a substantially reduced analysis time.
- Keywords
- Critical quality attributes, IMERs, Multi-dimensional characterization, Protein liquid chromatography, mAb,
- MeSH
- Cell Culture Techniques MeSH
- CHO Cells MeSH
- Chromatography, Affinity MeSH
- Chromatography, Reverse-Phase MeSH
- Cricetulus MeSH
- Humans MeSH
- Antibodies, Monoclonal * analysis chemistry MeSH
- Workflow MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Names of Substances
- Antibodies, Monoclonal * MeSH
Lipidated anorexigenic peptides are highly promising compounds for the treatment of obesity and related diseases. However, their exact mechanism of action still remains unknown. We labelled a lipidated analogue of an anorexigenic prolactin-releasing peptide (palm11-PrRP31) with an extremely stable ClickZip lanthanide tag, facilitating tracking of the peptide within the organism. We then employed a separation method based on liquid chromatography combined with inductively coupled plasma mass spectrometry (LC-ICP-MS). This technique involved the use of an unconventional mobile phase containing 5 % 1,2-hexanediol in H2O (v/v) with the addition of 2 % formic acid. Using a rapid. 6-min analysis, we were able to quantify the ClickZip tag - and thus indirectly the fate of the labelled peptides in the living organism - independently of free Ln3+ ions. The detection limits for the various lanthanide chemical forms were extremely low, ranging between 0.9 and 3.4 ng/L. We demonstrated the suitability of the method for analysing real biological samples like blood plasma, and confirmed the accuracy of our results. Prior to LC-ICP-MS analysis, we optimised a process involving the microwave-assisted digestion of liver samples to preserve the integrity of the ClickZip tag. We also identified several metabolites of the labelled peptides in the liver, urine, and blood plasma, highlighting the utility of the method for revealing the mechanism of action behind the labelled lipopeptides.
- Keywords
- 1,2-Hexanediol, Anorexigenic peptides, Method validation, Sample digestion,
- MeSH
- Chromatography, Liquid methods MeSH
- Click Chemistry MeSH
- Mass Spectrometry * methods MeSH
- Prolactin-Releasing Hormone chemistry MeSH
- Liver chemistry metabolism MeSH
- Lanthanoid Series Elements chemistry MeSH
- Mice MeSH
- Peptides chemistry analysis MeSH
- Animals MeSH
- Check Tag
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Names of Substances
- Prolactin-Releasing Hormone MeSH
- Lanthanoid Series Elements MeSH
- Peptides MeSH
BACKGROUND: A multicomponent meningococcal serogroups ABCWY vaccine (MenABCWY) could provide broad protection against disease-causing meningococcal strains and simplify the immunisation schedule. The aim of this trial was to confirm the effect of the licensed meningococcal serogroup B (MenB) vaccine, 4CMenB, against diverse MenB strains, and to assess the breadth of immune response against a panel of 110 MenB strains for MenABCWY containing the antigenic components of 4CMenB and licensed serogroups ACWY vaccine, MenACWY-CRM, the non-inferiority of the immune response with MenABCWY versus 4CMenB and MenACWY-CRM, safety, and MenABCWY lot-to-lot consistency. METHODS: We conducted a phase 3 randomised, controlled, observer-blinded trial of healthy adolescents and young adults (age 10-25 years) across 114 centres in Australia, Canada, Czechia, Estonia, Finland, Türkiye, and the USA. Exclusion criteria included previous vaccination with a MenB vaccine or (within the last 4 years) MenACWY vaccine. Participants were randomly allocated (5:5:3:3:3:1 ratio) via a central randomisation system using a minimisation procedure to receive 4CMenB at months 0, 2, and 6 (referred to as 4CMenB 0-2-6 hereafter); or 4CMenB at months 0 and 6 (referred to as 4CMenB 0-6 hereafter); or MenABCWY (three groups, each receiving one production lot of the MenACWY-CRM component) at months 0 and 6; or MenACWY-CRM at month 0. Demonstration in the per-protocol set of the consistency of three MenACWY-CRM component lots of the MenABCWY vaccine was a primary objective (demonstrated with two-sided 95% CIs for the ratio of human serum bactericidal antibody [hSBA] geometric mean titres against each serogroup within predefined criteria [0·5-2·0]). The primary endpoints (breadth of immune response) for the MenB component of MenABCWY and 4CMenB were measured using the endogenous complement hSBA (enc-hSBA) assay against a panel of 110 diverse MenB invasive disease strains. For each serum sample, 35 strains from the 110 MenB strain panel were randomly selected for testing. The 4CMenB breadth of immune response data have been published separately. For MenABCWY, breadth of immune response was assessed in two analyses: a test-based analysis of the percentage of samples (tests) without bactericidal serum activity against MenB strains 1 month after two MenABCWY doses versus the percentage after one MenACWY-CRM dose in the per-protocol set, and a responder-based analysis of the percentage of participants (responders) whose sera killed 70% or more strains at 1 month after two MenABCWY doses in the full analysis set. A lower limit of two-sided 95% CI above 65% would demonstrate breadth of immune response. Other primary outcomes included non-inferiority (5% margin) of two MenABCWY doses versus two 4CMenB doses by enc-hSBA assay in the per-protocol set, non-inferiority (10% margin) of two MenABCWY doses versus one MenACWY-CRM dose in MenACWY vaccine-naive participants by traditional hSBA assay in the per-protocol set, and safety in all vaccinated participants. This trial is registered with ClinicalTrials.gov, NCT04502693, and is complete. FINDINGS: Between Aug 14, 2020, and Sept 3, 2021, 3651 participants were enrolled and randomly allocated (900 in the 4CMenB 0-2-6 group and 908 in the 4CMenB 0-6 group, 1666 in the three MenABCWY groups combined, and 177 in the MenACWY-CRM group). All primary objectives for MenABCWY were met. Consistency of immune responses against the three production lots of the MenACWY component of MenABCWY was demonstrated since two-sided 95% CIs for the ratios of hSBA geometric mean titres against serogroups A, C, W, and Y for each pair of lots were within the predefined equivalence criteria. The lot data were pooled for the remainder of MenABCWY endpoints. By enc-hSBA assay, breadth of immune response against the MenB strain panel was 77·9% (95% CI 76·6 to 79·2) in the test-based analysis and 84·1% (81·4 to 86·5; 687 of 817 participants) in the responder-based analysis. Non-inferiority of MenABCWY to 4CMenB was demonstrated by enc-hSBA assay: the difference in percentage of samples with bactericidal serum activity between the MenABCWY group (82·5% [95% CI 82·1 to 83·0]; 21 222 of 25 715) and 4CMenB 0-2 group (83·1% [82·7 to 83·6]; 22 921 of 27 569) was -0·61% (-1·25 to 0·03). Non-inferiority of two-dose MenABCWY to one-dose MenACWY-CRM was demonstrated by traditional hSBA assay, with differences between the MenABCWY group and MenACWY group in percentages of participants with a four-fold rise in hSBA titres of 11·3% (5·9 to 19·0) for serogroup A, 47·2% (38·1 to 56·3) for serogroup C, 35·3% (26·9 to 44·5) for serogroup W, and 27·0% (19·4 to 35·8) for serogroup Y. MenABCWY reactogenicity was mostly of mild or moderate severity and transient, with similar frequencies of adverse events in the MenABCWY and 4CMenB groups and no safety concerns were identified. INTERPRETATION: This study demonstrates breadth of immune response against a panel of 110 MenB strains for the MenB component of the investigational MenABCWY vaccine, when administered as a 0-6 months schedule to the target population of adolescents and young adults, with predefined criteria for success met for both breadth of immune response endpoints and for non-inferiority versus 4CMenB. This investigational vaccine could provide broad meningococcal serogroup coverage in a simplified immunisation schedule, thus aiding the public health attempt in preventing invasive meningococcal disease due to five Neisseria meningitidis serogroups in adolescents and young adults. FUNDING: GSK.
- MeSH
- Child MeSH
- Adult MeSH
- Immunogenicity, Vaccine * MeSH
- Single-Blind Method MeSH
- Humans MeSH
- Meningococcal Infections * prevention & control immunology MeSH
- Meningococcal Vaccines * immunology adverse effects administration & dosage MeSH
- Adolescent MeSH
- Young Adult MeSH
- Neisseria meningitidis, Serogroup B immunology MeSH
- Neisseria meningitidis immunology MeSH
- Antibodies, Bacterial blood MeSH
- Healthy Volunteers MeSH
- Check Tag
- Child MeSH
- Adult MeSH
- Humans MeSH
- Adolescent MeSH
- Young Adult MeSH
- Male MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Clinical Trial, Phase III MeSH
- Multicenter Study MeSH
- Randomized Controlled Trial MeSH
- Names of Substances
- 4CMenB vaccine MeSH Browser
- Meningococcal Vaccines * MeSH
- Antibodies, Bacterial MeSH
BACKGROUND: Basic management for patients who have suffered a cardiac arrest and are admitted to an intensive care unit (ICU) after resuscitation includes setting targets for blood pressure and managing sedation and temperature. However, optimal targets and management are unknown. METHODS: The STEPCARE (Sedation, Temperature and Pressure after Cardiac Arrest and Resuscitation) trial is a multicenter, parallel-group, randomized, factorial, superiority trial in which sedation, temperature, and blood pressure strategies will be studied in three separate comparisons (SED-CARE, TEMP-CARE, and MAP-CARE). The trial population will be adults admitted to intensive care who are comatose after resuscitation from out-of-hospital cardiac arrest. The primary outcome will be all-cause mortality, and the secondary outcomes will be poor functional outcome (modified Rankin Scale 4-6), Health-Related Quality of Life using EQ-VAS, and specific serious adverse events in the intensive care unit predefined for each trial. All outcomes will be assessed at 6 months after randomization. The prognosticators, outcome assessors, statisticians, data managers, steering group, and manuscript writers will be blinded to treatment allocation. This statistical analysis plan includes a comprehensive description of the statistical analyses, handling of missing data, and assessments of underlying statistical assumptions. Analyses will be conducted according to the intention-to-treat principle, that is, all randomized participants with available data will be included. The analyses will be performed independently by two statisticians following the present plan. CONCLUSION: This statistical analysis plan describes the statistical analyses for the STEPCARE trial in detail. The aim of this predefined statistical analysis plan is to minimize the risk of analysis bias.
- Keywords
- STEPCARE trial, blood pressure, cardiac arrest, sedation, statistical analysis, temperature,
- MeSH
- Cardiopulmonary Resuscitation * methods MeSH
- Blood Pressure * MeSH
- Quality of Life MeSH
- Humans MeSH
- Randomized Controlled Trials as Topic MeSH
- Body Temperature MeSH
- Out-of-Hospital Cardiac Arrest * therapy mortality MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Multicenter Study MeSH
- Clinical Trial Protocol MeSH
Direct detection of DNA in complex biological samples bears several challenges regarding the selectivity and sensitivity of analyses. Therefore, DNA pre-extraction from bio-fluids is an emerging tool in biologically related fields. Specifically, a newly developing family of liquid biopsy techniques using PCR detection of circulating tumor DNA from blood serum or blood plasma could be significantly improved by harnessing fast and high-throughput DNA sample preparation. To address these needs, a 3D-printed device and a method based on gel electrophoresis combined with electrodialysis for the time-, cost- and labor-efficient preparative separation of DNA fragments from blood was developed. The proposed system also successfully eliminated large DNA fragments from the samples. Recovery for short DNA fragments was reaching up to 80 %. The method was tested on human genomic DNA and blood and blood serum spiked with DNA standards, and it significantly alleviated the signal of matrix DNA.
- Keywords
- Cancer, Circulating tumor DNA (ctDNA), Liquid biopsy, PCR, Preparative gel electrophoresis,
- MeSH
- Printing, Three-Dimensional * MeSH
- Time Factors MeSH
- DNA * blood isolation & purification chemistry analysis MeSH
- Humans MeSH
- Polymerase Chain Reaction MeSH
- Liquid Biopsy methods MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Names of Substances
- DNA * MeSH
OBJECTIVE AND BACKGROUND: Health-related quality of life (HRQoL) is reduced in narcolepsy type 1 (NT1), but proper information on HRQoL in narcolepsy type 2 (NT2) and idiopathic hypersomnia (IH) is lacking. This study examines HRQoL of NT1, NT2, IH, and healthy controls (HC) and assesses the HRQoL associates in these diseases. PATIENTS AND METHODS: 117 adults (64 NT1, 22 NT2, 31 IH; 61.5 % women; 38.3 ± 12.0 years; 71.8 % treated) and 41 HC (53.7 % women; 35.9 ± 9.6 years) completed questionnaires assessing sleepiness, fatigue, symptoms severity, sleep inertia, depressive and anxiety symptoms, HRQoL, and underwent a semi-structured interview. Data were analyzed using the Mann-Whitney and Kruskal-Wallis tests, Spearman's correlation coefficient, and regression analysis. RESULTS: HRQoL of NT1, NT2, and IH, separately, was poorer compared to HC (p < 0.001). According to the 36-Item Short Form Health Survey, the mental HRQoL was more impaired in NT2 and IH than NT1 (p < 0.05) in association with more pronounced depressive symptoms (p < 0.01; p < 0.05, respectively) and sleep inertia (p < 0.01; p < 0.01, respectively). Psychiatric disorders were more prevalent in NT2 and IH versus NT1 (p < 0.05). CONCLUSION: HRQoL is reduced in NT1, NT2, and IH, with this reduction being more pronounced in NT2 and IH. Poor mental HRQoL of NT2 and IH was associated both with the severity of depressive symptoms and more intense sleep inertia.
- MeSH
- Depression psychology MeSH
- Adult MeSH
- Idiopathic Hypersomnia * psychology MeSH
- Quality of Life * psychology MeSH
- Middle Aged MeSH
- Humans MeSH
- Narcolepsy * psychology MeSH
- Surveys and Questionnaires MeSH
- Severity of Illness Index MeSH
- Fatigue psychology MeSH
- Anxiety psychology MeSH
- Check Tag
- Adult MeSH
- Middle Aged MeSH
- Humans MeSH
- Male MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
