Neurotransmitters (NTs) and their metabolites play crucial roles in the regulation of the sleep-wake cycle. Thus, a comprehensive quantitative analysis of NTs would be useful in elucidating the ...potential mechanisms involved in sedative-hypnotic activities. In this study, we developed a high-throughput quantitative method based on a two-dimensional chromatography-mass spectrometry technique to simultaneously analyze 63 NTs and their metabolites in rat plasma, brain homogenate, and microdialysis samples from five different sleep-associated regions of the brain. Moreover, this method was used to study the neurochemical mechanism of an adenosine analog sedative-hypnotic candidate YZG-331. Most of the correlations between NTs were lost after the administration of the sedative, particularly in the caudate putamen (CPu) and dorsal raphe nucleus (DRN), indicating that the sleep-wake balance was affected. Administration of the adenosine analog YZG-331 could act similar as accumulation of adenosine, inducing adenosine and its metabolite adenine were decreased significantly in the CPu, accompanying with GABA, aspartate, and glutamate changed slightly by the communications between different neurons to further promote sleep. In addition, YZG-331 affected the metabolism of tryptophan and serotonin (5-HT) in the DRN and orbital frontal cortex (OFC). Melatonin and 5-hydroxyindole-3-acetic acid (a metabolite of 5-HT) were significantly increased in the OFC, and the levels of glutamate/glutamine, asparagine, and adrenaline were altered. Sleep homeostasis is a balance between the duration of sleep and wakefulness and is coordinated by all NTs. The high-throughput quantitative method introduced in this study may aid in revealing the temporal cohesion among NTs, evaluating sleep homeostasis, and determining the effects of sedative-hypnotic drugs.
Display omitted
•Quantify 63 NTs/NTs’ metabolites in plasma and brain•YZG-331 interrupts sleep homeostasis by acting similar as accumulation of adenosine in CPU
Method development in comprehensive two-dimensional liquid chromatography (LC×LC) is a challenging process. The interdependencies between the two dimensions and the possibility of incorporating ...complex gradient profiles, such as multi-segmented gradients or shifting gradients, make trial-and-error method development time-consuming and highly dependent on user experience. Retention modeling and Bayesian optimization (BO) have been proposed as solutions to mitigate these issues. However, both approaches have their strengths and weaknesses. On the one hand, retention modeling, which approximates true retention behavior, depends on effective peak tracking and accurate retention time and width predictions, which are increasingly challenging for complex samples and advanced gradient assemblies. On the other hand, Bayesian optimization may require many experiments when dealing with many adjustable parameters, as in LC×LC. Therefore, in this work, we investigate the use of multi-task Bayesian optimization (MTBO), a method that can combine information from both retention modeling and experimental measurements. The algorithm was first tested and compared with BO using a synthetic retention modeling test case, where it was shown that MTBO finds better optima with fewer method-development iterations than conventional BO. Next, the algorithm was tested on the optimization of a method for a pesticide sample and we found that the algorithm was able to improve upon the initial scanning experiments. Multi-task Bayesian optimization is a promising technique in situations where modeling retention is challenging, and the high number of adjustable parameters and/or limited optimization budget makes traditional Bayesian optimization impractical.
•A multi-task Bayesian optimization algorithm was developed to optimize LC×LC methods.•It uses both retention modeling and experimental data to guide the optimization.•The performance of multi-task and single-task Bayesian optimization was compared.•The separation of a complex pesticide sample was improved within a few iterations.•The algorithm can run unsupervised in a fully automated closed-loop environment.
In top‐down (TD) proteomics, efficient proteoform separation is crucial to reduce the sample complexity and increase the depth of the analysis. Here, we developed a two‐dimensional low pH/low pH ...reversed‐phase liquid chromatography separation scheme for TD proteomics. The first dimension for offline fractionation was performed using a polymeric reversed‐phase (PLRP‐S) column with trifluoroacetic acid as ion‐pairing reagent. The second dimension, a C4 nanocolumn with formic acid as ion‐pairing reagent, was coupled online with a high‐field asymmetric ion mobility spectrometry (FAIMS) Orbitrap Tribrid mass spectrometer. For both dimensions several parameters were optimized, such as the adaption of the LC gradients in the second dimension according to the elution time (i.e., fraction number) in the first dimension. Avoidance of elevated temperatures and prolonged exposure to acidic conditions minimized cleavage of acid labile aspartate–proline peptide bonds. Furthermore, a concatenation strategy was developed to reduce the total measurement time. We compared our low/low pH with a previously published high pH (C4, ammonium formate)/low pH strategy and found that both separation strategies led to complementary proteoform identifications, mainly below 20 kDa, with a higher number of proteoforms identified by the low/low pH separation. With the optimized separation scheme, more than 4900 proteoforms from 1250 protein groups were identified in Caco‐2 cells.
A new interface was designed to enable the coupling of reversed phase liquid chromatography (RPLC) and supercritical fluid chromatography (SFC). This online two-dimensional chromatographic system ...utilizing RPLC in the first dimension and SFC in the second was developed to achieve simultaneous achiral and chiral analysis of pharmaceutical compounds. The interface consists of an eight-port, dual-position switching valve with small volume C-18 trapping columns. The peaks of interest eluting from the first RPLC dimension column were effectively focused as sharp concentration pulses on small volume C-18 trapping column/s and then injected onto the second dimension SFC column. The first dimension RPLC separation provides the achiral purity result, and the second dimension SFC separation provides the chiral purity result (enantiomeric excess). The results are quantitative enabling simultaneous achiral, chiral analysis of compounds. The interface design and proof of concept demonstration are presented. Additionally, comparative studies to conventional SFC and case studies of the applications of 2D LC–SFC in pharmaceutical analysis is presented.
Display omitted
•The 2D LC–SFC enables simultaneous achiral-chiral analysis.•Primary dimension is reversed phase and secondary dimension is normal phase.•Small volume trapping columns are used to transfer fractions between dimensions.•Excellent linearity (R2>0.99) and sensitivity (~0.1% of target) were achieved.•Multiple trapping columns were used to analyze stereoisomers.
The biopharmaceutical industry is transitioning from currently deployed batch‐mode bioprocessing to a highly efficient and agile next‐generation bioprocessing with the adaptation of continuous ...bioprocessing, which reduces capital investment and operational costs. Continuous bioprocessing, aligned with FDA's quality‐by‐design platform, is designed to develop robust processes to deliver safe and effective drugs. With the deployment of knowledge‐based operations, product quality can be built into the process to achieve desired critical quality attributes (CQAs) with reduced variability. To facilitate next‐generation continuous bioprocessing, it is essential to embrace a fundamental shift‐in‐paradigm from “quality‐by‐testing” to “quality‐by‐design,” which requires the deployment of process analytical technologies (PAT). With the adaptation of PAT, a systematic approach of process and product understanding and timely process control are feasible. Deployment of PAT tools for real‐time monitoring of CQAs and feedback control is critical for continuous bioprocessing. Given the current deficiency in PAT tools to support continuous bioprocessing, we have integrated Infinity 2D‐LC with a post‐flow‐splitter in conjunction with the SegFlow autosampler to the bioreactors. With this integrated system, we have established a platform for online measurements of titer and CQAs of monoclonal antibodies as well as amino acid analysis of bioreactor cell culture.
This article demonstrates the potential to bridge the gap in online product quality assessments of biopharmaceutical drugs. With the integration of SegFlow interface with 2D–LC and a flow splitter, samples can be drawn from bioreactors at a scheduled intervals and purify the protein drugs using protein–A column on the 1st dimension of 2D–LC followed by product quality assessment on the 2nd dimension. In addition, this integrated platform serves as a valuable tool for online measurements of titer and amino acids.
A major challenge in the field of proteomics is obtaining high‐quality peptides for comprehensive proteome profiling by LC–MS. Here, evaluation and modification of a range of sample preparation ...methods using photosynthetically active Arabidopsis leaf tissue are done. It was found that inclusion of filter‐aided sample preparation (FASP) based on filter digestion improves all protein extraction methods tested. Ultimately, a detergent‐free urea‐FASP approach that enables deep and robust quantification of leaf and root proteomes is shown. For example, from 4‐day‐old leaf tissue, up to 11 690 proteins were profiled from a single sample replicate. This method should be broadly applicable to researchers working with difficult to process plant samples.
Pulmonary tuberculosis (TB) caused by Mycobacterium tuberculosis is a chronic disease. Currently, there are no sufficiently validated biomarkers for early diagnosis of TB infection. In this study, a ...panel of potential serum biomarkers was identified between patients with pulmonary TB and healthy controls by using iTRAQ‐coupled 2D LC‐MS/MS technique. Among 100 differentially expressed proteins screened, 45 proteins were upregulated (>1.25‐fold at p < 0.05) and 55 proteins were downregulated (<0.8‐fold at p < 0.05) in the TB serum. Bioinformatics analysis revealed that the differentially expressed proteins were related to the response to stimulus, the metabolic and immune system processes. The significantly differential expression of apolipoprotein CII (APOCII), CD5 antigen‐like (CD5L), hyaluronan‐binding protein 2 (HABP2), and retinol‐binding protein 4 (RBP4) was further confirmed using immunoblotting and ELISA analysis. By forward stepwise multivariate regression analysis, a panel of serum biomarkers including APOCII, CD5L, and RBP4 was obtained to form the disease diagnostic model. The receiver operation characteristic curve of the diagnostic model was 0.98 (sensitivity = 93.42%, specificity = 92.86%). In conclusion, APOCII, CD5L, HABP2, and RBP4 may be potential protein biomarkers of pulmonary TB. Our research provides useful data for early diagnosis of TB.
This study developed a two‐dimensional heart‐cutting LC method for the separation of amino acid enantiomers. Two approaches for achiral separation of amino acids, phenylalanine and tryptophan, were ...selected. Amino acids were separated on C18 or hydrophilic interaction liquid chromatography (HILIC) columns in first dimension after their enantiomer separation on a teicoplanin chiral column in second dimension. Mobile phases for both separation systems were optimized by testing different types of organic modifiers, concentrations of ion‐pair agent (sodium 1‐octanesulfonate), and ionic modifier (ammonium acetate). The resolution of enantiomers higher than 1.5 for both amino acids was achieved using a C18–teicoplanin coupled column separation system with mobile phases methanol/2 mM sodium 1‐octanesulfonate (10:90 and 75:25, step gradient between achiral and chiral columns, respectively). The lower resolution of amino acid enantiomers (RS ˃ 0.9), but higher column efficiency, was achieved on a HILIC–teicoplanin separation system with mobile phases acetonitrile/50 mM ammonium acetate (90:10 and 80:20, step gradient between achiral and chiral columns, respectively). The developed heart‐cutting 2D‐LC methods were validated in terms of linearity, limit of detection, limit of quantification, precision, and accuracy. The results suggested that the developed methods were applicable for the simultaneous determination of amino acid enantiomers in the dietary supplement. The sample contained l‐phenylalanine and l‐tryptophan.
•Sensitive and specific 2D-LC–MS/MS method for serum and plasma aldosterone.•Faster than published LC–MS/MS methods with no compromise to specificity.•Low sample volume requirement.•Immunoassays ...found to be overestimating aldosterone concentrations between 22% and 37%.•Linearity of the method will allow testing of urine and adrenal venous samples.
Accurate measurement of aldosterone is important for the standardized testing of primary hyperaldosteronism. Commercial immunoassays show substantial between-method variations resulting in significant clinical consequences. We developed a specific two dimensional (2D)-LC–MS/MS method for measuring aldosterone in human serum and plasma and compared it with three commercial immunoassays and an LC–MS/MS method.
250μL samples, controls and calibrators spiked with d4-aldosterone were subjected to liquid–liquid extraction. The samples were analyzed using negative mode electrospray and 2D-LC followed by MS detection using an ABSciex 5500 mass spectrometer and compared with immunoassays of Siemens (Coat-A-Count), DiaSorin (CLIA-LIAISON), and IBL (ELISA). Data was acquired using multiple reaction-monitoring mode.
LOQ and LOD of the method were 0.04 and 0.02nmol/L respectively. The assay was linear up to 166nmol/L. Inter and intra-assay imprecision at 0.13, 1.38 and 8.30nmol/L were <10%. Interferences were absent and no differences were observed between serum and plasma matrices. Method recovery ranged from 95% to 113%. Ion suppression was not observed. Evaluated immunoassays showed positive biases ranging between 22% and 37% when compared with the developed method.
We developed and validated an accurate method for measurement of aldosterone in human serum and plasma using 2D-LC–MS/MS which is suitable for clinical purposes. The method is faster than previously published LC–MS/MS methods, uses less sample, has adequate sensitivity while being able to preserve high specificity in a cost effective manner. Linearity of the assay makes it promising for urine and adrenal venous samples. Comparison with three commercial immunoassays demonstrates the advantages of the developed method.
PDF
