Peak-tracking algorithm for use in comprehensive two-dimensional liquid chromatography - Application to monoclonal-antibody peptides.

A peak-tracking algorithm was developed for use in comprehensive two-dimensional liquid chromatography coupled to mass spectrometry. Chromatographic peaks were tracked across two different chromatograms, utilizing the available spectral information, the statistical moments of the peaks and the relative retention times in both dimensions. The algorithm consists of three branches. In the pre-processing branch, system peaks are removed based on mass spectra compared to low intensity regions and search windows are applied, relative to the retention times in each dimension, to reduce the required computational power by elimination unlikely pairs. In the comparison branch, similarity between the spectral information and statistical moments of peaks within the search windows is calculated. Lastly, in the evaluation branch extracted-ion-current chromatograms are utilized to assess the validity of the pairing results. The algorithm was applied to peptide retention data recorded under varying chromatographic conditions for use in retention modelling as part of method optimization tools. Moreover, the algorithm was applied to complex peptide mixtures obtained from enzymatic digestion of monoclonal antibodies. The algorithm yielded no false positives. However, due to limitations in the peak-detection algorithm, cross-pairing within the same peaks occurred and six trace compounds remained falsely unpaired.

LC-HRMS and acetylcholinesterase affinity assay as a workflow for profiling alkaloids in Annona salzmannii extract.

Structure-based molecular networking is useful as a dereplication strategy to identify known molecules, unknown close analogues, or compound families. On the other hand, the ligand fishing assay is a remarkable alternative to accelerate the screening process and to overcome the drawbacks of laborious experiments usually adopted in natural product research. The combination of these approaches contributes to high productivity in disclosing active metabolites and a decrease in lead time identification. To provide a valuable data base for the alkaloids of A. salzmannii bark herein we disclose thirty-one isoquinoline alkaloids including benzyltetrahydroisoquinolines, aporphines, proaporphines, and protoberberines. Among these, twenty-six have not been described for A. salzmannii including the unprecedented alkaloid N,O-dimethylcoclaurine N-oxide. In addition, norcoclaurine (1), norreticuline (13), N,O-dimethylcoclaurine N-oxide (15), and N-acetylasimilobine (24) are now reported for the first time as ligand for acetylcholinesterase.

Rapid two-dimensional Protein-A size exclusion chromatography of monoclonal antibodies for titer and aggregation measurements from harvested cell culture fluid samples.

The success of monoclonal antibody (mAb) therapeutics have increased pharmaceutical investment in mAb production, which has led to a greater demand of technologies to efficiently characterize these biotherapeutics. The large size and heterogeneity of mAbs require the measurement of multiple critical quality attributes (CQAs) during production. The current workflow to measure CQAs of antibodies involves multiple one-dimensional liquid chromatography methods, including Protein-A (ProA), ion-exchange (IEX), reversed-phase, size exclusion (SEC), hydrophilic interaction, and hydrophobic interaction (HIC). Recent advances in commercial two-dimensional liquid chromatography (2D-LC) affords an opportunity to perform two separations at once to measure multiple CQAs in a single assay. Here, we describe the development of a 2D ProA-SEC method using entirely commercially available instrumentation. Each individual separation and the transfer of material between dimensions were optimized to develop a method that measures titer and aggregation of a target antibody from harvested cell culture fluid in under 5 min. We determined the effects of each parameter of the method on mAb recovery and stability, as well as speed, robustness, resolution, and accuracy of the aggregate amount detected in the second dimension (2D). While there are still sources of error caused by hardware limitations, our rapid ProA-SEC method is an effective screening tool with a significant throughput advantage over previously described methods. Additionally, this work serves as a basis for developing other 2D-LC methods with ProA as the first dimension (1D) separation coupled with different 2D separation, such as ProA-IEX and ProA-HIC.

Online Extraction Coupled to Liquid Chromatography Analysis (OLE-LC): Eliminating Traditional Sample Preparation Steps in the Investigation of Solid Complex Matrices.

Current methods employed for the analysis of the chemical composition of solid matrices (such as plant, animal, or human tissues; soil; etc.) often require many sample treatment steps, including an extraction step with exclusively dedicated solvents. This work describes an optimized analytical setup in which the extraction of a solid sample is directly coupled to its analysis by high-performance liquid chromatography. This approach avoids (i) the use of pumps and valves other than those comprising the HPLC instrument, (ii) the use of solvents other than those of the mobile phase, and (iii) the need to stop the mobile phase flow at any time during the full analytical procedure. The compatibility of this approach with the direct analysis of fresh tissues (leaves, stems, and seeds of four plant species with dissimilar chemical compositions) was successfully demonstrated, leading to the elimination of sample preparation steps such as drying, grinding, concentration, dilution, and filtration, among others. This work describes a new, simple, and efficient green approach to minimize or eliminate sample treatment procedures. It could be easily applied for quality control of plant materials and their derived products through chromatographic fingerprints and for untargeted metabolomic investigations of solid matrices, among other applications.