State-of-the-art in liquid chromatography-mass spectrometry.

Impressive progress has been made in the technology and application of combined liquid chromatography-mass spectrometry (LC-MS) in the past decennium. From a technique, that could only be used by a specialist, it has developed into a routinely applicable technique. LC-MS has become the method-of-choice of analytical support in many stages of drug development within pharmaceutical industries and has found its way into environmental, biochemical and other laboratories. This paper provides a perspective on the current technology, principles and applications of LC-MS.     

Racemates versus enantiomerically pure drugs: putting high-performance liquid chromatography to work in the selection process

In view of the current negative attitude towards pharmaceutical preparations containing two or more drug substances, it is not surprising that the stereoisomeric composition of drugs has become a key issue in their development, regulatory approval and marketing. As the requirements placed on the purity of drugs become more and more stringent, the question arises of whether a racemic mixture should be automatically considered as being 50% impure. The answer is complex and requires careful comparative evaluations of the activities, toxicities and pharmacokinetics of the two enantiomers. In addition to the risk-benefit factors, the cost-benefit factors should also be carefully weighed in order to reach a decision which will guarantee drug safety. Excessive development costs and an unnecessary regulatory burden resulting from systematic demands for enantiomerically homogeneous drugs can be avoided with very potent drugs administered at low dosages and low-potency drugs for which the "inactive" isomer is not considerably more toxic than the active isomer. Regardless of the final decision taken, during the preclinical and clinical drug development it is important to have analytical methods suitable for conducting pertinent studies. This paper discusses the importance of stereochemistry in drug development and the role of high-performance liquid chromatography as a pre-eminent tool for analytical and preparative enantioselective separations.     

Solid-phase microextraction in bioanalysis: New devices and directions

The primary objective of this review is to discuss recent technological developments in the field of solid-phase microextraction that have enhanced the utility of this sample preparation technique in the field of bioanalysis. These developments include introduction of various new biocompatible coating phases suitable for bioanalysis, such as commercial prototype in vivo SPME devices, as well as the development of sampling interfaces that extend the use of this methodology to small animals such as mice. These new devices permit application of in vivo SPME to a variety of analyses, including pharmacokinetics, bioaccumulation and metabolomics studies, with good temporal and spatial resolution. New calibration approaches have also been introduced to facilitate in vivo studies and provide fast and quantitative results without the need to achieve equilibrium. In combination with the drastic improvement in the analytical sensitivity of modern liquid chromatography–tandem mass spectrometry instrumentation, full potential of in vivo SPME as a sample preparation tool in life sciences can finally be explored. From the instrumentation perspective, SPME was successfully automated in 96-well format for the first time. This opens up new opportunities for high-throughput applications (>1000 samples/day) such as for the determination of unbound and total drug concentrations in complex matrices such as whole blood with no need for sample pretreatment, studies of distribution of drugs in various compartments and/or determination of plasma protein binding and other ligand–receptor binding studies, and this review will summarize the progress in this research area to date.     

Enhancing drug development and clinical studies with patient-centric sampling using microsampling techniques: Opportunities,challenges, and insights into liquid chromatography-mass spectrometry strategies

Microsampling has revolutionized pharmaceutical drug development and clinical research by reducing sample volume requirements, allowing sample collection at home or nontraditional sites, minimizing animal and patient burden, and enabling more flexible study designs. This perspective paper discusses the transformative impact of microsampling and patient-centric sampling (PCS) techniques, emphasizing their advantages in drug development and clinical trials. We highlight the integration of liquid chromatography-mass spectrometry (LC–MS) strategies for analyzing PCS samples, focusing on our research experience and a review of current literatures. The paper reviews commercially available PCS devices, their regulatory status, and their application in clinical trials, underscoring the benefits of PCS in expanding patient enrollment diversity and improving study designs. We also address the operational challenges of implementing PCS, including the need for bridging studies to ensure data comparability between traditional and microsampling methods, and the analytical challenges posed by PCS samples. The paper proposes future directions for PCS, including the development of global regulatory standards, technological advancements to enhance user experience, the increased concern of sustainability and patient data privacy, and the integration of PCS with other technologies for improved performance in drug development and clinical studies. By advancing microsampling and PCS techniques, we aim to foster patient-centric approaches in pharmaceutical sciences, ultimately enhancing patient care and treatment efficacy.     

金属氮杂环卡宾配合物的抗肿瘤活性研究进展

自1978年顺铂成功地被开发成癌症临床治疗药物以来,金属配合物作为小分子抗癌药物的开发成为人们的研究热点。其中,氮杂环卡宾能与多种过渡金属中心形成稳定的共价键,这种特殊的稳定性使得金属氮杂环卡宾配合物具有被开发成药物的潜能。近年来,金属氮杂环卡宾配合物被发现具有良好的抗癌活性,激发了广大无机药物化学研究者的研究热情。综合笔者课题组在金属氮杂环卡宾抗肿瘤配合物方面的前期研究,本文将对银、金、铑和铂氮杂环卡宾配合物的抗肿瘤活性及作用机制进行综述,以期为新型金属氮杂环卡宾抗肿瘤化合物的设计合成提供参考。     

Evaluation of the results from an inter-laboratory comparison study of the determination of acrylamide in crispbread and butter cookies

Analytical methods currently employed for determination of acrylamide (AA) in two carbohydrate-rich food samples, crispbread and butter cookies, obtained commercially, and native and spiked bread extract samples have been evaluated in a collaborative study. The objective of the study was to obtain information about the performance of the participating laboratories when analysing samples with an AA content close to the limit of quantification (LOQ) and at a higher AA level, and to investigate the influence of sample-preparation procedures on the results of the analysis. For this purpose an aqueous native extract of white bread crumb, a fortified extract, and AA standard solutions, the analyte content of which were not disclosed to the participants, were included in the study. A total of 62 laboratories, applying seven different measurement techniques and a broad spectrum of analyte extraction and sample-preparation procedures reported their analytical results. Because the measurement data were not normally distributed, they were evaluated by application of robust statistics. The relative performance of the laboratories was highlighted by calculation of z-scores. For the crispbread sample, especially, a large percentage of the calculated z-scores were outside the satisfactory range. From their distribution it became obvious that one of the analytical techniques might be biased, if not applied correctly. Consequently, the impact of the applied methods was examined in more detail. Information about the analytical technique, extraction solvent, quantity weighed, calibration method, clean-up, and the experience of the participating laboratories were extracted from the analytical protocols and transcribed into a data matrix which was evaluated by multifactor analysis of variance. The applied measurement technique seems to have a statistically significant influence on the analytical results.     

<Emphasis Type="Italic">In-silico</Emphasis> Screening using Flexible Ligand Binding Pockets: A Molecular Dynamics-based Approach

Summary In-silico screening of flexible ligands against flexible ligand binding pockets (LBP) is an emerging approach in structure-based drug discovery. Here, we describe a molecular dynamics (MD) based docking approach to investigate the influence on the high-throughput in-silico screening of small molecules against flexible ligand binding pockets. In our approach, an ensemble of 51 energetically favorable structures of the LBP of human estrogen receptor α (hERα) were collected from 3 ns MD simulations. In-silico screening of 3500 endocrine disrupting compounds against these flexible ligand binding pockets resulted in thousands of ER–ligand complexes of which 582 compounds were unique. Detailed analysis of MD generated structures showed that only 17 of the LBP residues significantly contribute to the overall binding pocket flexibility. Using the flexible LBP conformations generated, we have identified 32 compounds that bind better to the flexible ligand-binding pockets compared to the crystal structure. These compounds, though chemically divergent, are structurally similar to the natural hormone. Our MD-based approach in conjunction with grid–based distributed computing could be applied routinely for in-silico screening of large databases against any given target.     

Recent advances in the bioanalytical methods of polyethylene glycols and PEGylated pharmaceuticals

Polyethylene glycols are synthetic polymers composed of repeating oxyethylene subunits, which have been known for non‐toxic, non‐immunogenic, non‐antigenic, good solubility in water and therefore approved for pharmaceutical applications. Recently, attachment or amalgamation of polyethylene glycols to therapeutic small molecules, peptides, proteins, or nanoparticles has become a mature technology for the sake of improving their pharmacokinetic and pharmacological profiles, also referred to as PEGylation. By comparison, there are only a few PEGylated pharmaceuticals have been registered for further clinical trials and even less was approved for marketing. High failure rate of PEGylated pharmaceuticals in pre‐clinical and clinical trials could be majorly attributed to their unclear pharmacokinetic behaviors. Therefore, the in vivo fate of the PEGylated pharmaceuticals for the various routes of administration needs to be thoroughly investigated An accurate in vivo pharmacological study thereof highly depends on the precise detection of polyethylene glycols as well as their fragments in biological matrixes. The goal of this review is to highlight the analytical methods that were developed and applied to evaluate the polyethylene glycols in pharmaceutical ingredients and excipients, which bring us closer to bridging the gap between the development of polyethylene glycol‐based drug delivery systems and their clinical application.     

Application of Microdialysis for Pharmacokinetics of Traditional Chinese Medicine Studies

As a new and developing technique, microdialysis (MD) has extensive field application, and has become an important tool for the pharmacokinetics (PK) of traditional Chinese medicine (TCM) studies. This technique presents great value and good perspective on the study of PK of traditional Chinese medicine. In the present paper, the principle advantages of the MD sampling technique were reviewed, and its applications on the PK of traditional Chinese medicine studies were introduced.     

Front Cover: Can Cyanuric Acid and 2,4,6-Triaminopyrimidine Containing Ribonucleosides be Components of Prebiotic RNA? Insights from QM Calculations and MD Simulations (ChemPhysChem 11/2019)

As a step toward assessing their fitness as pre-RNA nucleobases, we employ DFT and MD simulations to analyze the noncovalent interactions of cyanuric acid (CA) and 2,4,6-triaminopyrimidine (TAP), and the structural properties of the associated ribonucleosides (rNs) and oligonucleotides. Our calculations reveal that the TAP : CA pair has a comparable hydrogen-bond strength to the canonical A : U pair. This strengthens the candidature of CA and TAP as prebiotic nucleobases. Further, the stacking between two canonical nucleobases is stronger than those between TAP or CA and a canonical base, as well as those between two TAP and/or CA, which indicates that enhanced stacking may have served as a driving force for the evolution from prebiotic to canonical nucleobases. Similarities in the DFT-derived anti/syn rotational barriers and MD-derived (anti) glycosidic conformation of the CA and TAP rNs and canonical rNs further substantiate their candidature as pre-RNA components. Greater deglycosylation barriers (as obtained by DFT calculations) for TAP rNs compared to canonical rNs suggest TAP rNs indicate higher resistance to environmental factors, while lower barriers indicate that CA rNs were likely more suitable for less-challenging locations. Finally, the tight packing in narrow CA:TAP-containing helices suggests that the prebiotic polymers were shielded from water, which would aid their evolution into self-replicating systems. Our calculations thus support proposals that CA and TAP can act as nucleobases of pre-RNA.     

Overview on advances in capillary electrophoresis-mass spectrometry of carbohydrates: a tabulated review

The increasing interest for carbohydrates as holder of essential bioinformations has boosted their full characterization through analytical techniques. The intent of this review is to summarize the recent trends regarding on-line and off-line CE-MS coupling for carbohydrate analysis. A statistical survey on the articles that use derivatizing agents as well as on the analyzer and type of instrument coupling (i.e. on- or off-line) is depicted. From a general overview it can be concluded that, whereas derivatization might be useful for the detection of neutral carbohydrates improving separation selectivity with volatile buffers and increasing sensitivity of the MS detection, relatively few works with derivatized carbohydrates were found; this was noticed in particular for glycosides and saccharides carrying ionizable groups, which are normally analyzed without any chemical modification. The most applied coupling is the on-line sheath-liquid interface; for on-line applications, ESI is the sole source used, whilst the most common analyzer is the IT. MS(n) is often exploited, as fragmentation increases the achieved structural information. CE-MS turned out to be mainly used for the analysis of carbohydrates in drug development (i.e. study of oligosaccharides from pathogens, carbohydrate-based drugs and drug metabolites), in nutrition and for characterization of glycans from glycoproteins. The reader will find elucidating tables regarding these recent CE-MS applications, including the main information on the analysis conditions. Comments are meant to help the immediate focus on the usefulness of the analytical technique and predict the difficulties found during analysis and, in case, their overcoming.     

Molecular dynamics and docking simulations as a proof of high flexibility in <Emphasis Type="Italic">E. coli</Emphasis> FabH and its relevance for accurate inhibitor modeling

Bacterial β-ketoacyl-acyl carrier protein synthase III (FabH) has become an attractive target for the development of new antibacterial agents which can overcome the increased resistance of these pathogens to antibiotics in clinical use. Despite several efforts have been dedicated to find inhibitors for this enzyme, it is not a straightforward task, mainly due its high flexibility which makes difficult the structure-based design of FabH inhibitors. Here, we present for the first time a Molecular Dynamics (MD) study of the E. colil FabH enzyme to explore its conformational space. We compare the flexibility of this enzyme for the unliganded protein and an enzyme-inhibitor complex and find a correspondence between our modeling results and the experimental evidence previously reported for this enzyme. Furthermore, through a 100 ns MD simulation of the unliganded enzyme we extract useful information related to the concerted motions that take place along the principal components of displacement. We also establish a relation between the presence of water molecules in the oxyanion hole with the conformational stability of structural important loops. Representative conformations of the binding pocket along the whole trajectory of the unliganded protein are selected through cluster analysis and we find that they contain a conformational diversity which is not provided by the X-ray structures of ecFabH. As a proof of this last hypothesis, we use a set of 10 FabH inhibitors and show that they cannot be correctly modeled in any available X-ray structure, while by using our set of conformations extracted from the MD simulations, this task can be accomplish. Finally, we show the ability of short MD simulations for the refinement of the docking binding poses and for MM-PBSA calculations to predict stable protein-inhibitor complexes in this enzyme.     

Challenges in implementing clinical liquid chromatography‐tandem mass spectrometry methods ‐ seeing the light at the end of the tunnel

The use of liquid chromatography–tandem mass spectrometry (LC–MS/MS) in the clinical setting is a relatively new application. One of the significant barriers hampering the transition of LC–MS/MS from the research lab into a clinical setting is the uncertainty of how to successfully develop and validate a method that meets guidelines for clinical applications. Here, we have taken this seemingly overwhelming process and broken it into five general stages for consideration: assessing the clinical validity of a new LC–MS/MS assay, determination of feasibility, assay development, assay validation and post‐implementation monitoring. Although various publications are available and serve as resources for determining development processes and acceptability criteria for specific LC–MS/MS assays, many of them are general recommendations or are specific to research applications that may not translate either practically or clinically. In this perspective special feature article, a resource is compiled that describes key differences between LC–MS/MS methods for research use versus clinical use. In addition, the challenges facing the expanding role of this technique in the clinical setting are discussed, including instrumentation/automation challenges, potential regulation of laboratory developed tests by the US Food and Drug Administration and standardization and harmonization of MS methods through the use of traceable materials and availability of guidance documents. Copyright © 2013 John Wiley & Sons, Ltd.     

Application of capillary electrophoresis to the analysis of metal-containing pharmaceuticals

The efficiency of the development and application of pharmaceuticals, including those based on metal compounds, essentially depends on the sophistication of the analytical procedure in use. The review is devoted to the state-of-the-art capillary electrophoresis (CE) as an analytical technique for studying drug preparations based on metal complexes. Examples are given to characterize the advantages of CE in the determination of the active principle and its metabolites, the evaluation of pharmacological properties, and the screening of compounds, as well as progress in the development of its methodology. In conclusion, the disadvantages of CE in this area and ways around these problems are critically considered in order to further develop this technique and to introduce it into clinical practice.     

Bioanalytical challenges and strategies for accurately measuring acyl glucuronide metabolites in biological fluids

Bioanalysis of unstable compounds such as acyl glucuronide metabolites represents a great analytical challenge owing to poor analyte stability in biological matrices. The primary goal for bioanalytical assay development is to minimize the breakdown of acyl glucuronide metabolite into its parent aglycone during sample collection, transportation, storage and analysis. Samples need to be stabilized ex vivo immediately after sample collection to minimize potential breakdown and thus to ensure accurate concentration measurement of both acyl glucuronide metabolite and its parent aglycone. In this review paper, formation of acyl glucuronide metabolites, the importance of establishing acyl glucuronide exposure measurement and safety coverage, optimization of sample pretreatment to stabilize the acyl glucuronide metabolites, current analytical strategy of assaying them as well as considerations for regulatory filings are discussed. It is important to identify acyl glucuronide metabolites that are capable of undergoing hydrolysis and pH-dependent intra-molecular migration as well as covalently binding to plasma and tissue proteins which can cause toxicity in vivo in the early stages of drug development. Carefully planning analytical experiments, identifying structures of acyl glucuronides and monitoring their concentrations in early drug development can help assess the risks associated with their exposures and potentially predict their concentrations in human circulation.     

Solid-phase microextraction. How far are we from clinical practice?

The current review briefly discusses the future development of solid-phase microextraction (SPME) and its potential application in the field of clinical medicine, including pharmacokinetic studies, therapeutic drug monitoring, biomarker discovery, and targeted and untargeted metabolomics. We also discuss aspects of automation and high-throughput analysis as major requirements of daily clinical practice. We give examples of clinically-validated applications of SPME and point out the regulatory restrictions limiting some in-vivo SPME studies. We briefly review the current state of progress in this extraction technique in the context of its future application in medical research and laboratory testing, including new directions (i.e. personalized medicine).     

Simultaneous electrochemical detection in paper-based analytical devices

The growing need for reliable analytical tools to perform measurements at the point-of-need has prompted the development of novel sensors that are low cost, portable, sensitive, easy to use, and capable of multiplexed analysis. Miniaturization of the sensors into microfluidic platforms has become a promising approach to achieve these self-contained sensors. However, traditional microfluidics often require relatively expensive and complicated pumping mechanisms that increase the cost and limit the portability of the sensors. From a material perspective, paper is an attractive substrate for constructing point-of-need sensors because of its affordability, vast availability, and self-pumping ability, particularly when combined with electrochemical detection. In this mini-review, we discuss various strategies to achieve multiplexing or simultaneous detection of multiple analytes in electrochemical paper-based devices and provide a brief guide on selecting the detection strategy based on the electrochemical property of the analytes.     

Differentiation of various kinds of Fructus schisandrae by surface desorption atmospheric pressure chemical ionization mass spectrometry combined with principal component analysis

Various kinds of Fructus schisandrae were studied by surface desorption atmospheric pressure chemical ionization mass spectrometry (DAPCI-MS) without any sample pretreatment. The volatile components in F. schisandrae were detected in the ambient environment and the analytical time for each sample was only 30 s. F. schisandrae are produced mainly in 5 different geographical regions (Elunchun, Mudanjiang, Tonghua, Tieling and Shangluo), and they could be successfully differentiated according to their chemical markers by Principal Component Analysis (PCA). A total of 8 components which gave more contribution for PCA analysis were unambiguously identified by comparison of the MS² data of chemical markers to the data of reference compounds as reported in the literature. Similarly, wild grown and cultivatable species of F. schisandrae were well separated by the above-mentioned method. In addition, raw and processed cultivatable F. schisandrae (steamed by water, alcohol, vinegar, or honey, and fried by honey) were found to be clustered at different location, respectively. Furthermore, the clustered degree of differently processed products was correlated with their clinical effects. Our results demonstrated that DAPCI-MS in combination with PCA was a feasible technique for high-throughput differentiation of various kinds of F. schisandrae. It is also possible that DAPCI-MS could become a powerful technology in the studies of traditional Chinese medicine studies and in situ analysis of Chinese herbs.     

On the analysis of the virulence nature of TIGR4 and R6 strains of Streptococcus pneumoniae using genome comparison tools

Comparative genome sequence analysis is a powerful technique for gaining insights into any genome of interest. Streptococcus pneumoniae is a human pathogen, which causes life-threatening diseases, such as pneumoniae, bacteremia, meningitis, etc. After the whole genome of two strains of S. pneumoniae, the virulent TIGR4 and non-pathogenic R6 were sequenced; there is a hope that comparing the genomes will allow an identification of the genes responsible for its virulence and thus the development of treatment and control. Many antimicrobial drugs have diminished the risk from pneumococcal disease because of its multi-drug resistance nature. Several pneumococcal proteins are also being investigated, as virulence factors as potential vaccine or drug targets. Structural and biochemical studies of these pneumococcal virulence factors have facilitated the development of novel antibiotics or protein antigen-based vaccines for the treatment of pneumococcal disease. Here we describe the comparison between the genomes of two strains of S. pneumoniae with few existing genomics databases and tools available in the public domain websites. By comparing nucleotide and protein sequences of the two strains, we investigate the existing differences and similarities. Mainly we focus on the virulence factors and its encoding genes in TIGR4 and how do they differ from R6 strain.