基于液质联用技术的代谢组学生物样本——血液及尿液的收集和处理

代谢组学是定性及定量描述内源性代谢物变化的科学，正广泛应用于疾病诊断和预后过程中。血液和尿液是代谢组学研究中常用的生物样本，但因其代谢物繁多、成分复杂，亟须使用灵敏度高、特异性强的分离分析技术。液相色谱-质谱联用（liquid chromatography-mass spectrometry， LC-MS）技术分析速度快、分离效率高，非常适用于代谢组学等方面的研究。生物样本的采集、处理过程是实验中最关键的一步，直接决定着实验结果的准确性，因此生物样本预处理过程的标准化显得尤为重要。本文综述了常用的生物流体—血液和尿液的收集、处理及储存方法，以期为有关代谢组学研究提供参考。     

质谱联用技术在生物样品分析中的应用

质谱分析具有分析范围广、灵敏度高、样品用量少、分析速度快、重复性好、操作简易、损耗低、分离与鉴定可同时进行等优点, 已广泛应用于环境科学、能源化工、生物医药等领域。本文综述了近3年国内外关于质谱联用技术在生物样品分析中应用的文献, 并着重介绍了质谱技术在药物代谢动力学和生物等效性、毒代动力学、药代动力学-药效学、群体药代动力学、药物及代谢物结构确证与裂解规律和代谢组学中的应用, 以期为生物样品分析的进一步发展提供参考。     

基于代谢组学的细胞内源性代谢物研究进展

代谢组学是继基因组学、转录组学和蛋白质组学之后新兴的一种定性和定量分析复杂生物样品中所有小分子代谢物的组学方法,也是目前组学研究领域的热点之一。代谢组学主要考察生物体系受刺激或扰动后内源性代谢物的变化,是系统生物学的有机组成部分。近年来在代谢组学研究中,细胞内源性代谢物的研究已取得了很大进展。本文结合代谢组学的基本含义,综述了细胞样品前处理方法及代谢靶向分析、代谢轮廓分析、代谢组学分析和代谢足迹分析在细胞方面的研究进展。     

脂质组学在疾病生物标记物研究应用现状

脂质组学通过研究脂质的结构和功能及其在体内的代谢变化,明确其对疾病诊断和治疗的作用,以期提高疾病风险预测的能力。常用脂质组学分析方法包括直接质谱注入法（鸟枪法）、色谱质谱联用法和核磁共振法等。脂质组学在疾病早期诊断、生物标志物的发现、新药研发以及系统研究方面都发挥了很大作用。本文旨在介绍脂质组学在疾病生物标志物发现中的应用及其研究方法。     

液相色谱-质谱法在生物样品药物定量分析中的基质效应

液相色谱-质谱法现已成为新药研发，尤其是药物代谢与药物动力学研究不可或缺的有力工具。液相色谱-质谱法由于其高分离效能、高选择性和高灵敏度，尤适于复杂生物样品中药物及其代谢产物的分析测定，其中以电喷雾电离(ESI)方式应用最为广泛。由于质谱检测的高选择性使人们认为样品制备过程可以简化，对组分间色谱分离的要求也相府降低。此外，为满足高通量分析的要求，     

慢性阻塞性肺病稳定期模型大鼠支气管肺泡灌洗液的代谢组学研究

目的：基于代谢组学方法研究慢性阻塞性肺病（chronic obstructive pulmonary disease，COPD）稳定期大鼠支气管肺泡灌洗液（bronchoalveolar lavage fluid，BALF）的潜在生物标志物。方法：采用烟熏联合细菌反复感染的方法制备COPD稳定期模型大鼠。采用超高效液相色谱-串联质谱技术采集大鼠的BALF数据，利用多元统计分析软件筛选差异代谢物，再结合数据库鉴定潜在生物标志物，并进行代谢通路分析。结果：多元统计分析结果显示，正常组与模型组BALF代谢物发生显著变化。通过比对数据库，共鉴定出30个差异性代谢物，主要涉及到体内的氨酰基-tRNA的生物合成，甘氨酸、丝氨酸和苏氨酸的代谢，鞘脂代谢，苯丙氨酸代谢和花生四烯酸代谢等8条代谢通路。结论：基于代谢组学研究COPD稳定期的BALF代谢物变化，寻找COPD稳定期相关代谢标志物，为治疗COPD提供理论参考。     

固相萃取在生物样品分析中的应用

生物样品中的药物浓度与药物的疗效有很大的相关性,尤其是血浆(或血清)药物浓度直接与药效相关。尿液中常常含有丰富的药物代谢产物,也被经常使用。唾液由于采集方便且有时和血浆游离药物浓度具有相关性而有时使用。其他脏器组织,除特别需要,较少用。生物样品的药物分析因其复杂多样性而对样品的预处理技术提出了较高的要求。在各种处理方法中,固相萃取由于其适用性广,实用性强而越来越受到广大科研工作者的重视。本文综述了固相萃取技术(SPE)及其在生物样品分析中应用的相关报道,以期对相关研究者提供理论指导和技术支持。     

人体生物等效性试验中生物样品分析的关键问题研究

采用体内生物等效性试验的方法开展一致性评价是仿制药申请的基础,生物等效性试验首选药动学研究的方法,而药动学中生物样品分析是最为重要的环节,它将直接影响药品的安全性和有效性,为生物等效性试验的结果做出关键性决定。目前我国的生物样品分析质量还有很大提升空间,基于此,本文根据相关指导原则,结合工作实际,针对生物等效性试验中生物样品分析方法的建立、验证、测试、记录和注意事项等,探讨生物等效性试验中生物样品分析的关键问题。     

代谢组学分析技术平台和数据处理的新进展

分析技术和生物计量学促进了代谢组学的飞速发展。代谢组学快速、灵敏、可定量、非侵入性以及系统性的特点,使其在新药研发、药物毒性筛选、疾病诊断等领域显示出广阔的前景。本文综述了代谢组学研究中的某些关键问题:样品处理方法,分析技术和数据处理的方法和原则,代谢组动态变化、生物标记物的鉴定和代谢途径的检索近年来的进展。评价了各种分析手段的优缺点,并展望代谢组学发展前景。     

基于LC-MS的代谢组学分析流程与技术方法

代谢组学是继基因组学、转录组学、蛋白质组学之后兴起的又一新的组学研究分支,但其研究技术方法一直以来都是影响代谢组学发展的瓶颈之一。超高效液相色谱-质谱联用（UPLC-MS）技术结合化学计量学比较好地实现了对大量样品和微量代谢物的快速定性、定量分析,极大地推动了代谢组学的发展。本文按照代谢组学分析流程综述了LC-MS与化学计量学相结合用于代谢组学研究的现状与进展,并对重要研究方向进行了简要展望。     

Harnessing metabolomics for nutrition research

Comprehensive analytical technologies are rapidly becoming a cornerstone of modern nutritional sciences. Two of these technologies, mass spectrometry (MS) and nuclear magnetic resonance (NMR), have proven highly informative for the global analysis of metabolites, commonly referred to as metabolomics. Metabolomics provides a powerful approach to study small molecules in order to better understand the implications and subtle perturbations in metabolism triggered by nutrients. By studying how dietary molecules can modulate the metabolome, researchers have begun to elucidate the molecular pathways by which nutrients affect health and disease, expand the current state of knowledge regarding how inter-individual variability contributes to differences in nutrient metabolism, and develop novel avenues of research for nutritional sciences. Although metabolomics has been more commonly used to study disease states, its use in the nutritional sciences is gaining momentum. The current review is written for the clinical researcher wishing to incorporate metabolomics into dietary intervention studies. This review will highlight the importance and benefit of identifying biomarkers that accurately reflect changes in nutrient intake and metabolism, and present numerous issues that can introduce variability into a dataset and confound a study's biological interpretation, including sample population demographics, the biological specimen selected, diurnal variation, collection methods, and sample storage parameters. Considering these important areas at the experimental design stage will ensure that metabolomics provides a comprehensive and accurate assessment of the molecular impact of a dietary intervention.     

Comparison of different serum sample extraction methods and their suitability for mass spectrometry analysis

Mass spectrometry has been widely used, particularly in pharmacokinetic investigations and for therapeutic drug monitoring purposes. Like any other analytical method some difficulties exist in employing mass spectrometry, mainly when it is used to test biological samples, such as to detect drug candidates in mammalian serum, which is rich in proteins, lipids and other contents that may interfere with the investigational drug. The complexity of the serum proteome presents challenges for efficient sample preparation and adequate sensitivity for mass spectrometry analysis of drugs. Enrichment procedures prior to the drug analysis are often needed and as a result, the study of serum or plasma components usually demands either methods of purification or depletion of one or more. Selection of the best combination of sample introduction method is a crucial determinant of the sensitivity and accuracy of mass spectrometry. The aim of this study was to determine the highest serum protein precipitation activity of five commonly used sample preparation methods and test their suitability for mass spectrometry. We spiked three small molecules into rabbit serum and applied different protein precipitation methods to determine their precipitation activity and applicability as a mass spectrometry introductory tool.     

A review of the analysis of tobacco-specific nitrosamines in biological matrices

Tobacco use constitutes a leading cause of mortality and morbidity worldwide. Tobacco-specific nitrosamines (TSNAs) are an important class of biomarkers for tobacco carcinogen uptake. The current review focuses on the issues and developments in analysis of these compounds in human biological matrices. The two most widely used techniques for TSNA bioanalysis are gas chromatography coupled with thermal energy analysis and liquid chromatography coupled with mass spectrometry, employing various sample preparation techniques. The review provides an overview of the tools and techniques currently available for TSNA bioanalysis that will help towards the ultimate goal of understanding the mechanisms of cancer caused by the use of tobacco products. A contrast and comparison of the important aspects of bioanalysis such as sample preparation, compound detection, and throughput is discussed for the thermal energy analysis– and mass spectrometry–based techniques. Complex sample extraction procedures, throughput, and the ability to validate are important issues of concern for the gas chromatography–thermal energy analysis–based methods. On the other hand, addressing ion suppression matrix effects remains an important challenge for hyphenated mass spectrometry–based methods. The review also provides an extensive summary of analytical procedures for various studies measuring tobacco-specific nitrosamines in different biological matrices.     

Practical metabolomics in drug discovery

Importance of the field: Metabolomics is increasingly becoming an important field in the pharmaceutical industry to support the discovery and development of therapeutic agents. It allows the comprehensive and simultaneous profiling of hundreds of discrete biologically important molecules, including amino acids, sugars, lipids and exogenous substances from biological fluids and tissues. Metabolomics is the 'omics' field that most represents the interplay of internal biological regulation and external environmental influences on disease, thereby being of particular importance to disease mitigation and management. Areas covered in this review: Technological advances in the experimental work flow, analytical detection strategies and bioinformatics tools have enabled metabolomics studies to become increasingly comprehensive, robust and informative for the understanding of disease, drug action and the development of biomarkers. This review will focus on the practical aspects of metabolomics studies as they have been applied to the study of mammalian biological systems, specifically targeted to the steps of experimental design with regard to sample preparation, sample analysis and data analysis of both polar and non-polar metabolites. What the reader will gain: The reader will gain an overview of the field of metabolomics as it applies to drug development and the practical issues involved with experimental design. We will discuss the various methods of sample preparation and analysis as they apply to different classes of metabolites and highlight recent advances in the field that illustrate these methods. Take home message: The field of metabolomics is a rapidly expanding discipline that is being applied to various aspects of drug development. The large diversity of metabolites found in nature dictates that different methods be developed for the investigation of different classes of metabolites. As the field of metabolomics continues to mature, it is likely that it will play an increasingly important role in the characterization of disease and the future development of biomarkers to assess drug efficacy and safety.     

A guide to recent trends in green applications of liquid phase microextraction for bioanalytical sample preparations

Liquid phase microextraction (LPME) techniques have become increasingly important components for bio-analytical sample preparation methods due to the versatility, adaptability, sensitivity, and conformity to green analytical chemistry (GAC) principles that they provide for the analyses of complex biological matrices. Despite having been used for sample preparations for some 25 years, LPME is not a static sample preparation field, but is continuously undergoing innovation and improvements and new research and applications for bio-analytical sample preparation are being published constantly. This review is meant to serve not only as a listing of recent literature, but as a guide to current trends in LPME. In order to fully understand the trends, advantages and limitations of this field in bio-analytical sample analyses, LPME based methods are compared with traditional sample preparation methods, the limitations and advantages of each LPME mode examined, and a variety of reviews and successful applications papers for LPME are included. To simplify searching for appropriate LPME methods these reviews and applications have been categorized to aid in searching for a needed application technique or publication. Following sections briefly cover the trends in LPME development, concentrating on bioanalytical sample preparation applications. The review continues with an examination of the trends for the use use of non-traditional green solvents for LPME based methods, and concludes with a discussion of recent innovations which have the potential for commercialization of high throughput automation for LPME based bio-analytical sample preparation and analysis methods.     

Bioanalysis of cisplatin analogues--a selective review

A variety of analytical procedures have been described for the determination of cisplatin and its analogues in biological fluids (plasma, plasma ultrafiltrate and urine), as well as in solid tissues. This paper attempts to review those methods which have been most commonly used in practice. These analytical methods may be conveniently divided into non-selective methods which detect only the platinum metal and selective methods which are capable of detecting the intact compounds. The non-selective methods include X-ray fluorescence, proton induced X-ray emission, flameless atomic absorption (FAA) and high-performance liquid chromatography (HPLC). The latter method requires pre-column derivatization with diethyldithiocarbamate. The selective methods generally employ a fractionation step using HPLC followed by either on-line or off-line detection. Off-line detection by FAA requires the collection of fractions from the HPLC column and is somewhat tedious. On the other hand, sample preparation is minimal and biological fluids may be injected directly onto the column. The most sensitive HPLC methods for the determination of cisplatin and its analogues in biological fluids employ on-line electrochemical detection or post column derivatization with bisulphite.     

Towards a universal automated and miniaturized sample preparation approach

Nowadays, analytical chemistry plays a fundamental role in developing strategies for monitoring residues and contaminants in complex matrices (e.g., biological, environmental, and food samples). Considering the significant number of interfering substances present, combined with the lower concentration levels in which the target analytes are commonly found, sample preparation is becoming a crucial step in the analytical workflow. For these reasons, automation and miniaturization of this process represent a current trend since by these means, several principles of Green Chemistry can be meet all together, including: (i) lower volumes of reagents and samples required; (ii) reduced waste generation; and (iii) high-throughput with lesser operators' handling. The employment of online sample preparation based on LC systems (e.g., online SPE and In-Tube SPME) can be considered promising tools to perform sample preparation in a more sustainable way than conventional approaches. For these reasons, herein, we present a review discussing the essential characteristics of miniaturized, automated systems, including column-switching modes and modern approaches based on chip-LC. These methodologies deserve attention mainly due to their greener character derived from automation and miniaturization of the process. Also, selected applications, mostly covering the last five years, are presented. A brief comparison between them and conventional LC-based approaches is carried out to bring an adequately discussed review to this journal audience.     

Metabolomics in clinical and forensic toxicology,sports anti-doping and veterinary residues

Metabolomics is a multidisciplinary field providing workflows for complementary approaches to conventional analytical determinations. It allows for the study of metabolically related groups of compounds or even the study of novel pathways within the biological system. The procedural stages of metabolomics; experimental design, sample preparation, analytical determinations, data processing and statistical analysis, compound identification and validation strategies are explored in this review. The selected approach will depend on the type of study being conducted. Experimental design influences the whole metabolomics workflow and thus needs to be properly assessed to ensure sufficient sample size, minimal introduced and biological variation and appropriate statistical power. Sample preparation needs to be simple, yet potentially global in order to detect as many compounds as possible. Analytical determinations need to be optimised either for the list of targeted compounds or a universal approach. Data processing and statistical analysis approaches vary widely and need to be better harmonised for review and interpretation. This includes validation strategies that are currently deficient in many presented workflows. Common compound identification approaches have been explored in this review. Metabolomics applications are discussed for clinical and forensic toxicology, human and equine sports anti-doping and veterinary residues.