Gas chromatography-mass spectrometry-based analytical strategies for fatty acid analysis in biological samples

Fatty acids play critical roles in biological systems. Imbalances in fatty acids are related to a variety of diseases, which makes the measurement of fatty acids in biological samples important. Many analytical strategies have been developed to investigate fatty acids in various biological samples. Due to the structural diversity of fatty acids, many factors need to be considered when developing analytical methods including extraction methods, derivatization methods, column selections, and internal standard selections. This review focused on gas chromatography-mass spectrometry (GC–MS)-based methods. We reviewed several commonly used fatty acid extraction approaches, including liquid–liquid extraction and solid-phase microextraction. Moreover, both acid and base derivatization methods and other specially designed methods were comprehensively reviewed, and their strengths and limitations were discussed. Having good separation efficiency is essential to building an accurate and reliable GC–MS platform for fatty acid analysis. We reviewed the separation performance of different columns and discussed the application of multidimensional GC for improving separations. The selection of internal standards was also discussed. In the final section, we introduced several biomedical studies that measured fatty acid levels in different sample matrices and provided hints on the relationships between fatty acid imbalances and diseases.     

Recent advances in column switching high-performance liquid chromatography for bioanalysis

Bioanalysis is a relevant area of analytical chemistry for clinical studies. Biological samples are complex and diverse, so sample preparation represents a challenge when chromatographic methods are developed. According to the principles of green analytical chemistry (GAC), recent trends in sample preparation include miniaturization, automation (online coupling to the analytical instrument), and high-throughput performance. In this context, column switching liquid chromatography stands out as a multidimensional chromatographic method in which an extraction column is directly coupled to high-performance liquid chromatography (HPLC) systems. This online method consists of two steps and involves two columns, the extraction and the chromatographic columns. In the former column, the analytes are isolated from the sample and preconcentrated; in the latter column, the analytes are separated. Online systems improve the sensitivity and accuracy of analytical methods, consume lower amounts of organic solvents, and minimize sample handling. This review summarizes state-of-the-art column switching liquid chromatography and focuses on selective stationary phases for preconcentration of analytes (first dimension), including reversed phases, monolithic phases, restricted access materials (RAMs), and molecularly imprinted polymers (MIP). Principles, instrumental aspects, applications in bioanalysis, and future trends in column switching liquid chromatography are also discussed.     

Fast separation of bacitracin on monolithic silica columns

The development of isocratic and gradient stability indicating HPLC methods for bacitracin (Bc) and bacitracin zinc (BcZn), which are complex mixture of several related polypeptides, is described. The methods are based on a new type of reversed phase (RP-18e) monolithic silicagel stationary phase. Chromatographic experimental conditions used on conventional column with microparticles were adopted and further modified to achieve efficient separation of Bc. The influence of methanol and acetonitrile in combination with phosphate buffer was thoroughly studied to separate microbiologically active components A, B1, B2, B3 and their oxidative degradation products F, H1, H2 and H3. Chromatographic peaks of all the mentioned components were identified using compounds isolated previously by preparative HPLC. Applying isocratic or gradient approach, highly efficient separation was achieved together with drastically reduced analysis times (ca. 6 min) compared to all published HPLC methods up to date. With thus developed HPLC methods, it is possible to evaluate not only the main degradation product F, but for the first time also several other oxidative degradation products of Bc (H1, H2 and H3). Such methods are also suitable for routine quality control and stability testing. Validation of both isocratic and gradient methods confirmed the selectivity and efficiency comparable to that on microparticulate columns, yet contrary to conventional columns with highly reduced analysis time.     

分子印迹聚合物的制备及其在临床药物分析中的应用进展

本文综述了近年来以分子印迹材料为基础的药物分析新方法及其在临床样本（血浆、尿液）中的应用，详细评述了基于分子印迹技术的固相萃取、固相微萃取、磁性固相萃取、传感器和酶联免疫分析等最新进展，为我国临床药物分析新方法研究提供参考。     

Historical study of separation technology innovation in the field of pharmaceutical science

The separation of organic compounds has been carried out using the method of distillation and recrystallization since the 17th century.1st separation technology innovation (1950s to 1960s). After World War II, the science of separating organic compounds evolved to the use of paper or column chromatography. 2nd separation technology innovation (1960s to 1980s). In the 1960s, thin-layer chromatograpy (TLC) was developed. TLC is a convenient analytical technique for organic compounds. Separation methods for organic compounds using column chromatography and preparative TLC were carried out as practical applications. Gas chromatography (GC) was also introduced in the 1960s. GC is a useful analytical method for a mixture of various volatile organic compounds. Many GC instruments with new kinds of sensors were developed. Gas chromatography of nonbenzenoid organometallic compounds and metal complexes was successfully conducted by the author. 3rd separation technology innovation (1980s to 1990s). In the 1980s, high-performance liquid-chromatography (HPLC) was developed. HPLC analytical methods were useful for various kinds of organic compounds. In the 1990s, LC-MS and LC-NMR instruments were introduced for biomedical products. 4th separation technology innovation (1990s to present). Several new ionization methods for mass-spectrometry (electrospray, matrix-aided lazer desorption ionization, etc) are applied for proteins and biomedical products. Several new separation technologies (supercritical field chromatography, capillary electrophoresis, field-flow fractionization, etc.) are now being developed.     

A study of some practical aspects of high temperature liquid chromatography in pharmaceutical applications

In the pharmaceutical industry fast and efficient separation techniques play an increasing role among analytical methods because the samples to be investigated grow both in complexity and number, and there is an increasing time pressure to complete the analysis. Reducing the analysis time without decreasing the efficiency is possible using higher pressures, elevated temperatures, smaller particle sizes, or a combination of these approaches. Recently developed chromatographic techniques such as the UHPLC (ultra high performance liquid chromatography) and HTLC (high temperature liquid chromatography) are highly promising in meeting these demands. In this study, high temperature liquid chromatography (HTLC) with a zirconia-based column and temperatures elevated up to 150°C was used. We investigated the chromatographic behaviour of a steroid active pharmaceutical ingredient (levonorgestrel) and its structurally related impurities as model compounds. The effect of the temperature in the range of 50-150°C and the flow-rate in the range of 0.5-3.0 ml/min, and using methanol as an organic modifier, were studied for optimisation of the separation method.     

Turbulent flow bioanalysis in drug metabolism and pharmacokinetics

Turbulent flow chromatography (TFC) as an analytical technique was introduced in the mid-1990s for online sample processing in bioanalysis. Turbulent flow columns are packed with large particles permitting the use of high mobile phase linear velocities. Solute molecules travel in a uniform concentrated band in which large matrix biomolecules are effectively separated from lower molecular weight analytes by differential mass transfer effects. In addition, the use of various bonded stationary phases enhances selectivity in TFC separations. Originally, the turbulent flow column was used for both online sample processing and the analytical separation. This mode of operation has largely been replaced with a dual column arrangement, in which sample processing is done online with the turbulent flow column and a second analytical column is employed for separation. Within the pharmaceutical industry, certain laboratories now use TFC routinely for bioanalytical support of in vivo drug discovery pharmacokinetic studies. Validated TFC methods are also used to support GLP-compliant preclinical toxicokinetic studies and clinical trials. TFC has been shown to simplify bioanalytical sample preparation and reduce sample processing costs when compared with offline techniques such as SPE. TFC may be combined with multiplexing up to four HPLC systems to a single mass spectrometer to increase analytical throughput.     

A simple and efficient approach to reversed-phase HPLC method screening

The development and utility of an efficient HPLC method screening strategy using only four columns for the separation of pharmaceutical compounds and related impurities is presented. The strategy established a two-column approach to enable rapid early method development, along with a four-column approach for commercial method development of the analytical methods utilized to verify the quality of drug substance or drug product. Mobile phases consisted of acetonitrile or methanol with aqueous trifluoroacetic acid for low pH screening, and ammonium hydroxide for high pH screening. Examples are provided to demonstrate the practicality and orthogonality of the method screening process. A unique system suitability check, using commercially available compounds, was incorporated as a tool for troubleshooting and for ensuring adequate system performance prior to screening. Initial testing of the strategy revealed that the columns chosen were successful in leading to assay and impurity methods for 40 pharmaceutical compounds.     

New stationary phases for enrichment and separation in the 'omics' era

'Omics' is a general term for a broad discipline of science and engineering concerned with analyzing the interactions of biological molecular components in various 'omes'. These include genome, proteome, metabolome, expressome and interactome. 'Ome' and 'omics' are very convenient handles for describing the holistic approach for looking at complex systems. 'Omics' will not only have an impact on our understanding of biological processes, but also on the prospect of more accurately diagnosing and treating disease. The development of these 'omics' has depended on, and has also driven, advances in chromatography and electrophoresis, as well as highly sensitive and specific analytical techniques to permit the handling of large numbers of samples with high selectivity and sensitivity. The development and design of novel stationary phases for selective enrichment and separation is one of the key points for establishing a successfully running 'omics' platform. Therefore, this review demonstrates the application of different new materials developed in our laboratory, such as chromatographic stationary phases for selective and sensitive high-speed purification, enrichment and separation in genomics, proteomics and metabolomics.     

A fast and efficient determination of amines and preservatives in cough and cold liquid and suspension formulations using a single isocratic ion-pairing high performance [correction of power] liquid chromatography method.

A single, highly selective ion-pairing reverse phase-high power liquid chromatography (RP-HPLC) method has been developed for the determination of amines and preservatives in a wide range of Tylenol((R)) liquid and suspension liquid products. As with many OTC products, the challenge is to quantitatively extract the analytes from difficult matrices and specifically analyze them in the presence of various excipients and flavors. Historically, separate analytical methods were used for each class of analytes (acids, bases and neutral compounds). In this method a mobile phase consisting of a buffered ion-pairing agent with acetonitrile, methanol and tetrahydrofuran was used to separate the charged amines from neutral and acidic compounds on a Phenomenex LUNA C8(2) 75 x 4.6 mm i.d. analytical column with a 3-microm particle size. The analytes include acids (benzoic acid), bases (pseudoephedrine, chlorpheniramine, dextromethorphan, doxylamine and diphenhydramine) and a neutral compound (butylparaben). The effects of pH, the chain length of the ion-pairing reagent, ionic strength and organic modifiers on the separation are discussed. The method is linear from 15 to 150% of the target amounts. The optimized method proves to be specific, robust and accurate for the analysis of the compounds.     

Green bioanalysis: some innovative ideas towards green analytical techniques

The key target of green chemistry is to make compounds and materials available to mankind, while causing no harm to the environment. In the 21st century analytical scientists are more concerned about green analytical method development. The number of literatures on green chemistry has undergone a dramatic increase in the new millennium. Green bioanalytical techniques aim to minimize or eliminate the hazardous waste associated with bioanalytical methods. An efficient and sincere approach towards bioanalytical method development has an enormous contribution towards green analysis. The selection of organic constituents of the mobile phase, choice of sample extraction process, adoption of an appropriate separation procedure and a few others, control the green chemistry approach of the bioanalytical method. In routine practice, UHPLC-MS can be the most suitable approach, while supercritical fluid chromatography is one of the best available techniques for green bioanalytical methods. Nevertheless, there always remains great scope of further research on green bioanalytical methods.     

Development of an on-site measurement system for salivary stress-related substances based on microchip capillary electrophoresis technology

Psychological stress is of major importance to all age groups in recent years, and may lead to mental disorder and various diseases. An objective and quantitative method for measuring salivary stress-related substances is highly desired because saliva collection is easy, stress free and noninvasive. We have developed a rapid and easy-to-use analytical tool for the measurement of cortisol and secretory immunoglobulin A (sIgA) based on microchip technology, immunoselectivity and electrophoretic separation technique. Performing immunoreaction and capillary electrophoresis (CE) separation on microchips is a promising technique for on-site determination of biogenic substances, and has a few advantages over conventional immunoassay methods: reduced sample size, shortening analysis times, high separation efficiency, reduced cost, and downsizing of analytical system. At this stage of our research, some preliminary prototypes of a high-sensitive microchip CE instrument were constructed to determine the stress-related substances in real saliva samples. However, there is not enough detection sensitivity for cortisol analysis. On the other hand, sIgA was successfully analyzed using a laboratory-built microchip CE system and optimal analytical conditions. The sIgA determination is rapid compared with a conventional immunoassay method, and provides an acceptable degree of repeatability and recovery. In the future, microchip technologies will enable total automation and integration of sample preparation. This research has widespread future potential for monitoring multiple stress-related markers within minutes from a trace of saliva, and can contribute to disease prevention and overall good health.     

Advances in the analysis of phenolic compounds in products derived from bees

Honey and propolis are rich in phenolic compounds, which act as natural antioxidants, and are becoming increasingly popular because of their potential role in contributing to human health. These compounds can also be used as indicators in studies into the floral and geographical origin of the honey and propolis themselves. We present here an overview of current analytical methods for measuring polyphenols in honey and propolis. The analytical procedure to determine individual phenolic compounds involves their extraction from the sample, analytical separation and quantification. The techniques reviewed are based on spectrophotometry as well as analytical separation techniques such as gas chromatography, high-pressure liquid chromatography and capillary electrophoresis.     

Biosensors for Deoxynivalenol and Zearalenone Determination in Feed Quality Control

Mycotoxin contamination of cereals used for feed can cause intoxication, especially in farm animals; therefore, efficient analytical tools for the qualitative and quantitative analysis of toxic fungal metabolites in feed are required. Current trends in food/feed analysis are focusing on the application of biosensor technologies that offer fast and highly selective and sensitive detection with minimal sample treatment and reagents required. The article presents an overview of the recent progress of the development of biosensors for deoxynivalenol and zearalenone determination in cereals and feed. Novel biosensitive materials and highly sensitive detection methods applied for the sensors and the application of these sensors to food/feed products, the limit, and the time of detection are discussed.     

Analysis of carbamazepine and its 10,11-epoxide in serum by direct sample injection using surfactant containing eluents and column switching

Column switching is used in conjunction with surfactant containing mobile phases and traditional reverse phase LC columns to provide a highly reproducible and accurate analytical procedure for carbamazepine and its 10,11-epoxide in serum. This approach eliminates the tedious sample preparation steps commonly used in the analysis of drugs by HPLC, while providing a high degree of protein removal prior to the final analysis. Various pre-columns were investigated and a pellicular reverse phase column was found suitable for optimum concentration of drugs and removal of serum proteins. A variety of standard reverse phase columns could be used for the analytical separation. The separation of the drugs could be accomplished with a high degree of reproducibility. Tandem pre-column operation was demonstrated to give a sample throughput of 10 h-1.     

Man-Made Synthetic Receptors for Capture and Analysis of Ochratoxin A

Contemporary analytical methods have the sensitivity required for Ochratoxin A detection and quantification, but direct application of these methods on real samples can be rarely performed because of matrix complexity. Thus, efficient sample pre-treatment methods are needed. Recent years have seen the increasing use of artificial recognition systems as a viable alternative to natural receptors, because these materials seem to be particularly suitable for applications where selectivity for Ochratoxin A is essential. In this review, molecularly imprinted polymers, aptamers and tailor-made peptides for Ochratoxin A capture and analysis with particular attention to solid phase extraction applications will be discussed.     

4-Bromomethyl-7-methoxycoumarin and analogues as derivatization agents for high-performance liquid chromatography determinations: a review.

A major part of modern analytical problem solving deals with the trace level determination of organic compounds and contaminants in biomedical, food and environmental samples. In the analysis of these samples chromatographic techniques play a predominant role. Unfortunately, however, even the combined force of an efficient separation plus a sophisticated mode of detection does not always create sufficient selectivity and/or sensitivity for the final goal to be attained. In such cases, special attention has to be devoted to derivatization or conversion of the analyte(s) of interest (for improved detection selectivity and/or sensitivity) and sample pretreatment (for trace enrichment and clean-up). The above is especially true when, as is often the case today, relatively polar drugs, endogenous compounds, additives or environmental pollutants and/or their (bio)-degradation products have to be determined. For such classes of compounds high-performance column liquid chromatography (HPLC) generally is the preferred method of separation. Reversed-phase HPLC with fluorescence detection is a powerful means of analysis for compounds which possess native fluorescence. They are, however, relatively few in number. In order to make the method useful for a much wider range of analytes, one can therefore resort to derivatization (labelling) or other means of analyte conversion to obtain highly fluorescent reaction products, which can then be detected with the required selectivity and sensitivity. 4-Bromomethyl-7-methoxycoumarin is often used as fluorescent label for the determination of compounds possessing a carboxylic group. About 8% of the biologically interesting analytes--ranging from polar amino acids and peptides to apolar fatty acids--possess such a group.(ABSTRACT TRUNCATED AT 250 WORDS)     

Recent advances in column switching sample preparation in bioanalysis

Column switching techniques, using two or more stationary phase columns, are useful for trace enrichment and online automated sample preparation. Target fractions from the first column are transferred online to a second column with different properties for further separation. Column switching techniques can be used to determine the analytes in a complex matrix by direct sample injection or by simple sample treatment. Online column switching sample preparation is usually performed in combination with HPLC or capillary electrophoresis. SPE or turbulent flow chromatography using a cartridge column and in-tube solid-phase microextraction using a capillary column have been developed for convenient column switching sample preparation. Furthermore, various micro-/nano-sample preparation devices using new polymer-coating materials have been developed to improve extraction efficiency. This review describes current developments and future trends in novel column switching sample preparation in bioanalysis, focusing on innovative column switching techniques using new extraction devices and materials.     

A novel high-throughput automated chip-based nanoelectrospray tandem mass spectrometric method for PAMPA sample analysis

Parallel artificial membrane permeability assay (PAMPA) has recently gained popularity as a novel, high-throughput assay capable of rapidly screening compounds for their permeability characteristics in early drug discovery. The analytical techniques typically used for PAMPA sample analysis are HPLC-UV, LC/MS or more recently UV-plate reader. The LC techniques, though sturdy and accurate, are often labor and time intensive and are not ideal for high-throughput. On the other hand, UV-plate reader technique is amenable to high-throughput but is not sensitive enough to detect the lower concentrations that are often encountered in early drug discovery work. This article investigates a novel analytical method, a chip-based automated nanoelectrospray mass spectrometric method for its ability to rapidly analyze PAMPA permeability samples. The utility and advantages of this novel analytical method is demonstrated by comparing PAMPA permeability values obtained from nanoelectrospray to those from conventional analytical methods. Ten marketed drugs having a broad range of structural space, physico-chemical properties and extent of intestinal absorption were selected as test compounds for this investigation. PAMPA permeability and recovery experiments were conducted with model compounds followed by analysis by UV-plate reader, UV-HPLC as well as the automated nanoelectrospray technique (nanoESI-MS/MS). There was a very good correlation (r(2) > 0.9) between the results obtained using nanoelectrospray and the other analytical techniques tested. Moreover, the nanoelectrospray approach presented several advantages over the standard techniques such as higher sensitivity and ability to detect individual compounds in cassette studies, making it an attractive high-throughput analytical technique. Thus, it has been demonstrated that nanoelectrospray analysis provides a highly efficient and accurate analytical methodology to analyze PAMPA samples generated in early drug discovery.