Determination of illicit and/or abused drugs and compounds of forensic interest in biosamples by capillary electrophoretic/electrokinetic methods

The application of capillary electrophoresis (CE) methods in forensic toxicology for the determination of illicit and/or misused drugs in biological samples is reviewed in the present paper. Sample pretreatments and direct injection modes used in CE for analysis of drugs in biological fluids are briefly described. Besides, applications of separation methods based on capillary zone electrophoresis or micellar electrokinetic chromatography with UV absorbance detection to (i) analysis of drugs of abuse, (ii) analysis of other drugs and toxicants of potential forensic interest and (iii) for metabolism studies are reviewed. Also, alternative CE methods are briefly discussed, including capillary isotachophoresis and separation on mixed polymer networks. High sensitivity detection methods used for forensic drug analysis in biological samples are then presented, particularly those based on laser induced fluorescence. A glimpse of the first examples of application of CE–mass spectrometry in forensic toxicology is finally given.     

手性药物分析方法研究进展

综述了近10 年来手性药物分离检测方法的发展,包括高效液相色谱法、气相色谱法、毛细管电泳法,以及超临界流体色谱法等,旨在为该领域的进一步发展提供参考。     

Separation and detection methods for covalent drug-protein adducts

Covalent binding of reactive metabolites of drugs to proteins has been a predominant hypothesis for the mechanism of toxicity caused by numerous drugs. The development of efficient and sensitive analytical methods for the separation, identification, quantification of drug-protein adducts have important clinical and toxicological implications. In the last few decades, continuous progress in analytical methodology has been achieved with substantial increase in the number of new, more specific and more sensitive methods for drug-protein adducts. The methods used for drug-protein adduct studies include those for separation and for subsequent detection and identification. Various chromatographic (e.g., affinity chromatography, ion-exchange chromatography, and high-performance liquid chromatography) and electrophoretic techniques [e.g., sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), two-dimensional SDS-PAGE, and capillary electrophoresis], used alone or in combination, offer an opportunity to purify proteins adducted by reactive drug metabolites. Conventionally, mass spectrometric (MS), nuclear magnetic resonance, and immunological and radioisotope methods are used to detect and identify protein targets for reactive drug metabolites. However, these methods are labor-intensive, and have provided very limited sequence information on the target proteins adducted, and thus the identities of the protein targets are usually unknown. Moreover, the antibody-based methods are limited by the availability, quality, and specificity of antibodies to protein adducts, which greatly hindered the identification of specific protein targets of drugs and their clinical applications. Recently, the use of powerful MS technologies (e.g., matrix-assisted laser desorption/ionization time-of-flight) together with analytical proteomics have enabled one to separate, identify unknown protein adducts, and establish the sequence context of specific adducts by offering the opportunity to search for adducts in proteomes containing a large number of proteins with protein adducts and unmodified proteins. The present review highlights the separation and detection technologies for drug-protein adducts, with an emphasis on methodology, advantages and limitations to these techniques. Furthermore, a brief discussion of the application of these techniques to individual drugs and their target proteins will be outlined.     

Evaluation of sample extraction methods for proteomic analysis of coniferous seeds

In this study, three methods of protein extraction from the seeds of the Chinese fir were compared by examining the quality (including the number of protein spots observed) in the two-dimensional gel electrophoresis (2-DE), obtained by isoelectric focusing and sodium dodecyl sulfate polyacrylamide gel electrophoresis of the total released protein. Three protein extraction methods were: TCA-acetone precipitation, SDS extraction/acetone precipitation, and phenol extraction methanol/ammonium acetate precipitation. The results showed that TCA-acetone precipitation was the most effective method for protein extraction; it gave the highest yield of total protein (8.9 mg protein per g seed weight) and the greatest number of proteins spots (1,034 spots) on the 2-DE gel. Further, several proteins were identified by liquid chromatography mass spectrometry (LC MS/MS), which are legumin-like storage protein, similar to AMP binding/acetate-CoA ligase, similar to 40S ribosomal protein S20, actin, ascorbate peroxidase, Similar to cysteine synthase, and unknown protein. These data demonstrates that TCA-acetone precipitation followed by 2-DE and LC MS/MS is a suitable method for proteomic analysis of coniferous species, such as Chinese fir and provides a valuable starting point for similar proteomic analysis of other coniferous tree species.     

Recent developments of enantioseparation techniques for adrenergic drugs using liquid chromatography and capillary electrophoresis: a review

This review provides the achievements of enantioseparation of adrenergic drugs and application of these methods in clinical and pharmaceutical analysis. The adrenergic agonist and antagonist drugs are analyzed in the direct and indirect modes by liquid chromatography (LC) and capillary electrophoresis (CE). Other chromatographic enantioseparation methods including super- and sub-critical fluid chromatography (SFC), and capillary electrochromatography (CEC) are presented likewise to analyse the cited compounds. The different separation processes for these drugs are briefly discussed and some applications are presented.     

Separation methods for pharmacologically active xanthones

Xanthones, as a kind of polyphenolic natural products with many strong bioactivities, are attractive for separation scientists due to the similarity and diversity of their structures resulting in difficult separation by chromatographic methods. High performance liquid chromatography (HPLC) and thin layer chromatography (TLC) are traditional methods to separate xanthones. Recently, capillary electrophoresis (CE), as a micro-column technique driven by electroosmotic flow (EOF), with its high efficiency and high-speed separation, has been employed to separate xanthones and determine their physicochemical properties such as binding constants with cyclodextrin (CD) and ionization constants. Since xanthones have been used in clinic treatment, the development of chromatographic and CE methods for the separation and determination of xanthones plays an essential role in the quality control of some herbal medicines containing xanthones. This article reviewed the separation of xanthones by HPLC, TLC and CE, citing 72 literatures. This review focused on the CE separation for xanthones due to its unique advantages compared to chromatographic methods. The comparison of separation selectivity of different CE modes including capillary zone electrophoresis (CZE), micellar electrokinetic chromatography (MEKC), microemulsion electrokinetic capillary chromatography (MEEKC) and capillary electrochromatography (CEC) was discussed. Compared with traditional chromatographic methods such as HPLC and TLC, CE has higher separation efficiency, faster separation, lower cost and more flexible modes. However, because of low sensitivity of UV detector and low contents of xanthones in herbal medicines, CE methods have seldom been applied to the analysis of real samples although CE showed great potential for xanthone separation. The determination of xanthones in herbal medicines has been often achieved by HPLC. Hence, how to enhance CE detection sensitivity for real sample analysis, e.g. by on-line preconcentration and CE-MS, would be a key to achieve the quantitation of xanthones.     

Laboratory database to manage electrophoresis and chromatography separations and the associated samples

A database was developed to store, organize, and retrieve the data associated with electrophoresis and chromatography separations. It allows laboratories to store extensive data on separation techniques (analytical and preparative). The data for gel electrophoresis includes gel composition, staining methods, electric fields, analysis, and samples loaded. The database stores data on chromatography conditions, the samples used, and the fractions collected. The data structure of this database was designed to maintain the link between samples (including fractions) from chromatography separations and their analysis by gel electrophoresis. The database will allow laboratories to organize and maintain a large amount of separation and sample data in a uniform data environment. It will facilitate the retrieval of the separation history of important samples and the separation conditions used.     

Improved mutation detection in GC-rich DNA fragments by combined DGGE and CDGE.

Denaturing gradient gel electrophoresis (DGGE) has proven to be a powerful pre-screening method for the detection of DNA variants. If such variants occur, however, in DNA fragments that are very rich in G and C, they may escape detection. To overcome this limitation, we tested a novel gel system which combines DGGE and constant denaturant gel electrophoresis (CDGE), as it might have the advantages of both methods. Indeed, this combination had the advantages of both methods, good separation of hetero-duplex molecules and prevention of total strand dissociation, and it proved successful in the detection of DNA variants in several GC-rich fragments.     

DNA指纹图谱技术在土壤微生物多样性研究中的应用

土壤中的微生物多样性是十分丰富的,传统培养方法对土壤微生物多样性的研究有很大局限性。近年来,各种基于16S rDNA基因的指纹图谱分析技术取得了长足的进步,并广泛应用于土壤微生物多样性的研究。这些技术主要有变性梯度凝胶电泳(DGGE)/温度梯度凝胶电泳(TGGE)、单链构象多态性(SSCP)、随机引物扩增多态性DNA(RAPD)、限制性片段长度多态性(RFLP)和扩增核糖体DNA限制性分析(ARDRA)等。对这些技术近年来在土壤微生物多样性研究领域的应用予以简短综述,并初步探讨未来几年土壤微生物分子生态学发展的方向。     

The use of micropreparative electrophoresis of protein/peptide isolations for primary structure determinations

High-performance electrophoresis chromatography (HPEC) is a recent development that features continuous-elution gel electrophoresis for isolating proteins or peptides in range of 1 to 300 microgram quantities. Column gel electrophoresis is conducted under thermostated conditions, and the field voltage can be varied within a run with a programmable power supply. Applications of this apparatus in protein purification are presented to demonstrate the utility of the (Model 230A) HPEC. These examples include on-line detection with direct analyte recovery of highly purified sample, which mimics high-performance liquid chromatography, for subsequent structure-function characterization. A method to remove salts from sodium dodecyl sulfate electrophoresed samples for subsequent sequencing or amino acid analysis is described. This desalting procedure recovers from 90%-95% of the sample and employs a low molecular weight cut-off membrane during sample centrifugation onto a polyvinylidene difluoride membrane. Subsequent washings are performed to efficiently remove salts, free amino acids and detergents that are known to interfere with sequence analysis. Sequence information such as initial recovery, repetitive yields and chromatogram comparisons are presented to demonstrate the utility of this procedure when used following isolation of sample with HPEC.     

Hippophae rhamnoides L.: chromatographic methods to determine chemical composition, use in traditional medicine and pharmacological effects

There is an increasing interest in the usage of chromatographic methods on the analysis of chemical compounds present in Hippophae rhamnoides L. In this paper, the chromatographic techniques applied for the determination, separation and identification of chemical compounds of H. rhamnoides L. are reviewed. We examined the existing chromatographic methods based on separations by paper and thin-layer chromatography, gas chromatography, high-performance liquid chromatography and capillary electrophoresis and also methods of detection by ultraviolet absorption, fluorescence, refractive index, electrochemical and mass spectrometry. Biological properties of the plant and its pharmacological effects and use in traditional medicine have also been reviewed.     

Enzyme-linked bridging assay method for the quantification of oligonucleotide-based drugs in biological matrices

With ongoing efforts to develop oligonucleotide-based (ODN-based) therapeutics, there is a need for a sensitive, high-throughput method of quantification of ODN-based drugs in biological matrices. To overcome the insufficient sensitivity and time-consuming sample extraction procedures involved in conventional capillary gel electrophoresis (CGE) and high-performance liquid chromatography (HPLC), we developed a nucleic acid hybridization-based enzyme-linked bridging assay (ELBA), which shows significant advantages over CGE methods in evaluating ODN-based drugs in plasma and tissue: (1) It has higher sensitivity; (2) it involves easier sample extraction procedures; (3) it is suitable for many ODN-based drugs, even those with different secondary structures and modifications, including phosphorothioate oligonucleotide (PSODN), mixed backbones with 2'-O-Me (MBO), locked nucleic acid (LNA) modifications, and B- and C-type CpG sequences; and (4) it is highly selective, even during simultaneous quantification, with regard to intact ODNs and their 3'-metabolites. This universal design produces a rapid, sensitive, specific assay with minimal method development time. It is well suited to high-throughput analysis of various ODN-based drugs.     

Recent development of multi-dimensional chromatography strategies in proteome research

As a complementary approach to two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), multi-dimensional chromatography separation methods have been widely applied in all kinds of biological sample investigations. Multi-dimensional liquid chromatography (MDLC) coupled with bio-mass spectrometry (MS) is playing important roles in proteome research due to its high speed, high resolution and high sensitivity. Proteome analysis strategies mainly include bottom-up and top-down approaches which carry out biological sample separation based on peptide and protein levels, respectively. Electrophoretic methods combined with liquid chromatography like IEF-HPLC and HPLC-SDS-PAGE have been successful applied for protein separations. As for MDLC strategy, ion-exchange chromatography (IEX) together with reversed phase liquid chromatography (RPLC) is still a most widely used chromatography in proteome analysis, other chromatographic methods are also frequently used in protein pre-fractionations, while affinity chromatography is usually adopted for specific functional protein analysis. Recent MDLC technologies and applications to variety of proteome analysis have been achieved great development. A digest peptide-based approach as so-called "bottom-up" and intact protein-based approach "top-down" analysis of proteome samples were briefly reviewed in this paper. The diversity of combinations of different chromatography modes to set up MDLC systems was demonstrated and discussed. Novel developments of MDLC techniques such as high-abundance protein depletion and chromatography array were also included in this review.     

A comparison of rapid electrophoretic and chromatographic methods for the investigation of common histological dyes

Summary The compositions of fifty-nine common histological dyes, as well as duplicate samples of several dyes from different suppliers, have been studied by agar gel electrophoresis, agarose gel electrophoresis, paper electrophoresis, paper chromatography and thin layer chromatography. Tables are presented to show the number of components present in each dye as disclosed by the different methods; the cases where duplicate samples were available are summarised in a separate table.On the basis of effectiveness and convenience agar gel electrophoresis and thin layer chromatography were by far the best methods. The Chromatographic method was of slightly wider applicability but as electrophoretic methods gave information on dye charge, agar gel electrophoresis was the best single method.     

Assay methods of modified lipoproteins in plasma

Modified lipoproteins, especially oxidatively modified low-density lipoprotein (Ox-LDL), are present in the plasma of patients with atherosclerosis and related diseases. The modification of LDL is believed to play an important role in the development of atherosclerosis. Thus, measurement of plasma Ox-LDL is essential not only for investigating its relevance to atherosclerotic diseases, but also for diagnosis. Chromatographic methods are effective for indirectly measuring the oxidatively modified state of LDL or directly measuring the modified LDL. Indirect determination can be done by estimating the LDL subfraction, LDL particle size, oxidized amino acids in apolipoprotein B, lipid hydroperoxide or F(2)-isoprostane in LDL. Direct determination of the modified LDL in plasma can be done with chromatographic methods such as anion-exchange chromatography and size-exclusion chromatography. Other methods for estimating the modified state of LDL include electromigration methods such as agarose gel, polyacrylamide gradient gel and capillary electrophoresis. Recently, enzyme-linked immunosorbent assay methods of malondialdehyde (MDA)-LDL and autoantibodies against Ox-LDL have been developed to assess Ox-LDL in plasma. This review article summarizes the detection and assay methods of modified lipoproteins in plasma.     

Principles and methods for the analysis and purification of synthetic deoxyribonucleotides by high-performance liquid chromatography

The need for high-purity oligodeoxyribonucleotides for various applications has resulted in the development of novel synthesis, purification, and analytical techniques, A diversity of methods, including polyacrylamide slab gel electrophoresis, capillary gel electrophoresis, as well as high-performance liquid chromatography (HPLC), have been successfully used to aid in the characterization and isolation of these synthetic compounds. The information contained in this review article primarily details both the theoretical and practical aspects related to the use of HPLC for the analysis and purfication of synthetic DNA. In addition, a variety of postsynthesis sample preparation protocols, commonly employed prior to and after HPLC, are described.     

Strategy for analysis and screening of bioactive compounds in traditional Chinese medicines

Traditional Chinese medicines (TCMs), due to their long time clinic test and reliable therapeutic efficacy, are attracting increased global attention served as excellent pools of bioactive compounds for the discovery of new drugs. However, hundreds or even thousands of components are usually contained in traditional Chinese medicines and only a few compounds are responsible for the pharmaceutical and/or toxic effects. The large numbers of other components in traditional Chinese medicines make the screening and analysis of the bioactive components extremely difficult. By the way, the combination effect of bioactive components on the pharmacological activity makes it very difficult to clear the therapeutic mechanism of TCMs. Therefore, some strategies have to design for screening of bioactive compounds in traditional Chinese medicines, which further leads to disclose the therapeutic mechanism of TCMs in molecular level. The review will summarize the present state of the art of screening strategy for active compounds in traditional Chinese medicines, and the chromatography methods for screening and analysis of bioactive compounds in traditional Chinese medicines will be emphasized.     

Selenoprotein P analysis in human plasma

Several recent analytical methods for determination of Se and selenoprotein P have involved high-performance liquid chromatography (HPLC) using heparin-affinity columns coupled to inductively coupled plasma-mass spectrometry (ICP-MS) for Se detection. HPLC-ICP-MS chromatography using tandem HPLC columns with ICP-MS detection was used to detect the major selenium-containing proteins in plasma (glutathione peroxidase, albumin, and selenoprotein P). The efficiency of HPLC separation of plasma selenoprotein P was investigated by analyzing HPLC fractions using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with immunoblot analysis. The HPLC fraction corresponding to selenoprotein P contained 25.1% of total selenoprotein P as measured by immunoblot analysis. The majority (74.9%) of total selenoprotein P found by immunoblot analysis was contained in the early HPLC fractions, consistent with either poor heparin affinity, which was not evident based on the HPLC-ICP-MS technique alone or nonspecific binding of the antibody. Immunoblot analysis of selenoprotein relies on antibodies binding to a selenoprotein P epitope, which might be preserved when selenoprotein P is broken down to release selenocysteine residues. Immunoblot methods overestimate selenoprotein P and are not suitable for determinations of intact selenoprotein P.     

Capillary affinity gel electrophoresis

Capillary affinity gel electrophoresis is a new technique for the recognition of the specific DNA base and/or sequence. This technology is also applicable to the characterization of binding properties of DNA-based drugs, chiral separation, and the selective separation of antibody mimetics using imprinted polymers. This article reviews the present state of studies on the capillary affinity gel electrophoresis, including the principle, theory, methods, and applications of this technology. The great potential of capillary affinity gel electrophoresis for the detection of the mutation onDNA is illustrated.     

Analytical methods to determine phytoestrogenic compounds

The analytical methods for the determination of phytoestrogenic compounds in edible plants, plant products and biological matrices are reviewed. The detection, qualitative and quantitative methods based on different chromatographic separations of gas chromatography (GC), high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE) coupled with various detections by ultraviolet absorption (UV), electrochemical detection (ED), fluorescence detection, mass spectrometry (MS) and nuclear magnetic resonance spectroscopy (NMR), as well as non-chromatographic immunoassay are each extensively examined and compared. An overview on phytoestrogen chemistry, bioactivities and health effects, plant precursors, metabolism and sample preparation is also presented.