Assay for taurine conjugates of bile acids in serum by reversed-phase high-performance liquid chromatography

The purpose of this study was to develop a new high-performance liquid chromatographic (HPLC) procedure for quantifying taurine conjugates of bile acids in serum. The technique involved three basic steps. The first removed free amino acids via solid-phase extraction of the serum. The second step involved the reaction of the extracted serum with the enzyme choloylycine hydrolase, which liberated the taurine from the conjugated bile acids. The third step was the reversed-phase HPLC separation of o-phthalicdicarboxaldehhyde derivatives of taurine. The assay provides a simple technique for determination of the total amount of taurine-conjugated bile acids in serum.     

Gas chromatographic determination of malonaldehyde formed by lipid peroxidation

Gas chromatographic (GC) methods for the determination of malonaldehyde (MA) all require formation of a stable derivative of MA since free MA is not suitable for direct GC analysis. Most reported GC methods give a total measure of free MA and its bound forms because their assay conditions are sufficient to hydrolyze or decompose bound MA during sample preparation. In this paper, GC methods that provide a measure of total MA (free plus bound) are reviewed. A recently developed capillary GC method that allows determination of free MA apart from other forms is also discussed. The method involves derivatization of MA to N-methylpyrazole under milder reaction conditions than with other GC methods. The method represents an advantage over existing techniques for free MA determination because capillary GC offers the highest efficiency of separation among chromatographic methods thus allowing a more specific and accurate measure of MA.     

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.     

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.     

High-performance liquid chromatographic separation of bile acid pyrenacyl esters with cyclodextrin-containing mobile phase

The high-performance liquid chromatographic separation of bile acid pyrenacyl esters with cyclodextrin-containing mobile phase is presented. Compared with conventional methods, inclusion chromatography gives much more satisfactory separation of derivatized bile acids in a short time. The application of this method to the separation of glycine-conjugated bile acids in human bile is also described.     

Glyco-7 alpha, 12 alpha-dihydroxy-5 beta-cholanic acid as internal standard for high-pressure liquid chromatographic analysis of conjugated bile acids

Glyco-7 alpha, 12 alpha-dihydroxy-5 beta-cholanic acid was tested as internal standard for high-pressure liquid chromatographic analysis of the five main glycine- and taurine-conjugated bile acids present in adult human serum and bile. When the standard is added to the samples before extraction, the recovery rate throughout the procedure is similar to that of other bile acids. For all bile acids studied, the response, relative to the internal standard, is linear at 205 nm. Baseline separation is observed between the internal standard and all other bile acids, both in artificial mixtures and extracts of biological samples. Thus, glyco-7 alpha, 12 alpha-dihydroxy-5 beta-cholanic acid is a reliable internal standard for HPLC analysis of conjugated bile acids in serum and bile.     

Sensitive high-performance liquid chromatographic method for the determination of a benzonaphthazepine antipsychotic agent, SCH 39166, and its active metabolite, SCH 40853, in human plasma and its cross-validation with a gas chromatographic method

A sensitive high-performance liquid chromatographic (HPLC) method was developed for the determination of a benzonaphthazepine antipsychotic agent, SCH 39166, and its active metabolite, SCH 40853. The HPLC method required a single-step organic extraction at alkali pH followed by HPLC analysis utilizing a CN column with UV detection at 205 nm. The limit of quantitation was 1 ng/ml for SCH 39166 and 0.5 ng/ml for SCH 40853. The HPLC method was cross-validated with a previously reported GC method by the analysis of 73 plasma samples spiked with various concentrations of SCH 39166 and SCH 40853. The correlation coefficient was 0.9969 for SCH 39166 and 0.9984 for SCH 40853. Both GC and HPLC methods were used for the determination of plasma concentrations and yielded similar pharmacokinetic parameters for SCH 39166 and SCH 40853 in man following oral administration of SCH 39166 (100 mg).     

Biochemical and physiological evidence that bile acids produced and released by lake char (Salvelinus namaycush) function as chemical signals

It has been hypothesized that faeces of juvenile lake char (Salvelinus namaycush) may contain chemical cues that mediate behaviour of conspecifics. However, our knowledge of bile acids naturally produced and released by fish is limited. Using HPLC, we fractionated bile acids produced and released by lake char and examined their stimulatory effectiveness using electro-olfactogram recordings. Taurocholic acid, taurochenodeoxycholic acid, taurooxocholanic acid, taurooxodeoxycholic acid 3alpha-sulphate, trace amounts of taurolithocholic acid and an unidentified sulphated bile steroid were found in bile and faeces. Bile acids were either taurine amidated or sulphated, or both. Lake char released an average of 4 nmol min(-1) bile acids per kilogram of body weight into their tank water. Urinary bile acids accounted for only a small portion of total bile acids released into water. Water and faeces contained higher proportion of taurochenodeoxycholic acid and sulphated bile acids (relative to taurocholic acid) than bile. The electro-olfactogram recordings demonstrated that bile acids released by lake char were detectable by their olfactory system at nanomolar concentrations, which is well below the levels of bile acids released into water. The exquisite olfactory sensitivity of lake char to water-borne bile acids released by their conspecifics is consistent with a role for these compounds as important chemical signals.     

High-performance liquid chromatographic determination of the magnetic resonance imaging contrast agent gadobenate ion in plasma, urine, faeces, bile and tissues

The gadobenate ion is an intravascular paramagnetic contrast agent for magnetic resonance imaging. An HPLC method for assaying gadobenate ion in plasma, urine, faeces, bile and tissue samples is described. The analysis is based on the reversed-phase chromatographic separation of gadobenate ion from the endogenous components of biological matrices and detection by UV absorption at 210 nm. The selectivity of the method was satisfactory. The mean absolute recovery was greater than 95%. The precision and accuracy of the analytical methods were in the range 0.1–6.5% and −12 to +9.3%, respectively. The detection limits in plasma (0.1 ml), urine (0.05 ml), dried faeces (200 mg suspended in 4 ml water), bile (0.5 ml), and dried liver tissue (100 mg suspended in 1 ml water) were, respectively, 0.24, 0.47, 2.6, 0.63 and 2.8 nmol ml⁻¹ (corresponding to 0.16, 0.31, 1.7, 0.42 and 1.9 μg ml⁻¹).     

Separation of drugs by packed-column supercritical fluid chromatography

Packed-column supercritical fluid chromatography (pSFC) has been expected to analyze various kinds of compounds. Many researchers have expected a new chromatographic technique that overcomes the limitations of other techniques, HPLC and GC. In pharmaceutical development, chromatography plays an important role in the evaluation of safety and efficacy of a new compound. This article provides an overview of the separation of drugs by pSFC. The effects of the chromatographic parameters were studied for the separation of steroids. In chiral separation, the successful results were shown and compared with HPLC.     

Separation methods for antibacterial and antirheumatism agents in plant medicines

Traditional oriental medicines (TOM), with a very long history and many remarkable features, are very popular in Asian countries, especially in China, Japan and Korea. With the development of advanced analytical techniques, the modernization of traditional medicine has become a hot area in recent years and some herbal medicines have been increasingly accepted in western countries. Separation and determination of active components in various herbal medicines are considered to be critical for the modernization process. Antibacterial and antirheumatism agents are widely distributed in many medical plants and commonly used in clinical treatment. Therefore, the development of effective separation methods for the quality control of herbal medicines is absolutely important. In this article, the separation methods for the analysis of antibacterial and antirheumatism compounds in TOM were reviewed, including thin layer chromatography (TLC), gas chromatography (GC), supercritical fluid chromatography (SFC), high-performance liquid chromatography (HPLC), capillary electrophoresis (CE) and related hyphenation techniques. Sample preparation procedures and further development of these methods were also discussed.     

Gas chromatography of bile acids

Bile acids, the end products of cholesterol metabolism in the liver, are of vital importance in the tissue distribution of cholesterol. Abnormalities in cholesterol biosynthesis or metabolism are often reflected in the proportions, concentrations and conjugation of bile acids in various tissues and determination of bile acids in these tissues is important in the diagnosis of hepatobiliary diseases. Several methods for quantitative determination of bile acids in biological fluids are known and have been reviewed. In this review, we have discussed the gas-chromatographic method for determination of bile acids with special reference to bile acid quantitation in plasma, bile, urine and stool.     

Evaluation of fecal mutagenicity and colorectal cancer risk

Colorectal cancer is one of the most common internal malignancies in Western society. The cause of this disease appears to be multifactorial and involves genetic as well as environmental aspects. The human colon is continuously exposed to a complex mixture of compounds, which is either of direct dietary origin or the result of digestive, microbial and excretory processes. In order to establish the mutagenic burden of the colorectal mucosa, analysis of specific compounds in feces is usually preferred. Alternatively, the mutagenic potency of fecal extracts has been determined, but the interpretation of these more integrative measurements is hampered by methodological shortcomings. In this review, we focus on exposure of the large bowel to five different classes of fecal mutagens that have previously been related to colorectal cancer risk. These include heterocyclic aromatic amines (HCA) and polycyclic aromatic hydrocarbons (PAH), two exogenous factors that are predominantly ingested as pyrolysis products present in food and (partially) excreted in the feces. Additionally, we discuss N-nitroso-compounds, fecapentaenes and bile acids, all fecal constituents (mainly) of endogenous origin. The mutagenic and carcinogenic potency of the above mentioned compounds as well as their presence in feces, proposed mode of action and potential role in the initiation and promotion of human colorectal cancer are discussed. The combined results from in vitro and in vivo research unequivocally demonstrate that these classes of compounds comprise potent mutagens that induce many different forms of genetic damage and that particularly bile acids and fecapentaenes may also affect the carcinogenic process by epigenetic mechanisms. Large inter-individual differences in levels of exposures have been reported, including those in a range where considerable genetic damage can be expected based on evidence from animal studies. Particularly, however, exposure profiles of PAH and N-nitroso compounds (NOC) have to be more accurately established to come to a risk evaluation. Moreover, lack of human studies and inconsistency between epidemiological data make it impossible to describe colorectal cancer risk as a result of specific exposures in quantitative terms, or even to indicate the relative importance of the mutagens discussed. Particularly, the polymorphisms of genes involved in the metabolism of heterocyclic amines are important determinants of carcinogenic risk. However, the present knowledge of gene-environment interactions with regard to colorectal cancer risk is rather limited. We expect that the introduction of DNA chip technology in colorectal cancer epidemiology will offer new opportunities to identify combinations of exposures and genetic polymorphisms that relate to increased cancer risk. This knowledge will enable us to improve epidemiological study design and statistical power in future research.     

Determination of polycyclic aromatic hydrocarbons in smoked meat products and smoke flavouring food additives

Formation, factors affecting concentrations, legal limits and occurrence of polycyclic aromatic hydrocarbons in smoked meat products and smoke flavour additives are briefly reviewed. The most used techniques such as thin-layer chromatography (TLC), gas chromatography (GC) and high-performance liquid chromatography (HPLC) are evaluated. Also, sample preparation, pre-separation procedures, separation and detection systems being used for determination are discussed with emphasis to latest development in applied food analysis and the chosen data regarding the concentration of polycyclic aromatic hydrocarbons in smoked meat products and smoke flavour additives are summarised.     

Chromatographic behavior of oxygenated derivatives of cholesterol

Oxygenated derivatives of cholesterol have important functions in many biochemical processes. These oxysterols are difficult to study because of their low physiological concentrations, the facile formation of cholesterol autoxidation artifacts, and lack of information on their chromatographic behavior. Focusing on metabolites and autoxidation products of cholesterol, we have documented the chromatographic mobilities of 35 oxysterols under a variety of conditions: eight solvent systems for thin-layer chromatography on silica gel, several mobile phases for reversed-phase high-performance liquid chromatography (HPLC), and two types of stationary phase for capillary gas chromatography (GC) using trimethylsilyl derivatives. Notable differences in selectivity could be obtained by modifying the stationary or mobile phases. Separations of oxysterol pairs isomeric at side-chain carbons or C-7 were achieved on normal-phase, reversed-phase, chiral, or silver-ion HPLC columns. Chromatographic behavior is also described for side-chain hexadeuterated and heptafluorinated oxysterols, which are useful as standards in isotope dilution analyses and autoxidation studies, respectively. The overall results are relevant to many problems of oxysterol analysis, including the initial separation of oxysterols from cholesterol, determination of highly polar and nonpolar oxysterols, separation of isomeric pairs, selection of derivatization conditions for GC analysis, and quantitation of the extent of cholesterol autoxidation.     

Liquid chromatography of active principles in Sophora flavescens root

Herbal medicines were one of the major resources for healthcare in earlier stages, and some traditional herbal medicines have been in use for more than 2000 years. Currently, they are attracting more and more attention of the modern pharmaceutical industry, as scientists has become aware that herbs have almost infinite resources for medicine development. This review provides an overview of the analytical approaches applied in the researches concentrated on various aspects of the matrine-type alkaloids in Sophora flavescens root. Emphasis will be laid on the analytical processes of high-performance liquid chromatography (HPLC), capillary electrophoresis (CE), as well as gas chromatography (GC) methods. The sample extraction, separation and detection have been summarized. In addition, the applications of chromatographic determinations are introduced for the main matrine-type alkaloids in S. flavescens root, such as matrine, sophoridine, sophocarpine, lehmannine, sophoramine, oxymartine, oxysophocarpine, cytosine and aloperine. The advantages and limitations of HPLC, CE and GC methods in the analytical applications of the alkaloids are also discussed.     

Determination of enantiomerization barriers by dynamic and stopped-flow chromatographic methods

In recent years, dynamic chromatography and stopped-flow chromatographic techniques have become versatile tools for the determination of enantiomerization and isomerization barriers. Increasing demands for the stereochemical safety of chiral drugs contributed to the rapid development of new techniques. New computer-aided evaluation systems allow the on-line determination of interconversion barriers from the experimental chromatograms. Both dynamic chromatography and stopped-flow chromatography have been applied to the entire range of chromatographic methods (GC, SFC, HPLC, CE).     

Alkaline solvent systems for thin-layer chromatography of bile acids

Thin-layer chromatographic separation of the common bile acids and their taurine and glycine conjugates in chloroform-methanol-ammonia is reported. An alkaline system offers advantages for the separation and nondestructive staining of bile acid conjugates.     

Chromatographic separation of 2,3-benzodiazepines

A review of chromatographic methods for the determination of 2,3-benzodiazepines (2,3-BZs) is presented. The determinations are performed to investigate the presence of potential impurities in drug substances and to study their pharmacokinetic profile in biological samples, either in animals or in humans. Several methods dealt with a pretreatment of samples, i.e., liquid–liquid extraction by using a variety of solvents, solid-phase extraction, direct injection of specimens into the chromatographic apparatus. Different chromatographic techniques have been used. High-performance liquid chromatography allows optimal sensitivity and specificity by using ultraviolet or diode array detection methods. Gas chromatography-mass spectrometry and gas chromatography with nitrogen-phosphorous or electron-capture detectors have been also reported. Suitable methods for the separation of enantiomers of 2,3-BZs have been described. Thin-layer chromatography has been shown to be capable to isolate analytes from biological samples as urine or faeces. The reported chromatographic techniques are currently applied to define the metabolic pathways of 2,3-BZs in experimental and clinical studies.