Simultaneous high-performance liquid chromatographic determination of visoltricin, acuminatopyrone and chlamydosporols in Fusarium cultures on maize

Visoltricin (VIS), acuminatopyrone (ACP), clamydosporol (CL), isochlamydosporol (ICL) and chlamydospordiol (DIOL), recently characterized Fusarium metabolites, were separated on a polymeric RP-18 column eluted with acetonitrile-0.01% ammonia solution (35:65) at 1 ml/min and detected with a diode-array UV detector. The presence of ammonia in the mobile phase improved the shape of the CL and VIS peaks. The use of a polymeric column was required owing to the basic pH of the mobile phase. Maize cultures of several strains of F. tricinctum and F. chlamydosporum were analysed with this procedure after extraction with aqueous methanol, partitioning with methylene chloride and clean-up with a C₁₈ minicolumn. VIS was produced only by F. tricinctum, whereas ACP and chlamydosporols were produced by both Fusarium species.     

Microbial transformation of (+)-adrenosterone

The microbial transformation of (+)-adrenosterone (1) by Cephalosporium aphidicola afforded three metabolites identified as androsta-1,4-diene-3,11,17-trione (2), 17beta-hydroxyandrost-4-ene-3,11-dione (3) and 17beta-hydroxyandrosta-1,4-diene-3,11-dione (4). The fermentation of 1 with Fusarium lini also produced metabolites 2 and 4, while the fermentation with Trichothecium roseum afforded metabolite 3. The structures of transformed products were determined by spectroscopic methods.     

Potential of HILIC-MS in quantitative bioanalysis of drugs and drug metabolites

One fundamental requirement for many lead optimization processes is the need for bioanalytical support within pharmaceutical drug discovery and development. Currently, most bioanalytical methods for pharmaceutical analysis employ HPLC coupled with MS/MS. The combination of HPLC and MS/MS detection frequently offers the complete resolution of the dosed compounds from their metabolites and the endogenous interferences to avoid extra efforts for chemical separation and sample clean-up procedures resulting in higher-throughput assays for a series of new chemical entities (NCEs). Hydrophilic interaction chromatography (HILIC) has been demonstrated to be a powerful technique for the retention of polar analytes offering a difference in selectivity compared to traditional RP chromatography. This review summarizes the HILIC-MS/MS methods for the trace quantitative determinations of the drug compounds and their metabolites to support both in vitro and in vivo experiments. The challenges on performing HILIC-MS/MS assays such as matrix ionization suppression and the potential for endogenous interferences are also presented.     

Determination of fumonisins B1 and B2 in Portuguese maize and maize-based samples by HPLC with fluorescence detection

Fumonisins B₁ (FB₁) and fumonisin B₂ (FB₂) are the main members of a family of mycotoxins produced by Fusarium verticillioides, Fusarium proliferatum, and other fungi species of the section Liseola. The present work shows the results of comparative studies using two different procedures for the analysis of fumonisins in maize and maize-based samples. The studied analytical methods involve extraction with methanol/water, dilution with PBS, and clean-up through immunoaffinity columns. Two reagents (o-phthaldialdehyde and naphthalene-2,3-dicarboxaldehyde) were studied for formation of fluorescent derivatives. The separation and identification were carried out by high-performance liquid chromatography with fluorescence detection. The optimized method for analysis of fumonisins in maize involved extraction with methanol/water (80:20), clean-up with an immunoaffinity column, and derivatization with naphthalene-2,3-dicarboxaldehyde (NDA). The limit of detection was 20 μg kg⁻¹ for FB₁ and 15 μg kg⁻¹ for FB₂. Recoveries of FB₁ and FB₂ ranged from 79% to 99.6% for maize fortified at 150 μg kg⁻¹ and 200 μg kg⁻¹, respectively, with within-day RSDs of 3.0 and 2.7%. The proposed method was applied to 31 samples, and the presence of fumonisins was found in 14 samples at concentrations ranging from 113 to 2,026 μg kg⁻¹. The estimated daily intake of fumonisins was 0.14 μg kg⁻¹ body weight per day.     

Determination of methandrostenolone and its metabolites in equine plasma and urine by coupled-column liquid chromatography with ultraviolet detection and confirmation by tandem mass spectrometry

Monitoring steroid use requires an understanding of the metabolism in the species in question and development of sensitive methods for screening of the steroid or its metabolites in urine. Qualitative information for confirmation of methandrostenolone and identification of its metabolites was primarily obtained by coupled-column high-performance liquid chromatography-tandem mass spectrometry. The steroids and a sulphuric acid conjugate were isolated and identified by their daughter ion mass spectra in the urine of both man and the horse following administration of methandrostenolone. Spontaneous hydrolysis of methandrostenolone sulphate gave 17-epimethandrostenolone and several dehydration products. This reaction had a half-life of 16 min in equine urine at 27 degrees C. Mono- and dihydroxylated metabolites were also identified. Several screening methods were evaluated for detection and confirmation of methandrostenolone use including thin-layer chromatography and high-performance liquid chromatography. Coupled-column liquid chromatography was used for automated clean-up of analytes difficult to isolate by manual methods. The recovery of methandrostenolone was 101 +/- 3.3% (mean +/- S.D.) at 6.5 ng/ml and both methandrostenolone and 17-epimethandrostenolone were quantified in urine by ultraviolet detection up to six days after a 250-mg intramuscular dose to a horse. The utility of on-line tandem mass spectrometry for confirmation of suspected metabolites is also shown.     

Identification of metabolites of 4,4'-diaminodiphenylmethane (methylene dianiline) using liquid chromatographic and mass spectrometric techniques.

The in vitro metabolism of 4,4'-diaminodiphenylmethane (methylene dianiline, MDA) was investigated using rabbit liver microsomes. Minimal clean-up of the microsomal incubations was carried out using zinc sulphate precipitation followed by solid-phase extraction on Sep-Pak C18 cartridges. Three metabolites were detected in hepatic microsomal incubations, namely the azodiphenylmethane (azo) azoxydiphenylmethane (azoxy) and 4-nitroso-4'-aminodiphenylmethane (nitroso) compounds. The azo and azoxy metabolites were produced enzymatically whereas the nitroso compound may have been formed via a non-enzymatic process. Reversed-phase high-performance liquid chromatography-plasma spray mass spectrometry was used to initially detect these metabolites. Fast atom bombardment mass spectrometry and fast atom bombardment tandem mass spectrometry were utilized to further structurally characterise these compounds. Comparison of mass spectral data obtained from synthesised standards with data obtained on the putative metabolites substantiated the characterisation of these compounds.     

The state-of-the-art in the analysis of type-A and -B trichothecene mycotoxins in cereals

The aim of this review is to describe the state-of-the-art in the analysis of A- and B-trichothecene mycotoxins in cereals and to support knowledge and experience exchange between laboratories in the field of Fusarium mycotoxin analysis. Current screening tests and quantitative methods for the most prevalent type-A and -B trichothecenes, HT-2 and T-2-toxin, and deoxynivalenol (DON) are reviewed. This includes the extraction and clean-up procedures and chromatographic methods (TLC, HPLC, GC) applied and the immunochemical methods, especially enzyme-linked immunosorbent assay (ELISA), employed for the determination of these mycotoxins. Results from recent intercomparison studies of the determination of DON are also discussed. Experience gained during these intercomparisons clearly shows the need for further improvement in the determination of trichothecenes, to obtain more accurate and comparable results. This also indicates there is a strong need for the development of further certified reference materials (CRM) which would enable comparison of measurement results between different European laboratories for several A- and B-trichothecenes. For both A- and B-trichothecenes there is still a lack of simple and reliable screening methods enabling the rapid detection of these mycotoxins at low cost.     

Analytical methods to determine phosphonic and amino acid group-containing pesticides

A comprehensive view on the possibilities of the most recently developed chromatographic methods and emerging techniques in the analysis of pesticides glyphosate, glufosinate, bialaphos and their metabolites is presented. The state-of-the-art of the individual pre-treatment steps (extraction, pre-concentration, clean-up, separation, quantification) of the employed analytical methods for this group of chemicals is reviewed. The advantages and drawbacks of the described analytical methods are discussed and the present status and future trends are outlined.     

Trace analysis of benzodiazepine drugs in blood using deactivated amberlite XAD-7 porous polymer beads and silica capillary column gas chromatography with electron-capture detection

A general method for the trace analysis of benzodiazepine drugs and their major metabolites at single dose therapeutic levels in 0.2 ml blood samples is described. The method involves solvent extraction of blood with toluene, isolation of the analytes using deactivated Amberlite XAD-7 porous polymer beads, and analysis of the cleaned-up extracts by capillary column gas chromatography with electron-capture detection. The clean-up technique eliminates lipids and other interfering material, enabling routine analysis of blood extracts to be carried out with no significant deterioration in column or detector performance over a period of many months. The use of fused-silica capillary columns coated with SE-52 and the correct choice of chromatographic conditions permits underivatised benzodiazepines of widely differing volatilities and polarites to be analysed. Data for 26 benzodiazepines and metabolites are presented.     

Studies on the determination of chlorotestosterone and its metabolites in bovine urine.

Chlorotestosterone and its metabolites were determined in urine samples from bovine animals treated with chlorotestosterone acetate by oral and intramuscular routes. Sample preparation, involving enzymatic deconjugation and solid-phase extraction, was optimised. The effect of different enzyme preparations, pH, and time of incubation were studied. An extraction/clean-up procedure based on solid-phase extraction (C18 cartridge) and liquid-liquid clean-up was developed. Determination of chlorotestosterone and its metabolites was by enzyme immunoassay and GC-MS. Metabolites were converted into their TMS-enol-TMS-ether and TMS-oxime-TMS-ether forms before GC-MS (EI) analysis.     

One-step isolation and identification of hydroxylamino-dinitrotoluenes, unstable products from 2,4,6-trinitrotoluene metabolites, with thin-layer chromatography and laser time-of-flight mass spectrometry

Two kinds of hydroxylamino-dinitrotoluenes (HADNTs), 2-hydroxylamino-4,6-dinitrotoluene (2HADNT) and 4-hydroxylamino-2,6-dinitrotoluene (4HADNT), are known to be major metabolites produced from 2,4,6-trinitrotoluene (TNT) by bacteria. These chemicals could not be identified as TNT metabolites produced by Pseudomonas sp. strain TM15 because the mass spectra of these chemicals could not be obtained by liquid chromatography-mass spectrometry (MS) or gas chromatography-MS, which are the classic methods for identifying the metabolites of xenobiotics. However, these problems are overcome by isolating 2HADNT and 4HADNT from TNT metabolites with one-step thin-layer chromatography using dichloromethane as the developing solvent, and individually extracting them into acetonitrile by collecting spots of 2HADNT and 4HADNT. The purity of each HADNT was approximately 98%, based on the results of high-performance liquid chromatographic analyses. 2HADNT and 4HADNT are identified by obtaining their mass spectra with laser time-of-flight MS. 2HADNT and 4HADNT dissolve in distilled water and are spontaneously broken down with time. Also, heat treatment (increasing temperatures) and dissolved oxygen accelerate the destruction of HADNTs. This technique may be applicable for the identification and exact quantitative analysis of unstable and fragile compounds such as HADNTs.     

Effect of chemical compounds on amino acid content of some Fusarium species and its significance to fungal chemotaxonomy

The cellular amino acid profiles of nine species of Fusarium; namely, Fusarium anthophilum, Fusarium avenaceum, Fusarium cerealis, Fusarium graminearum, Fusarium graminum, Fusarium oxysporum f. sp. conglutinans, Fusarium pseudograminearum, Fusarium roseum, and Fusariumsacchari var. elongatum growing on malt extract medium were determined. The amino acid profiles of the investigated fungi were varied and could be used for identification and characterisation of certain Fusarium species. Addition of certain chemical compounds including aspartic acid, glutamic acid, methionine, selenium, and urea to the growth medium affected the amino acid profiles. However, susceptibility of amino acid content to environmental conditions increased the variation of amino acid profiles among all the investigated Fusarium species. Some amino acids were only produced when certain chemical compounds were added to the growth medium. Valine was produced by F. anthophilum only in the presence of aspartic acid or selenium, while serine was produced in the presence of aspartic acid, glutamic acid, or methionine. Also, cysteine was produced by F. avenaceum in the presence of glutamic acid or urea. F. cerealis produced tryptophan only in the presence of aspartic acid or urea, while F. graminearum produced leucine in glutamic, methionine or urea. Similarly, many different amino acids were produced by each Fusarium species only in the presence of certain chemical compounds. The results revealed that the amino acid profiles will be more useful for characterisation and identification of fungi if they are determined under different conditions.     

Solid phase extraction of food contaminants using molecular imprinted polymers

Food contamination from natural or anthropogenic sources poses severe risks to human health. It is now largely accepted that continuous exposure to low doses of toxic chemicals can be related to several chronic diseases, including some type of cancer and serious hormonal dysfunctions.Contemporary analytical methods have the sensitivity required for contamination detection and quantification, but direct application of these methods on food samples can be rarely performed. In fact, the matrix introduces severe disturbances, and analysis can be performed only after some clean-up and preconcentration steps. Current sample pre-treatment methods, mostly based on the solid phase extraction technique, are very fast and inexpensive but show a lack of selectivity, while methods based on immunoaffinity extraction are very selective but expensive and not suitable for harsh environments. Thus, inexpensive, rapid and selective clean-up methods, relaying on “intelligent” materials are needed. Recent years have seen a significant increase of the “molecularly imprinted solid phase extraction” (MISPE) technique in the food contaminant analysis. In fact, this technique seems to be particularly suitable for extractive applications where analyte selectivity in the presence of very complex and structured matrices represents the main problem. In this review, several applications of MISPE in food contamination analysis will be discussed, with particular emphasis on the extraction of pesticides, drugs residua, mycotoxins and environmental contaminants.     

Critical study of and improvements in chromatographic methods for the analysis of type B trichothecenes

Various analytical methods used in the analysis of type B trichothecenes (deoxynivalenol, nivalenol, 3- and 15-acetyldeoxynivalenol) in cereals were compared and optimised in this work. These methods use either GC-electron-capture detection (ECD) of trimethylsilyl, trifluoroacetyl and heptafluorobutyryl derivatives or HPLC with UV or photodiode array detection of analytes. A new HPLC procedure using fluorescence detection prior derivatisation with coumarin-3-carbonyl chloride has been also tested. Five extraction solvents and two solid-phase extraction cartridges (silica, Florisil) plus a especial clean-up column (MycoSep 225) were compared in order to obtain the best recovery of the mycotoxins with minimal presence of coextractives in the chromatograms. The chosen extraction solvent was a mixture of acetonitrile-water (84:16, v/v). The MycoSep 225 column was chosen as the best alternative for clean-up of grain samples. For GC-ECD analysis, derivatisation of analytes with heptafluorobutyric anhydride prior the final determination was chosen as the most suitable procedure. HPLC-photodiode array (at 221 nm) analysis was more suitable for determination of type B trichothecenes than HPLC of the fluorescent coumarin-3-carbonyl derivatives. Recoveries obtained in spiked corn, rice and wheat are reported. The utility of the proposed methodology was assayed in cereal cultures of various Fusarium strains.     

Automated on-line multi-dimensional high-performance liquid chromatographic techniques for the clean-up and analysis of water-soluble samples

The application of an automated on-line multi-dimensional liquid-liquid chromatographic technique for the clean-up and analysis of water-soluble samples was investigated. The use of microparticulate aqueous-compatible steric exclusion columns as the primary separation step coupled to either reversed-phase, normal-phase or ion-exchange columns as the secondary step allowed the direct injection of complex samples without prior clean-up. The entire operation was automatically controlled by a microprocessor-based liquid chromatograph with time-programmable events which allowed precise switching of high-pressure pneumatically operated valves. Both heart-cutting and on-column concentration methods were used. The heart-cutting technique had the advantage of selectivity but lacked sensitivity; more successful was the on-column concentration technique, which, by the concentration of the solute from a larger volume of exclusion column effluent on to the secondary column, gave better sensitivity. The technique was applied to the analysis of theophylline and caffeine in biological fluids, catecholamines in urine, vitamins in a protein food supplement and sugars in molasses and candy bars.     

Isothermal gas chromatographic determination of nanogram amounts of chlorimipramine, chlorpromazine and their N-desmethyl metabolites in plasma using nitrogen-selective detection

A gas chromatographic method using nitrogen-selective detection for the quantitative determination of nanogram amounts of chlorimipramine, chlorpromazine and their nor1 and nor2 derivatives in plasma is described. Derivatization with trifluoroacetic anhydride of nor1 and nor2 metabolites allowed the chromatographic separation of these compounds. A three-step solvent extraction procedure was performed using n-heptane containing 1% isoamyl alcohol and n-hexane and compared with a plasma clean-up procedure using C18 Sep-Pak cartridges. The two procedures were characterized by similar degrees of precision. The use of C18 Sep-Pak cartridges, however, produced a significant time and material saving over the conventional extraction method.     

Immuno-affinity columns versus conventional clean-up: a method-comparison study for the determination of zearalenone in corn

The efficiency of a modern analytical method employing immuno-affinity columns (IACs) is compared to a well established traditional technique with respect to the determination of zearalenone (ZON) in corn in the μg/kg range. Despite of a constant error of about 4 μg/kg in the examined working range of 10–200 μg/kg, analytical data obtained from the analysis of spiked and naturally contaminated samples showed good correspondence for the compared methods. The performance characteristics of immuno-affinity-chromatography as a new clean-up technique for the determination of ZON in corn is reported for the first time and compared to a conventional clean-up procedure     

Fast mass spectrometry-based methodologies for pharmaceutical analyses

Historically, most bioanalytical methods for drug analysis in pharmaceutical industry were developed using HPLC coupled with UV or fluorescence detection. However, there is a trend toward interfacing separation technologies with more sensitive tandem mass spectrometry (MS/MS)-based systems. MS/MS detection offers complete resolution of the parent compounds from their first pass metabolites to avoid extra efforts for separation and sample clean-up procedures resulting in shorter run times. With the increasing demand for ever faster screening, there is a continuing demand for bioanalytical methods possessing higher sample throughput for both in vitro and in vivo drug metabolism and pharmacokinetic evaluations to accelerate the discovery process. This review focuses on the current approaches for fast MS-based assays (cycle-time less than 5 min) of pharmaceuticals and their metabolites that have been reported in the peer-reviewed publications.     

Different quantitation approaches for xenobiotics in human urine samples by liquid chromatography/electrospray tandem mass spectrometry

The potential of liquid chromatography combined with tandem mass spectrometry (LC/MS/MS) for the determination of pesticide metabolites in human urine at the sub-ppb level is explored. Metabolites from two organophosphorous pesticides, 4-nitrophenol (from parathion and parathion-methyl) and 3-methyl-4-nitrophenol (from fenitrothion), are taken as model analytes to conduct this study. After direct injection of the urine sample (10 microL), different approaches were evaluated in order to achieve correct quantitation of analytes using an electrospray ionisation (ESI) interface. Thus, the feasibility of using external calibration was checked versus the use of different isotope-labeled internal standards. The advantages of applying coupled-column liquid chromatography (LC/LC) as an efficient clean-up without any type of sample manipulation are also discussed. The combination of LC/LC with ESI-MS/MS allows the direct analysis of free metabolites in urine, as the automated clean-up performed by the coupled-column technique is sufficient for the removal of interferences that suppress the ionisation of analytes in the ESI source. Using this procedure with external calibration, good precision and recoveries, and detection limits below 1 ng/mL are reached with analysis run times of around 8 min. The hyphenated technique LC/LC/ESI-MS/MS is proved to be a powerful analytical tool, allowing the rapid, sensitive and selective determination of 4-nitrophenol and 3-methyl-4-nitrophenol in human urine without any sample treatment.     

An overview of sample preparation procedures for LC-MS multiclass antibiotic determination in environmental and food samples

Antibiotics are a class of pharmaceuticals that are of great interest due to the large volumes of these substances that are consumed in both human and veterinary medicine, and due to their status as the agents responsible for bacterial resistance. They can be present in foodstuffs and in environmental samples as multicomponent chemical mixtures that exhibit a wide range of mechanisms of action. Moreover, they can be transformed into different metabolites by the action of microorganisms, as well as by other physical or chemical means, resulting in mixtures with higher ecotoxicities and risks to human health than those of the individual compounds. Therefore, there is growing interest in the availability of multiclass methods for the analysis of antimicrobial mixtures in environmental and food samples at very low concentrations. Liquid chromatography (LC) has become the technique of choice for multiclass analysis, especially when coupled to mass spectrometry (LC-MS) and tandem MS (LC-MS²). However, due to the complexity of the matrix, in most cases an extraction step for sample clean-up and preconcentration is required before analysis in order to achieve the required sensitivities. This paper reviews the most recent developments and applications of multiclass antimicrobial determination in environmental and food matrices, emphasizing the practical aspects of sample preparation for the simultaneous extraction of antimicrobials from the selected samples. Future trends in the application of LC-MS-based techniques to multiclass antibiotic analysis are also presented.