Derivatization procedures for the detection of estrogenic chemicals by gas chromatography/mass spectrometry

This work evaluated derivatization procedures for detecting both natural and synthetic estrogenic chemicals by gas chromatography/mass spectrometry (GC/MS). Different silylating agents, mainly trimethylsilylating (TMS) agents, were compared, and the roles of various content of trimethylchlorosilane (TMCS, as a stimulator) were investigated. The difference in the abundances of the derivatives was caused by the steric hindrance of multiple hydroxyl groups and ethynyl groups in the structures of estrogenic chemicals. The use of TMCS produces an increase in the derivatization yield, especially for the compounds with multiple hydroxyl groups (i.e., 17beta-estradiol (E(2)) and estriol (E(3))). Mass spectra of O-TMS derivatives and tentative fragmentation profiles are proposed. Molecular ions were the base peaks for all the derivatives, and were used as the quantitation ions to obtain maximum detection sensitivity and specificity. Sample enrichment was achieved by Oasis HLB solid-phase extraction cartridges. The quantitation limits of these compounds ranged from 5 to 10 ng/L in 1000-mL water samples. Recovery of the estrogenic chemicals in spiked various water samples ranged from 78 to 102% while relative standard deviation (RSD) ranged from 1 to 15%.     

Derivatization of azaspiracid biotoxins for analysis by liquid chromatography with fluorescence detection

Azaspiracids (AZAs) are an important group of regulated lipophilic biotoxins that cause shellfish poisoning. Currently, the only widely available analytical method for quantitation of AZAs is liquid chromatography-mass spectrometry (LC-MS). Alternative methods for AZA analysis are needed for detailed characterization work required in the preparation of certified reference materials (CRMs) and by laboratories not equipped with LC-MS. Chemical derivatization of the amine and carboxyl groups on AZAs was investigated for the purpose of facilitating analysis by LC with fluorescence detection (FLD). Experiments towards chemical modification of AZA1 at the amine achieved only limited success. Derivatization of the carboxyl group, on the other hand, proved successful using the 9-anthryldiazomethane (ADAM) method previously applied to the okadaic acid (OA) group toxins. Extraction and clean-up methods were investigated for shellfish tissue samples and a post-reaction solid phase extraction procedure was developed for the AZA ADAM derivatives. Chromatographic separations were developed for the LC-FLD analysis of derivatized AZAs alone or in the presence of other derivatized toxins. This new analytical method for analysis of AZAs enabled verification of AZA1-3 concentrations in recently certified reference materials. The method demonstrated good linearity, repeatability and accuracy showing its potential as an alternative to LC-MS for measurement of AZAs.     

Determination of psychotropic phenylalkylamine derivatives in biological matrices by high-performance liquid chromatography with photodiode-array detection.

Several procedures using high-performance liquid chromatography with photodiode-array detection have been developed to create phytochemical and toxicological profiles of phenylalkylamine derivatives in biological samples (e.g. plant materials and urine). Mescaline-containing cactus samples were extracted with basic methanol, using methoxamine as internal standard; the extraction and clean-up of urine samples were performed on cation-exchange solid-phase extraction columns. The extracts were separated on a 3-micron ODS column with acetonitrile-water-phosphoric acid-hexylamine as the mobile phase. Peak detection was performed at 198 or 205 nm; peak identity and homogeneity were ascertained by on-line scanning of the UV spectra from 190 to 300 nm. The detection limit of phenylalkylamine derivatives in urine and cactus material was 0.026-0.056 micrograms/ml and 0.04 micrograms/mg, respectively. Following a single oral dose of 1.7 mg/kg methylenedioxymethylamphetamine (MDMA) the concentrations found in urine ranged from 1.48 to 5.05 micrograms/ml MDMA and 0.07-0.90 micrograms/ml methylenedioxyamphetamine (a metabolite of MDMA). The mescaline content of the cactus Trichocereus pachanoi varied between 1.09 and 23.75 micrograms/mg.     

High-performance liquid chromatographic procedures for the determination of temafloxacin in biological matrices.

A simple and precise high-performance liquid chromatographic procedure has been developed for the determination of temafloxacin and its trace level metabolites in biological matrices. Plasma samples are ultrafiltered after addition of an internal standard in a displacing reagent containing sodium dodecyl sulfate and acetonitrile. Plasma ultrafiltrates or diluted urines are chromatographed on a reversed-phase analytical column, using an ion-pair chromatographic mobile phase and fluorescence detection. The chromatographic system allows resolution and quantitation of temafloxacin's oxidative metabolites, which collectively account for less than 2% of the administered dose. The mean intra-assay coefficient of variation for determination of temafloxacin concentrations in plasma ranging from 0.05 to 10.0 micrograms/ml was 0.7%. The procedure was implemented at four laboratories for the analysis of over 12,000 samples from clinical studies. Inter-assay coefficients of variation estimated from routine analyses of quality control samples in these studies averaged 4% or lower for concentrations in the 0.15-10 micrograms/ml range. The limit of quantitation of the procedure is approximately 10 ng/ml; inter-assay coefficients of variation at 15 ng/ml averaged under 9%. Calibration curves were reproducible and highly linear, with correlation coefficients typically averaging over 0.9995. An alternative, more complex procedure, involving methylene chloride extraction, which extends the detection limits to below 1 ng/ml, is also described.     

Two-dimensional liquid chromatography/mass spectrometry set-up for structural elucidation of metabolites in complex biological matrices

For absorption, distribution, metabolism and excretion (ADME) studies of drug candidates, mass spectrometry (MS) has become an indispensable tool for the characterization of biotransformation pathways. Samples from in vivo animal studies such as plasma, tissue extracts or excreta contain vast amounts of endogenous compounds. Therefore, the generation of metabolite patterns requires dedicated sample pre-treatment and sophisticated separation methods. Methodologies used for metabolite separation are often inappropriate for structure elucidation. Therefore, a two-dimensional liquid chromatography (LC) approach in combination with MS was developed. Study samples were analyzed using high-performance liquid chromatography (HPLC) for the generation of a qualitative and quantitative metabolite pattern (first dimension) with high reproducibility and recovery without extensive sample pre-treatment. Selected radioactive metabolite fractions were then applied to micro-HPLC with off-line radioactivity monitoring and subsequent MS detection (second dimension). Applying the two-dimensional HPLC/MS approach not only major metabolites could be identified, even minor and trace metabolites were characterized. The usage of sampled metabolite fractions allowed also the re-analysis of specific metabolites for additional investigations (e.g. H/D exchange experiments or product ion scanning experiments). It could be clearly shown that the two-dimensional HPLC/MS approach showed mass spectra with higher sensitivity and selectivity significantly improving the characterization of minor and trace metabolites in in vivo ADME studies.     

Prediction of clozapine metabolism by on-line electrochemistry/liquid chromatography/mass spectrometry

Combining electrochemical conversion, liquid chromatography and electrospray ionization mass spectrometry (EC/LC/ESI-MS) on-line allows the rapid identification of possible oxidation products of clozapine (CLZ) in the absence and in the presence of glutathione. CLZ is, depending on the applied potential, oxidized to various products in an electrochemical flow-through cell using a porous glassy carbon working electrode. Several hydroxylated and demethylated species are detected on-line using LC/MS. While hydroxy-CLZ is most abundant at a potential of 400 mV, demethylation occurs more readily at higher potentials (at around 700 mV versus Pd/H₂ reference). In the presence of glutathione (GSH), various isomeric glutathione adducts and respective products of further oxidation can be identified. The thioadducts are characterized by tandem MS. Mono-GSH and bis-GSH derivatives can be seen in the chromatograms. The results correlate well with the cyclic voltammetric profile of CLZ. The data are relevant from a pharmacological point of view, since similar metabolites (phases I and II) have been reported in the literature. The EC/LC/MS and EC/MS methods should be valuable tools that can be used to anticipate and understand the metabolization patterns of molecules of pharmacological interest and to point out reactive intermediates.     

Study of the tautomerization of quinonoid dihydropterins by liquid chromatography/electrochemistry

An investigation of the tautomerization of quinoid dihydropterins by dual-electrode liquid chromatography/electrochemistry (l.c.e.c.) is presented. The effect of various side-chains in the 6-position of the pterin revealed that wide variations in the rate of tautomerization occur with different pterin species. The effects of two buffers (phosphate and acetate) with low pK_a values were also studied over the pH range 2–8. The rate of tautomerization was much faster for quinonoid dihydrobiopterin in the phosphate buffer than in the acetate buffer whereas the rate of rearrangement of the other quinonoid dihydropterins was little affected by the type of buffer. This report also illustrates the advantages of l.c.e.c. for this type of investigation. Variations in pH are readily studied without changes in experimental conditions. Species that cannot be resolved chromatographically are shown to be resolvable by the electrochemical detector. Stability to rearrangement can be compared to stability to oxidation. Finally, only very little sample is required.     

Comparison of different liquid chromatography methods for the determination of corticosteroids in biological matrices

Various extraction techniques can be combined with column liquid chromatography (LC) and ultraviolet (UV) or mass spectrometric (MS) detection for the determination of synthetic corticosteroids in biological matrices. Target analysis of low concentrations of 25 microg/kg of dexamethasone in feed can be performed by combining immunoaffinity chromatography (IAC) and LC with UV detection. A straightforward multi-analyte procedure is obtained by tandem solid-phase extraction (SPE) and subsequent LC-UV. However, the limits of detection for feed samples are then relatively poor, viz. 100 microg/kg. A multi-analyte method which meets modern demands of about 5 microg/kg detection limit requires one-step SPE combined with LC-MS analysis. As regards urine corticosteroids can be determined down to a level of 0.5 microg/l by either SPE-LC-MS- MS or SPE(IAC)-LC-MS.     

Coupled high-performance liquid chromatography-gas chromatography for the determination of pesticide residues in biological matrices

A fully automated high-performance liquid chromatography-gas chromatography (HPLC-GC) network is described. A ten-port valve set up as a loop type LC-GC interface allowed the transfer of large LC effluent fractions into the gas chromatograph by concurrent solvent evaporation. The system performed highly efficient sample enrichment and clean up by LC and on-line GC separation with sensitive electron-capture detection. The efficiency of the system was demonstrated by application to the trace analysis of N-(3-chloro-2,6-dimethylphenyl)-N-(2-oxotetrahydrofuranyl)-2-me thoxyacetamide (CGA 80000) in various crops and soil samples. The residue level determined was 0.02 mg/kg for crop samples and 0.01 mg/kg for soil samples. The relative standard deviations of the calibration graphs were in the range 2-5%; the mean recovery was greater than 85%.     

High-performance liquid chromatography packing materials for the analysis of small molecules in biological matrices by direct injection

The increasing demand on high-performance liquid chromatography to resolve mixtures of closely related components in complex biological matrices in less time with higher precision has led to the development of a variety of new high-performance liquid chromatography columns, which eliminate the need for sample preparation. These packings isolate small molecules from biological macromolecules on direct sample injection by exerting two separation mechanisms. They allow elution of all sample macromolecules with high recovery in one peak at the extraparticulate void, because of size-exclusion interactions with hydrophilic outer particulate surfaces. Simultaneously, these packings allow permeation and partitioning of small molecules on bonded-phases which are protected from contamination by macromolecules. The names given to these new packings include "internal surface reversed-phase", "shielded hydrophobic phase", "semipermeable surface", "dual zone material" and "mixed-functional phases". The fundamental principles behind each of the design concepts are reviewed, and applications are cited.     

Derivatization reagents in liquid chromatography/electrospray ionization tandem mass spectrometry

Liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) is one of the most prominent analytical techniques owing to its inherent selectivity and sensitivity. In LC/ESI-MS/MS, chemical derivatization is often used to enhance the detection sensitivity. Derivatization improves the chromatographic separation, and enhances the mass spectrometric ionization efficiency and MS/MS detectability. In this review, an overview of the derivatization reagents which have been applied to LC/ESI-MS/MS is presented, focusing on the applications to low molecular weight compounds.     

Determination of prostaglandins and thromboxane in whole blood by high-performance liquid chromatography with fluorimetric detection

A highly sensitive and specific assay for the quantitation of prostaglandins (PGs) such as PGE1, PGE2, PGF1 alpha, PGF2 alpha, 6-keto-PGF1 alpha, and including thromboxane B2, is described. The method involves the addition of PGF1 alpha and PGE1 as the internal standards, extraction from whole blood and purification by silica gel column chromatography. Following conversion into the methoximes, purification by reversed-phase chromatography and esterification with panacyl bromide, samples are analysed by high-performance liquid chromatography with fluorimetric detection. The lower limit of detection of the eicosanoids 6-keto-PGF1 alpha, thromboxane B2 and PGF2 alpha in blood is ca. 50 pg/ml and that of PGE2 is 100 pg/ml. Assay linearity is demonstrated over a range from 60 pg to 60 ng of eicosanoid injected. The method allows simultaneous assessment of prostaglandins and thromboxane extracted from complex biological fluids at picogram levels.     

Analysis of leukotrienes by liquid chromatography/electrochemistry after conversion to dinitrobenzoate derivatives

A sensitive liquid chromatography method has been developed using electrochemistry for the determination of leukotrienes in biological fluids. Biological specimens are treated with 3,5-dinitrobenzoyl chloride in acetonitrile which undergoes rapid reaction with hydroxyl groups of non-peptidic leukotrienes in the presence of pyridine and with amino groups of peptidic leukotrienes in the presence of potassium tetraborate buffer. The resulting dinitrobenzoate derivatives of leukotrienes are highly electroactive, suitable for reduction or oxidation at moderate potentials by an electrochemical detector. In reductive mode at -0.7 V or oxidative mode at +1.15 V potentials, the lower limits of detection for leukotriene derivatives were approximately 8 +/- 3 pg and 70 +/- 16 pg respectively, with a signal-to-noise ratio of 5 to 1. This method was applied to the detection of leukotrienes in plasma, nasal and bronchial fluids of patients with asthma.     

Voltammetric/amperometric detection for liquid chromatography

A voltammetric/amperometric detector based on a dual-electrode electrochemical detector is described for liquid chromatography. The detector combines the advantages of both voltammetric and amperometric detection. A three-dimensional data array of current response as a function of both time (chromatographic domain) and potential (electrochemical domain) is obtained. From the chromatographic point of view, this allows post-experimental choice of the optimal detection potential. Different detection potentials can even be chosen for each chromatographic peak. Having the voltammetric data as well as the chromatographic data provides ready identification of chromatographically unresolved compounds and the ability to resolve such co-eluting compounds voltammetrically. The voltammetric data also provide a second method of peak identification for greater certainty in peak assignments. Voltammetric detection limits of less than 10 pmol of material injected on the column were achieved with this detection method. From the electrochemical perspective, voltammetric/amperometric detection provides a technique for obtaining hydrodynamic voltammograms with small amounts or small volumes of sample. Voltammograms can also be obtained for the individual components of complex mixtures without the need for isolation steps.     

On-line electrochemistry/liquid chromatography/mass spectrometry for the simulation of pesticide metabolism

On-line electrochemistry/liquid chromatography/mass spectrometry (EC/LC/MS) was employed to mimic the oxidative metabolism of the fungicide boscalid. High-resolution mass spectrometry and MS/MS experiments were used to identify its electrochemical oxidation products. Furthermore, the introduction of a second electrochemical cell with reductive conditions provided important additional information on the oxidation products. With this equipment, hydroxylation, dehydrogenation, formation of a covalent ammonia adduct, and dimerization were detected after initial one-electron oxidation of boscalid to a radical cation. On-line reaction with glutathione yielded different isomeric covalent glutathione adducts. The results of the electrochemical oxidation are in good accordance with previously reported in vivo experiments, showing that EC/LC/MS is a useful tool for studying biotransformation reactions of various groups of xenobiotics.     

Determination of phenols in environmental samples by liquid chromatography – electrochemistry

A reverse-phase high-performance liquid chromatography procedure with gradient elution and electrochemical detection is described for the determination of phenolic compounds, including several priority pollutant chlorophenols, in sea-water and sediments. In addition, a method for concentrating phenols from sea-water was examined. A solid-phase extraction using a derivatized poly(styrene-divinylbenzene) copolymer is discussed. The recovery, repeatability and detection limits are shown. Electrochemical detection provided selectivity as well as sensitivity. Phenols at the ng/l level were detected. The method was applied to the analysis of the most important phenolic compounds in sea-water and marine sediments.     

Determination of glutathione in biological material by high-performance liquid chromatography with electrochemical detection

Glutathione in biological samples is extracted by perchloric acid and separated by ion-paier chromatography on a RP-18 phase. In a post-column reaction, glutathione is converted to an isoindole derivative by reaction with o-phthalaldehyde and detected at a galssy carbon electrode at 800 mV v. Ag/AgCl/3 M KCl. The detection limit is 40 pmol of glutathione injected.     

Analysis of erythromycin in biological fluids by high-performance liquid chromatography with electrochemical detection

A simple and sensitive high-performance liquid chromatographic assay was developed for the quantitative determination of major erythromycin components and their potential metabolites or degradation products in plasma and urine. An ether extract of alkalized plasma sample was chromatographed on a reversed-phase column and the components in the column effluent were monitored by an electrochemical detector. The recovery of the drug from extraction was virtually 100%. The detection limits for erythromycin A in plasma were 5-10 ng/ml and 30 ng/ml using 1 and 0.2 ml of sample, respectively. For urine samples, a simple one-step deproteinization with two volumes of acetonitrile was satisfactory for analysis. The method has been evaluated in plasma and urine from dogs receiving oral or intravenous erythromycin A. The standard curves for potential metabolites or degradation products were not constructed due to the lack of sufficient samples.