Biomedical applications of thermospray liquid chromatography-mass spectrometry

Thermospray liquid chromatography-mass spectrometry has been applied in the solution of a number of problems of biological and biomedical interest. These include the analysis of phenazines from the Gram negative bacterium Pseudomonas aeruginosa, steroids released by rat adrenals and eicosanoids generated by human inflammatory cells. The application of the technique to leukotrienes in blood is discussed. Isotopic labelling prior to analysis, to facilitate identification and structure elucidation is outlined with reference to the steroids.     

Glutathione oxidation in real time by thermospray liquid chromatography-mass spectrometry

The oxidation and reduction of glutathione and oxidized glutathione were studied in real time by liquid chromatography-mass spectrometry during exposure to hydrogen peroxide and mercaptoethanol. By mass spectrometry mixed disulfides and both reversible and irreversible oxidations of sulfur to higher states (sulfinic and sulfonic acids) were directly observed during exposure to hydrogen peroxide. The irreversible oxidation of glutathione to glutathione sulfonic acid could be detected after 30 min exposure of glutathione to 40 mM H2O2 at 20 degrees C. A peak consistent with glutathione-sulfinic acid was transiently present, suggesting this compound behaved as an oxygen consuming antioxidant. Liquid chromatography-mass spectrometry appears to be an excellent method to study oxidation and reductions of sulfur containing peptides and amino acids.     

Structure elucidation of drug metabolites using thermospray liquid chromatography-mass spectrometry

Thermospray liquid chromatography-mass spectrometry (LC-MS) has been used to provide structural information both from in vitro and in vivo experiments. This paper will describe the more salient aspects of the technique that have emerged. The ability of the interface to handle gradients was essential for its successful application to metabolism studies, owing to the wide range of compound polarity involved. The examples discussed in this paper include the use of LC-MS in the analysis of in vitro incubations of drugs with hepatocyte cell cultures and the direct analysis of plasma samples from in vivo studies in the dog.     

Evaluation of eluents in thermospray liquid chromatography-mass spectrometry for identification and determination of pesticides in environmental samples.

The influence of different eluents in positive and negative ion mode thermospray liquid chromatography-mass spectrometry was studied with several groups of pesticides, including carbamates, chlorotriazines, phenylureas, phenoxy acids and organophosphorus and quaternary ammonium compounds, and the corresponding degradation products. Using the positive ion mode in combination with reversed-phase eluents the base peaks generally corresponded either to [M + H]+ for the chlorotriazines and their hydroxy metabolites or to [M + NH4]+ for the carbamates, the phenylureas, the organophosphorus pesticides and their oxygen analogues. In the negative ion mode different processes such as (dissociative) electron-capture and anion attachment mechanisms occurred. Fragment ions such as [M - CONHCH3]- for the carbamates, [M - H]- for the chlorotriazines, phenylureas and chlorinated phenoxy acids and [M].-, [M - R]- (R being a methyl or ethyl group) for organophosphorus pesticides were usually formed. Depending on the eluent additive used (ammonium acetate, ammonium formate and/or chloroacetonitrile), three different adduct ions were formed: [M + CH3COO]-, [M + HCOO]- and [M + Cl]-. Normal-phase eluents with cyclohexane, n-hexane and/or dichloromethane provided more structural information and enhanced the response of several compounds. The positive ion mode was useful for the detection of chlorinated phenoxy acids and chlorophenols which could not be detected in the positive ion mode using reversed-phase systems. The base peaks generally corresponded to [M].+, [M + H]+ or [M - Cl]+. For the characterization of difenzoquat, a quaternary ammonium pesticide of which trace level analysis is troublesome, a post-column ion-pair extraction system was used. An aqueous mobile phase with a sulphonate-type counter ion was applied and an extraction solvent containing cyclohexane-dichloromethane-n-butanol (45:45:10) was used in thermospray liquid chromatography-mass spectrometry. Illustrative examples of the determination of residue levels of pesticides in soil matrices are shown.     

Analysis of tropane alkaloids with thermospray high-performance liquid chromatography-mass spectrometry

A thermospray high-performance liquid chromatography-mass spectrometry method for analysis of hyoscyamine and scopolamine in plant cell culture samples is described. The alkaloids were separated on a polymeric reversed-phase column with an alkaline ammonium acetate buffer-acetonitrile eluent. Selected-ion recording of the protonated molecular ions was used for quantitation of the compounds. The compounds were fragmented by discharge-assisted ionization and elevated thermospray capillary temperatures or ion repeller potentials.     

Evaluation of the potential of thermospray liquid chromatography-mass spectrometry in neurochemistry

Optimized operating conditions previously developed for the determination of neuroactive indoleamines and metabolites were adapted to meet the requirements of thermospray liquid chromatography-mass spectrometry (LC-MS) in terms of the ammonium acetate buffer system needed in this technique. Mass spectra were obtained for nineteen indolic compounds in both the positive and negative ion modes. The positive thermospray mass spectra of indoles with a free primary amino group are characterized by the base peak at [M + H]+, whereas the alcohol and acid metabolites show the base peak at [M + NH4]+. In the negative mode only amino acids and acids give good mass spectra with base peaks at [M - H + ACOOH]-. Detection limits by selected ion monitoring were of the order of 50-100 pg SIM on-column, allowing the direct determination of endogenous serotonin in an extract from rat hypothalamus. Quantitation was performed by isotope dilution MS. In the same way 5-hydroxyindoleacetic, indoleacetic, indolepropionic and indolelactic acids in urine were directly determined in an ethyl acetate extract from acidified urine samples. Likewise, gamma-aminobutyric acid and tricyclic antidepressants gave detection limits of 10 pg whereas only nanogram sensitivity could be achieved with catecholamines.     

Analysis of candidate anticancer drugs by thermospray high-performance liquid chromatography-mass spectrometry

Thermospray high-performance liquid chromatography-mass spectrometry was used to confirm the identity of five bulk anticancer drugs, and in some cases, to identify drug impurities. Analysis resulted in both molecular weight and structural (fragment ions) information obtained from the full scan spectra of as little as 50 ng of each drug. The technique was also used to evaluate the chromatographic specificity of corresponding ultraviolet or refractive index high-performance liquid chromatographic detection in the presence of drug degradation products.     

Characteristic fragmentation of thromboxane B2 in thermospray high-performance liquid chromatography-mass spectrometry

Cyclo- and lipo-oxygenase metabolites of arachidonic acid have been reported to have very simple thermospray mass spectra. However, thromboxane B2 (TXB) in ammonium acetate-methanol and at interface temperatures below the point of total vaporization shows a mass spectral pattern characterized by abundant ions at low masses. The more abundant TXB fragment ions have been characterized as adducts from four principal fragments with molecular masses of 156, 170, 196 and 326. Positive- and negative-ion mass spectra, and mass spectra obtained with an alternative thermospray buffer (butylammonium acetate) support the molecular masses of these fragments. A tentative assignment of the fragments can be made by comparison of the thermospray mass spectra of TXB, 2,3-dinor-TXB and some of their methyl ester and methyl oxime derivatives. Interface temperature and solvent composition effects on the fragmentation, as well as in the electron-capture processes observed when working in the filament-on mode, are discussed. Fragmentation mechanisms can be related to those observed for monosaccharides, and imply retro-Diels-Alder as well as retroaldolic condensation-type rearrangements.     

Identification of by-products in phenylisocyanate pre-column derivatization by thermospray liquid chromatography-mass spectrometry

Summary Thermospray liquid chromatography-mass spectrometry has been applied to the identification of by-products in precolumn derivatization by phenylisocyanate. These byproducts are eluted early in the same chromatographic region as the low molecular weight derivatives and were located by chromatographic analysis of a blank sample. Their identification would offer further qualitative information in the use of phenylisocyanate as a derivatizing agent. Five compounds resulted from the reaction of phenylisocyanate and the reaction medium were identified: two from a reaction between phenylisocyanate and methanol, two from the reaction between phenylisocyanate and water, and one from the polymerisation of phenylisocyanate.     

Determination of eicosanoids, phospholipids and related compounds by thermospray liquid chromatography-mass spectrometry

Thermospray mass spectrometry has proven to be a useful technique for analyzing various biological compounds including eicosanoids and phospholipids. Molecular ions as well as fragment ions which reveal useful structural information are produced for underivatized eicosanoids and phospholipids using filament-off or filament-on thermospray mass spectrometry, respectively. In conjunction with on-line chromatographic separation, complex mixtures of biological samples can be rapidly analyzed with great reliability. Data will be presented concerning the analysis of prostaglandins, other eicosanoids and molecular species of phospholipids as well as the application of these methodologies to complex biological samples.     

Analysis of candidate anticancer drugs by thermospray high-performance liquid chromatography-mass spectrometry and by high-resolution mass spectrometry

Thermospray high-performance liquid chromatography-mass spectrometry (TSP-HPLC-MS) and direct probe high-resolution MS was used to analyze four candidate anticancer drugs. The techniques were used to confirm the identity of the bulk drug and to identify impurities. Analysis by TSP-HPLC-MS resulted in molecular weight information from the separated components using as little as 50 ng of each drug. The high-resolution direct probe MS analysis provided additional structural information and possible empirical formulas for the parent drugs and their impurities. The use of both of these complimentary techniques proved to be very specific for the detection of the anticancer drugs and for postulating the identity of impurities.     

Planar solid phase extraction--a new clean-up concept in multi-residue analysis of pesticides by liquid chromatography-mass spectrometry

Efficient clean-up is indispensable for preventing matrix effects in multi-residue analysis of pesticides in food by liquid and gas chromatography coupled to mass spectrometry. As a completely new approach, highly automated planar chromatographic tools were applied for powerful clean-up, called high-throughput planar solid phase extraction (HTpSPE). Thin-layer chromatography (TLC) was used to completely separate pesticides from matrix compounds and to focus them into a sharp zone, followed by extraction of the target zone by the TLC-MS interface. HTpSPE resulted in extracts nearly free of interference and free of matrix effects, as shown for seven chemically representative pesticides in four different matrices (apples, cucumbers, red grapes, tomatoes). Regarding the clean-up step, quantification by LC-MS provided mean recovery (against solvent standards) of 90-104% with relative standard deviations of 0.3-4.1% (n=5) for two spiking levels of 0.1 and 0.5 mg/kg. Clean-up of one sample was completed in a manner of minutes, while running numerous samples in parallel at reduced costs, with very low sample and solvent volumes.     

Direct analysis of the major human seminal prostaglandins by thermospray high-performance liquid chromatography-mass spectrometry

Thermospray high-performance liquid chromatography-mass spectrometry (HPLC-MS) can be a powerful tool for characterizing eicosanoids in complex biological samples. The positive ion spectra obtained from primary prostaglandins such as PGE1 PGE2, 19-OHPGE1, 19-OHPGE2, PGF2 alpha, PGD2, 6-keto-PGF1 alpha and from leukotriene B4 are very simple, with base peaks corresponding to ions arising from the loss of H2O from the (M + H)+ and (M + NH4)+ ions, except for PGB2 and PGF2, where the latter two ions predominate. The application of this technique to the concurrent determination of the E1 and E2 prostaglandins and their 19-hydroxylated derivatives in human semen is described. The technique affords a moderate level of sensitivity (5-20 ng on-column) and excellent specificity so that virtually no sample manipulation is required other than dilution in acetone and centrifugation. The clear supernatant is injected directly into the HPLC-MS system. A similar analysis by either gas chromatography (GC) or GC-MS would need multi-step derivatization, thus increasing the sample manipulation required and the total analysis time.     

Detection of ribose-methylated nucleotides in enzymatic hydrolysates of RNA by thermospray liquid chromatography-mass spectrometry.

A procedure has been developed for the detection and characterization of ribose-methylated dinucleotides of the type NmpN' in enzymatic digests of RNA. Differences in reaction products from hydrolysis using RNase T2, which will not cleave NmpN', and hydrolysis by nuclease P1 are analyzed by thermospray liquid chromatography-mass spectrometry. The method is applicable to dinucleotides present in nanogram range quantities and is suited for the characterization of new or unexpected ribose-methylated nucleotides for which chromatographic mobilities are not known.     

Identification of trichothecenes by thermospray, plasmaspray and dynamic fast-atom bombardment liquid chromatography-mass spectrometry

Thermospray, plasmaspray and dynamic fast-atom bombardment liquid chromatography-mass spectrometry are compared for the identification of six trichothecenes. Thermospray spectra of the trichothecenes exhibit only a very abundant ammonium adduct ion. Plasmaspray, which provides a more energetic ionization process than thermospray, produces some fragment ions in addition to an abundant ammonium adduct ion. The spectra obtained by dynamic fast-atom bombardment exhibit a protonated molecule, a glycerol adduct ion and numerous fragment ions formed by the losses of functional groups as neutrals in various combinations. Thermospray and plasmaspray are suitable only for monitoring of the trichothecenes, whereas dynamic fast-atom bombardment is suitable for monitoring and for structure characterization.     

Application of thermospray liquid chromatography-mass spectrometry for the analysis of chloramphenicol and three related compounds

The thermospray (TS) liquid chromatographic-mass spectrometric analysis of the antibiotic chloramphenicol and three related compounds is presented. The three additional compounds are dehydrochloramphenicol, aminodehydrochloramphenicol, and nitrophenylaminopropanediol. Baseline separation of the four compounds is achieved. The TS mass spectrum of each of the four compounds includes a prominent [MH]+ ion plus some fragment ion peaks.     

Evaluation of an automated thermospray liquid chromatography-mass spectrometry system for quantitative use in bioanalytical chemistry.

An automated thermospray liquid chromatography-mass spectrometry system is described, including an autosampler and a gradient liquid chromatography system controlled from the mass spectrometer data system. The performance and reliability of the equipment during unattended operation were evaluated by repeated injections of standard solutions of some antiasthmatic drugs, using deuterium-labelled analogues as internal standards. High sensitivity and reproducibility were achieved during a 19-hour run, incorporating gradient elution and a total of 54 injections. The relative standard deviation of the peak area measurement of the internal standards was in the range of 6.5-8.2%. The corticosteroid budesonide can be routinely measured in plasma down to 0.1 nmol/l. Direct injection of a small plasma volume into the thermospray liquid chromatography-mass spectrometry system could be used to monitor drug plasma levels during a toxicity study in dogs.     

Influence of reversed- and normal-phase liquid chromatographic eluents in the fragmentation of selected chlorinated herbicides in thermospray liquid chromatography-mass spectrometry

The effect of two completely different mobile phase compositions, reversed-phase acetonitrile-water + ammonium acetate and normal-phase cyclohexane, were compared in filament-on thermospray liquid chromatography-mass spectrometry (LC-MS) for the determination of selected chlorinated herbicides such as chloroatrazines and chlorinated phenoxyacetic acids. By using acetonitrile-water + 0.05 M ammonium acetate mixtures in positive ion mode thermospray LC-MS, the chloroatrazine herbicides showed the acetonitrile adduct ion [M + (CH₃CN)H]⁺ as the base peak, whereas the chlorinated phenoxyacetic acids showed no signal. In contrast, when cyclohexane, which is reported for the first time as an eluent in the thermospray technique, was used as the mobile phase the chlorinated phenoxyacetic acid herbicides exhibited [M – H]⁺, [M – Cl]⁺ and M⁺˙ as the main ions. Negative ion mode thermospray LC-MS showed [M – H]^? as the base peak for the chloroatrazines in the different mobile phases, whereas the chlorinated phenoxyacetic acids exhibited [M + H]^?, [M + Cl]^? or [M – HCl]^? as the base peaks in cyclohexane and [M + acetate]^? in acetonitrile-water-ammonium acetate.     

Determination of pesticides in compost by pressurized liquid extraction and gas chromatography-mass spectrometry

A gas chromatographic-mass spectrometric method was developed for the determination of pesticides in compost. The investigated pesticides included two fungicides, two herbicides and 10 insecticides. The pesticides were extracted from the compost by pressurized liquid extraction. The extract was cleaned up by a partition between hexane and acetonitrile followed by a dispersive solid-phase extraction using a porous carbon made from Moso bamboo (Phyllostachys pubescens). The overall recoveries were 81-104% and the relative standard deviations (RSDs) ranged from 2.4 to 12%. The minimum detectable concentrations were 0.02-0.04 microg g(-1). This method was successfully applied to a compost sample from food waste as well as commercial compost.     

Comparison of microextraction procedures to determine pesticides in oranges by liquid chromatography-mass spectrometry

A liquid chromatographic–mass spectrometric method has been developed for the determination of bitertanol, carbendazim, fenthion, flusilazole, hexythiazox, imidacloprid, methidathion, methiocarb, pyriproxyfen and trichlorfon. Two procedures, based on stir bar sorptive extraction (SBSE) and matrix solid-phase dispersion (MSPD), have been evaluated for the extraction of these compounds in oranges. Their respective advantages and disadvantages are also discussed. The recoveries obtained by MSPD ranged from 47 to 96% and the relative standard deviations (RSDs) ranged from 1 to 15%, whereas with the SBSE method the recoveries were between 8 and 84% and the RSDs between 4 and 16%. Although, the limits of quantitation of most compounds are much better (0.001–0.05 mg kg⁻¹) by SBSE, it is not suitable to determine some polar pesticides as carbendazim, imidacloprid and trichlorfon. Results obtained by both methods were compared, in terms of sensitivity and selectivity, with a classical ethyl acetate extraction method, and the three methods were applied to analyze real samples. As MSPD is easier to perform, faster than the organic solvent extraction, and shows equal accuracy and resolution, its application for analyzing pesticides in oranges is recommended.