Thermospray Liquid Chromatography-mass Spectrometry Research Articles

Confirmation of Residual Oxolinic Acid, Nalidixic Acid and Piromidic Acid in Fish by Thermospray Liquid Chromatography-Mass Spectrometry

Abstract A thermospray high-performance liquid chromatographic-mass spectrometric (TSP LC-MS) method has been developed for analysis of pylidonecarboxyl ic acid (quinolone) antibacterials such as nalidixic acid (NA). oxolinic acid (OXA) and piromidic acid (PMA). The LC separation was carried out on an Inertsil ODS-2 (150 × 4.6m i.d.) using 0. 1M ammonium acetate buffer(pH 4. 5)-acetonitrile (6:4) as the mobile phase at a flow rate of 0.8 ml/min. The vaporizer temperature was 165 °C and the ion source block temperature was 260 °C. The mass spectra obtained from NA, OXA and PMA were very simple, with base peaks corresponding to the protonated molecule, MH+. The calibration curve for NA was rectilinear from 1 to 20 ng with a detection limit by selected ion monitoring (SIM) of about 100 pg (signal to noise ratio of 2).

Determination of ceftiofur and its metabolite desfuroylceftiofur in bovine serum and milk by ion-paired liquid chromatography

A simple and sensitive liquid chromatographic method has been developed for the simultaneous determination of ceftiofur and its metabolite desfuroylceftiofur in bovine serum and milk. The method involved an ultrafiltration of diluted serum/milk with an equal volume of 50% acetonitrile through a 10 000 dalton molecular mass cut-off filter. Separation of ceftiofur and desfuroylceftiofur from the other serum/milk components was performed by ion-paired (octane and dodecanesulfonate) liquid chromatography using a reversed-phase column eluted with acetonitrile—water solution. The ultraviolet—visible absorbance of the column effluent was monitored in 200–350 nm range of a photodiode-array detector or at λ max 289.6 nm for ceftiofur, λ max 265.8 nm for desfuroylceftiofur and λ max 271.4 nm for dimer of desfuroylceftiofur. Recoveries of ceftiofur from bovine milk spiked with 1 and 10 μg/ml were 95.9 and 97.0% with coefficients of variation of 3.69 and 2.51%, respectively. Recovery of ceftiofur from bovine serum spiked with 10 μg/ml was 90.4% with a coefficient of variation of 5.29%. A correlation coefficient of 0.9992 occurred with ceftiofur in aqueous solutions ( n = 5, in duplicates). The limit of detection was estimated to be approximately 50 ppb (ng/ml). Additionally, this paper documents the presence of a ceftiofur metabolite in bovine serum under in vitro and in vivo conditions. The metabolite was identified as desfuroylceftiofur together with its dimer 3,3'-desfuroylceftiofur disulfide by thermospray liquid chromatography—mass spectrometry.

Determination of aquatic photodegradation products of selected pesticides by gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry

The photodegradation of the organophosphorus pesticide fenitrothion and the chlorotriazine herbicide propazine has been investigated under different light conditions. The photodegradation of propazine was carried out using a xenon lamp in the suntest apparatus whereas a high-pressure mercury lamp was used for fenitrothion. For propazine, experiments were carried out in distilled water and artificial seawater containing humic acids at 44°c, whereas for fenitrothion a water-methanol (5:1) solution at 20°C was employed. Identification of the sufficiently volatile photodegradation products was achieved by gas chromatography-mass spectrometry (GC-MS) with electron impact (El), whereas for the direct characterization of the hydroxy metabolite of propazine thermospray liquid chromatography-mass spectrometry (LC-MS) is required. The photodegradation pathways of the pesticides in water included dealkylation, deamination, dehalogenation, hydroxylation, isomerization and solvolysis.

A thermospray mass spectrometric assay for Fe-induced 4-hydroxynonenal in tissues.

A method is presented for the determination of 4-hydroxynonenal (HNE) in tissue homogenates following in vitro lipid peroxidation induced by iron (Fe++). HNE is measured as the pentafluorobenzyl oxime derivative using liquid chromatography thermospray mass spectrometry. In vitro metabolism of HNE via the glutathione/glutathione-S-transferase pathway was inhibited using iodoacetic and iodobenzoic acids. The assay has been used as an indicator of the peroxidizability of tissue samples from animals both adequate in and depleted of alpha-tocopherol. The concentrations of HNE produced in tissues taken from animals depleted of alpha-tocopherol were found to be up to 8 times higher than those taken from animals supplemented with alpha-tocopherol.

Determination of paraquat and diquat by liquid chromatography-thermospray mass spectrometry

Thermospray liquid chromatography-mass spectrometry can be used for identifying and determining both paraquat (C 12H 14N 2+ 2) and diquat (C 12H 12N 2+ 2). Reversed-phase liquid chromatography was performed using a 15-cm Shim-pack CLCODS column, with methanol-water (80–20) + 0.1 M ammonium acetate (adjusted to pH 5 with trifluoroacetic acid) as buffers, at a flow-rate of 1.0 ml/min. The mass spectral sensitivity was best when the temperatures of the vaporizer, block and tip heater of the ion source block were set at paraquat and diquat, respectively. Detection limits by selected ion monitoring were of the order of 20 ng ( S/ N = 3.5). The mass spectra are influenced by temperature and therefore, precise temperature contorl is essential.

An investigation of the compounds produced by spray-drying an aqueous solution of glucose and glycine

Spray-drying an aqueous solution of glucose and glycine results in the production of a brown powder that has an aroma reminiscent of chocolate powder. In this study, volatile components of the brown powder were extracted by Grob headspace stripping or solvent extraction techniques and analysed by gas chromatography-mass spectrometry (GCMS) or, in specific instances, gas chromatography fourier transform infrared detection (GC-FTIR). Solvent extracts were also derivatised with diazomethane to increase the amenability of many compounds to study by gas chromatography (GC) techniques. These approaches yielded the identification of a range of oxygen and nitrogen heterocyclic compounds. In further experiments, coloured compounds that were extracted into an organic solvent were separated by high performance liquid chromatography (HPLC) and detected at a wavelength of 430 nm. Thermospray liquid chromatography-mass spectrometry (LCMS) techniques were also employed to measure the molecular mass of detected coloured species. Furthermore, desorption mass spectrometry was used to record an electron impact spectrum of a coloured compound that had been fractionated by HPLC. These data indicated the detection of this compound in GCMS and GC-FTIR studies and have led to a postulated identity of this and other related coloured species detected during these investigations.

High-performance liquid chromatography—thermospray mass spectrometry of ten sulfonamide antibiotics: Analysis in milk at the ppb level

Ten sulfonamide antibiotics including sulfanilamide (SNL), sulfamethazine (SMZ), sulfamethizole (SMTZ), sulfachloropyridazine and sulfaquinoxaline (SQX), were analyzed by thermospray (TSP) mass spectrometry on-line with a high-performance liquid chromatography—UV detection system. Except for the pairs SMZ—SMTZ and sulfadimethoxine—SQX, the standards were resolved in both the UV and TSP profiles. Co-eluting compounds could be differentiated in TSP by their different relative molecular masses. The [M + H]+ ion was the base peak for all the standards except SNL, which showed an [M + NH4]+ ion. Collision-induced dissociation of the [M + H]+ ions afforded daughter ion spectra characterized by common ions at m·z 92, 108 and 156, and ions derived from the amine substituent ([MH − 155]+). TSP detection limits [signal-to-noise ratio (S N) > 3] were below 20 ng (scan mode), 2 ng (selected reaction monitoring, daughter ions from [M + H]+) and 400 pg (selected ion monitoring). UV detection limits were ca. 2 ng (S N > 5). Results obtained from the multi-residue analysis of spiked cow milk samples at the low ng ml level are presented.

Determination of a new calcium antagonist and its main metabolite in plasma by thermospray liquid chromatography—mass spectrometry

A highly sensitive thermospray liquid chromatographic—mass spectrometric method has been developed for the simultaneous determination of FRC-8653 (I), a new calcium antagonist, and its main metabolite (M-4) in plasma. A deuterated analogue of I was added to the plasma as the internal standard. After the purification and concentration of the plasma sample on bonded-phase disposable columns, the extract was injected into the thermospray liquid chromatograph and analysed by selected-ion monitoring mass spectrometry. The calibration curves obtained were linear over the concentration range 0.5–100 ng/ml. The limits of quantification are 0.5 ng/ml for I and 1 ng/ml for M-4 in plasma, which are sufficient to evaluate plasma concentrations after oral administration to rats.

High-performance liquid chromatography—thermospray mass spectrometry of hydroperoxy polyunsaturated fatty acid acetyl derivatives

A method for the analysis of hydroperoxy polyunsaturated fatty acids was developed. The hydroperoxy groups were acetylated by acetic anhydride, and the mixture was partially purified on a Sep-Pak C 18 cartridge and analysed by high-performance liquid chromatography with thermospray mass spectrometry. Generally, the base ion, [M + H − n(60)] + or [M + H − n(60) − n(H 2O)] +, is produced through elimination of acetic acid or water ( n = number of hydroperoxy groups). The detection limit for these derivatives was ca. 1 pmol at concentrations of hydroperoxy polyenoic acids prior to derivatization. Using this method, many hydroxy and hydroperoxy polyunsaturated fatty acid derivatives could be detected simultaneously within 30 min on a selected-ion monitoring detection chromatogram without a gradient system. The assay was successfully applied to hydroxy and hydroperoxy polyunsaturated fatty acids from an incubation mixture of rat brain homogenate to which polyunsaturated fatty acids had been added.

Thermospray liquid chromatographic—mass spectrometric analysis of anti-AIDS nucleosides: quantification of 2',3'-dideoxycytidine in plasma samples

Thermospray liquid chromatography—mass spectrometry was investigated as a method for quantification of 2',3'-dideoxycytidine (DDC) from human plasma. A stable isotope analog of DDC ([ 15N 2, 2H 2]DDC) was used as an internal standard. Selected ion monitoring of the protonated molecular ions for DDC and the internal standard was used to record mass chromatograms. The areas of the peaks in the mass chromatograms were used for quantification. The detection limit of DDC in this assay was 50 pg on-column. The calibration curve was linear over the desired range, 0.25–20 ng/ml. The major advantages of this assay over others are: no derivatization, high sensitivity, high specificity and short assay time.

High-performance liquid chromatography-thermospray mass spectrometry of gibberellins

Seven tetracyclic monocarboxylic acid gibberellins (GA 1, GA 3, GA 4, GA 5 GA 7, GA 9 and GA 30 and the methyl ester derivatives GA 1, GA 3, GA 4, GA 7 and GA 9 were analyzed using combined high-performance liquid chromatography-thermospray mass spectrometry or direct injection thermospray mass spectrometry. The seven free acid GAs were resolved using a 25-min water-acetonitrile (0.1 M ammonium acetate) gradient mobile phase and a 5 μ, 150 mm × 4.6 mm, ODS column. Positive-ion thermospray mass spectra of these compounds typically showed intense [M + NH 4] + ions and few, if any, fragment ions. In the negative-ion mode (filament on) the free acid GAs showed [M - H] − or [M + HCO 2] − ions as the base peaks. Negative-ion spectra of the methyl ester GAs showed the [M + HCO 2] − ions, however, the base peaks were [M] −· or [M + HCO 2H] − ions attributed to electron capture processes. Thermos tandem mass spectra were obtained for GA 1, GA 3 and GA 30 from the collisionally activated dissociation (CAD) of their [M + NH 4] + ions. The CAD mass spectra show differences which allow the differentiation of the isomers GA 3 and GA 30 without the chromatographic separation. The daughter ions resulting from fragmentation processes corresponded to varying losses of CO, CO 2, ammonia and water.

Determination of ceftazidime in dolphin serum by liquid chromatography with ultraviolet—visible detection and confirmation by thermospray liquid chromatography—mass spectrometry

A simple and sensitive liquid chromatographic method has been developed for the determination of therapeutic levels of ceftazidime in dolphin serum. The method involved an ultrafiltration of diluted serum with an equal amount of acetonitrile—ethanol—water (40:40:20, v/v/v) through a 10 000 daltons molecular mass cut-off filter. Separation of ceftazidime from the other serum components was performed by ion-paired (dodecanesulfonate) liquid chromatography using a reversed-phase column eluted with acetonitrile—water solution. The ultraviolet absorbance of the column effluent was monitored in the 200–340 nm range of a photodiode-array detector or at 258.8 nm on a variable-wavelength ultraviolet—visible detector. Recoveries of ceftazidime from dolphin serum spiked with 20 and 2 μg/ml were 92.9 and 91.1% with coefficients of variation of 5.5 and 5.7%, respectively. A correlation coefficient of 0.9994 occurred with ceftazidime in aqueous solutions ( n = 6, in duplicates). The limit of detection for this antibiotic was estimated to be approximately 50 ppb (ng/ml). The unbound ceftazidime concentrations in dosed dolphin serum were determined to calculate the protein bindings of this antibiotic which yielded 32 ± 2%. The ceftazidime peak identity in dosed dolphin serum was confirmed by thermospray liquid chromatography—mass spectrometry. The thermospray mass spectrum of ceftazidime exhibited only the fragment ions, involving the opening of the β-lactam ring, at m/z 237, 255 and 315 when positive-ion detection mode was employed and the fragment ions at m/z 235, 253 and 313 when negative-ion detection mode was used.

Solvent degradation of cloxacillin in vitro: Tentative identification of degradation products using thermospray liquid chromatography-mass spectrometry

The determination of cloxacillin and the other penicillin antibiotics at trace levels in pharmaceutical samples and biological fluids and tissues is difficult. Degradation of penicillins occurs during exposure to chemicals and solvents used in sample extraction, storage of extracts and chemical analysis. One of the most important factors for the determination of cloxacillin in bovine milk and tissues is the choice of solvent used for extraction. Cloxacillin stored in different solutions underwent two types of degradation: hydrolysis with decarboxylation to yield cloxacillinpenilloic acids when water or aqueous solutions of acetonitrile or 2-propanol were used, and alcoholysis to form cloxacillinpenicilloic acid ester when methanol or ethanol was used. Cloxacillin stored in aqueous methanol or acetonitrile solution underwent faster degradation when stored in water or aqueous solutions of 2-propanol or ethanol. The solvent combinations that resulted in minimum sample degradation of cloxacillin were acetonitrile-ethanol-water (25:25:50) or ethanol-water (50:50). Degradation of cloxacillin was faster at the lower levels (500 and 50 ng/ml) of the antibiotic for all solvents tested. Nearly complete degradation of cloxacillin was observed at the 50 ng/ml level after 2 h at 20°C. Degradation was nearly non-existent at the 1 mg/ml level of the antibiotic in each solvent.

High-performance liquid chromatography—thermospray mass spectrometry of hydroxy-polyunsaturated fatty acid acetyl derivatives

A method for the determination of hydroxy-polyenoic acids has been developed. Hydroxy groups of hydroxy-polyunsaturated fatty acid were acetylated by acetic anhydride, and the mixture was partially purified on a Sep-Pak C 18 cartridge and analysed by high-performance liquid chromatography combined with thermospray mass spectrometry. Using this method, fifteen kinds of hydroxy-polyunsaturated fatty acid derivative could be detected simultaneously within 30 min on a selected-ion monitoring detection chromatograph without a gradient system. Generally, the base ion, [M + H − n(60)] +, is produced through elimination of acetic acid ( n = number of the hydroxy group of hydroxy-polyunsaturated fatty acid). The detection limit for these derivatives was ca. 0.2 pmol at the levels of hydroxy-polyenoic acids prior to derivatization. They could be analysed in the range 0.5–25 pmol. The assay was successfully applied to hydroxy-polyunsaturated fatty acids from an incubation mixture of rat brain homogenate to which polyunsaturated fatty acid was added.

Determination of taxol by high-performance liquid chromatography-thermospray mass spectrometry

A high-performance liquid chromatography-thermospray mass spectrometry method has been developed for the determination of taxol, found in the Taxus species. The compound is chromatographed by isocratic elution in 14 min and is quantitated by selected-ion recording of the protonated molecule. The method is linear over the range 1–1000 ng (1.2 pmol-1.2 nmol) of taxol per injection. The standard deviation of replicate bark samples ( n = 6) was 12.8%.

Synthesis and analysis of O,O'-dicarboxylate (dibenzyl) bispilocarpates as possible prodrugs of pilocarpine.

As a part of a series of studies to develop prodrug derivatives of pilocarpine, the O,O'-succinyl (dibenzyl), O,O-adipoyl (dibenzyl), O,O-fumaryl (dibenzyl), and O,O-terephthaloyl (dibenzyl) bispilocarpate fumarates were synthesized as a new class of pilocarpine prodrugs. The compounds were prepared from pilocarpic acid benzyl monoester by coupling two pilocarpic acid benzyl monoesters together with spacer chains by usual esterification methods. Liquid chromatography, thermospray liquid chromatography-mass spectrometry, high-resolution mass spectrometry, and NMR spectroscopy were applied to the identification and the purity evaluation of the synthetic products.

Bispilocarpic acid monoesters as prodrugs of pilocarpine: I. Preparation and identification

Several alkyl and aralkyl bispilocarpic acid monoesters were synthesized as a new class of pilocarpine prodrugs in order tc minimize the formation of pro-moiety per mole of active pilocarpine. The compounds were prepared from pilocarpic acid by the usual esterification methods. The yield of the synthesis varied from 34 to 95%. Liquid chromatography (LC), thermospray-liquid chromatography-mass spectrometry (TSP-LC-MS), electron ionization-mass spectrometry (EI-MS) and NMR spectroscopy were applied for the identification and purity evaluation of the synthetic products.

Reversed-phase high-performance liquid chromatography—thermospray mass spectrometry of radiation-induced decomposition products of thymine and thymidine

High-performance liquid chromatography-thermospray mass spectrometry was applied to the analysis of various radiation-induced decomposition products of thymidine including N-(2-deoxy-β- d- erythro-pentofuranosyl)formamide and the various diastereomers of 5,6-dihydroxy-5,6-dihydrothymidine, 5-hydroxy-5,6-dihydrothymidine and 5,6-dihydrothymidine. This method combines high sensitivity and product resolution, rendering it particularly useful for monitoring the formation of radiation-induced base damage within DNA.

The use of thermospray-liquid chromatography/mass spectrometry for the verification of chemical warfare agents

Thermospray-liquid chromatography mass spectrometry (TSP-LC-MS) is a relatively new analytical technique which proved to be useful for the verification of chemical warfare agents and their polar degradation products in aqueous solutions. The principles of the technique are described and comparisons are made with other forms of mass spectrometric analysis. A survey is presented of the results obtained so far at the Prins Maurits Laboratory TNO. The analysis of organophosphorus nerve agents and their hydrolysis products (organophosphorus acids) in various types of water is described. Special attention is paid to the nerve agent VX. Direct analysis of vesicants in water by TSP-LC-MS is limited. However, analysis of their hydrolysis products, as well as related compounds such as adducts of mustard gas with nucleosides and peptides, is possible. Finally, the use of TSP-LC-MS for the analysis of other compounds of chemical warfare interest (toxins) is indicated.

Separation et identification de l'acide formique et d'acides plurifonctionnels par chromatographie liquide couplée à la spectrométrie de masse avec détection d'ions négatifs

Thermospray liquid chromatography-mass spectrometry in the negative ion mode was used to identify the organic acids that could be formed by sugar degradations. The use of 0.01 M hydrochloric acid solution as a mobile phase allowed us to obtain (M − 1) − ions or (M + Cl) − ions, depending on the organic acid. It is important to emphazise that (M + Cl) − ions are particularly characteristic of the formic acid and lactoned polyhydroxy acids.