Thermospray Mass Research Articles

Determination of imidacloprid in vegetables by high-performance liquid chromatography with diode-array detection

An HPLC method is described for the determination of imidacloprid residues in vegetables at levels ranging from 0.01 to 0.60 mg/kg. The selection of the extraction and clean-up procedure is discussed. Spectral data obtained with diode-array detection allow the identification of imidacloprid residues. Thermospray mass spectrometric studies were carried out in combination with HPLC. The mean recoveries and standard deviations were 95% and 4.7%, respectively, in the various crops tested. Registration of the analytical results for a control sample in quality control charts demonstrated the performance of the method. Data for incurred residues of imidacloprid in vegetable samples routinely applying the proposed method are also presented.

Determination of Fumonisins B1 and B2 and Their Major Hydrolysis Products in Corn, Feed, and Meat, Using HPLC

A high-performance liquid chromatograph coupled to a fluorescence detector (excitation, 229 nm; emission, 442 nm) was used to analyze o-phthaldialdehyde-derivatized fumonisin B1 (FB1), fumonisin B2 (FB2), hydrolyzed FB1 (HFB1), hydrolyzed FB2 (HFB2), and partially hydrolyzed FB1 (PHFB1) from corn (1848 ng/g FB1 and 1092 ng/g FB2), feed (756 ng/g FB1 and 252 ng/g FB2), and spiked meat (720 ng/g FB1, 600 ng/g FB2, 960 ng/g HFB1, 840 ng/g HFB2) samples. Thermospray mass spectrometry was used to confirm the base hydrolysis products of FB1 and FB2. Chromatography was achieved on a C18, reversed-phase column. The mobile phases were acetonitrile/water/acetic acid mixtures set at A (40:59:1) and B (60:39:1), pumped in a gradient of 100% A to 100% B (9 min), and followed by a stepup to 100% acetonitrile (C) at the end of 17 min. The flow rate was 1 mL/min. The HPLC method has a retention factor (k) between 2 and 10, a selectivity coefficient (α) between 1.13 and 1.65, and a lower detection limit of 20 ng/mL at a signal to noise ratio of 5:1. Keywords: Fumonisins; HPLC; mass spectrometry; metabolites; hydrolysis

Identification of nonvolatile components in lemon peel by high-performance liquid chromatography with confirmation by mass spectrometry and diode-array detection

High-performance liquid chromatography (HPLC) with diode-array detection and thermospray mass spectrometry (LC-TSP-MS) were used to identify nonvolatile compounds in a lemon peel extract. In this way, the retention time characteristics, the UV-Vis spectra and the mass spectra provide structural information without the necessity of isolating the individual compounds. The lemon peel extract was separated into petroleum ether, chloroform, ethyl acetate and butanol fractions. The chloroform fraction was found to contain mainly the limonoids; the petroleum ether fraction contained coumarins, phenyl propanoid glycosides and flavones-C-glucosides; and the ethyl acetate fraction which had the best antioxidant activity, was found to contain flavonols, flavones-O-glycosides and flavonones.

Characterization ofC-glycosylflavones fromDissotis rotundifolia by liquid chromatography — UV diode array detection — tandem mass spectrometry

By high-performance liquid chromatography (HPLC) coupled with UV diode-array detection (DAD) and thermospray mass spectrometry (TSP-MS), four main constituents of a polar, whole plant extract fromDissotis rotundifolia T. were characterized. The fourC-glycosylflavones, isoorientin, orientin, vitexin and isovitexin were detected in the methanolic and hydroalcoholic extract of the plant as well as in the commercial drug preparation ‘Sirop de Dissotis’. Although the UV data and TSP mass spectra allowed rapid characterisation of all fourC-glycosylflavones, exact attribution of the peaks to their structures could not be achieved as neither the UV spectra nor the TSP mass spectra enabled differentiation of one position isomer from the other. Therefore a successful attempt was made to distinguish the 6-C from the 8-C-glycosylflavones by thermospray tandem mass spectrometry (TSP-MS-MS). The collision induced dissociation (CID) spectra of the particular ion [M+H-120]+ gave fragments which permitted differentiation of position isomers. To confirm the accuracy of on-line results, reference compounds were included in the HPLC study.

Liquid chromatographic-thermospray mass spectrometric analysis of sesquiterpenes of Armillaria (Eumycota: Basidiomycotina) species

Conventional HPLC analysis of the sesquiterpene aryl esters present in the crude extracts of the pathogenic basidiomycete Armillaria is complicated by difficulties in identification solely on the basis of retention time. On-line coupling of liquid chromatography with thermospray mass spectrometry (LC-TSP-MS) provides a means for their direct detection. Crude extracts of seven Armillaria species were analysed providing a semi-quantitative survey of sesquiterpene aryl ester production within the genus. The distribution of these metabolites and its significance with regard to species differentiation and pathogenicity are discussed. Unknown components were also detected by on-line LC-TSP-MS screening of extracts, facilitating a targeted isolation route.

Reaction of mucochloric acid with adenosine: formation of 8-(N6-adenosinyl)ethenoadenosine derivatives.

The bacterial mutagen mucochloric acid was reacted with adenosine in aqueous solutions at 37 degrees C. In the HPLC chromatograms of the reaction mixtures two peaks of unidentified products were observed at longer retention times than the previously characterized "etheno" and "ethenocarbaldehyde" adducts. Following isolation and purification with chromatographic methods, the products were characterized by UV absorbance, 1H and 13C NMR spectroscopy, and thermospray mass spectrometry. The products were found to consist of ethenoadenosine derivatives which bonded an additional adenosine unit to C-8 in the etheno bridge. In one of the products a formyl group and in the other an oxalo group was localized at C-7 in the etheno bridge. The yield of the products was about 0.04 mol% (calculated from the original amount of adenosine) in the reaction mixture held for 4 days at pH 7.4. It was concluded that mucochloric acid acts as an oxidative agent during the course of formation of the products.

Determination of Degradation Products Occurring in Acidic Solutions of a 21-Aminosteroid (Tirilazad) Using a Gradient Hplc Method

Abstract A high performance liquid chromatographic (HPLC) method quantitating degradation products of a 21-aminosteroid (tirilazad mesylate) in acidic solutions is described. Mobile phase elution consists of a 58 minute gradient from 5 to 95% mobile phase A, where mobile phases A and B are (81:9:10:0 v/v) and (5.4:0.6:10:84 v/v) acetonitrile, tetrahydrofuran, 0.2 M ammonium formate/ formic acid buffer (pH 3.5) and water. A base deactivated octadecyl silane column thermostatted at 42°C, and fixed wavelength detection at 254 nm are used for this method. The assay is compatible with thermospray mass spectrometry, offering several advantages for the identification of impurities. Validation of the method, along with the use of mass spectrometry for peak identity and purity is discussed.

ω-Hydroxylation of docosahexaenoic acid or arachidonic acid in human colonic well differentiated adenocarcinoma homogenate

Human colonic well differentiated adenocarcinoma homogenate was incubated with NADPH and docosahexaenoic acid (22:6( n − 3)) or arachidonic acid (20:4( n − 6)) as a substrate. On a selected ion monitoring chromatogram obtained with reversed phase-high-performance liquid chromatography thermospray mass spectrometry, ω-hydroxydocosahexaenoic acid (ω-HDHE) or ω-hydroxyeicosatetraenoic acid (ω-HETE) from an incubation mixture of the homogenate was detected in significant amount, compared to that from a colonic region remote from the carcinoma. In contrast, epoxydocosapentaenoic acids and the dihydroxy derivatives from 22:6( n − 3) or epoxyeicosatrienoic acids and the dihydroxy derivatives from 20:4( n − 6) were detected in low amounts, compared to that from a colonic region remote from the carcinoma. The results suggest that highly active NADPH-dependent ω-oxidations of polyunsaturated fatty acids occur in colonic adenocarcinoma homogenate.

Pteridines. Part CVI. Isolation and characterization of limipterin (1‐O‐(L‐erythro‐biopterin‐2′‐yl)‐β‐N‐acetylglucosamine) and its 5,6,7,8‐tetrahydro derivative from green sulfur bacterium Chlorobium limicola f. thiosulfatophilum NCIB 8327

AbstractA new pteridine compound was isolated from green sulfur photosynthetic bacteria, Chlorobium limicola f. thiosulfatophilum NCIB 8327. The structure of this pterin derivative was established to be 1‐O‐(L‐erythro‐5,6,7,8‐ tetrahydropterin‐2′‐yl)‐β‐N‐acetylglucosamine (1) from 1H‐NMR and CD spectra as well as from various mass spectrometric techniques and chemical‐cleavage techniques. Upon acid hydrolysis of 1, equimolar amounts of biopterin (2) and N‐acetylglucosamine were produced. The structure of the hydrolysis product 2 was confirmed by comparing its NMR, UV, CD, and MS and its chromatographical behavior with those of an authentic specimen. N‐Acetylglucosamine was identified by an enzymatic hydrolysis experiment as well as by NMR and thin layer chromatography. Electrospray (ES), fast‐atom‐bombardment (FAB), and thermospray (TS) mass spectrometry of 1 yielded an MH+ at m/z 441. Periodate‐oxidation experiments of the intact molecule 1 and of its hydrolysis product 2 are consistent with the proposed structure. Differential I2 oxidation experiments with the native compound showed that the in vivo oxidation state of this pterin is its tetrahydro form. We propose the trivial name ‘limipterin’ for this new compound.

Evaporative light scattering and thermospray mass spectrometry: Two alternative methods for detection and quantitative liquid chromatographic determination of ginkgolides and bilobalide in Ginkgo biloba leaf extracts and phytopharmaceuticals

AbstractEvaporative light scattering and thermospray mass spectrometry have been investigated as two alternative methods for the liquid chromatographic detection of ginkgolides and bilobalide. Both techniques were used to quantify these metabolites in leaf extracts and in some phytopharmaceuticals. Only a minimum of sample pre‐purification was required. Results were compared with those obtained by gas chromatography (flame ionization detection), which is one of the most suitable methods for routine determinations of such compounds. The isolation of ginkgolides A, B, C, and J and bilobalide from leaves of Ginkgo biloba is also described.

Determination of Sulfadimethoxine and Sulfamethazine Residues in Animal Tissues by Liquid Chromatography and Thermospray Mass Spectrometry

A simple, sensitive, and rapid method for simultaneous determination of sulfamethazine and sulfadimethoxine residues in animal tissues by liquid chromatography (LC) with on-line confirmation by thermospray mass spectrometry is reported. Tissue extracts, after cleanup on C18 cartridges, were injected into a reversed-phase C18 column, and the sulfa drugs were determined by ultraviolet (UV) detection at 265 nm. On-line confirmation of sulfamethazine and sulfadimethoxine in the extracts by mass spectrometry was obtained by feeding the sulfa drugs eluting from the chromatographic column after UV detection directly into the ion source of a mass spectrometer with a thermospray ionization LC interface. Analytical results obtained with the LC method, which has detection limits of 2 and 10 ng/g of tissue, for sulfamethazine and sulfadimethoxine, respectively, compared favorably with those obtained with the official thin-layer chromatographic-densitometric method.

Development of the On-Line High-Performance Liquid-Chromatography Thermospray Mass Spectrometry Method for the Analysis of Phospholipid Molecular Species in Rat Brain

An on-line thermospray liquid chromatography/mass spectrometry method was developed to rapidly analyze phosphoglyceride molecular species in biological fluid. After total lipid extraction, the extract was subjected to the analysis using on-line reversed-phase high-performance liquid chromatography (HPLC) and filament-on thermospray mass spectrometry. Using nonconventional HPLC conditions, partial separation of individual phospholipid class (phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine, and phosphatidylcholine) and partial separation of molecular species within each class were achieved. In addition, good peak shape was maintained throughout the analysis. By monitoring characteristic fragment ions (diacylglycerol ions) formed in the filament-on thermospray process and according to the retention time, individual molecular species in each phospholipid class can be identified. Using this method, we observed significant differences in the molecular species composition of each phospholipid class in rat whole-brain lipid. Although nonlinear calibration curves were observed for all the diacylglycerol ions monitored, even in the presence of internal standard, semiquantitative and quantitative results still could be obtained for a mixture of phospholipids.

Membrane with controllable permeability for drugs

Membranes find application in medicine for the continuous delivery of drugs. The control of the delivered amount would be of great advantage. Therefore a composite membrane comprising a conductive polymer as an active separation layer is developed. By applying various potentials to the membrane system, a controlled permeability through the membrane is achieved. Depending on the redox state of the conducting polymer different rates of permeation for water soluble substances are observed. Dopamine, a neurotransmitter is chosen for testing this new composite membrane. The amount of permeated dopamine is detected on-line with a thermospray mass spectrometer. Changes in the rate of permeation up to 40 % are attained.

Trace analysis of impurities in 3′-azido-3′-deoxythymidine by reversed-phase high-performance liquid chromatography and thermospray mass spectrometry

An analytical method has been developed for the detection of trace amounts of impurities in 3′-azido-3′-deoxythymidine referred to herein as AZT (Zidovudine). A sample extract was preconcentrated by normal-phase high-performance liquid chromatography (HPLC) with subsequent on-line reversed-phase HPLC-thermospray mass spectrometry (TSP-MS). During the sample extraction and concentration step, carried out by semipreparative normal-phase chromatography, the preliminary separation of the impurities from the AZT takes place. The organic solvent (dichloroethane-acetonitrile, 40:60) is evaporated from the collected fractions and the compounds are redissolved in a smaller volume of the reversed-phase mobile phases for a further degree of concentration. The collected fractions are then subjected to reversed-phase HPLC-TSP-MS. The influence of acetonitrile concentration and pH on the reversed-phase separation together with the sensitivity of the TSP-MS detection have been examined to maximise detection levels. The 3′-azido-3′-deoxy-5′-O-tritylthymidine, triphenyl methanol and 3′-chloro-3′-deoxythymidine, which are route-indicative impurities formed during the synthesis can be detected in the 50–100 ppb (w/w) range.

Disposition of Ceftiofur Sodium in Swine following Intramuscular Treatment

Ceftiofur displays a broad spectrum of bactericidal activity against both Gram-positive and Gram-negative bacteria including β-lactamase-producing strains. Three to five milligrams of ceftiofur per kilogram of body weight injected intramuscularly (im) on three consecutive days at 24-h intervals is recommended for bacteria-associated respiratory disease in swine. Metabolism studies in 12 swine given 5 mg of [ 14 C]ceftiofur/kg of body weight im for 3 consecutive days showed that 61.8±4.7% of the dose was excreted in urine and 10.8±5.1% of the dose was excreted in feces. The highest blood residues of about 15 ppm of [ 14 C]ceftiofur free acid equivalent were observed at 2 h after treatments, which declined to ∼2 ppm at 24 h. Almost all of the radioactivity in the blood was found in the plasma, suggesting that the drug did not penetrate the erythrocytes. Most of the radioactivity was bound to macromolecules in the plasma and tissues. Desfuroylceftiofur glutathione disulfide was found only in the liver and 3,3'-desfuroylceftiofur disulfide (dimer) only in the urine. Desfuroylceftiofur cysteine disulfide was present in plasma, tissues, and urine. The structures of the metabolites were deduced from HPLC comparison with synthetic standards, proton NMR, and thermospray mass spectrometry. Polar metabolites are present in urine and tissues after 12 h of treatment, indicating β-lactam and cepham ring opening. Details of disposition of ceftiofur in swine are presented and compared with rats from multiple oral dosing

Determination of levamisole in animal tissues using liquid chromatography–thermospray mass spectrometry

A method is described for the determination of the anthelmintic levamisole in liver, kidney and muscle using liquid chromatography–thermospray mass spectrometry. Samples are extracted by homogenizing with ethyl acetate, applied to Bakerbond CN solid-phase extraction columns, washed with chloroform–hexane and eluted with methanol. The extracts are evaporated to dryness under nitrogen and the residues are dissolved in mobile phase and injected into the LC–MS system. The positive M + 1 ion at m/z 205 is monitored. The detection limit for the assay is 5 ng g–1. Mean recoveries were about 93% for liver, 85% for kidney and 79% for muscle. The assay has been used for statutory testing purposes and for measuring levamisole concentrations in liver, kidney and muscle from sheep dosed with a commercial drench containing levamisole.

High-performance liquid chromatography—thermospray mass spectrometry of ω-hydroxy polyunsaturated fatty acids from rat brain homogenate

A method for the analysis of ω-hydroxy polyunsaturated fatty acids (ω-HPUFAs) in rat tissue homogenate, supplemented with NADPH and homo-γ-linolenic acid [20:3( n − 6)], arachidonic acid [20:4( n − 6)], eicosapentaenoic acid [20:5( n − 3)] or docosahexaenoic acid [22:6( n − 3)] as a substrate was developed. By ion analysis of chromatograms obtained with reversed-phase HPLC—thermospray MS, many ω-HPUFAs corresponding to each precursor fatty acid could be characterized by the high intensity of the molecular ion (MH +) and quasimolecular ion (MNH + 4, MNa +), while other common HPUFAs were characterized by the high intensity of the base ion of MH + − H 2O. On a selected-ion monitoring chromatogram of rat brain homogenate, significant amounts of ω-HPUFA from each precursor fatty acid, especially from 22:6( n − 3), were detected compared with the amounts found in rat large intestine homogenate. Based on these results, a highly active NADPH-dependent ω-oxidation system is suggested for rat brain homogenate resulting in extensive oxidation of 22:6( n − 3).

Liquid chromatography and mass spectrometry determination of aromatic amines in hydrolysed urine from workers exposed to thermal degradation products of polyurethane

An LC-MS method is described for the determination of 4,4′-MDA (4,4′-methylene dianiline) in hydrolysed urine as a biomarker for exposure to 4,4′-MDI (4,4′-methylene diphenylisocyanate). Dideuterated 4,4′-MDA (MDDA) was used as the internal standard. Urine was hydrolysed using sulphuric acid. and 4,4′-MDA was then derivatised with pentafluoro propionic anhydride (PFPA). Thermospray (TSP) and plasmaspray (discharge assisted thermospray, PSP) mass spectrometry monitoring both positive and negative ions were investigated. LC combined with negative ion PSP-MS gave a highly selective and sensitive method of analysis. Quantification was performed monitoring the (M-20)− ions of 4,4′-MDA-PFPA and the MDDA-PFPA. The instrumental detection limit was 0.1 pg/μl of 4,4′-MDA-PFPA. A linear calibration curve was obtained with a The overall precision for human urine spiked to a concentration of 0.5 μg/L was 10% (n=5). The detection limit was about 0.2 μg/l of urine. In technical grade MDA, the precursor of technical grade MDI, several isomers and oligomers with 2–5 aromatic rings were found. In pooled urine samples from ten workers exposed to thermal degradation products of a MDI based polyurethane (PUR) the concentration of 4,4′-MDA was 4 μg/litre. Several MDA isomers and oligomers were observed in the samples.

Separation and characterization of the main methylated nucleobases from nuclear, cytoplasmic and poly (A) + RNA by high-performance liquid chromatography and mass spectrometry

We were able to detect nine methylated nucleobases (3-methyluracil, 1-, 2-, 3- and 7-methylguanine, 1-, 2-, 3- and 6-methyladenine) in RNA from rat and calf liver, baker's yeast, Torula and Euglena cells by using reversed-phase high-performance liquid chromatography and thermospray mass spectrometry. Total cellular, nuclear, cytoplasmic and poly (A) + RNA from rat liver showed marked methylation, mainly of 1- and 3-methylguanine, and 3- and 2-methyladenine. These bases were especially abundant in nuclear RNA and, to a lesser extent, in poly (A) + RNA. In contrast, 7-methylguanine and 6-methyladenine were poorly represented in poly (A) + RNA.

Ion formation of N-methyl carbamate pesticides in thermospray mass spectrometry: The effects of additives to the liquid chromatographic eluent and of the vaporizer temperature

The effects of three additives—ammonium acetate, ammonium formate, and nicotinic acid—to the liquid chromatographic (LC) eluent and of the vaporizer temperature on the ion formation of N-methyl carbamate pesticides in thermospray (TSP) mass spectrometry was investigated by using filament- or discharge-assisted ionization. Nineteen carbamates and 12 of their known environmental degradation products were used as model compounds. The additives cause a strong reduction in the abundance of the characteristic fragment ions [M + H − CH 3NCO] + and [M − CH 3NCO] − for some of the carbamates. The addition of nicotinic acid reduces the quasimolecular ion intensity and, in most cases, produces nicotinic acid adduct ions. The addition of ammonium acetate or ammonium formate increases the intensity of the quasimolecular ion and in most cases produces a base peak for the ammonium adduct ion. The combination of a suppression of fragmentation and an enhancement of quasimolecular ion formation produces an overall gain in sensitivity. As to more specific effects, the addition of the ammonium salts reduces the intensity of M −• with the chlorinated carbamate barban and suppresses the formation of “odd” adduct ions in the TSP mass spectra of most other carbamates. Monitoring the intensity of the fragment and the quasimolecular ion signal as a function of the probe stem temperature, and the related probe tip temperature, proved to be an easy method to study the thermal degradation of the carbamates. This monitoring procedure showed that methiocarb and its sulfone already suffer from thermal degradation at a stem temperature of 90 °C and that these compounds will therefore present problems in quantitation with LC/TSP mass spectrometry.